Download Immunogenicity of B16 melanoma cells after

Immunogenicity of B16 melanoma cells after
radiochemoimmunotherapy and cell death modulation
by zVAD-fmk
Immunogenität von B16 Melanomzellen nach
Radiochemotherapie und Zelltodesmodulation durch
zVAD-fmk
Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr. rer. nat.
vorgelegt von
Nina Werthmöller
aus Ibbenbüren
Als Dissertation genehmigt von der Naturwissenschaftlichen
Fakultät der Friedrich-Alexander-Universität
Erlangen-Nürnberg
Tag der mündlichen Prüfung: 24.08.2015
Vorsitzende/r des Promotionsorgans: Prof. Dr. Wilms
Gutachter/in: Prof. Dr. Nimmerjahn
Prof. Dr. Fietkau
Contents
Contents ................................................................................................................. iii
Abbreviations ........................................................................................................ viii
List of figures ............................................................................................................x
List of tables ........................................................................................................... xv
Abstract ................................................................................................................... 1
Zusammenfassung ................................................................................................... 3
1
Introduction ..................................................................................................... 6
1.1
Metastatic Melanoma ......................................................................................... 6
1.2
Anti-tumor immune response ............................................................................. 7
1.2.1
1.3
Immunosurveillance and immunoevasion .............................................................. 7
Cell death ........................................................................................................... 9
1.3.1
Apoptosis ................................................................................................................. 9
1.3.2
Necrosis ................................................................................................................. 10
1.3.3
Necroptosis............................................................................................................ 11
1.3.4
zVAD-fmk ............................................................................................................... 12
1.3.5
Immunogenicity of dying and dead cells ............................................................... 13
1.4
Danger signals .................................................................................................. 13
1.4.1
HMGB1 .................................................................................................................. 13
1.4.2
HSP70 .................................................................................................................... 14
1.4.3
ATP......................................................................................................................... 14
1.5
Radiotherapy .................................................................................................... 15
1.6
Chemotherapy .................................................................................................. 16
iii
2
1.7
Hyperthermia ................................................................................................... 16
1.8
Scope of this thesis ........................................................................................... 18
Material and Methods .................................................................................... 19
2.1
Reagents and laboratory materials .................................................................... 19
2.2
Cell culture ....................................................................................................... 24
2.3
Treatment of B16-F10 cells ................................................................................ 24
2.4
Cell death determination .................................................................................. 25
2.5
Analysis of cell cycle .......................................................................................... 26
2.6
Detection of danger signals ............................................................................... 26
2.7
Isolation of peritoneal macrophages ................................................................. 27
2.8
Generation of bone marrow-derived dendritic cells ........................................... 27
2.9
Analysis of expression of activation markers on dendritic cells and macrophages 28
2.10
Detection of inflammatory cytokines secreted by DCs and macrophages ............ 28
2.11
Phagocytosis assay............................................................................................ 29
2.12
Migration assay ................................................................................................ 29
2.13
Analysis of presentation of the model tumor antigen OVA ................................. 30
2.14
Cultivation of murine NK cells ........................................................................... 30
2.15
Analysis of the maturation status of NK cells ..................................................... 30
2.16
Kill of B16-F10 cells by NK cells .......................................................................... 31
2.17
Detection of secreted INFγ by NK cells after activation with treated melanoma
cells
31
2.18
Mixed lymphocyte reaction with CD4+ or CD8+ T cells ....................................... 32
2.19
Analysis of proliferation of OVA-specific CD8+ T cells ......................................... 32
2.20
Detection of various cytokines secreted by T cells after activation ..................... 33
2.21
Induction of B16-F10 melanomas in C57/BL6, MyD88 KO or RAG KO mice .......... 33
iv
2.22
Treatment of B16-F10 melanomas with ionizing irradiation, hyperthermia,
dacarbazine, zVAD-fmk, Apyrase and NK cell depleting antibody.................................... 34
2.23
Analysis of immune cell infiltration into the tumor by flow cytometry................ 35
2.24
Analysis of immune cell infiltration into draining lymph nodes by flow cytometry
36
2.25
Analysis of OVA-specific T cell activation by using OT1 mice and B16-OVA
melanoma cells ............................................................................................................. 36
3
2.26
Analysis of in vivo proliferation of OVA-specific CD8+ T cells .............................. 37
2.27
Statistical analysis ............................................................................................. 37
Results ........................................................................................................... 38
3.1
In vitro immunogenic potential of melanoma cells after treatment with RT, DTIC
and/or HT in absence or presence of zVAD-fmk ............................................................. 38
3.1.1
Melanoma cell apoptosis after combination of DTIC with HT .............................. 38
3.1.2
Melanoma cell necrosis especially after RT and HT .............................................. 39
3.1.3
Necroptosis is inducible in B16 melanoma cells ................................................... 40
3.1.4
zVAD-fmk induces a timely-restricted G2 cell cycle arrest ................................... 42
3.2
Analysis of the in vitro immunogenic potential of B16 cells ................................ 43
3.2.1
The release of HMGB1 but not that of Hsp70 is enhanced by zVAD-fmk ............. 43
3.2.2
Increased surface expression of MHCII and CD86 on macrophages induced by
supernatants of melanoma cells whose cell death was modulated with zVAD-fmk ......... 44
3.2.3
zVAD-fmk increased the secretion of certain inflammatory cytokines by
peritoneal macrophages .................................................................................................... 48
3.2.4
Increased surface expression of MHCII and CD86 on DCs induced by supernatants
of RT, DTIC and HT treated melanoma cells whose cell death was modulated with zVADfmk
49
v
3.2.5
zVAD-fmk increased the secretion of certain inflammatory cytokines by bone
marrow derived DCs ........................................................................................................... 53
3.2.6
Single and multimodal melanoma treatments in the absence or presence of
zVAD-fmk do not impair phagocytosis of the tumor cells and migration of macrophages
and DCs towards SN of the tumor cells .............................................................................. 55
3.2.7
Presentation of tumor antigen by DCs is not influenced by SN of treated
melanoma cells................................................................................................................... 57
3.2.8
SN of treated melanoma cells do not impair activation status, maturation status,
or kill activity of NK cells..................................................................................................... 58
3.2.9
SN of melanoma cells do not impact on DC-induced T cell proliferation, but on
cytokine secretion when gathered from melanoma cells treated with RT, DTIC and HT in
combination with zVAD-fmk .............................................................................................. 60
3.3
In vivo impact of zVAD-fmk on tumor growth retardation induced by RT or
combination of RT, DTIC and HT .................................................................................... 62
3.3.1
Combination of fractionated RT DTIC and HT reduces tumor growth significantly
and addition of zVAD-fmk further retards it ...................................................................... 62
3.3.2
Combination of fractionated RT, DTIC and HT with zVAD-fmk impacts on immune
cell infiltration into the draining lymph nodes and the presentation of tumor antigen ... 66
3.3.3
Combination of fractionated RT, DTIC and HT with zVAD-fmk do not influence the
proliferation, but affects the expression of immune stimulatory cytokines of T cells ...... 68
3.3.4
Combination of fractionated RT, DTIC and HT with zVAD-fmk retards tumor
growth in a T cell dependent manner ................................................................................ 70
3.3.5
Nucleotides and HMGB1 contribute to anti-tumor effects induced by zVAD-fmk in
combination with fractionated RT, DTIC and HT ................................................................ 72
4
Discussion ...................................................................................................... 74
4.1
B16-F10 cells as mouse model for malignant melanoma .................................... 74
vi
4.2
Immune therapy as a treatment option against melanoma ................................ 74
4.3
Immunogenic cell death .................................................................................... 75
4.4
Necrosis and necroptosis in melanoma cells ...................................................... 76
4.4.1
Impact of multimodal treatments on melanoma cell death forms....................... 76
4.4.2
Induction of melanoma necroptosis by zVAD-fmk................................................ 77
4.5
Effects of zVAD-fmk on the immunogenic potential of B16 melanoma cells in vitro
77
4.5.1
Effects of zVAD-fmk on the cell cycle .................................................................... 77
4.5.2
Effects of zVAD-fmk on the secretion of DAMPs .................................................. 78
4.5.3
Effects of zVAD-fmk on macrophages and DCs ..................................................... 78
4.5.4
Effects of zVAD-fmk on T cells ............................................................................... 80
4.5.5
Effects of zVAD-fmk on NK cells ............................................................................ 81
4.6
zVAD-fmk induced B16-F10 tumor growth retardation in vivo ............................ 81
4.6.1
Immune stimulatory effects of zVAD-fmk in vivo.................................................. 82
4.6.2
Infiltration of immune cells into the tumor .......................................................... 82
4.6.3
Tumor growth retardation in immune deficient mice .......................................... 83
4.7
Conclusion ........................................................................................................ 84
4.8
Outlook ............................................................................................................ 86
References ............................................................................................................. 87
Publications ........................................................................................................... 96
vii
Abbreviations
APCs: Antigen presenting cells
ASC: Apoptosis associated speck-like protein
ATP: Adenosin-5´-triphosphate
BRAF: v-raf murine sarcoma viral oncogene homolog B1
Caspase: Cysteine aspartyl-specific proteases
CD: cluster of Differentiation
CFSE: carboxyfluorescein succinimidyl ester
CT: Chemotherapy
CTL: cytotoxic T lymphocyte
CTLA4: cytotoxic T-lymphocyte-associated Protein 4
CYLD: Cylindromatosis
DAMP: Damage associated molecular pattern
DCs: Dendritic cells
DMSO: Dimethyl sulfoxide
DNA: Deoxyribonucleic acid
DTIC: Dacarbazine
FADD: Fas associated protein with death domain
FBS: fetal bovine serum
FDA: Food and Drug Administration
FITC: Fluorescein isothiocyanate
Gy: Gray
HLA: Human Leukocyte Antigen
HMGB1: High mobility group protein B1
HSPs: Heat shock proteins
HT: Hyperthermia
IDO: Indoleamine 1,2-dioxygenase
IL: Interleukin
i.p.: Intraperitoneal
i.v.: Intravenous
INF: Interferon
LPS: Lipopolysaccarid
MAPK: BRAF mitogen-activated protein kinase pathway
viii
MDSCs: myeloid derived suppressor cells
MFI: mean fluorescence intensity values
MHC: Major Histocompatibility Complex
Nec-1: Necrostatin-1
NK cells: Natural killer cells
NKT cells: Natural killer T cells
NLRP3: NOD-like receptor family, pryrin domain containing-3 protein
P2RX7: Purinergic receptor P2x
PAMP: Pathogen associated molecular pattern
PCR: Polymerase Chain Reaction
PGE2: Prostaglandin E2
PRR: Pattern recognition receptor
PS: Phosphotidylserin
RAGE: receptor for advanced glycation end product
RHIM: RIP homotypic interaction motif
RIP: Receptor interacting protein
RT: Radiotherapy
SN: Supernatant
TAA: tumor associated antigens
TAM: Tumor associated macrophages
TGF: Transforming growth factor
TH: T helper cells
TLR: Toll like receptor
TNF: Tumor necrosis factor
TNFR: Tumor necrosis factor receptor
TRADD: TNF receptor associated death domain
TRAF: TNF receptor associated factor
TRAIL: Tumor necrosis factor related apoptosis inducing ligand
Tregs: Regulatory T cells
X-ray : ionizing irradiation
zVAD-fmk: Z-Val-Ala-Asp-fluoromethylketone
ix
List of figures
Figure 1: Radio(chemo)therapies are capable of inducing various cell death
forms that differ in their immunogenicity. .......................................................... 10
Figure 2: Necroptosis as an alternative form of programmed cell death. ......... 12
Figure 3: Dot plot of an Anx5-FITC/PI staining of B16 melanoma cells 24h after
treatment with 2Gy. .......................................................................................... 25
Figure 4: Histogram of the cell cycle phase of B16 melanoma cells 24h after
treatment with 2Gy. .......................................................................................... 26
Figure 5: Dot plot of a CFSE/ F4/80 staining of Macrophages and B16
melanoma cells after phagocytosis assay. ....................................................... 29
Figure 6: Histogram of a CFSE staining of CD4+ T cells after incubation with
LPS activated DCs for 5 days .......................................................................... 32
Figure 7: Pictures of local irradiation with the linear accelerator PRIMART of
C57/BL6 mice with B16-F10 tumors................................................................. 34
Figure 8: A schematic diagram and pictures of the local hyperthermia of a local
hyperthermia treatment of a C57/BL6 mouse with a B16-F10 tumor. .............. 35
Figure 9: Apoptosis of B16 melanoma cells after single and combined treatment
with RT, DTIT and/or HT. ................................................................................. 38
Figure 10: Necrosis of B16 melanoma cells after single and combined treatment
with RT, DTIC and/or HT. ................................................................................. 39
Figure 11: Necrosis of B16 melanoma cells after single and combined treatment
with RT, DTIC and/or HT in the absence or presence of nec-1 or zVAD-fmk. . 40
Figure 12: Impact of necrostatin-1 on zVAD-fmk-induced B16 necrosis. ......... 41
Figure 13: G2 cell cycle arrest of B16 cells after single and combined treatment
with RT, DTIC and/or HT in the absence or presence of zVAD-fmk................. 42
x
Figure 14: Release of the dangers signals HMGB1 and HSP70 of melanoma
cells after single or multimodal treatments with RT, DTIC and/or HT in the
absence and presence of zVAD-fmk. ............................................................... 43
Figure 15: Representative histograms of the expression of MHCII or CD86 on
macrophages after contact with supernatants of treated melanoma cells in the
absence or presence of zVAD-fmk................................................................... 44
Figure 16: Surface expression of activation markers on macrophages after
contact with supernatants of melanoma cells in the absence or presence of
zVAD-fmk. ........................................................................................................ 45
Figure 17: Representative histograms of the expression of MHCII or CD86 on
macrophages after contact with supernatants of treated melanoma cells in the
absence or presence of zVAD-fmk................................................................... 46
Figure 18: Surface expression of activation markers of macrophages after
contact with supernatants of melanoma cells in the absence or presence of
zVAD-fmk. ........................................................................................................ 47
Figure 19: Secretion of inflammatory cytokines by macrophages after contact
with supernatants of melanoma cells in the absence or presence of zVAD-fmk.
......................................................................................................................... 48
Figure 20: Representative histograms of the expression of MHCII or CD86 on
DCs after contact with supernatants of treated melanoma cells in the absence
or presence of zVAD-fmk. ................................................................................ 49
Figure 21: Surface expression of activation markers of dendritic cells after
contact with supernatants of melanoma cells in the absence or presence of
zVAD-fmk. ........................................................................................................ 50
xi
Figure 22: Representative histograms of the expression of MHCII or CD86 on
DCs of MyD88KO mice after contact with supernatants of treated melanoma
cells in the absence or presence of zVAD-fmk. ................................................ 50
Figure 23: Representative histograms of the expression of MHCII or CD86 on
DCs of C57/BL6 mice after contact with supernatants of treated melanoma cells
in the absence or presence of zVAD-fmk. ........................................................ 51
Figure 24: Surface expression of activation markers of dendritic cells after
contact with supernatants of melanoma cells in the absence or presence of
zVAD-fmk. ........................................................................................................ 52
Figure 25: Impact of anti-HMGB1 antibody on the surface expression of
activation markers on DCs after contact with supernatants of treated melanoma
cells in the presence of zVAD-fmk. .................................................................. 53
Figure 26: Secretion of inflammatory cytokines by dendritic cells after contact
with supernatants of melanoma cells in the absence or presence of zVAD-fmk.
......................................................................................................................... 54
Figure 27: Impact of nucleotides and Toll-like receptor signaling on the
secretion of the inflammatory cytokine TNFα by dendritic cells after contact with
supernatants of melanoma cells....................................................................... 55
Figure 28: Phagocytosis of melanoma cells by macrophages and dendritic cells
and the migration of those towards supernatants of the tumor cells. ............... 56
Figure 29: Presentation of the model tumor antigen OVA by dendritic cells after
contact with B16-OVA melanoma cells. ........................................................... 57
Figure 30: Phenotype of NK cells after contact with SN of treated melanoma
cells in absence or presence of zVAD-fmk. ...................................................... 58
Figure 31: Impact of treatment of melanoma cells on killing activity of NK cells.
......................................................................................................................... 59
xii
Figure 32: Release of INFγ by NK cells after contact with SN of treated
melanoma cells. ............................................................................................... 59
Figure 33: Proliferation of T cells after co-incubation with allogeneic or
syngeneic DCs pre-incubated with SN of treated melanoma cells. .................. 61
Figure 34: Secretion of cytokines by T cells after contact with dendritic cells
after activation by SN of treated melanoma cells. ............................................ 62
Figure 35: Timetable of treatment and in vivo growth of B16-F10 tumors in
C57/BL6 mice after fractionated RT, DTIC and HT in absence or presence of
zVAD-fmk. ........................................................................................................ 63
Figure 36: Infiltration of cells of the innate immune system into B16-F10 tumors
in C57/BL6 mice after fractionated RT, DTIC and HT in absence and presence
of zVAD-fmk. .................................................................................................... 64
Figure 37: Infiltration of cells of the adaptive immune system into B16-F10
tumors in C57/BL6 mice after fractionated RT, DTIC and HT in absence and
presence of zVAD-fmk. .................................................................................... 65
Figure 38: Immune cell infiltration into draining lymph nodes of B16-OVA tumor
bearing OT-1 mice after fractionated RT, DTIC and HT in the absence or
presence of zVAD-fmk. .................................................................................... 67
Figure 39: Immune cell infiltration into draining lymph nodes of B16-OVA tumor
bearing OT-1 mice after fractionated RT, DTIC and HT in the absence or
presence of zVAD-fmk. .................................................................................... 68
Figure 40: In vivo proliferation of specific CD8+ T cells in B16-OVA tumor
bearing OT-1 mice after fractionated RT, DTIC and HT in the absence or
presence of zVAD-fmk. .................................................................................... 69
xiii
Figure 41: Intracellular expression of INF y and IL2 of CD8+ T cell of B16-OVA
tumor bearing OT-1 mice after fractionated RT, DTIC and HT in the absence or
presence of zVAD-fmk and ex vivo restimulation with OVA peptide. ............... 69
Figure 42: In vivo growth of B16-F10 tumors in RAG KO mice after fractionated
RT, DTIC and HT in the absence or presence of zVAD-fmk. ........................... 71
Figure 43: In vivo growth of B16-F10 tumors in C57/BL6 mice after fractionated
RT, DTIC and HT in absence or presence of zVAD-fmk and in presence of
Apyrase. ........................................................................................................... 72
Figure 44: In vivo growth of B16-F10 tumors in MyD88 KO mice after
fractionated RT, DTIC and HT in absence or presence of zVAD-fmk. ............. 73
Figure 45: Combination of RT, DTIC, HT with zVAD-fmk is capable of activating
both innate and adaptive immune cells against the tumor cells. ...................... 84
xiv
List of tables
Table 1: Cell lines..............................................................................................19
Table 2: Laboratory materials............................................................................19
Table 3 : Instruments ........................................................................................20
Table 4: Reagents .............................................................................................21
Table 5: Antibodies ...........................................................................................22
xv
Abstract
Standard tumor therapies like radiotherapy (RT) or chemotherapy (CT) do not
only kill the tumor cells directly to destroy the primary tumor but can also induce
anti-tumor immune responses against the primary tumor and metastasis. The
latter is achievable by rendering the tumor cells immunogenic. Tumor cell death
forms like necrosis or its programmed form necroptosis are immunogenic and
one basis for the induction of anti-tumor immunity. These cell death forms can
be induced by distinct combinations of stress stimuli or by inhibition of apoptosis
with the pan-caspase inhibitor zVAD-fmk. Since melanomas are very resistant
to ionizing radiation applied in radiotherapy (RT) or chemotherapeutics, the
identification of multimodal therapies that induce immunogenic melanoma cell
death is a big challenge.
Since hints exist that additional immune stimulation by hyperthermia (HT)
augments the efficacy of melanoma therapies and that tumors can be sensitized
for RT with zVAD-fmk, we asked whether combinations of RT with the
chemotherapeutic agent dacarbazine (DTIC) and/or HT induce immunogenic
melanoma cell death and how this is influenced by zVAD-fmk. We revealed that
necroptosis is inducible in poorly immunogenic B16-F10 melanoma cells with all
standard therapies. The highest percentage of necrosis was detected after
treatments including RT and HT. zVAD-fmk generally increased melanoma
necrosis independent of the treatment, concomitantly with an increased release
of danger signals like ATP and especially HMGB1. Supernatants of melanoma
cells whose cell death was modulated with zVAD-fmk induced an up-regulation
of the activation markers CD86 and MHCII on macrophages. The same was
seen on dendritic cells (DCs), but here only when zVAD-fmk was added to
multimodal tumor treatments including DTIC. zVAD-fmk also led to an
enhanced release of inflammatory cytokines like TNFα or IL-6 by both,
macrophages and DCs. DCs or macrophages of MyD88 KO mice and mice
treated with apyrase did not increase CD86 and MHCII on their surface. This
suggests that nucleotides like ATP and HMGB1 might induce these effects. The
presence of zVAD-fmk in combination with RT, DTIC and HT further activated
NK cells to produce and secrete INFγ and CD4+ T cells to produce IL-6 and
INFγ.
1
In in vivo experiments we revealed that zVAD-fmk added to a combined
treatment of the tumor with fractionated RT, DTIC and HT retards the tumor
growth significantly and results in significantly reduced infiltration of regulatory T
cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Further it
increased the infiltration of DCs, NK cells and CD8+ T cells. Concomitantly, a
significantly increased NK cell, DCs and CD8+ T cell infiltration into the draining
lymph nodes was induced. Especially CD8+ DCs were found that are
predestinated for cross presentation of tumor antigen. Furthermore an
increased secretion of INF-γ by CD8+ T cells after ex vivo re-stimulation was
observed when taking the cells of tumor bearing mice that had been treated
with fractionated RT, DTIC and HT in combination with zVAD-fmk. Of note is
that zVAD-fmk did not reduce tumor growth in RAG KO mice, MyD88 KO mice
and mice treated with Apyrase, suggesting that HMGB1 and nucleotides like
ATP mediate the anti-tumor immune reaction by zVAD-fmk and that cells of the
adaptive immune systems, especially T cells, are the main effector cells.
We assume that melanoma cell death modulation with zVAD-fmk results in an
enhanced release of the danger signals ATP and HMGB1 by inducing
necroptosis. These danger signals activate especially DCs which then activate
CD8+ T cells that are capable of killing the tumor cells. We conclude that
HMGB1, nucleotides and CD8+ T cells mediate zVAD-fmk induced antimelanoma immune reactions in multimodal therapy settings.
2
Zusammenfassung
Standard Tumor Therapien wie die Radiotherapie (RT) oder Chemotherapie
(CT) können nicht nur die Tumorzellen direkt töten, um vor allem dem
Primärtumor zu zerstören, sondern auch das Immunsystem gegen den Tumor
aktivieren. Auf diese Weise kann Metastasen und Rezidiven vorgebeugt
werden. Dies kann zum Beispiel durch immunogene Zelltodesformen wie
Nekrosen oder Nekroptosen erreicht werden. Der nekrotische Zelltod kann
durch großen Stress oder durch Inhibierung von Apoptosen wie durch den PanCaspase-Inhibitor zVAD-fmk induziert werden. Melanome sind normalerweise
sowohl
sehr
strahlenresistent
als
auch
sehr
resistent
gegenüber
Chemotherapeutika. Aus diesem Grund ist es für die Onkoimmunologie sehr
wichtig multimodale neue Therapien zu finden, die einen immunogenen Zelltod
induzieren und dadurch das Immunsystem gegen den Tumor aktivieren können.
Da es erste Hinweise darauf gibt, dass Hyperthermie (HT) immunstimulierend
wirkt und dadurch die Effizienz der Tumortherapie erhöht werden kann und
anderseits zVAD-fmk Tumore für die Strahlentherapie sensibilisieren kann,
wurde in dieser Arbeit untersucht, ob und wie Kombinationen von RT, CT und
HT immunogene Zelltodesformen induzieren können. Des Weiteren wurde
untersucht wie das Immunsystem durch diese Therapien aktiviert wird und wie
dies mittels zVAD-fmk modifiziert werden kann.
Es konnte gezeigt
werden,
dass Nekroptose in den sehr schwach
immunogenen B16-F10-Zellen durch alle Standard-Therapien induziert werden
kann. Vor allem Kombinationen aus Strahlentherapie und Hyperthermie führten
zu den höchsten Anteilen an Nekroptosen. Der Zelltod konnte mit zVAD-fmk
moduliert werden. Eine zusätzliche Inkubation der Melanomzellen mit diesem
Inhibitor führte zu einer Erhöhung von Nekrosen. Dieser Effekt war unabhängig
von der Behandlung. Die erhöhte Nekrose korrelierte mit der Freisetzung von
Gefahrensignalen wie ATP und vor allem HMGB1. Die Überstände von B16F10-Zellen, die mit zVAD-fmk behandelt wurden, führen zu einer erhöhten
Expression von MHCII und CD86 auf der Oberfläche von Makrophagen und auf
DCs. Bei letzteren allerdings nur, wenn zVAD-fmk mit RT, CT und HT
kombiniert wurde. Der Pan-Caspase-Inhibitor führte auch zu einer erhöhten
Freisetzung verschiedener inflammatorischer Zytokine wie TNFa und IL-6.
3
Diese Zytokine wurden nach der Kombinationsbehandlung sowohl von
Makrophagen als auch von DCs vermehrt sezerniert. Wurden die DCs oder
Makrophagen aus MyD88 KO-Mäusen generiert oder wurden die Überstände
der behandelten Tumorzellen mit Apyrase behandelt, konnte keine erhöhte
Expression von MHCII oder CD86 auf der Oberfläche der Immunzellen mehr
festgestellt werden. Dies deutet darauf hin, dass HMGB1 und ATP diese Effekte
initiieren. Zusätzlich führte es dazu, dass auch keine erhöhte Sekretion der
inflammatorischen
Zytokine
detektierbar
war.
Außerdem
führte
eine
Kombination von zVAD-fmk mit RT, CT und HT zu einer Aktivierung von NKZellen. Der Reifungszustand blieb unverändert, aber die NK Zellen sezernierten
vermehrt INFγ nach dieser Behandlung. Vergleichbar dazu produzierten CD4+
T-Zellen nach einer „mixed lymphocyte reaction“ (MLR) mehr INFγ und IL-6,
wenn die Tumorzellen mit zVAD-fmk, RT, CT und HT behandelt wurden.
In in vivo Experimenten konnte gezeigt werden, dass zVAD-fmk in Kombination
mit RT, CT und HT das Tumorwachstum signifikant verzögern konnte und
außerdem zu einer reduzierten Infiltration von Tregs und MDSCs in den Tumor
führte. Im Gegensatz dazu wurde durch dieses Behandlungsschema die
Infiltration von DCs, NK-Zellen und CD8+ T-Zellen in den Tumor signifikant
erhöht. Zusätzlich konnte auch eine erhöhte Infiltration dieser Zelltypen in den
Lymphknoten beobachtet werden, wobei signifikant mehr der infiltrierenden
DCs
auch
CD8+
und
somit
prädestiniert
für
Kreuzpräsentation
von
Tumorantigenen waren. zVAD-fmk in der Kombinationsbehandlung führte auch
zu einer erhöhten Aktivierung der infiltrierenden CD8+ T-Zellen. Ex vivo
produzierten diese T-Zellen vermehrt INFγ nach Restimulierung. Dahingegen
führte zVAD-fmk in Kombination mit RT, CT und HT in RAG KO Mäusen,
MyD88 KO Mäusen oder in Mäusen, die zusätzlich mit Apyrase behandelt
wurden, nicht mehr zu einem verzögerten Tumorwachstum. Dies lässt darauf
schließen, dass auch in vivo HMGB1 und Nukleotide wie ATP die Mediatoren
für die Anti-Tumor-Immunantwort und T Zellen die Haupt-Effektorzellen sind.
Zusammenfassend deuten die Versuche darauf hin, dass zVAD-fmk in
Kombination mit RT, CT und HT den Zelltod der Melanomzellen moduliert. Die
Tumorzellen sterben vermehrt nekrotisch bzw. nekroptotisch, wodurch es zu
einer erhöhten Freisetzung von HMGB1 und ATP kommt. Die Gefahrensignale
aktivieren NK Zellen, Makrophagen und DCs. DCs wiederum aktivieren T
4
Zellen, vor allem CD8+ T Zellen, die daraufhin die Tumorzellen effektiv
zerstören können. HMGB1, Nukleotide und CD8+ T Zellen sind somit für die,
durch zVAD-fmk induzierten, Immunreaktionen in multimodalen Therapien
verantwortlich.
5
1 Introduction
1.1 Metastatic Melanoma
Metastatic melanoma is the most aggressive form of skin cancer with an overall
survival of 8 to 18 months. The incidence rate of metastatic melanoma has
increased over the past decades [1]. Every year, more than 132.000 patients
were diagnosed with melanoma internationally. The rates have increased by
28% in men and 21% in woman [2, 3]. The highest incidences are within the
white population, especially in Australia and New Zealand. Here, more than 60
cases per 100.000 inhabitants are reported every year [4]. The highest risk
factors for melanoma are the genetic background and environmental factors.
Above all, excessively exposure to sunlight is the main risk factor. People with
green or blue eye color, red or blond hair color and the presence of freckles
have a higher risk to get a melanoma [5]. About 20% of all patients develop a
metastasis and have therefore very poor prognosis [6]. The usual treatment
scheme is the surgical resection. Until 2011 only two drugs were approved for
metastatic melanoma, the chemotherapeutic agent dacarbazine (DTIC) and
high dose IL-2. But both therapies cannot dramatically increase the overall
survival. The response rate of DTIC is only 10-15% and leads to an overall
survival of eight months [2]. In comparison, high dose IL-2 induces T cell
activation and proliferation and has response rates of 15% but only 6-10% of
the patients have complete remissions. But high dose IL-2 induces severe
toxicity [3, 7]. Both the toxicity and the absence of randomized phase III trials
are the reasons that high dose IL-2 is not a standard form of therapy for
metastatic melanoma [1].
In
2011,
the
Food
and
Drug
Administration
(FDA)
approved
the
immunotherapeutic agent ipilimumab, an anti-CTLA4 antibody and also
vemurafenib, a v-raf murine sarcoma viral oncogene homolog B1 (BRAF)
inhibitor, as treatment against melanoma. This form of cancer is a very
heterogenous disease but 40-60% of the tumors have mutations in the gene
coding for BRAF and 90% of these mutations results in the substitution of valine
for glutamine (V600E) [8, 9]. Due to the mutated form of BRAF, mitogenactivated protein kinase pathway (MAPK) is constitutive activated and leads to
6
an increased cellular proliferation and oncogenic activity. The BRAF inhibitor
vemurafenib is selective for BRAF with the V600E mutation. A phase III trial
compared patients with vemurafenib to dacarbazine and demonstrated
improvements in response rates (48% to 5%) and in progression free survival
(5.3 to 1.6 month) [9].
But the therapy is limited by a short duration that
averages only 6 month [1].
The melanoma is considered as “immunogenic” since it has the ability for
spontaneous regressions [10-12]. For that reason the immune therapy is an
innovative and promising approach for treatment of melanoma. Due to the
immunogenicity of melanoma, they are often associated with infiltration of
lymphocytes. The infiltration correlates with areas of histologic regression [11].
CTLA4 is a negative regulator of the immune system. Up-regulation of CTLA4
on the surface of cytotoxic T cells (CTLs) results in termination of proliferation
and activation of these cells. Owing to the fact that CTLs are the key players in
the immune response against the tumor, suppression of CTLA4 can be an
effective treatment against melanomas [13, 14]. Different clinical trials could
show that response rates of the anti-CTLA4 antibody ipilimumab are between 5
and 20%. Studies with higher concentrations of ipilimumab could show better
response rates but higher toxicities have been observed [15-17].
1.2 Anti-tumor immune response
Efficient anti-tumor therapies aim to stop the proliferation of the tumor cells, kill
them and induce a systemic anti-tumor immunity. This leads not only to the
deletion of the primary tumor but also to the prevention of metastasis and
recurrences. For an immune response against the tumor, the immune cells
have to recognize the tumor cells. Activated immune cells, which recognized the
tumor as foreign, can fight the tumor. An immune memory forms which can
protect against metastasis and recurrences. However, tumors can also
efficiently escape immune surveillance when being established and during
development.
1.2.1 Immunosurveillance and immunoevasion
Tumor immune escape consists of three steps. The first step is the elimination
phase and the surveillance by immune cells [18]. It starts with recruitment of
7
innate immune cells such as NK cells, NKT cells, γδ T cells, macrophages and
dendritic cells (DCs) [19]. Especially NK cells kill tumor cells, which results in
the release of tumor specific antigens. The antigens are phagocytosed and
processed by DCs. The DCs migrate to the next lymph node while presenting
the tumor antigen via MHCII or cross present them via MHCI. This leads to
activation of naïve CD4+ and CD8+ T cells [20]. The activated T cells migrate
back to the tumor and attack and kill tumor cells [21]. Immunosurveillance
results in destroying of premalignant cells so that tumors do not develop. But
the anti-tumor immune response might be too weak to prevent cancerogenesis.
The cells enter the equilibrium phase which can last for years. In this phase,
cells and mediators of the adaptive immune system prevent the outgrowth of
tumor cells but cannot destroy them completely. The main actors are CD4+ and
CD8+ T cells and the cytokines INFγ and IL-2. In the escape phase, tumor cells
begin to expand in an uncontrolled manner [19]. The escape may result from
the enrichment of immunosuppressive immune cells, mediators and cytokines in
the tumor microenvironment. Responsible are production of mediators like
galectin-1 or indoleamine 1,2-dioxygenase (IDO) or the immunosuppressive
cytokines IL-10 or TGFβ as well as the recruitment of regulatory immune cells
as regulatory T cells (Tregs) or myeloid derived suppressor cells (MDSCs) [22].
The secreted products of the tumor can also obviate the differentiation,
maturation and migration of DCs and therefore the effector function of these
cells [23]. Tumor cells do not only escape from innate and adaptive immune
system but also change processing and presentation of tumor associated
antigens (TAA) by altering expression of MHCI or HLA molecules [24-26].
Additionally, vesicles with tumor antigens – tumor exosomes – are able to
induce tolerance against the tumor [27]. The type of macrophages in the tumor
has
also
effects
on
the
tumor
microenvironment.
Tumor associated
macrophages (TAM) mainly are M2 macrophages which results in TH2
responses through the production of prostaglandin E2 (PGE2), TGFβ and IL-10.
In contrast, M1 macrophages results in TH1 responses and therefore in antitumor immunity by production of IL-12 (summarized in [18, 28]). Different
mediators induce the type of macrophages. TNFα, LPS or INFγ lead to a shift to
M1. In contrast IL-4, IL-10 or TGFβ favor M2 macrophages [29]. A chronic
inflammatory environment and hypoxic conditions promote tumor development.
8
It decreases the secretion of IL-12 and TNFα from macrophages and increases
IL-10 secretion by these cells [30, 31]. Therefore tumor development can be a
consequence of a chronic inflammation [32, 33]. But also the form of tumor cell
death impacts on induction of anti-tumor immunity.
1.3 Cell death
It has become evident that cancer cells can be rendered visible to the immune
system by standard therapies such as chemotherapy (CT) or radiotherapy (RT),
either alone or in combination with (further) immune stimulation (e.g. with
hyperthermia (HT)). The resulting tumor microenvironment determines which
immune cells get recruited and triggers the activation or suppression of DCs. It
has become more and more evident that besides the induction of tumor cell
apoptosis by RT, CT and/or immune therapy, other tumor cell death forms exist
that bear high immunogenic potential.
1.3.1 Apoptosis
It was thought for long time that apoptosis is the only programmed death
mechanism in animals and therefore absolutely essential for development and
tissue homeostasis. In addition, apoptosis impacts on immune reactions. Cells
express Fas or TNF receptors via that immune cells can induce apoptosis which
is characterized by distinct morphological changes. During the early phase of
apoptosis, cell shrinkage and chromatin condensation (pyknosis) takes place
[34]. Because of cell shrinkage, the cells become smaller which is in
combination with chromatin condensation one of the most characteristic feature
of apoptosis. In later apoptosis, membrane blebbing and destructive
fragmentation of the nucleus (karyorrhexis) occurs. This is followed by
separation of cell fragments into apoptotic bodies which is called “budding”
(summarized in [35]). Mainly apoptotic cell death is non- or even antiinflammatory since apoptotic cells do not release their cellular constituents into
the surrounding tissue. Immune cells, especially macrophages, quickly
phagocytose most apoptotic cells which prevent secondary necrosis. The
engulfing cells do not produce inflammatory cytokines [36, 37]. But apoptosis is
not non- or even anti-inflammatory in general. Under specific conditions,
apoptosis can be immunogenic. Mostly immunogenic apoptosis is characterized
9
by expression of calreticulin, especially on early apoptotic cells [38], [39].
Additionally, apoptotic cell death is necessary for induction of an efficient antitumor immune response since apoptotic cells recruit immune cells to the tumor
[13].
However, mice with defects in key elements of apoptosis have an almost
normal development. One possible and perhaps the only explanation for this
fact is the existence of other programmed death mechanisms which can
balance defects of apoptosis [40]. Those death forms are of main interest in
cancer therapy, since often resistances to apoptosis are observed [41].
1.3.2 Necrosis
Figure 1: Radio(chemo)therapies are capable of inducing various cell death forms that
differ in their immunogenicity.
The tumor cells die after treatment with chemotherapeutics, hyperthermia and ionizing
irradiation. Dependent on the treatment the tumor cells can die apoptotic which results in a nonor even an anti-inflammatory response. In contrast cells can die necrotic which results in
release of DAMPs and therefore in an activation of both, the innate and adaptive immune
system. Modified after [42].
In contrast to apoptosis, necrosis was considered as a passive and uncontrolled
cell death form. It is induced by extremely cellular stress like heat stress or by
toxic agents.
10
However, these stimuli can also induce apoptosis. Mostly the energy level
determines the cell death form. It could be shown that low levels of ATP lead to
necrotic cell death [43, 44]. Necrotic cells are morphological clearly different
from apoptotic ones. The plasma membrane becomes permeable, organelles
can dilate and ribosomes dissociate from the endoplasmic reticulum. In the late
necrosis, the nucleus also disintegrates [45].
1.3.3 Necroptosis
Nowadays, evidences have come up that also a programmed form of necrosis
exist, the so called necroptosis [46]. It is characterized by similar changes as
they occur in classical necrosis like rounding of the cell, cytoplasmic swelling,
lack of DNA fragmentation and plasma membrane rupture. Necroptosis can be
induced by activation of pattern recognition receptors (PRRs) like Toll like
receptors (TLR). The PRRs are expressed on almost all cells of the innate
immune system and recognize pathogen associated molecular patterns
(PAMPs) [47]. PAMPs have been shown to induce necroptosis in some cell
types. For example, Lipopolysaccharide (LPS) can induce necroptosis in
macrophages when caspase 8 activity is inhibited [48]. Necroptosis can be
further activated by TNFα, Fas Ligand, TRAIL, respectively by ligation of the
death receptors (CD95, TNFR1, TNFR2, TRAIL1 or TRAILR2). Of note is that
the same triggers can also induce apoptosis [49]. However, necroptosis is
independent of caspases and mainly occurs when the caspases (especially
caspase 8) are not activated or inhibited [50]. It has to be regarded as a cell
death backup mechanism when apoptosis is inhibited [51]. The best
characterized pathway for the induction of necroptosis is the ligation of TNFR1.
Of note is that addition of TNFα can cause cell survival, apoptosis and
necroptosis (Figure 2). If the so called complex I is formed, cell survival results.
Complex I consists of TNF receptor associated death domain (TRADD),
receptor interacting protein1 (RIP1), TNF receptor associated factor (TRAF2)
and TRAF5. The apoptotic pathway starts after formation of complex IIa. This
complex consists of caspase 8, FADD and RIP1. The deubiquitinilation of RIP1
by CYLD results in the activation of the RIP1 and RIP3. Caspase 8 activates the
classical caspase cascade and can further inactivate RIP1 and RIP3 by
cleaving. If caspase 8 is inhibited or deleted, the apoptotic pathway is disturbed
resulting in activation of the necroptotic pathway [52]. RIP1 and RIP3 are
11
therefore the key factors for necroptosis. They interact via their RIP homotypic
interaction motif (RHIM).
Nec-1
zVAD-fmk
Figure 2: Necroptosis as an alternative form of programmed cell death.
After binding of TNFα to the receptor, complex I is building. This can result in three ways, in
activation of NF-κB, in apoptosis or necroptosis. Thereby necroptosis in inhabitable with
necrostatin-1 (nec-1) and can be induced by blocking apoptosis with zVAD-fmk.
Modified after [40].
RIP1 is a serine-threonine kinase and exists in two forms, the open and the
closed form. The catalytically cleft is blocked in the closed form and the protein
is inactive. Due to the autophosphorylation on Ser161 in the activation loop, the
protein is in the open form and the catalytically cleft is accessible for the
substrate [53]. This can be inhibited by necrostatin-1 (nec-1). Therefore, nec-1
is an effective inhibitor of necroptosis.
1.3.4 zVAD-fmk
Another possibility to induce necroptosis is inhibition of apoptosis by zVAD-fmk.
It has been shown that zVAD-fmk is capable of inducing this programmed form
of cell death in a selected group of cells, e.g. in mouse fibrosarcoma L929 cells
[54]. This pan-caspase inhibitor blocks apoptosis by inhibiting caspase 8 and
therefore may foster necroptosis by triggering the induction of the autocrine
production of TNFα and simultaneously by inhibiting the apoptotic pathway.
12
Moretti et al. showed that inhibition of caspase 8 by zVAD-fmk enhances the
efficacy of RT in solid tumors. It decreases the tumor outgrowth and increase
extracellular HMGB1 in the tumor microenvironment [55].
1.3.5 Immunogenicity of dying and dead cells
Independent of the way of necrosis induction, this cell death form is generally
more immunogenic compared to apoptotic cell death. Specialized phagocytes,
especially macrophages, swiftly take up apoptotic cells. This results in non- or
even anti-inflammatory events. The activated macrophages secret antiinflammatory cytokines like IL-10 or TGFβ after contact with apoptotic cells [56].
In contrast, necrotic cells have immune stimulatory potential. Due to the necrotic
cell death, the plasma membrane of the cells becomes permeable. This leads to
the release of damage associated molecular pattern (DAMPs) [57].
1.4 Danger signals
Most danger signals are present in every cell and are necessary for survival of
the cells. They are required intracellular but act as danger signals when being
extracellular. The presence of DAMPs outside the cell is a sign for danger that
activates the immune system [58].
1.4.1 HMGB1
One of the most prominent danger signals is HMGB1. It is ubiquitously
expressed in the nucleus of mammalian cells and highly conserved between
different species. It act as a non-histone chromatin-associated protein, it binds
to DNA and facilitates the binding of transcription factors [59]. Another function
is it’s the role in recognition of DNA damages in the process of mismatch repair
[60]. Since necrosis leads to plasma membrane rupture, intracellular HMGB1
gets passively released [61] [62], but inflammatory cells even secrete HMGB1
actively [63]. Standard tumor therapies like RT or CT have been shown to
induce the release of HMGB1 [64], which then can act as immune activating
DAMP. Such therapies induce both, apoptosis and necrosis. HMGB1 can bind
several receptors but it mostly binds to the receptor for advanced glycation end
products (RAGE) and to toll like receptors (TLR), especially TLR2 and TLR4
13
[65]. HMGB1 thereby represents a strong activator of DCs [66]. It fosters
antigen cross presentation by DCs and consecutive activation of naïve T cells.
1.4.2 HSP70
Other important DAMPs are heat shock proteins (HSPs), especially HSP70 [67].
Inside the cell, HSPs protect the cells of cellular stress. They act as chaperones
and thereby stabilize proteins or can ubiquitinate damaged proteins which leads
to their degradation in the proteasome. Of note is that outside the cell, HSPs
can efficiently activate the immune system. Many HSPs like HSP70 chaperon
tumor proteins. When HSP70 gets released, it delivers the bound antigens to
APCs. The latter internalize the HSPs and thereby also the antigens by receptor
mediated endocytosis. Finally, the tumor antigens get cross-presented via
MHCI molecules and can stimulate the CD8+ T cell response (cytotoxic T cells;
CTL) in this way [68]. Another immune stimulatory effect of HSPs is the
enhanced secretion of proinflammatory cytokines by APCs like DCs when
binding to PPR. Taken together, extracelluar HSPs act as danger signals
resulting in maturation and activation of APCs. But HSPs do not only act on
APCs they also stimulate and activate NK cells. HSP70 in presence of
proinflammatory cytokines stimulate the cytotoxic activity of NK cells [69].
1.4.3 ATP
Another DAMP is Adenosin-5´-triphosphate (ATP). In contrast to the other
DAMPs that are mostly recognized by PRRs, there is another sensor for ATP,
the inflammasome. The NOD-like receptor family pryrin domain containing-3
protein (NLRP3) inflammasome detects bacterial products or endogenous
damage signals like ATP, uric acid cristals or alum [70]. Usually, the intracellular
concentration of ATP is relatively high (3-10mM) but the extracellular
concentration is pretty low (400-700nM) [71]. Different sorts of stress induce the
release of ATP by the cells. ATP acts on purinergic receptors especially on
P2RX7 [72]. ATP binds the P2RX7 receptor on DCs which leads to the
activation of the NLPR3 inflammasome [73]. ATP stimulation results in the
aggregation of NLRP3 with apoptosis-associated speck-like protein (ASC) and
caspase-1. The mature caspase-1 cleaved pro-IL-1β and IL-1β gets released.
Active IL-1β is important for priming of CD8+ T cells. Therefore, the activation of
the inflammasome establishes a link between the innate and the adaptive
14
immune system and is an important part of the anticancer immunity [74]. The
release of ATP is stress- induced and controlled by different mechanism. There
are several mechanisms known like maxo-anion channel [75], volume sensitive
chloride channel [76] and P2X7 receptor channels dependent ones [77]. Some
studies already have shown that ionizing radiation also influences the ATP
release. Gamma irradiation induced P2X7 receptor dependent ATP release
from B16-F10 melanoma cells [78]. A big challenge is to identify multimodal
tumor therapies that lead to activation of the immune systems by triggering the
release of DAMPs.
1.5 Radiotherapy
Radiotherapy (RT) is defined as the medical use of ionizing radiation and is
used for treatment of acute and chronic inflammatory diseases with low doses
(single dose ≤1Gy) or for local tumor control in cancer patients with high dose
(single dose >1Gy) [79]. RT in general is an important tool in cancer treatment
and applied in over 50% of all tumor therapies. The exposure to ionizing
radiation induces DNA damage. Chemical bounds within the nucleic acid are
disturbed and DNA double strand breaks may result. Many, but not all, double
strand DNA breaks can be fixed by repair systems of the cell. The photons of
the ionizing radiation can directly or indirectly ionize atoms and break covalent
molecular bonds in this way. The indirect effects of ionizing radiation are mainly
induced by free oxygen radicals that induce ionizations and DNA damage [80,
81]. Besides induction of DNA double strand breaks, also modifications of
bases and DNA cross linking occurs. The radiation-induced damage of the cell
is partly dependent of the phase of its cell cycle [82]. Thereby the G2 phase is
the most radio-sensitive cell cycle phase [83].
Unfortunately,
melanomas
are
very
radioresistant
so
that
standard
radiotherapies are not an effective treatment option for melanoma. But in the
last years, the radiation techniques have become better and better.
Consequently, there is the possibility to use higher doses of single radiation
fractions. Hypofractionation is coming more in the focus for innovative RT. It has
been shown that hypofractionated RT is a possibility to treat melanoma [84].
Nonetheless, RT is not a common treatment for melanoma. Only unresectable
15
and metastatic melanomas are treated with a combination of radiotherapy and
immune therapy with ipilimumab. . Radiotherapy can increase the anti-tumor
effects of ipilimumab [85, 86]. Also in a preclinical murine model it was shown
that CTLA4 blockade alone does not affect the tumor growth or survival of the
animals at all. Only the combination with radiotherapy improved the overall
survival significantly. The effect is dependent on CD8+ T cells and NK cells [85].
This gives first hints that radiotherapy can modulate and increase the anti-tumor
immune response in melanoma and that multimodal therapies consisting of
standard tumor therapies in combination with immunotherapy are important
tools in the cancer therapy of this tumor entity.
1.6 Chemotherapy
Chemotherapy is one of the most used cancer therapies and is often combined
with radiotherapy, surgery, and/or hyperthermia. Chemotherapeutic agents are
cytotoxic. They kill cells that divide very rapid, which is a characteristic of cancer
cells. It can work by different mechanism. There are alkylating agents,
antimetabolites, anti-microtubule agents, topoisomerase inhibitors and cytotoxic
antibiotics (summarized in [87]).
Dacarbazine (DTIC) is an alkylating agent and the only chemotherapeutic agent
which is approved by the United States Food and Drug Administration (FDA) for
the treatment of melanoma. The response rate of patients with monotherapy is
very poor. Only 1-2% of patients have complete response rates and less than
2% are alive after 6 years [88]. The half-life of dacarbazine is only 19 minutes
and it has a terminal half-life of 5h [89]. As all alkylating agents, DTIC destroys
cells and especially cancer cells by adding an alkyl group in their DNA. This
prevents the growing of tumor cells. Additionally DTIC cause apoptosis as cell
death form [90].
1.7 Hyperthermia
As additive to RT and CT, hyperthermia (HT) is more and more used for
heatable tumors. Mild HT is defined as heating of the tumor up to 40°C-44°C.
There are various techniques used such as local, interstitial or deep regional HT
16
[2]. It cannot replace other standard treatments but can sensitize tumor cells for
them. Therefore the main aim of HT is to enhance the efficiency of RT and CT
[67]. Tumors are more heat sensitive than healthy tissue which leads to a higher
damage in the tumor cells [91]. There are two main forms of HT, whole body HT
or regional HT. For whole body HT, the temperature of large areas or even the
whole body is increased. Especially the whole body hyperthermia is used to
treat metastatic cancer that has spread. Like fever, whole body hyperthermia
has general immune stimulatory effects. As already seen in the 19 th century by
Busch and later by Coley fever induced by bacteria can be an effective tool in
cancer therapy [92, 93]. Higher temperatures increase the activity of different
immune cells [94]. The local/regional HT is used as an adjuvant for treatment
with
chemotherapeutics
or
radiation.
Local
HT
acts
synergistic
with
chemotherapeutics since it increases the blood flow and thereby delivers the
chemotherapeutics more efficiently into the tumor [95]. HT can also boost the
effectiveness of RT. It inhibits the DNA repair of irradiation-induced double
strand breaks by e.g. denaturation of DNA protein kinases that repair DNA
damage [96]. HT can sensitize the cells for irradiation when it is delivered
before RT or it can aggravate the irradiation induced cellular stress when it is
applied after RT. Another effect of HT relevant for the induction of anti-tumor
immunity is its capability to increase the expression and especially the release
of HSPs. This is correlated with the tumor`s immunogenicity [67]. The
effectiveness of HT in addition to CT or RT has been proven in several clinical
studies. In most therapies, HT is performed one or two times per week. It was
shown to be effective against various cancer entities like breast [97], bladder
[98] but also against malignant melanoma [99]. Another positive effect is that
HT does not induce severe side effects or toxicities [2]. For melanoma it is
known that human melanoma cells are sensitive for heat [100]. A randomized
trial of RT with or without HT could show that the addition of hyperthermia to RT
increased the complete response rate from 35 to 62 percent and two-year local
control rates from 28 to 46 percent in this tumor entity [99].
17
1.8 Scope of this thesis
The activation of the immune system is essential for an efficient cancer therapy,
especially for attacking small tumor masses, recurrent tumors or metastases.
Standard tumor therapies such as RT and CT aim of inducing proliferation stop
of the tumor cells and consecutively tumor cell death, thereby modifying the
tumor cells phenotype. Apoptosis is a non- or even an anti-inflammatory cell
death form, while necrotic cells activate the immune system by the release of
danger signals like HMGB1, HSPs or ATP. Especially for melanoma systemic
anti-tumor
responses
are
clinically
observed
after
combination
of
radiochemotherapy with distinct immune therapies.
Nevertheless, few preclinical data exist about melanoma cell death forms that
are induced by combinations of standard tumor therapies alone or especially in
addition with further immune stimulation by e.g. hyperthermia and how these
cell death forms modulate the immune activation against the tumor. Especially
the knowledge whether immunogenic necrosis or necroptosis is inducible by
distinct combinations of RT, CT and HT and how this might be fostered by
blocking tumor cell apoptosis by pan caspase inhibitors such as zVAD-fmk is
scarce.
Therefore, the first aim of this thesis was to analyze melanoma tumor cell death
forms which were induced by distinct clinically relevant treatment schemes
containing RT, CT and HT and to examine how these cell death forms can be
modulated with zVAD-fmk. Especially melanoma is susceptible for immune
tumor therapies in multimodal settings, as recent clinical data have proven. The
main focus of this work was therefore set on how the therapy-modulated
melanoma cells and their supernatants impact on activation and functionality of
immune cells like macrophages, DCs, T cells and NK cells as well as on the
immune system related mechanisms of therapy-induced tumor growth
retardation in vivo, analyzed with the syngeneic B16/C57/BL6 mouse model.
18
2 Material and Methods
2.1 Reagents and laboratory materials
Table 1: Cell lines
Cell line
Source
Location
B16-F10
ATCC
USA, Manassas
B16-OVA
ATCC
USA, Manassas
Material
Source
Location
BD Falcon tubes Polysterol
BD Falcon
New York, USA
Cell culture plates Microtest
BD Falcon
New York, USA
Cell strainer (70µm)
BD Falcon
New York, USA
Centrifuge Tubes
Greiner Bio-One
Frickenhausen, Germany
Culture flask (25, 75, 175
Greiner Bio-One
Frickenhausen, Germany
BD Falcon
New York, USA
Miltenyi
Bergisch Gladbach,
Table 2: Laboratory materials
(96-well, U-Boden)
cm2)
Disposable pipettes (5ml,
10ml, 50ml)
Gentle MACS C Tubes
Germany
MACS Separation MS
Miltenyi
Bergisch Gladbach,
Columns
Germany
Multiwell plates 96 well
Thermo Scientific
Waltham, USA
Multiwell plates Cellstar 6
Greiner Bio-One
Frickenhausen, Germany
BD Falcon
New York, USA
Reaction tubes safelock
Eppendorf AG
Hamburg, Germany
Syringes (5ml)
BD Falcon
New York, USA
Syringes Soft-Ject (1ml)
Henke Sass Wolf
Tuttlingen, Germany
well
Needles Microlance 3
(0,4x19mm)
19
Table 3: Instruments
Instrument
Source
Location
Anaesthesia Unvet Porta
Groppler
Deggendorf, Germany
Balance CS200
OHaus
Kirchheim, Germany
Balance Mettler P1200N
Mettler Toledo
Giessen, Germany
BSD50
BSD Medical
Munich, Germany
Cell culture incubator RBP
Heraeus Instruments
Hanau Germany
Centrifuge 420R
Hettich
Tuttlingen, Germany
Centrifuge 5804R
Eppendorf
Hamburg, Germany
Centrifuge Megafuge 1.0 RS
Heraeus Instruments
Hanau, Germany
Clean bench Hera safe
Heraeus Instruments
Hanau, Germany
Beckman Coulter, Inc.
Brea, USA
Freezer (-20°C)
Liebherr
Ochsenhausen, Germany
Freezer (-80°C)
Heraeus Instruments
Hanau, Germany
Gallios® Flow Cytometer.
Beckman Coulter, Inc.
Brea, USA
Gentle MACS Dissociator
Miltenyi Biotek
Bergisch Gladbach,
Isotec4
6220
Heraeus Instruments Hanau,
Germany
Flow cytometer Epics XL
MCL
Germany
Hyperthermia device
Home-made, Medical
Erlangen, Germany
Physics
Laminar flow Laminair LFM
Heraeus Instruments
Hanau, Germany
linear accelerator PRIMART
Siemens
Munich, Germany
Microplatewasher
Biosan
Riga, Lithuania
Minishaker MS1
IKA
Staufen, Germany
pH-Meter C761 Calimatic
Knick
Berlin, Germany
Plate reader HTS 7000
Perkin Elmer
Waltham USA
Refrigerator
Liebherr
Ochsenhausen, Germany
Thermomixer 5436
Eppendorf
Hamburg, Germany
Water bath
GFL
Burgwedel, Germany
X-Ray tube Isovolt Titan
GE Inspection
Hürth, Germany
24486
Inteliwasher 3D
Technologies
20
Table 4: Reagents
Reagent
Source
Location
Accutase
Sigma-Aldrich
Munich, Germany
Apyrase
Sigma-Aldrich
Munich, Germany
Brewer`s thioglycollate
Fluka
Munich, Germany
Carboxyl-Fluorescein-
Molecular Probes/life
Carlsbad, USA
Diacetat-Succinimidyl-Ester
technologies
(CFSE)
CD4 Isolations kit
Miltenyi Biotek
Bergisch Gladbach,
Germany
CD8 Isolations kit
Miltenyi Biotek
Bergisch Gladbach,
Germany
Dacarbazine
abcr
Karlsruhe, Germany
DMSO
Carl Roth
Karlsruhe, Germany
Dulbecco´s Phosphate Buffer
Sigma-Aldrich
Munich, Germany
Easycoll seperating solution
Biochrom
Berlin, Germany
FBS
Biochrom
Berlin, Germany
Granulozyten-Monozyten
Miltenyi Biotek
Bergisch Gladbach,
Saline
stimulierender Faktor (GM-
Germany
CSF)
HMGB1 ELISA
IBL International
Hamburg, Germany
HSP70 ELISA
R&D Systems
Minneapolis, USA
IL-1β ELISA
BioLegend
San Diego, USA
IL-2
Sigma-Aldrich
Munich, Germany
IL-6 ELISA
BioLegend
San Diego, USA
INFγ ELISA
R&D Systems
Minneapolis, USA
Isofluran
Abbott
Wiesbaden, Germany
L-Glutamin (200 mM)
GIBCO
Carlsbad, USA
Lipopolysaccharid ultrapure
Sigma Aldrich
Munich, Germany
Nec-1
Sigma-Aldrich
Munich, Germany
NK cells isolation kit
Miltenyi Biotek
Bergisch Gladbach,
Germany
Non essential aminoacids
Biochrom
Berlin, Germany
Penicillin/Streptomycin
GIBCO
Carlsbad, USA
(10000U/mL; 10000 µg/mL)
21
PenStrep
Gibco Life
Karlsruhe, Germany
Technologies
Propidiumiodid
Sigma-Aldrich
Munich, Germany
Ringer’s solution
Braun Melsungen AG
Melsungen, Germany
RPMI 1640
Sigma-Aldrich
Munich, Germany
Greiner
Frickenhausen, Germany
TM
ThinCert
Tissue culture
inserts
Bio-one
TNF ELISA
BioLegend
San Diego, USA
Triton-X100
Merck
Darmstadt, Germany
Trypsin (10x)
GIBCO
Carlsbad, USA
Tumor dissociation kit
Miltenyi Biotec
Bergisch Gladbach, GER
zVAD-fmk
Bachem AG
Bubendorf, Switzerland
β-Mercaptoethanol
Gibco
Carlsbad, USA
Antibody
Source
Location
Anti-HMGB1 antibody
LSBio
Nottingham, UK
B220- PCC5.5
eBioscience
Frankfurt, Germany
Beats INFγ, IL-6
eBioscience
Frankfurt, Germany
CD11b-PE
BD Pharmingen,
New York, USA
CD11c-FITC
BD Pharmingen,
New York, USA
CD11c-PE
BD Pharmingen,
New York, USA
CD25-AF488
eBioscience
Frankfurt, Germany
CD25-Pe-Cy7
eBioscience
Frankfurt, Germany
CD3-e450
BD Pharmingen,
New York, USA
CD45.2-PCC5.5
eBioscience
Frankfurt, Germany
CD4-PerCP-Cy5.5
BD Pharmingen,
New York, USA
CD4-Vioblue
Miltenyi Biotec
Bergisch Gladbach, GER
CD40-APC
eBioscience
Frankfurt, Germany
CD80-PE
BD Pharmingen,
New York, USA
CD86-Alexa Fluor®700
BD Pharmingen,
New York, USA
CD8a-APC
BD Pharmingen,
New York, USA
Table 5: Antibodies
22
CD8a-PE
BD Pharmingen,
TM
New York, USA
F4/80-AF647
Invitrogen
Carlsbad, Kalifornien, USA
F4/80-e450
eBioscience
Frankfurt, Germany
FoxP3-APC
Miltenyi Biotec
Bergisch Gladbach, GER
MHCII-e450
eBioscience
Frankfurt, Germany
NK1.1-APC
eBioscience
Frankfurt, Germany
OVA257-264 (SIINFEKL)
eBioscience
Frankfurt, Germany
23
2.2 Cell culture
Mouse melanoma cells (B16-F10 and B16-OVA) derived from C57/BL6 mice
were cultured in RPMI 1640 medium containing stable Glutamin and
supplemented with 10% heat inactivated (30min at 56°C) fetal bovine serum
(FBS), 100U/ml penicillin and 100µg/ml streptomycin. Both cell lines were
tested negatively for mycoplasma sc. by PCR detection kit and were maintained
under conditions of 37°C and 95% relative humidity in a 5% CO 2 atmosphere.
The cells were used when they reached 90% confluence.
2.3 Treatment of B16-F10 cells
The B16-F10 cells were irradiated with a clinically relevant single dose of 2Gy
with a X-Ray Generator (120 kV, 12.2mA, 0.5min). The only FDA-approved
chemotherapeutic drug for metastatic melanoma and mostly used therapeutic
agent dacarbazine (DTIC) was used in the concentration of 250µM and added
immediately after the irradiation. For hyperthermia (HT), the cells were treated
with an in-house developed chamber which was placed in a cell incubator as
described previously [101]. The temperature was controlled all the time during
the experiments. Its variation was less than 0.2°C. The cells remained at 41.5°C
for 1h. For combined applications, the tumor cells were stored at 37°C for 4h
between RT and HT. A 4h break between irradiation and hyperthermia is the
maximum time allowed in clinical settings. The pan-caspase inhibitor zVAD-fmk
was used in the concentration of 50µM and the RIP1 kinase inhibitor
necrostatin-1 (nec-1) in the concentration of 10µM. Both inhibitors were solved
in dimethyl sulfoxide and were added directly after HT. For degradation of
extracellular ATP and other nucleotides, the cells were incubated with 10U/ml
medium Apyrase right after HT.
24
2.4 Cell death determination
Figure 3: Dot plot of an Anx5FITC/PI staining of B16 melanoma
cells 24h after treatment with 2Gy.
An exemplary dot plot of the staining
is shown and the following cell
populations can be distinguished:
Anx5- PI-: viable; Anx5+ PI-:
apoptotic; Anx5+ PI+: necrotic (PI+
cells are referred to secondary
necrotic cells, while PI++ cells are
primary necrotic ones with still
complete intact DNA).
The FITC labelled Anx5 binds
Ca2+
dependent
to
phosphatidylserine (PS) which
resides in the inner membrane
leaflet within viable cells and is exposed on the surface of apoptotic cells. For
this reason, Anx5 is a marker for apoptosis. However, Anx5 can also bind
necrotic cells since necrotic cells lose their membrane integrity. Therefore a
second marker is needed to discriminate between apoptosis and necrosis. To
determine between this two cell deaths forms, PI was used. It intercalates into
DNA and can pass the membrane of necrotic cells since it is disturbed. With this
staining viable cells (Anx5-, PI-), apoptotic cells (Anx5+, PI-) and necrotic cells
(Anx5+, PI+) can be distinguished. Further necrosis can be divided in primary
and secondary necrosis (Figure 3). Secondary necrosis follows after apoptosis
which results in a slight degradation of DNA. In contrast primary necrotic cells
have a complete intact DNA. For the analysis, 1*105 cells were solved in 400µl
Ringer´s solution containing 0.2µg Anx5-FITC and 0.4µg PI and incubated for
30min at 4°C in the dark. The cells were then analyzed by flow cytometry using
Flow cytometer Epics XL MCL.
25
2.5 Analysis of cell cycle
Figure 4: Histogram of the cell cycle
phase of B16 melanoma cells 24h
after treatment with 2Gy.
An exemplary histogram of a cell cycle
analysis of B16 melanoma cells is
shown. Because of different DNA
contents subG1, G1, S and G2 phases
can be distinguished.
To analyze the phases of cell
cycle, the cells were stained with
PI in the presence of detergent
and then also analyzed by flow
cytometry. As detergent, TritonX100 was used to facilitate the
entering of PI into the cells. PI
intercalates into the DNA and enables conclusions regarding the DNA content
of a cell. The DNA duplicates during S Phase of the cell cycle. Therefore cells in
the S phase have higher DNA contents than cells in the G1 phase. Cells in the
G2 phase have a double content of DNA as shown in (Figure 4) with the
doubled PI signal monitored in the FL3 channel. The staining was performed
according to the method described by Riccardi and Nicoletti et al [102]. Shortly,
a maximum of 5*105 cells were fixed in 70% ethanol for a minimum of 20min at
-20°C. The cells were permeabilized with a solution containing 192 ml 0.2 M
Na2HPO4 and 8 ml 0.1%(v/v) Triton X-100 at pH 7.8 and incubated at room
temperature for 5min. After that the cells were stained with 2µg/ml PI and
200µg/ml RNase for 30min. The cells were analyzed by flow cytometry using
Flow cytometer Epics XL MCL.
2.6 Detection of danger signals
To analyze the presence of extracellular danger signals, supernatants (SN) of
B16-F10 cells were collected 24h after treatment (1ml). SN were frozen at
-80°C and thawed once for analysis of HMGB1 and HSP70. The ELISAs were
performed according to the manufacturer’s instructions.
26
2.7 Isolation of peritoneal macrophages
To isolate peritoneal macrophages of C57/BL6 or MyD88 KO mice, 2.5ml of 4%
(w/v) Brewer`s thioglycollate broth were injected into the peritoneum of older
mice (minimum 20 weeks) as described previously by Schleicher and Bogdan
[103]. 4 days after injection, the mouse was killed and the peritoneum was
washed with 10ml PBS. The macrophages were cultured in RPMI 1640 medium
containing 10% heat inactivated FBS (30min at 56°C), stable Glutamin, 100U/ml
penicillin
and
100μg/ml
streptomycin
(Gibco,
Carlsbad,
USA).
The
macrophages were characterized by F4/80 staining and used for activation
analysis or functional assays.
2.8 Generation of bone marrow-derived dendritic cells
Generation of bone marrow-derived dendritic cells with GM-CSF was performed
as described before [104]. In brief, bone marrow cells were isolated from femur
and tibiae of C57/BL6 or MyD88 KO mice which are between eight and ten
weeks old by flushing the bones with RPMI 1640 medium. The bone marrow
cells were then cultured in DCs medium consisting of RPMI 1640 medium
containing 10% heat inactivated FBS (30min at 56°C), 100U/ml penicillin and
100μg/ml streptomycin, 0.1% β-mercaptoethanol (50mM) and freshly added
200U/ml mouse GM-CSF. 2 x 106 cells were suspended in 10ml DCs medium
and seeded in 100mm petri dishes. At day three, 10ml of DCs medium
containing fresh GM-CSF were added. At day six, half of the SN was collected
and centrifuged (350 g, 5 min, and room temperature). Thereafter, the cell pellet
was re-suspended in 10ml fresh DCs medium and returned to the plate. The
DCs were harvested at day eight and characterized by CD11c staining and
used for activation analysis or functional assays.
27
2.9 Analysis of expression of activation markers on dendritic cells
and macrophages
To analyze the expression of the activation markers MHCII and CD86 on DCs
and macrophages, 5 x 105 isolated cells were co-incubated in 6-Well
suspension cell plates with 1ml SN obtained from mock-treated or treated B16F10 tumor cells for 16h. The SN was collected 24h after the respective
treatment. After solving the cells with accutase they were incubated for 10 min
at 4°C with Fc-blocking reagent to avoid unspecific binding of the staining
antibodies to Fc-receptors. Both DCs and macrophages were then stained for
30min at 4°C with the fluorescence antibodies MHCII-e450, CD11c-FITC and
CD86-Alexa Fluor®700. DCs were additional stained with CD40-APC, CD80PE, CD83-PE and CD11b-PE. Incubation of the cells with medium, medium with
LPS, medium with DTIC, or zVAD-fmk served as controls. Multicolor flow
cytometry was performed with the Gallios® Flow Cytometer.
2.10 Detection of inflammatory cytokines secreted by DCs and
macrophages
Various mouse cytokines secreted by activated macrophages or DCs were
analyzed with specific ELISA kits according to the manufacturer’s instructions.
For this, SN of DCs or macrophages were collected 16h after coincubation with
SN of treated B16-F10 cells. SN was tested for secreted IL-1β, TNFα and IL-6.
28
2.11 Phagocytosis assay
Figure 5: Dot plot of a CFSE/ F4/80
staining of Macrophages and B16
melanoma cells after phagocytosis
assay.
An exemplary dot plot of the staining of
a phagocytosis assay is shown. B16
melanoma cells are positive for CFSE
(FL1) and macrophages for F4/80
(FL9). Macrophages which have
phagocytosed B16 cells are double
positive.
For
the
phagocytosis
assay,
B16-F10 melanoma cells were
labeled with carboxy-fluorescein
succinimidyl ester (CFSE) 24h
before the respective treatments.
For this, the cells were adjusted
to 2 x 106 cells per ml in PBS and 1µg CFSE was added per ml cell suspension.
The cells were incubated for 20min at 37°C, washed with RPMI 1640 and
consecutively sown. The peritoneal macrophages or DCs (10 5) were then coincubated with the CFSE labelled tumor cells for 1h at 37°C at a ratio of 1 to 5.
Afterwards, the macrophages or DCs were solved with accutase and incubated
for 10min at 4°C with Fc-blocking reagent to avoid unspecific binding of the
staining antibodies to Fc-receptors and then stained with F4/80-e450
(macrophages) or MHCII-e450 (DC). After that the cells were analyzed by twocolor flow cytometry. As previously described [105] and again verified by
confocal microscopy, the double positive cells are macrophages or DCs that
have phagocytosed the tumor cells (Figure 5).
2.12 Migration assay
For transwell migration assays the ThinCertTM Tissue culture inserts system with
pore sizes of 3 µm was used. The isolated peritoneal macrophages or bone
marrow-derived DCs were re-suspended in medium and 800µl of the cell
suspension containing 500.000 cells were placed on each of the 6-well inserts.
Supernatants of mock-treated or treated B16-F10 cells were placed in the 6-well
29
plates. After incubation for 16h at 37°C, the migrated cells were collected and
the number was determined by flow cytometry using the Gallios® Flow
Cytometer.
2.13 Analysis of presentation of the model tumor antigen OVA
For analysis of presentation of the tumor model antigen OVA, bone marrow
derived DCs were co-incubated with treated B16-OVA cells for 16h. After
solving the cells with accutase they were incubated for 10min at 4°C with Fcblocking reagent to avoid unspecific binding of the staining antibodies to Fcreceptors. The DCs was then stained for 30min at 4°C with the fluorescence
antibodies CD11c-FITC and OVA257-264 (SIINFEKL)-PE. Multicolor flow
cytometry was performed with the Gallios® Flow Cytometer.
2.14 Cultivation of murine NK cells
NK cells were isolated from spleens of C57/BL6 mice by using the NK cell
isolation kit. The isolation was performed according to the manufacturer`s
instructions. Afterwards, the cells were cultured in 12 well cell culture plates.
5*105 cells per well were seeded in RPMI 1640 containing 10% heat inactivated
FBS (30min at 56°C), 100U/ml penicillin, 100μg/ml streptomycin, 0.1% βmercaptoethanol (50mM) and 10µg/ml IL-2.
2.15 Analysis of the maturation status of NK cells
For analysis of the maturation status, the isolated NK cells were cultured in 12
well culture cell plates. After 24h, the cells were co-incubated with 500µl SN of
treated melanoma cells. Further 24h later, the cells were incubated for 10min at
4°C with Fc-blocking reagent to avoid unspecific binding of the staining
antibodies to Fc-receptors. The NK cells were then stained for 30min at 4°C
with the fluorescence antibodies NK1.1-APC, CD3e-V450, CD11b-FITC and
CD27-PE-Cy7. The detected mean fluorescence intensity values (MFI) after
antibody staining was relativized to mock-treated NK cells.
30
2.16 Kill of B16-F10 cells by NK cells
Isolated NK cells were cultured in 12 well cell culture plates for 24h. B16-F10
cells were treated as described in 2.3. 24h after treatment the B16-F10 cells
were harvested and co-incubated with NK cells for 24h. The cells were then
incubated for 10 min at 4°C with Fc-blocking reagent to avoid unspecific binding
of the staining antibodies to Fc-receptors. To exclude NK cells, they were
stained for 30min at 4°C with the fluorescence antibodies NK1.1-APC and to
analyze the cell death of B16-F10 cells, they were stained with Anx5-FITC/PI.
Multicolor flow cytometry was performed with the Gallios® Flow Cytometer.
2.17 Detection of secreted INFγ by NK cells after activation with
treated melanoma cells
NK cells were isolated of from spleens of C57/BL6 mice and cultured in 12 well
cell culture plates for 24h. B16-F10 cells were treated as described in 2.3. 24h
after treatment the B16-F10 cells were harvested and co-incubated with the
isolated NK cells. SN were collected after 24h. INFγ secreted by activated NK
cells was analyzed with specific ELISA kits, according to the manufacturer`s
instructions.
31
2.18 Mixed lymphocyte reaction with CD4+ or CD8+ T cells
Figure 6: Histogram of a CFSE
staining of CD4+ T cells after
incubation with LPS activated DCs
for 5 days
An exemplary histogram of CD4+ T
cells is shown after MLR assay.
Unactivated and unproliferated T cells
are highly positive for CFSE. With
proliferation, the CFSE staining
becomes lower.
Dendritic cells were generated
of bone marrow of C57/BL6
mice and incubation of the
isolated cells with GM-CSF.
Afterwards, DCs were activated
with SN of treated B16-F10 as
described in 2.8 and 2.9. CD4+ or CD8+ T cells were isolated of spleens of
Balb/c mice to obtain allogeneic T cells. The T cells were isolated using CD4 or
CD8 isolation kits, according to the manufacturer`s instructions. The isolated T
cells were stained with CFSE (1.5µg/ml) for 10min at RT. The activated DCs
(2.5*104) were co-incubated with the CFSE stained T cells (5*105) in 96 well
round bottom plates. After 5 days at 37°C, the cells were solved and then
incubated for 10min at 4°C with Fc-blocking reagent to avoid unspecific binding
of the staining antibodies to Fc-receptors. After that the T cells were stained for
30min at 4°C with the fluorescence antibodies CD3-e450, CD4-PerCP-Cy5.5,
CD8-APC and CD25-Pe-Cy7. Multicolor flow cytometry was performed with the
Gallios® Flow Cytometer.
2.19 Analysis of proliferation of OVA-specific CD8+ T cells
Dendritic cells were generated of bone marrow of C57/BL6 mice with GM-CSF
and then activated with SN of treated B16-OVA cells as described in 2.8. and
2.9.. CD8+ T cells were isolated of spleens of OT1 mice. The CD8 T cells were
isolated using a CD8 T cell isolation kit according to the manufacturer`s
instructions. CD8+ T cells of OT1 mice are specific for OVA257-264 grouped
with MHCI of C57/BL6 mice (SIINFEKL). Isolated T cells were stained with
32
CFSE (1.5µg/ml) for 10min at RT. The activated DCs (2.5*10 4) were coincubated with the CFSE stained T cells (5*105) in 96 well round bottom plates.
After 5 days at 37°C, the cells were solved and then incubated for 10 min at 4°C
with Fc-blocking reagent to avoid unspecific binding of the staining antibodies to
Fc-receptors. After that the T cells were stained for 30min at 4°C with the
fluorescence antibodies CD3-e450, CD4-PerCP-Cy5.5, CD8-APC and CD25Pe-Cy7. Multicolor flow cytometry was performed with the Gallios® Flow
Cytometer.
2.20 Detection of various cytokines secreted by T cells after
activation
Different cytokines were analyzed in SN of isolated CD4+ or CD8+ T cells after
co-incubation with activated allogeneic DCs by using a beat based assay of
eBioscience which was performed according to the manufacturer`s instructions
and measured with multicolor flow cytometry with the Gallios® Flow Cytometer.
2.21 Induction of B16-F10 melanomas in C57/BL6, MyD88 KO or RAG
KO mice
For in vivo analysis of tumor growth after the respective treatments, eight to ten
week old female C57/BL6, MyD88 KO or RAG KO mice were used. 10 6 B16F10 cells, solved in 200µl Ringer`s solution, were injected at day zero into the
right, shaved flank of the mice. The tumor volumes were followed every day.
For this, width and length were measured using a digital caliper and tumor
volume was calculated according to following formula: volume (mm³) = 0.5 ×
width² (mm²) × length (mm) [106].
33
2.22 Treatment of B16-F10 melanomas with ionizing irradiation,
hyperthermia, dacarbazine, zVAD-fmk, Apyrase and NK cell
depleting antibody
Figure 7: Pictures of local irradiation with the linear
accelerator PRIMART of C57/BL6 mice with B16-F10
tumors.
For irradiation the mice were narcotized with isofluran
and positioned in a Plexiglas® box. The irradiation was
performed locally at the tumor with a clinically relevant
single dose of 2Gy.
At day 8, 9 and 10 after tumor induction, the
tumors were locally irradiated with a clinically
relevant single dose of 2Gy. To irradiate the tumor bearing mice, we
manufactured a Plexiglas® box which allows the irradiation of three mice at
once. The mice were anesthetized before placing them into the box. For the
irradiation procedure, the mice were kept under Isoflurane anesthesia. The
tumors were locally irradiated at the indicated days with a clinically relevant
single dose of 2Gy using a linear accelerator. The planning of the irradiation
was conducted using a computer tomography image of the irradiation box and
tumor bearing mice with Philips pinnacle software to obtain an optimal target
volume. To further protect normal tissue, the gantry of the 6 MV linear
accelerator was drifted to 340 degree. Two hours after the irradiation,
dacarbazine (2mg/mouse at day 8 and 10) and zVAD-fmk (2mg/kg at day 8, 9
and 10) were injected i.p.. Apyrase was injected i.v. one hour after irradiation
(25U at day 8). Hyperthermia was performed 4 hours after irradiation at day 8
and 10. For hyperthermia, the tumors were heated locally to 41.5°C for 30min
34
using BSD50 hyperthermia system that was adjusted to the mouse
experiments. Heating of the tumor is performed with a microwave probe,
comparable with clinical settings. This heats the tumor locally but takes care for
normal tissue. With temperature sensors we control the temperature of the
tumor but also of the body temperature of the mice. During the whole treatment
the mice are narcotized with isofloran and are lying on a heat plate to prevent a
cool down.
2.23 Analysis of immune cell infiltration into the tumor by flow
cytometry
Figure 8: A schematic diagram and pictures of the local hyperthermia of a local
hyperthermia treatment of a C57/BL6 mouse with a B16-F10 tumor.
For hyperthermia the mice were narcotized with isofluran and positioned on a heat plate to
control the body temperature. Heating of the tumor is performed with a microwave probe.
During the whole time the temperature of the tumor is controlled. The tumor is heated to 41.5°C
for 30min.
For analysis of immune cell infiltration into the tumor, the tumor was prepared
with a tumor dissociation kit at day 15 according to the manufacturer`s
instructions. After the dissociation, the tumor cells were centrifuged with
35
easycoll separating solution to discard dead cells. The cells were incubated for
10 min at 4°C with Fc-blocking reagent and then stained for 30min at 4°C with
the following fluorescence labeled antibodies: CD4-PCC5.5, CD8-PE, CD3V450, CD11c-PE, NK1.1-APC, B220- PCC5.5, F4/80-AF647 and CD45.2PCC5.5. Determination of Tregs was performed with FoxP3 Staining Buffer Set
and the antibodies CD4-Vioblue, CD25-AF488 and FoxP3-APC. Multicolor flow
cytometry was performed with the Gallios® Flow Cytometer.
2.24 Analysis of immune cell infiltration into draining lymph nodes
by flow cytometry
For analysis of immune cell infiltration into the draining lymph nodes, the axillary
lymph nodes were prepared. The cells were incubated for 10 min at 4°C with
Fc-blocking reagent and then stained for 30min at 4°C with the following
fluorescence labeled antibodies: CD4-PCC5.5, CD8-PE, CD3-V450, CD11c-PE,
NK1.1-APC, CD45.2-PCC5.5 and OVA257-264 (SIINFEKL)-PE. Multicolor flow
cytometry was performed with the Gallios® Flow Cytometer.
2.25 Analysis of OVA-specific T cell activation by using OT1 mice
and B16-OVA melanoma cells
106 B16-OVA cells were solved in 200µl Ringer's solution and injected into the
right shaved flank of the mice at day zero. At day 8, 9 and 10 the irradiation was
performed. The tumors were locally irradiated with a single dose of 2Gy using a
linear accelerator (PRIMART). Two hours after the irradiation, dacarbazine (day
8 and 10) and zVAD-fmk (day 8, 9 and 10) were injected i.p.. Hyperthermia was
performed 4 hours after irradiation at day 8 and 10. The tumors were heated
locally to 41.5°C for 30min. At day 14, draining lymph nodes (axillary) were
removed. T cell activation was measured by analyzing intracellular INFγ and
intracellular IL-2 after restimulation. Therefore 2*106 cells were restimulated
with OVA peptide (10-7M) and Golgi Plug for 5 hours. Extracellular markers
were stained with CD3-V450 and CD8-PE by incubating for 30min at 4°C. After
washing, the cells were permeabilized by addition of Cytofix/Cytoperm and
incubated for 20min at 4°C. For intracellular staining, the antibody INFγ-Pe-Cy7
36
was added and the cells were incubated for another 30min at 4°C. The cells
were washed and analyzed by multicolor flow cytometry with the Gallios® Flow
Cytometer.
2.26 Analysis of in vivo proliferation of OVA-specific CD8+ T cells
106 B16-OVA cells were solved in 200µl Ringer`s solution and injected into the
right, shaved flank of the C57/BL6 mice at day zero. At day 8, 9 and 10 the
irradiation was performed. The tumors were irradiated with a single dose of 2Gy
using a linear accelerator (PRIMART). Two hours after the irradiation,
dacarbazine (day 8 and 10) and zVAD-fmk (day 8, 9 and 10) were injected i.p..
Hyperthermia was performed 4 hours after irradiation at day 8 and 10. The
tumors were heated locally to 41.5°C for 30min. At day 11 OVA specific CD8+ T
cells of OT1 mice were isolated using a CD8 T cell isolation kit. These T cells
were stained with CFSE (1.5µg/ml) for 10min at room temperature. After
washing of the cells they were solved in Ringer’s solution and injected i.v. (10 6
in 200µl) in tumor bearing mice. 5 days after injection, the tumors were
prepared with a tumor dissociation kit. After the dissociation, the tumor cells
were centrifuged with easycoll separating solution to discard death cells. The
cells were incubated for 10 min at 4°C with Fc-blocking reagent and then
stained for 30min at 4°C with the following fluorescence labeled antibodies:
CD4-PCC5.5, CD8-PE, CD3-V450. The cells were analyzed by multicolor flow
cytometry with the Gallios® Flow Cytometer.
2.27 Statistical analysis
For all experiments Graph pad prism (Version 5.04) was applied for statistical
analysis with the Mann-Whitney-U test. Results were considered statistically
significant for P < 0.05 (* or #) and highly significant for P < 0.01 (** or ##).
37
3 Results
3.1 In vitro immunogenic potential of melanoma cells after
treatment with RT, DTIC and/or HT in absence or presence of
zVAD-fmk
3.1.1 Melanoma cell apoptosis after combination of DTIC with HT
The form of tumor cell death is important for activation of the immune system
and therefore for an efficient anti-tumor treatment (summarized in [107]).
Because of this we first analyzed the B16 melanoma death in vitro, induced by
different single and combinatory treatments.
A)
B)
C)
D)
Figure 9: Apoptosis of B16 melanoma cells after single and combined treatment with RT,
DTIC and/or HT.
The tumor cell death forms were analyzed with flow cytometry after staining of the cell
suspension with Anx5-FITC and PI. Anx5-FITC+ and PI- cells were defined as apoptotic ones.
Shown are different time points after the respective treatment(s) (A: 24h, B: 48h, C: 72h, D:
96h). Representative data of one out of four experiments, each performed in triplicates, are
presented as mean ± S.D. * P < 0.05; ** P < 0.01 related to untreated (mock) cells. DTIC:
dacarbazine at a concentration of 250µM; Gy: Gray; HT: hyperthermia with 41.5°C for 1h;
zVAD-fmk: pan-caspase inhibitor. The order of irradiation with 2Gy and HT indicated on the xaxis determines the chronology of the treatment. mock: untreated tumor cells.
38
Single treatment of melanoma cells by RT, DTIC or HT did not induce higher
tumor cell apoptosis, both at early and late time points after treatment (Figure
9). It is already known that melanoma cells are very radio- and chemoresistant
[108]. HT has been shown to be capable to boost the efficacy of CT and RT
treatments, even in melanomas [109, 110]. For that reason, we analyzed
whether combination of RT and/or DTIC with HT can enhance apoptosis
induction in melanoma cells. Combination of HT and the chemotherapeutic
agent DTIC increased apoptosis in melanoma cells. Further addition of RT
resulted in even 15% of apoptotic melanoma cells 96h after the treatment, but in
dependence on the order of RT and HT application. More cells died apoptotic
when HT was performed before RT. To summarize, only combinatory
treatments including HT significantly induced apoptotic cell death in B16
melanoma cells (Figure 9).
3.1.2 Melanoma cell necrosis especially after RT and HT
Figure 10: Necrosis of B16 melanoma cells after single and combined treatment with RT,
DTIC and/or HT.
The tumor cell death forms were analyzed with flow cytometry after staining of the cell
suspension with Anx5-FITC and PI. Anx5-FITC+ and PI+ cells were defined as necrotic ones.
Shown are different time points after the respective treatment (A: 24h, B: 48h, C: 72h, D: 96h).
Representative data of one out of four experiments, each performed in triplicates, are presented
as mean ± S.D. * P < 0.05; ** P < 0.01 related to untreated (mock) control samples. DTIC:
dacarbazine at a concentration of 250µM; Gy: Gray; HT: hyperthermia with 41.5°C for 1h;
zVAD-fmk: pan-caspase inhibitor. The order of irradiation with 2Gy and HT indicated on the xaxis determines the chronology of the treatment. mock: untreated tumor cells. mock: untreated
tumor cells.
39
Single treatment of B16 cells with DTIC did also not induce necrotic cell death in
melanoma cells, even 96h after treatment. However, single treatment with 2Gy
of RT was capable of inducing necrosis. This was seen 24h and 48h after
irradiation. Not only irradiation alone but above all, combinatory treatments
induced necrotic cell death. The highest rates are detectable 48h after
treatment. Here, more than 40% of necrotic B16 cells were present after
treatment with RT, DTIC and HT (Figure 10). Again, the order of RT and HT
application is important. Here, more cells died necrotic when RT was performed
before HT.
3.1.3 Necroptosis is inducible in B16 melanoma cells
A)
B)
Figure 11: Necrosis of B16 melanoma cells after single and combined treatment with RT,
DTIC and/or HT in the absence or presence of nec-1 or zVAD-fmk.
The cell death forms were analyzed with flow cytometry after staining of the cell suspension with
AnxA5-FITC and PI, 72 h (A) or 24 h (B) after the respective treatment. Anx5-FITC+ and PI+
cells were defined as necrotic ones. The cells were further incubated with the necroptosis
inhibitor nec-1 (A) or zVAD-fmk (B). Representative data of one out of four experiments, each
performed in triplicates, are presented as mean ± S.D. * P < 0.05; ** P < 0.01 related samples
without (w/o) inhibitor. DTIC: dacarbazine at a concentration of 250µM; Gy: Gray; HT:
hyperthermia with 41.5°C for 1h; zVAD-fmk: pan-caspase inhibitor. The order of irradiation with
2Gy and HT indicated on the x-axis determines the chronology of the treatment. mock:
untreated tumor cells.
40
We then analyzed whether necroptosis is inducible in B16-F10 melanoma cells.
The treatment with the potent small-molecule inhibitor of necroptosis,
necrostatin-1 (nec-1), reduced the amounts of necrotic tumor cells. This was
independent of the death stimuli (Figure 11A).
Figure 12: Impact of necrostatin-1 on zVAD-fmk-induced B16 necrosis.
Necrosis of B16 mouse melanoma cells was analyzed with two color flow cytometry by staining
the cells with AnxA5-FITC and PI 24h after irradiation with 2Gy in the presence or absence of
zVAD-fmk or zVAD-fmk plus nec-1. Representative data of one out of three experiments, each
performed in duplicates, are presented as mean ± S.D. ** P < 0.01; nec-1: necrostatin-1, Gy:
Gray
Further, the pan caspase inhibitor zVAD-fmk (50 µM) was capable of inducing
necrotic B16-F10 melanoma cell death. The addition of zVAD-fmk increased the
percentage necrotic cells in combination with all treatments (Figure 11B).
Mostly, zVAD-fmk doubles the percentage of necrotic cells. To further prove
that zVAD-fmk induced necroptosis, we additionally added nec-1. The addition
of this necroptosis inhibitor blocked the zVAD-fmk induced B16 tumor cell
necrosis (Figure 12).
41
3.1.4 zVAD-fmk induces a timely-restricted G2 cell cycle arrest
A)
B)
C)
Figure 13: G2 cell cycle arrest of B16 cells after single and combined treatment with RT,
DTIC and/or HT in the absence or presence of zVAD-fmk.
The cell cycle phases were analyzed by PI staining after incubation of the cells with the
detergent Triton-X100. The G2 arrest was determined 24h (A), 48h (B) and 72h (C) after the
indicated treatment(s). Representative data of one out of four experiments, each performed in
triplicates, are presented as mean ± S.D. ** P < 0.01 related to samples without (w/o) inhibitor.
DTIC: dacarbazine at a concentration of 250µM; Gy: Gray; HT: hyperthermia with 41.5°C for 1h;
zVAD-fmk: pan-caspase inhibitor. mock: untreated tumor cells
The cell cycle of melanoma cells was not influenced neither 24h nor 48h or 72h
after the respective treatment(s). The amount of cells in the more radiosensitive
42
G2 phase was always around 15% (Figure 13A-C). However, addition of zVADfmk induced a timely restricted G2 arrest independent of the death stimulus
(Figure 13B).
3.2
Analysis of the in vitro immunogenic potential of B16 cells
3.2.1 The release of HMGB1 but not that of Hsp70 is enhanced by zVADfmk
Figure 14: Release of the dangers signals HMGB1 and HSP70 of melanoma cells after
single or multimodal treatments with RT, DTIC and/or HT in the absence and presence of
zVAD-fmk.
The concentration of HMGB1 (A, B) and of HSP70 (C, D) in the supernatants of B16-F10
mouse melanoma cells was determined by ELISA technique 24h hours after the indicated
treatments. The tumor cells were treated with ionizing irradiation with 2Gy alone or in
combination with the chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia
(HT; 41.5°C for 1 hour), in each case in the absence (w/o) or presence of the pan-caspase
inhibitor zVAD-fmk (50 µM) Representative data of one out of three experiments, each
performed in duplicates, are presented as mean ± S.D. * P < 0.05; ** P < 0.01 related to mock
(A, C) or samples without (w/o) inhibitor (B, D). Gy: Gray; zVAD-fmk: pan-caspase inhibitor.
mock: untreated tumor cells.
To get first hints about the immunogenic potential of the treated melanoma cells
the release of the danger signals HSP70 and HMGB1 was analyzed. HT alone
and in combination with RT or DTIC enhanced the release of both danger
signals HMGB1 and HSP70. RT or DTIC neither alone nor in combination
impacted on the release of one of these danger signals (Figure 14A and 14C).
43
But fostering necrosis with the cell death modulator zVAD-fmk increased the
amount of HMGB1 in the SN of the tumor cells significantly. The increased
release correlated with the induction of necrosis by zVAD-fmk, but was
independent of the treatment with RT, DTIC and/or HT (Figure 14B). In
contrast, the pan caspase inhibitor zVAD-fmk did not increase the release of
HSP70 by B16 melanoma cells (Figure 14D).
3.2.2 Increased surface expression of MHCII and CD86 on macrophages
induced by supernatants of melanoma cells whose cell death was
modulated with zVAD-fmk
A)
B)
2Gy+DTIC+HT
2Gy+DTIC+HT+zVAD
-fmk
Figure 15: Representative histograms of the expression of MHCII or CD86 on
macrophages after contact with supernatants of treated melanoma cells in the absence
or presence of zVAD-fmk.
The expression of the activation markers MHCII (A) and CD86 (B) on the surface of peritoneal
mouse macrophages of C57/BL6 mice was analyzed by multicolor flow cytometry after contact
with supernatants (SN) of B16 mouse melanoma cells obtained 24h after treatment with ionizing
irradiation with 2Gy alone or in combination with the chemotherapeutic agent dacarbazine
(DTIC; 250 µM) and hyperthermia (HT; 41.5°C for 1 hour), in each case in the absence (grey) or
presence of the pan-caspase inhibitor zVAD-fmk (50 µM; black)
44
Because of the inhibition of caspases and consecutive induction of programmed
necrosis, zVAD-fmk has been suggested to be a promising therapeutic strategy
A)
B)
Figure 16: Surface expression of activation markers on macrophages after contact with
supernatants of melanoma cells in the absence or presence of zVAD-fmk.
The expression of the activation markers MHCII (A) and CD86 (B) on the surface of peritoneal
mouse macrophages was analyzed by multicolour flow cytometry after contact with
supernatants of the B16-F10 mouse melanoma cells obtained 24 h after the respective
treatments. The tumor cells were treated with ionizing irradiation with RT (2Gy) alone or in
combination with the chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia
(HT; 41.5°C for 1 hour), in each case in the absence (w/o) or presence of the pan-caspase
inhibitor zVAD-fmk (50 µM) (A, B). Representative data of one out of 3 experiments, each
performed in triplicates, are presented as mean ± S.D. ** P < 0.01 related to samples without
(w/o) inhibitor. MFI: mean fluorescence intensity. Gy: Gray. mock: SN of untreated tumor cells.
to enhance the efficacy of RT in solid tumors [55]. Therefore we next
investigated whether SN of melanoma cells that have been exposed to zVADfmk in addition to RT, DTIC, and/or HT influence the activation and function of
macrophages and dendritic cells (DCs). SN of neither mock treated melanoma
cells nor of treated ones altered the expression of the activation markers MHCII
45
Figure 17: Representative histograms of the expression of MHCII or CD86 on
macrophages of WT mice in the presence of apyrase or of MyD88 KO mice after contact
with supernatants of treated melanoma cells in the absence or presence of zVAD-fmk.
The expression of the activation markers MHCII (A, C) and CD86 (B, D) on the surface of
peritoneal mouse macrophages of C57/BL6 mice (A, B) or MyD88 ko mice (C,D) was analyzed
by multicolor flow cytometry after contact with supernatants (SN) of B16 mouse melanoma cells
obtained 24h after treatment with ionizing irradiation with 2Gy in combination with the
chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia (HT; 41.5°C for 1 hour),
in the absence (grey) or presence of the pan-caspase inhibitor zVAD-fmk (50 µM; black). SN of
treated melanoma cells were additionally incubated with Apyrase (10U/ml) (A, B).
and CD86 on macrophages (Figure 16). However, the modulation of melanoma
cell death by zVAD-fmk resulted in tumor cell SN that induced a significant
increased expression of these activation markers on macrophages (Figure 15
and 16). This was seen in every case when zVAD-fmk was present, even in
untreated cells (Figure 16B). To prove that this is not a direct effect of zVADfmk on the immune cells, zVAD-fmk was directly added to macrophages or
DCs. It thereby did not impact on the expression of the activation markers
(Figure 16 and 21). Because the upregulation of MHCII and CD86 is not
directly induced by zVAD-fmk, other molecules which were released by the
46
tumor cells are responsible for the upregulation of these activation markers on
macrophages and DCs.
Figure 18: Surface expression of activation markers of macrophages after contact with
supernatants of melanoma cells in the absence or presence of zVAD-fmk.
The expression of the activation markers MHCII (A, C) and CD86 (B, D) on the surface of
peritoneal macrophages of C57/BL6 mice (A, B) or MyD88 KO mice (C, D) was analyzed by
multicolor flow cytometry, after contact with the supernatants (SN) of the B16-F10 mouse
melanoma cells obtained 24 h after the respective treatments. The tumor cells were treated with
ionizing irradiation with 2Gy alone or in combination with the chemotherapeutic agent
dacarbazine (DTIC; 250 µM) and hyperthermia (HT; 41.5°C for 1 hour), in each case in the
absence or presence of the pan-caspase inhibitor zVAD-fmk (50 µM) SN of melanoma cells
were additionally incubated with Apyrase (10U/ml)(A, B). Data of three experiments, each
performed in triplicates, are presented as mean ± S.D. ** P < 0.01 related to samples without
(w/o) inhibitor. MFI: mean fluorescence intensity. DTIC: dacarbazine; Gy: Gray; HT:
hyperthermia; zVAD-fmk: pan-caspase inhibitor. mock: SN of untreated tumor cells.
To analyze two possible effector molecules which may induce the upregulation
of MHCII or CD86 on peritoneal macrophages, Apyrase was added to the SN or
macrophages of MyD88 KO mice were used for the analyses. Apyrase is a
calcium-activated plasma membrane-bound enzyme that degrades nucleotides.
It catalyzes the hydrolysis of ATP to AMP and inorganic phosphate. MyD88 KO
mice have defects in TLR signaling. The increased expression of MHCII and
CD86 was is dependent on nucleotides and ligands such as HMGB1 for TLR
47
signaling, since no upregulation was observed when using macrophages of
MyD88 KO mice or incubating the SN of the treated tumor cells with Apyrase
(Figure 17 and Figure 18).
3.2.3 zVAD-fmk increased the secretion of certain inflammatory cytokines
by peritoneal macrophages
Figure 19: Secretion of inflammatory cytokines by macrophages after contact with
supernatants of melanoma cells in the absence or presence of zVAD-fmk.
Supernatants of the B16-F10 mouse melanoma cells were obtained 24 h after the respective
treatment(s). The tumor cells were treated with ionizing irradiation with 2Gy alone or in
combination with the chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia
(HT; 41.5°C for 1 hour), in each case in the absence or presence of the pan-caspase inhibitor
zVAD-fmk (50 µM) The peritoneal macrophages were coincubated with the obtained SN for
16h. Supernatants of these macrophages were then analyzed for TNFα (A), IL-6 (B) or IL-1β (C)
by ELISA. The fold increase of the cytokine release of macrophages after incubation with SN of
treated B16 melanoma cells related to macrophages after incubation with media is displayed.
Data of three experiments, each performed in triplicates, are presented as mean ± S.D. **
P < 0.01 related to samples without (w/o) inhibitor. MFI: mean fluorescence intensity. Gy: Gray;
mock: SN of untreated tumor cells.
Activated macrophages increase the expression of activation markers on their
surface but also produce and secrete inflammatory cytokines. Incubation of
macrophages with SN of irradiated tumor cells whose cell death was modulated
with zVAD-fmk resulted in a significant increased secretion of TNFα, IL-6 and
IL-1 β (Figure 19). Neither the single treatment with RT nor the combinatory
48
treatment containing RT, DTIC and HT increased the release of one of these
inflammatory cytokines significantly.
3.2.4 Increased surface expression of MHCII and CD86 on DCs induced
by supernatants of RT, DTIC and HT treated melanoma cells whose
cell death was modulated with zVAD-fmk
Figure 20: Representative histograms of the expression of MHCII or CD86 on DCs after
contact with supernatants of treated melanoma cells in the absence or presence of
zVAD-fmk.
The expression of the activation markers MHCII (A) and CD86 (B) on the surface of bone
marrow derived dendritic cells of C57/BL6 mice was analyzed by multicolor flow cytometry after
contact with supernatants (SN) of B16 mouse melanoma cells obtained 24h after treatment with
ionizing irradiation with 2Gy in combination with ionizing irradiation with 2Gy in combination with
the chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia (HT; 41.5°C for 1
hour), in the absence (grey) or presence of the pan-caspase inhibitor zVAD-fmk (50 µM; black).
In contrast to macrophages, the expression of the activation markers MHCII and
CD86 on the surface of bone marrow derived DCs was not generally increased
by incubation with SN of tumor cells whose cell death had been modulated with
zVAD-fmk. Only the cell death induction with RT in combination with DTIC and
HT together with zVAD-fmk generated SN that consecutively increased the
expression of both activation markers on DCs significantly (Figures 20 and 21).
As already observed for macrophages, cell death induction with only RT or the
combination of RT, DTIC and HT did not increase the expression of these
activation markers.
49
Figure 21: Surface expression of activation markers of dendritic cells after contact with
supernatants of melanoma cells in the absence or presence of zVAD-fmk.
The expression of the activation markers MHCII (A) and CD86 (B) on the surface of bone
marrow derived DCs was analyzed by multicolour flow cytometry, after contact with the
supernatants of the B16-F10 mouse melanoma cells obtained 24 h after the respective
treatment(s). The tumor cells were treated with ionizing irradiation with 2Gy alone or in
combination with the chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia
(HT; 41.5°C for 1 hour), in each case in the absence or presence of the pan-caspase inhibitor
zVAD-fmk (50 µM) Representative data of one out of 3 experiments, each performed in
triplicates, are presented as mean ± S.D. ** P < 0.01 related to samples without (w/o) inhibitor.
MFI: mean fluorescence intensity. Gy: Gray; mock: SN of untreated tumor cells.
Figure 22: Representative histograms of the expression of MHCII or CD86 on DCs of
MyD88KO mice after contact with supernatants of treated melanoma cells in the absence
or presence of zVAD-fmk.
The expression of the activation markers MHCII (A) and CD86 (B) on the surface of bone
marrow derived DCs of MyD88 ko mice was analyzed by multicolor flow cytometry after contact
with supernatants (SN) of B16 mouse melanoma cells obtained 24h after treatment with ionizing
irradiation with 2Gy in combination with the chemotherapeutic agent dacarbazine (DTIC;
250 µM) and hyperthermia (HT; 41.5°C for 1 hour) in the absence (grey) or presence (black) of
the pan-caspase inhibitor zVAD-fmk (50 µM).
50
Figure 23: Representative histograms of the expression of MHCII or CD86 on DCs of
C57/BL6 mice after contact with Apyrase-treated supernatants of treated melanoma cells
in the absence or presence of zVAD-fmk.
The expression of the activation markers MHCII (A) and CD86 (B) on the surface of bone
marrow derived DCs of C57/BL6 mice was analyzed by multicolor flow cytometry after contact
with supernatants (SN) of B16 mouse melanoma cells obtained 24h after treatment with ionizing
irradiation with 2Gy in combination with the chemotherapeutic agent dacarbazine (DTIC;
250 µM) and hyperthermia (HT; 41.5°C for 1 hour) in the absence (grey) or presence (black) of
the pan-caspase inhibitor zVAD-fmk (50 µM). SN of treated melanoma cells were additionally
incubated with Apyrase (10U/ml).
51
A)
B)
C)
D)
Figure 24: Surface expression of activation markers of dendritic cells after contact with
supernatants of melanoma cells in the absence or presence of zVAD-fmk.
The expression of the activation markers MHCII (A, C) and CD86 (B,D) on the surface bone
marrow derived DCs (C57/BL6; A, B) or MyD88 KO mice (C, D) was analyzed by multicolour
flow cytometry, after contact with the supernatants of the B16-F10 mouse melanoma cells
obtained 24 h after the respective treatment(s). The tumor cells were treated with ionizing
irradiation with 2Gy alone or in combination with the chemotherapeutic agent dacarbazine
(DTIC; 250 µM) and hyperthermia (HT; 41.5°C for 1 hour), in each case in the absence or
presence of the pan-caspase inhibitor zVAD-fmk (50 µM)SN of treated melanoma cells were
additionally incubated with Apyrase (10U/ml) (A, B). Data of three experiments, each performed
in triplicates, are presented as mean ± S.D. MFI: mean fluorescence intensity. Gy: Gray; zVADfmk: pan-caspase inhibitor. mock: SN of untreated tumor cells.
Again, the increased expression of MHCII and CD86 is dependent on TLR
signaling since bone marrow derived DCs of MyD88 KO mice did not respond to
the tumor cell SN (Figure 22C, D and 24C, D). HMGB1 acts through TLR2 and
TLR4, but there are other molecules which act through these receptors. To
confirm that these effects are mostly dependent on HMGB1, the SN of the
treated tumor cells were incubated with a HMGB1 depleting antibody before
adding them to DCs of C57/BL6 mice (Figure 25). No increased expression of
MHCII or CD86 on DCs was detectable anymore when incubating the SN with
the anti-HMGB1 antibody. As performed for macrophages, we also analyzed
the effects of nucleotides on the expression of the activation markers and
revealed that their upregulation is dependent on it since no increased
expression of MHCII and CD86 was detectable when incubating the SN with
Apyrase (Figure 23A, B and Figure 24A, B).
52
Figure 25: Impact of anti-HMGB1 antibody on the surface expression of activation
markers on DCs after contact with supernatants of treated melanoma cells in the
presence of zVAD-fmk.
The expression of the activation markers MHCII (A) and CD86 (B) on the surface of bone
marrow derived dendritic cells (DCs) of C57/BL6 wild type mice was analyzed by multicolor flow
cytometry after contact with supernatants (SN) of B16 mouse melanoma cells obtained 24h
after the treatments. The tumor cells were treated with ionizing irradiation with 2Gy in
combination with the chemotherapeutic agent dacarbazine (DTIC; 250 µM), hyperthermia (HT;
41.5°C for 1 hour), and the pan-caspase inhibitor zVAD-fmk (50 µM) in the absence or presence
of a neutralizing antibody against HMGB1 (A, B). Representative data of one out of 3
experiments, each performed in duplicates, are presented as mean ± S.D. * P < 0.05 related to
treated tumor cells in the absence of anti-HMGB1 antibody; MFI: mean fluorescence intensity.
Gy: Gray; mock: SN of untreated tumor cells.
3.2.5 zVAD-fmk increased the secretion of certain inflammatory cytokines
by bone marrow derived DCs
As performed for macrophages the activation status of DCs was also
determined by analyzing the released cytokines by DCs after incubation with
SN of treated melanoma cells. Neither a single treatment with RT nor the
combination RT, DTIC and HT impacted on the release of TNFα, IL-6 or IL-1β.
But contact of DCs with SN of melanoma cells whose cell death was modulated
with zVAD-fmk resulted in an increased release of TNFα and IL-6, but not of IL1β (Figure 26).
53
Figure 26: Secretion of inflammatory cytokines by dendritic cells after contact with
supernatants of melanoma cells in the absence or presence of zVAD-fmk.
Supernatants of the B16-F10 mouse melanoma cells were obtained 24 h after the respective
treatment(s). The tumor cells were treated with ionizing irradiation with 2Gy alone or in
combination with the chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia
(HT; 41.5°C for 1 hour), in each case in the absence or presence of the pan-caspase inhibitor
zVAD-fmk (50 µM) DCs were coincubated with the obtained SN for 16h. Supernatants of these
DCs were then analyzed for TNFα (A), IL-6 (B) or IL-1β (C) by ELISA. The fold increase of the
cytokine release of DCs after incubation with SN of treated B16 melanoma cells related to DCs
after incubation with media is displayed. Data of three experiments, each performed in
triplicates, are presented as mean ± S.D. ** P < 0.01 related to samples without (w/o) inhibitor.
Gy: Gray; mock: SN of untreated tumor cells.
To analyze the effects of the danger signals HMGB1 and nucleotides such as
ATP on the secretion of inflammatory cytokines by DCs, bone marrow derived
DCs of MyD88 KO mice were used or SN that had been incubated with Apyrase
to degrade extracellular nucleotides. Both prevented the enhanced secretion of
TNFα by DCs (Figure 27).
54
Figure 27: Impact of nucleotides and Toll-like receptor signaling on the secretion of the
inflammatory cytokine TNFα by dendritic cells after contact with supernatants of
melanoma cells.
Supernatants of the B16-F10 mouse melanoma cells were obtained 24 h after the respective
treatment(s). The tumor cells were treated with ionizing irradiation with 2Gy alone or in
combination with the chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia
(HT; 41.5°C for 1 hour), in each case in the absence or presence of the pan-caspase inhibitor
zVAD-fmk (50 µM) DCs were coincubated with the obtained SN for 16h. Supernatants of these
DCs were analyzed for TNFα by ELISA. The SN of melanoma cells were treated with Apyrase
(10U/ml) (A) or DCs were obtained of MyD88 KO mice (B). The fold increase of the cytokine
release of DCs after incubation with treated B16 melanoma cells related to DCs after incubation
with media is displayed. Data of three experiments, each performed in triplicates, are presented
as mean ± S.D. MFI: mean fluorescence intensity. Gy: Gray; mock: SN of untreated tumor cells.
3.2.6 Single and multimodal melanoma treatments in the absence or
presence of zVAD-fmk do not impair phagocytosis of the tumor
cells and migration of macrophages and DCs towards SN of the
tumor cells
One prerequisite for starting an adaptive immune response against cancer cells
is the presentation of tumor derived antigens by antigen presenting cells (APCs)
such as macrophages and DCs. Before antigen presentation it has to be taken
up by the immune cells. Therefore, the capability of macrophages and DCs to
migrate towards SN of treated B16 cells was analyzed as well as the
phagocytosis of the tumor cells. Compared to mock treated tumor cells all
treatments and cell death modulation with zVAD-fmk did not impact on
phagocytosis of the tumor cells by macrophages or DCs (Figure 28A and C).
The percentage of DCs that had phagocytosed melanoma cells was higher
compared to that of macrophages.
55
Figure 28: Phagocytosis of melanoma cells by macrophages and dendritic cells and the
migration of those towards supernatants of the tumor cells.
Phagocytosis of CFSE-labelled B16-F10 mouse melanoma cells by F4/80 + peritoneal mouse
macrophages (A) or MHCII + dendritic cells (C) was analyzed by two-colour flow cytometry. The
migration of macrophages (B) or dendritic cells (D) towards supernatants of the melanoma cells
was also analyzed by flow cytometry. The B16-F10 mouse melanoma cells used for the
phagocytosis assays (A, C) were treated with ionizing irradiation with 2Gy alone or in
combination with the chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia
(HT; 41.5°C for 1 hour), in each case in the absence or presence of the pan-caspase inhibitor
zVAD-fmk (50 µM). For the migration assays (B, D), supernatants of the respective treated
tumour cells were used. The fold increase of migration of DCs to SN of treated B16 melanoma
cells related to migration of DCs to media is displayed. Representative data of one out of three
experiments, each performed in triplicates, are presented as mean ± S.D. * P < 0.05 related to
samples without (w/o) inhibitor. Gy: Gray; mock: SN of untreated tumor cells.
After acquisition of tumor antigens by immature DC, the migration of the
immune cells to lymph nodes and appropriate co-activation is mandatory. As
outlined above, especially SN of melanoma cells whose cell death was
modulated with zVAD-fmk induced an up-regulation of activation markers on
APCs. We then tested if the migration of macrophages and DCs to SN of tumor
cells is dependent on distinct treatment combinations and/or cell death
modulation by zVAD-fmk. The migration of the immune cells was not
significantly influenced by irradiation of the tumor cells or combined treatment
with RT, DTIC and HT (Figure 28B and D). However, even cell death
modulation of melanoma cells with zVAD-fmk did not impact on the migration of
macrophages (Figure 28B), an increased migration of DCs to SN of melanoma
56
cells which were treated with RT, DTIC and HT and zVAD-fmk was observed
(Figure 28D).
3.2.7 Presentation of tumor antigen by DCs is not influenced by SN of
treated melanoma cells
To start an adaptive immune response, DCs have to (cross-)present tumor
antigens after migration to the tumor and phagocytosis of tumor cells. We
therefor next analyzed the presentation of the model tumor antigen OVA by
DCs after phagocytosis of B16-OVA cells which overexpress the OVA peptide.
Analysis of the model antigen presentation with flow cytometry revealed that
irradiation slightly, but not significantly increased it. Cell death modulation with
zVAD-fmk had no impact on it (Figure 29).
Figure 29: Presentation of the model tumor antigen OVA by dendritic cells after contact
with B16-OVA melanoma cells.
Presentation of SIINFEKL (OVA+MHCII) by bone marrow derived DCs was analyzed by
multicolour flow cytometry, after co-incubation of DCs with B16-OVA mouse melanoma cells
24h after the respective treatment(s). The tumor cells were treated with ionizing irradiation with
2Gy alone or in combination with the chemotherapeutic agent dacarbazine (DTIC; 250 µM) and
hyperthermia (HT; 41.5°C for 1 hour), in each case in the absence or presence of the pancaspase inhibitor zVAD-fmk (50 µM). Data of three experiments, each performed in triplicates,
are presented as mean ± S.D. ** P < 0.01 related to samples without (w/o) inhibitor. Gy: Gray;
mock: untreated tumor cells.
57
3.2.8 SN of treated melanoma cells do not impair activation status,
maturation status, or kill activity of NK cells
A)
C)
B)
D)
Figure 30: Phenotype of NK cells after contact with SN of treated melanoma cells in
absence or presence of zVAD-fmk.
24h after isolation, murine NK cells were coincubated with SN of treated melanoma cells. The
tumor cells were treated with ionizing irradiation with 2Gy alone or in combination with the
chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia (HT; 41.5°C for 1 hour),
in each case in the absence or presence of the pan-caspase inhibitor zVAD-fmk (50 µM). The
maturation status of NK cells was analyzed 16 hours afterwards by multicolor flow cytometry.
Four maturation states were distinguished: CD11b and CD27 negative (A), CD27 single positive
(B), CD11b and CD27 double positive (C) and CD11b single positive (D). The fold increase of
NK cells after incubation with SNs of B16 melanoma cells related to NK cells after incubation
with media is displayed. Data of three experiments, each performed in triplicates, are presented
as mean ± S.D. Gy: Gray; mock: SN of untreated tumor cells.
For anti-tumor immune responses, NK cells play an important role as cells of
the innate immune system. Mature NK cells can directly induce tumor cell death
or can release proinflammatory cytokines, especially INFγ, and therefore
enhance the immune response against the tumor. Only mature NK cells initiate
anti-tumor responses. We therefore analyzed the maturation status of NK cells
after contact with SN of treated B16 cells. Immature NK cells neither express
CD11b nor CD27 on their surface. With ongoing maturation, NK cells express
first CD27 then both CD11b and CD27 and fully mature NK cells express
CD11b but no CD27 anymore [111]. Neither the irradiation nor the combinatory
combined treatment with RT, DTIC and HT, nor the modulation of cell death
with zVAD-fmk had effects on the maturation status of NK cells (Figure 30).
58
Figure 31: Impact of treatment of melanoma cells on killing activity of NK cells.
24h after isolation, murine NK cells were coincubated with pre-treated melanoma cells. The
tumor cells were treated with ionizing irradiation with 2Gy alone or in combination with the
chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia (HT; 41.5°C for 1 hour),
in each case in the absence or presence of the pan-caspase inhibitor zVAD-fmk (50 µM).The kill
of the B16-F10 melanoma cells by NK cells was then analyzed by multicolor flow cytometry.
The cell death is relativized to treated B16-F10 cells without NK cells. Data of three
experiments, each performed in triplicates, are presented as mean ± S.D. Gy: Gray; mock:
untreated tumor cells.
Figure 32: Release of INFγ by NK cells after contact with SN of treated melanoma cells.
24h after isolation, murine NK cells were coincubated with SN of treated melanoma cells. The
tumor cells were treated with ionizing irradiation with 2Gy alone or in combination with the
chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia (HT; 41.5°C for 1 hour),
in each case in the absence or presence of the pan-caspase inhibitor zVAD-fmk (50 µM). 16
hours later, the SN of the NK cells was analyzed for INFy by ELISA-Data of three experiments,
each performed in triplicates, are presented as mean ± S.D. Gy: Gray; mock: untreated tumor
cells.
SN of treated melanoma cells did not change the expression of activation
markers in NK cells that were already expressed directly after isolation.
59
However, the age of mice impacted on the maturation status of the isolated NK
cells. The younger the mice the more mature NK cells were present. Similar
effects were seen when NK cells were not incubated with SN of treated
melanoma cells but directly with the treated cells. The functionality of NK cells is
as important as their maturation and activation status for anti-tumor response.
Therefore we next analyzed the ability to kill untreated or treated B16 cells and
measured the release of the immune stimulatory factor INFγ in the SN of NK
cells after contact with the melanoma cells. Treated melanoma cells were
coincubated with NK cells for 16h and the cell death of B16-F10 cells was then
analyzed with Anx5-FITC and PI staining. The cell death was relativized to
treated B16-F10 cells without NK cells. The NK cells were all functional and
killed B16-F10 cells. They killed almost 30% of the melanoma cells. However,
the amount of killed cells was not dependent on the treatment of the B16 cells
(Figure 31). Combination of RT, DTIC and HT decreased the release of INFγ
by NK cells, but this could be reversed by zVAD-fmk (Figure 32).
3.2.9 SN of melanoma cells do not impact on DC-induced T cell
proliferation, but on cytokine secretion when gathered from
melanoma cells treated with RT, DTIC and HT in combination with
zVAD-fmk
T cells and especially CD8+ T cells are the most effective cells in killing tumor
cells when they are activated. One result of their activation of is induction of
proliferation. This can be measured in vitro with a mixed lymphocyte reaction
(MLR). This method assumes that T cells are activated by allogeneic DCs
comparable as after specific activation which results in proliferation of the T
cells. Only immune suppressive factors inhibit proliferation after coincubation of
T cells with allogeneic DCs. CD4+ or CD8+ T cells were isolated from spleens
of BalbC mice, stained with CFSE and consecutively coincubated with
allogeneic bone marrow derived DCs of C57/BL6 mice which had been
activated with SN of treated melanoma cells before.
60
Figure 33: Proliferation of T cells after co-incubation with allogeneic or syngeneic DCs
pre-incubated with SN of treated melanoma cells.
T cells were isolated of spleens of Balb/C (A, B) or C57/BL6 (C) mice and co-incubated with
bone marrow derived dendritic cells of C57/BL6 mice after activation of the latter with SN of
treated B16-F10 (A, B) or B16-OVA (C) cells. The tumor cells were treated with ionizing
irradiation with 2Gy alone or in combination with the chemotherapeutic agent dacarbazine
(DTIC; 250 µM) and hyperthermia (HT; 41.5°C for 1 hour), in each case in the absence or
presence of the pan-caspase inhibitor zVAD-fmk (50 µM).Proliferation of CFSE-stained T cells
was analyzed by multicolor flow cytometry. Data of three experiments, each performed in
triplicates, are presented as mean ± S.D. Gy: Gray; mock: SN of untreated tumor cells.
When performing the experiments with CD4+ T cells, almost 50% of the T cells
had proliferated after contact with allogeneic DCs. No significant changes in
proliferation rates were induced by pre-incubation of the DCs with the SN of
treated melanoma cells (Figure 33A). Similar effects were observed when
using CD8+ T cells. In general, the proliferation rate of CD8+ T cells was higher
than that of CD4+ T cells (Figure 33B). T cells proliferate not only after
incubation with allogeneic DCs, but also after incubation with syngeneic DCs
that present a specific antigen. Bone marrow derived DCs of C57/BL6 mice
were co-incubated with treated B16-OVA cells to get activated. Afterwards,
these DCs were coincubated with CD8+ T cells origination from spleen of OT-1
mice. CD8+ T cells of OT-1 mice specifically recognize DCs of C57/BL6 mice
presenting the model antigen OVA257-264. A lower basal proliferation rate in
comparison to the allogeneic situation was observed (Figure 33C). But again,
no impact of melanoma cell treatment with by RT or RT, DTIC and HT was
observed. Of note is that combination treatment of B16-OVA cells in the
61
presence of zVAD-fmk slightly increased the proliferation of specific CD8+ T
cells (Figure 33C).
Figure 34: Secretion of cytokines by T cells after contact with dendritic cells after
activation by SN of treated melanoma cells.
T cells were isolated of spleens of C57/BL6 mice and coincubated with bone marrow derived
dendritic cells of C57/BL6 mice after activation with SN of treated B16-OVA cells. The tumor
cells were treated with ionizing irradiation with 2Gy alone or in combination with the
chemotherapeutic agent dacarbazine (DTIC; 250 µM) and hyperthermia (HT; 41.5°C for 1 hour),
in each case in the absence or presence of the pan-caspase inhibitor zVAD-fmk (50 µM).
Supernatants of T cells were analyzed for IL6 and INFy by a beat based assay of eBioscience
and flow cytometry. The fold increase of the respective cytokine related to SNs of T cells after
incubation with media is displayed. Data of three experiments, each performed in triplicates, are
presented as mean ± S.D. ** P < 0.01 related to samples without (w/o) inhibitor. Gy: Gray;
mock: SN of untreated tumor cells.
Activated T cells do not only proliferate and are capable of killing tumor cells,
but also secrete immune stimulatory cytokines to further enhance the anti-tumor
immune response. Therefore, we next analyzed SN of activated T cells after
coincubation with DC that had been pre-incubated with SN of treated melanoma
cells for IL-6 and INFγ. An enhanced secretion of IL-6 and INFγ was only
observed after combined treatment of B16 cells with RT, DTIC and HT with
zVAD-fmk (Figure 34).
3.3 In vivo impact of zVAD-fmk on tumor growth retardation
induced by RT or combination of RT, DTIC and HT
3.3.1 Combination of fractionated RT DTIC and HT reduces tumor growth
significantly and addition of zVAD-fmk further retards it
Melanomas are highly radio- and chemoresistant. Therefore it is important to
identify multimodal tumor treatments that result in efficient and long lasting
tumor control. To prove the effectiveness of RT in combination with DTIC and
62
HT and further cell death modulation with zVAD-fmk against the melanomas
also in vivo, 1*106 B16-F10 cells were injected into the right, shaved flank of
approximately eight week old C57/BL6 mice. After one week, a tumor had
established and the treatment was started. One group of mice stayed untreated,
one got zVAD-fmk, one was fractionated irradiated with a single dose of 2Gy,
one was fractionated irradiated and got zVAD-fmk, one group was treated with
fractionated RT, DTIC and HT and the last group was treated with combination
of fractionated RT, DTIC, HT and zVAD-fmk.
Figure 35: Timetable of treatment and in vivo growth of B16-F10 tumors in C57/BL6 mice
after fractionated RT, DTIC and HT in absence or presence of zVAD-fmk.
The tumor growth of syngeneic B16-F10 tumors in wild type C57/BL6 mice after treatment is
displayed (B). The tumors were locally irradiated at days 8, 9 and 10 with a clinically relevant
single dose of 2Gy using a linear accelerator. Two hours after the irradiation, DTIC (2mg/mouse
at day 8 and 10) and zVAD-fmk (2mg/kg at day 8, 9 and 10) were injected i.p.. HT (41.5°C for
30min) was performed 4 hours after irradiation at day 8 and 10. For this, the mice were
anesthetized and the tumors were heated locally under temperature control to 41.5°C for 30min
using the BSD50 hyperthermia system (A). The tumor volume was monitored with an electronic
caliper. Joint data of three independent experiments, each with 3 mice per group, are presented
as mean ± S.D. * P < 0.05 ; ** P < 0.01 related to tumors treated with 2Gy+DTIC+HT; # P <
0.05 ; ## P < 0.01 related to untreated tumors (mock). DTIC: dacarbazine; Gy: Gray; HT:
hyperthermia; zVAD-fmk: pan-caspase inhibitor.
Treatment of tumor bearing mice with fractionated RT with a single dose of 2Gy
did not affect the tumor growth significantly. The same was seen when the mice
63
were treated with zVAD-fmk or combination of RT with zVAD-fmk. Combination
of fractionated RT with DTIC and HT reduced the tumor growth significantly.
Addition of zVAD-fmk to it further decreased the tumor growth significantly and
resulted in the most pronounced tumor growth inhibition (Figure 35B).
Figure 36: Infiltration of cells of the innate immune system into B16-F10 tumors in
C57/BL6 mice after fractionated RT, DTIC and HT in absence and presence of zVAD-fmk.
The infiltration of immune cells [Mphs (A), DCs (B), NK cells (C) and MDSCs (D)] was analyzed
by multicolor flow cytometry, 96h after the last treatment of the tumors. The tumors were locally
irradiated at days 8, 9 and 10 with a clinically relevant single dose of 2Gy using a linear
accelerator. Two hours after the irradiation DTIC (2mg/mouse at day 8 and 10) and zVAD-fmk
(2mg/kg at day 8, 9 and 10) were injected i.p.. HT (41.5°C for 30min) was performed 4 hours
after irradiation at day 8 and 10. Data of three experiments, each performed in triplicates, are
presented as mean ± S.D. * P < 0.05 ; ** P < 0.01 related to tumors treated without (w/o) zVADfmk; # P < 0.05 ; ## P < 0.01 related to untreated tumors (mock). DTIC: dacarbazine; Gy: Gray;
HT: hyperthermia; zVAD-fmk: pan-caspase inhibitor.
An efficient tumor therapy does not only kill tumor cells but also activates the
immune system to prevent metastasis and recurrences. One indication for
activation of the immune system is the infiltration of immune cells into the tumor.
Important immune cells for tumor control are DCs, NK cells and CD8+ T cells.
64
Figure 37: Infiltration of cells of the adaptive immune system into B16-F10 tumors in
C57/BL6 mice after fractionated RT, DTIC and HT in absence and presence of zVAD-fmk.
The infiltration of immune cells [CD8+ T cells (A), CD4+ T cells (B), and Tregs (C)] was
analyzed by multicolor flow cytometry, 96h after the last treatment of the tumors. The tumors
were locally irradiated at days 8, 9 and 10 with a clinically relevant single dose of 2Gy using a
linear accelerator. Two hours after the irradiation DTIC (2mg/mouse at day 8 and 10) and
zVAD-fmk (2mg/kg at day 8, 9 and 10) were injected i.p.. HT (41.5°C for 30min) was performed
4 hours after irradiation at day 8 and 10. Data of three experiments, each performed in
triplicates, are presented as mean ± S.D. ** P < 0.01 related to tumors treated without (w/o)
zVAD-fmk; # P < 0.05 ; ## P < 0.01 related to untreated tumors (mock). DTIC: dacarbazine; Gy:
Gray; HT: hyperthermia; zVAD-fmk: pan-caspase inhibitor.
Negative influences on the tumor treatment have the immune suppressive
Tregs and MDSCs. Fractionated RT alone or in combination with DTIC and HT
increased the infiltration of NK cells into the tumor. zVAD-fmk negatively
impacted on it when combined with fractionated RT. In contrast, in combination
with fractionated RT, DTIC and HT, it further slightly enhanced the infiltration of
NK cells (Figure 36C). DCs infiltrated into the tumor only after combined
treatments with zVAD-fmk (Figure 35B). All treatments decreased the
infiltration of macrophages and MDSCs into the tumor (Figure 36A and D).
Figure 36D).
Fractionated RT alone or in combinatory treatments increased the infiltration of
CD4+ T cells (Figure 37B). The same was seen for CD8+ T cells. Additional
injection of zVAD-fmk boosted the infiltration of CD8+ T cells (Figure 37A). In
comparison, the number of immune suppressive cells like Tregs was decreased
by single treatment with zVAD-fmk alone and by the combination of fractionated
65
RT and zVAD-fmk slightly. It was significantly decreased after fractionated RT
and the combination of fractionated RT, DTIC and HT in absence or presence
of zVAD-fmk (Figure 37C).
3.3.2 Combination of fractionated RT, DTIC and HT with zVAD-fmk
impacts on immune cell infiltration into the draining lymph nodes
and the presentation of tumor antigen
The immune cells have to be present, but also activated for destroying tumor
cells. For activation of cells of the adaptive immune system, the antigen has to
be presented or cross presented by DCs. Here not only the number but also the
phenotype and activation status of the DCs are important. Cross presentation is
mainly performed by CD8+ DCs [112, 113]. This takes place in the draining
lymph nodes. After capturing of antigens, DCs migrate to the lymph node and
present the antigens to T cells. We therefore also analyzed the infiltration of the
immune cells into the draining lymph nodes. Fractionated RT alone or
combination of fractionated RT, DTIC and HT did not influence the number of
CD4+ T cells in the lymph nodes significantly (Figure 38A). However,
combination of fractionated RT, DTIC and HT in presence zVAD-fmk even
decreased the number of CD4+ T cells significantly. The infiltration of CD8+ T
cells into the lymph nodes was not influenced by any of the treatments (Figure
38B). Fractionated RT or combination of fractionated RT, DTIC and HT did
further not impact on NK cell infiltration, but this was significantly enhanced by
adding zVAD-fmk (Figure 38C). Similar effects were seen when analyzing the
infiltration of DCs into the lymph nodes (Figure 39). Combination of fractionated
RT, DTIC and HT with zVAD-fmk increased the infiltration of DCs significantly
and especially that of cross presenting CD8+ DCs (Figure 39A, B).
66
Figure 38: Immune cell infiltration into draining lymph nodes of B16-OVA tumor bearing
OT-1 mice after fractionated RT, DTIC and HT in the absence or presence of zVAD-fmk.
The infiltration of immune cells [CD4+ T cells (A), CD8+ T cells (B) and NK cells (C)] was
analyzed by multicolor flow cytometry, 96h after the last treatment. The tumors were locally
irradiated at days 8, 9 and 10 with a clinically relevant single dose of 2Gy using a linear
accelerator. Two hours after the irradiation DTIC (2mg/mouse at day 8 and 10) and zVAD-fmk
(2mg/kg at day 8, 9 and 10) were injected i.p.. HT (41.5°C for 30min) was performed 4 hours
after irradiation at day 8 and 10. Data of three experiments, each performed in triplicates, are
presented as mean ± S.D. ** P < 0.01 related to tumors treated without (w/o) zVAD-fmk. DTIC:
dacarbazine; Gy: Gray; HT: hyperthermia; zVAD-fmk: pan-caspase inhibitor. Mock: untreated
tumors.
Although zVAD-fmk only enhanced the infiltration of cross presenting CD8+
DCs, all treatment schemes in presence or absence of zVAD-fmk enhanced the
cross presentation of a model tumor antigen. This was shown with an enhanced
presentation of OVA peptide by DCs in draining lymph nodes of OT-1 mice
bearing a B16-OVA tumor. Already zVAD-fmk or fractionated RT alone
enhanced the presentation of the model antigen by DCs (Figure 39C).
67
Figure 39: Immune cell infiltration into draining lymph nodes of B16-OVA tumor bearing
OT-1 mice after fractionated RT, DTIC and HT in the absence or presence of zVAD-fmk.
The infiltration of immune cells into the draining lymph nodes [DCs (A, B)] and presentation of
SIINFEKL (C) was analyzed by multicolor flow cytometry, 96h after the last treatment. The
tumors were locally irradiated at days 8, 9 and 10 with a clinically relevant single dose of 2Gy
using a linear accelerator. Two hours after the irradiation DTIC (2mg/mouse at day 8 and 10)
and zVAD-fmk (2mg/kg at day 8, 9 and 10) were injected i.p.. HT (41.5°C for 30min) was
performed 4 hours after irradiation at day 8 and 10. Data of three experiments, each performed
in duplicates, are presented as mean ± S.D. # P < 0.05 related to untreated tumors (mock).
DTIC: dacarbazine; Gy: Gray; HT: hyperthermia; zVAD-fmk: pan-caspase inhibitor. mock:
untreated mice.
3.3.3 Combination of fractionated RT, DTIC and HT with zVAD-fmk do not
influence the proliferation, but affects the expression of immune
stimulatory cytokines of T cells
Activated T cells proliferate and produce and secrete immune stimulatory
cytokines such as INFγ and the T cell growth factor IL-2. B16-OVA tumor
bearing mice were treated as described above and CFSE stained CD8+ T cells
which were isolated of spleens of OT-1 mice were injected i.v.. 96h afterwards,
the in vivo proliferation of these CD8+ T cells and the expression of intracellular
INFγ and IL-2 after ex vivo re-stimulation was analyzed. Neither the
68
combination treatment with fractionated RT, DTIC and HT nor the combination
100
80
60
40
20
2G
y+
D
TI
C
+H
T+
2G
y+
D
TI
zV
A
D
C
m
+H
-fm
k
T
0
oc
k
percentage of proliferated cells [%]
in vivo Proliferation of OVA-specific CD8+ T cells
Figure 40: In vivo proliferation of specific CD8+ T cells in B16-OVA tumor bearing OT-1
mice after fractionated RT, DTIC and HT in the absence or presence of zVAD-fmk.
CFSE stained CD8+ T cells were isolated of draining lymph nodes from OT-1 mice and
analyzed by multicolor flow cytometry, 96h after the last treatment of the tumors. The latter were
locally irradiated at days 8, 9 and 10 with a clinically relevant single dose of 2Gy using a linear
accelerator. Two hours after the irradiation DTIC (2mg/mouse at day 8 and 10) and zVAD-fmk
(2mg/kg at day 8, 9 and 10) were injected i.p.. HT (41.5°C for 30min) was performed 4 hours
after irradiation at day 8 and 10. Data of three experiments, each performed in duplicates, are
presented as mean ± S.D. DTIC: dacarbazine; Gy: Gray; HT: hyperthermia; zVAD-fmk: pancaspase inhibitor. mock: untreated mice.
Figure 41: Intracellular expression of INF y and IL2 of CD8+ T cell of B16-OVA tumor
bearing OT-1 mice after fractionated RT, DTIC and HT in the absence or presence of
zVAD-fmk and ex vivo restimulation with OVA peptide.
Intracellular INFγ (A) and IL-2 (B) of CD8+ T cells of B16-OVA tumor bearing OT-1 mice after
treatment and restimulation with OVA peptide was analyzed by multicolor flow cytometry, 96h
after the last treatment. The latter were locally irradiated at days 8, 9 and 10 with a clinically
relevant single dose of 2Gy using a linear accelerator. Two hours after the irradiation DTIC
(2mg/mouse at day 8 and 10) and zVAD-fmk (2mg/kg at day 8, 9 and 10) were injected i.p.. HT
(41.5°C for 30min) was performed 4 hours after irradiation at day 8 and 10. Data of three
experiments, each performed in duplicates, are presented as mean ± S.D. * P < 0.05 related to
tumors in the absence (w/o) of zVAD-fmk. DTIC: dacarbazine; Gy: Gray; HT: hyperthermia;
zVAD-fmk: pan-caspase inhibitor. mock: untreated mice.
69
with zVAD-fmk affected the proliferation of CD8+ T cells in vivo. Almost 90% of
the CD8+ T cells had proliferated, even in mock treated mice (Figure 40).
Additionally, none of the treatment(s) influenced the intracellular expression of
IL-2 after ex vivo restimulation of the T cells (Figure 41B). However, T cells of
mice whose tumor was treated with fractionated RT, DTIC and HT in
combination with zVAD-fmk expressed higher amounts of INFγ after
restimulation (Figure 41A).
3.3.4 Combination of fractionated RT, DTIC and HT with zVAD-fmk
retards tumor growth in a T cell dependent manner
The enhanced infiltration of CD8+ T cells after fractionated RT, DTIC and HT
and cell death modulation with zVAD-fmk gave first hints that the therapyinduced tumor growth retardation is dependent on T cells. To prove this,
analogue experiments were performed with tumor bearing RAG-2-deficient
(RAG KO) mice. zVAD-fmk in combination with fractionated RT, DTIC and HT
did not significantly further decrease the tumor growth induced by fractionated
RT, DTIC and HT (Figure 42). The latter treatment kills tumor cells directly or
activates other immune cells against the tumor, since it was not significantly
dependent on T cells
70
Figure 42: In vivo growth of B16-F10 tumors in RAG KO mice after fractionated RT, DTIC
and HT in the absence or presence of zVAD-fmk.
The tumor growth of syngeneic B16-F10 tumors in RAG KO mice is displayed. The tumors were
locally irradiated at days 8, 9 and 10 with a clinically relevant single dose of 2Gy using a linear
accelerator. Two hours after the irradiation DTIC (2mg/mouse at day 8 and 10) and zVAD-fmk
(2mg/kg at day 8, 9 and 10) were injected i.p.. HT was performed 4 hours after irradiation at day
8 and 10. For this, the mice were anesthetized and the tumors were heated locally under
temperature control to 41.5°C for 30min using the BSD50 hyperthermia system. The tumor
volume was monitored with an electronic caliper. Joint data of three independent experiments,
each with 3 mice per group, are presented as mean ± S.D. # P < 0.05 ; ## P < 0.01 related to
untreated tumors (mock). DTIC: dacarbazine; Gy: Gray; HT: hyperthermia; zVAD-fmk: pancaspase inhibitor.
71
3.3.5 Nucleotides and HMGB1 contribute to anti-tumor effects induced by
zVAD-fmk in combination with fractionated RT, DTIC and HT
Figure 43: In vivo growth of B16-F10 tumors in C57/BL6 mice after fractionated RT, DTIC
and HT in absence or presence of zVAD-fmk and in presence of Apyrase.
The tumor growth of syngeneic B16-F10 tumors in wild type C57/BL6 mice is displayed. The
tumors were locally irradiated at days 8, 9 and 10 with a clinically relevant single dose of 2Gy
using a linear accelerator. Two hours after the irradiation DTIC (2mg/mouse at day 8 and 10)
and zVAD-fmk (2mg/kg at day 8, 9 and 10) were injected i.p.. Apyrase was injected i.v. one
hour after irradiation (25U/mouse at day 8). HT was performed 4 hours after irradiation at day 8
and 10. For this, the mice were anesthetized and the tumors were heated locally under
temperature control to 41.5°C for 30min using the BSD50 hyperthermia system. The tumor
volume was monitored with an electronic caliper. Joint data of three independent experiments,
each with 3 mice per group, are presented as mean ± S.D. * P < 0.05 related to tumors treated
with 2Gy+DTIC+HT; ## P < 0.01 related to untreated tumors (mock). DTIC: dacarbazine; Gy:
Gray; HT: hyperthermia; zVAD-fmk: pan-caspase inhibitor. mock: untreated mice.
To analyze the influence of the danger signals ATP as nucleotide and HMGB1
on the therapy-induced tumor growth retardation in vivo, the experiments were
additionally performed by injecting Apyrase (Figure 43) or with MyD88 KO mice
(Figure 44). B16-F10 tumor bearing C57/BL6 mice, were treated with
fractionated RT, DTIC, HT and zVAD-fmk and additional i.v. injection of Apyrase
after the multimodal treatment to induce degradation of extracellular present
nucleotides in vivo. Nucleotides seem not to be the main players in therapyinduced tumor growth retardation, since still significant further tumor growth
retardation was observed when adding zVAD-fmk to treatment with fractionated
RT, DTIC and HT in the presence of Apyrase (Figure 43). Nevertheless, the
72
tumor growth was still stronger compared to an environment with nucleotides
being present (Figure 35).
Regarding the impact of TLR signaling, zVAD-fmk did not further impact on
therapy-induced tumor growth retardation in MyD88 KO mice (Figure 44). The
tumor growth retardation after treatment with fractionated RT, DTIC and HT was
similar in the absence or presence of zVAD-fmk (Figure 44).
MyD88 ko
tumor volume [mm3]
2000
mock
2Gy+DTIC+HT
2Gy+DTIC+HT+zVAD-fmk
1500
1000
#
500
#
0
n.s.
##
0
7
8
9
##
10
days
11
12
13
Figure 44: In vivo growth of B16-F10 tumors in MyD88 KO mice after fractionated RT,
DTIC and HT in absence or presence of zVAD-fmk.
The tumor growth of syngeneic B16-F10 tumors in MyD88 KO mice is displayed. The tumors
were locally irradiated at days 8, 9 and 10 with a clinically relevant single dose of 2Gy using a
linear accelerator. Two hours after the irradiation DTIC (2mg/mouse at day 8 and 10) and
zVAD-fmk (2mg/kg at day 8, 9 and 10) were injected i.p.. HT was performed 4 hours after
irradiation at day 8 and 10. For this, the mice were anesthetized and the tumors were heated
locally under temperature control to 41.5°C for 30min using the BSD50 hyperthermia system.
The tumor volume was monitored with an electronic caliper. Joint data of three independent
experiments, each with 3 mice per group, are presented as mean ± S.D. # P < 0.05, ## P < 0.01
related to untreated tumors (mock). DTIC: dacarbazine; Gy: Gray; HT: hyperthermia; zVAD-fmk:
pan-caspase inhibitor.
73
4 Discussion
4.1 B16-F10 cells as mouse model for malignant melanoma
The B16 mouse melanoma cell line is one of the most used tumor models for
melanoma over the last half century and is a spontaneous C57/BL6-derived
melanoma [114]. The B16 melanomas are defined as poorly immunogenic since
they express rather low levels of MHCI molecules [115]. Nevertheless tumor
regression
can
be
induced
by
immunotherapeutic
treatments
which
demonstrate the immunogenic potential of these cells [15]. Therefore, we
decided to use the B16 melanoma cells as model system for melanoma to test
the efficacy and mode of action of combined therapies in absence or presence
of further cell death modulation with zVAD-fmk.
4.2 Immune therapy as a treatment option against melanoma
Melanomas are often highly radio- and chemoresistant [108]. Because of this,
RT or CT alone is not an efficient treatment option for most of the melanomas.
Primary tumors are often removed by surgical resection. Since this therapy form
does not affect the immune system it cannot prevent recurrences or metastasis.
Furthermore, metastasis often grew in places where a surgical resection is not
possible. For this an additional immune therapy is necessary. Due to the fact
that existing immune therapies against melanomas cause high side effects or
does only lead to responses for short time [12], there is a strong need for
additional and/or alternative immune therapies or immune modulating drugs.
Ipilimumab, an anti-CTLA4 antibody is the most used immune modulating drug
for melanoma. Blocking CTLA4, a negative regulator of the immune system and
terminator of T cell activation and proliferation, leads to an enhanced and long
lasting activation of T cells. Clinical trials could show that response rates of the
anti-CTLA4 antibody ipilimumab are between 5 and 20%. Studies with higher
concentrations of ipilimumab could show better response rates, but also higher
toxicities have been observed [15-17]. New blocking antibodies for melanoma
treatments are anti-PD-1 or anti-PD-1L. These antibodies aim to enhance the T
cell immune response in a tumor specific manner by blocking the interaction of
the inhibitory receptor PD-1 on T cells with PD-L1 expressed on tumor cells.
74
First results of clinical trials are promising. The response rate of nivolumab is
over 2 years. The antibody is well tolerated and toxicities were mild, less
frequent and less severe than those observed with ipilimumab [13, 14]. Another
immune activating strategy for melanoma treatment is the use of vemurafenib, a
v-raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitor. 40-60% of
the tumors have mutations in the gene coding for BRAF and 90% of these
mutations results in the substitution of valine for glutamine (V600E) [8, 9]. The
mutated form of BRAF results in a constitutive activated mitogen-activated
protein kinase pathway (MAPK) constitutive activated which leads to an
increased cellular proliferation and oncogenic activity. The BRAF inhibitor
vemurafenib is selective for BRAF with the V600E mutation. A phase III trial
demonstrated improvements in response rates and in progression free survival
[9]. But the limiting factor of this therapy form is the short duration that in
average is only approximately 6 month [1].
Beside the importance of an immune therapy, the standard therapies should not
be neglected. Under certain clinical conditions, RT, especially in multimodal
settings, should be considered as treatment option. New approaches of
radiotherapy like stereotactic radiosurgery or brachytherapy have been shown
to be an effective treatment option [116]. Hyperthermia acts as an adjuvant for
RT and has been proven in clinical trials to improve local control of malignant
melanoma [117]. Further, chemoresistance of malignant melanoma can be
overcome in a tumor cell-selective manner by interfering with anti-apoptotic Bcl2 family members [118]. Therefore, cell death modulators might be beneficial in
multimodal settings. Targeting apoptotic pathways and modulation of apoptotic
cell death are therefore promising strategies to combat cancer and inflammatory
diseases in general. Here caspases represent key targets for drug development
since they are central in initiation and execution of cell death and in maturation
of inflammatory cytokines [4].
4.3 Immunogenic cell death
Innovative melanoma therapies should therefore be multimodal ones and aim to
induce immunogenic tumor cell death forms. The latter modify the tumor
microenvironment by releasing danger signals (DAMPs). Immune cells might
75
get recruited and activated. Tumor cell death is defined as immunogenic when
the cells express calreticulin on their surface and/or release DAMPs like
HMGB1 and ATP [119]. Calreticulin is mostly expressed on pre- or early
apoptotic cells. In contrast, DAMPs are mainly released by necrotic cells.
Necrosis in general is the more immunogenic cell death form. One possible new
therapeutic approach is to manipulate the cell death to be more immunogenic.
The tumor and its microenvironment should be modified to release danger
signals which results in recruiting and activation of immune cells [16].
4.4 Necrosis and necroptosis in melanoma cells
4.4.1 Impact of multimodal treatments on melanoma cell death forms
Since malignant melanoma is resistant to standard therapies, only combinatory
therapies might result in sufficient tumor cell death. The in vitro experiments
revealed that combination of HT with DTIC leads to apoptosis and in
combination with RT to necrosis. The triple combination consisting of HT, RT
and DTIC resulted to the highest percentages of apoptotic and necrotic
melanoma cell death. This mixture is beneficial for induction of anti-tumor
immune responses, since cells dying via apoptosis release chemotactic factors
to attract immune cells [13] and also together with the necrotic ones release
DAMPs to activate them [17]. In this context it is also important to consider the
order of therapies. Hyperthermia can sensitize the cells for irradiation when it is
delivered before RT or it can aggravate the irradiation induced cellular stress
when it is applied after RT [96]. Irradiation first and HT afterwards induced
significantly more cell death than the other way around (Figure 9 and 10).
However, in the clinics mostly HT is performed before RT due to logistic
reasons. To improve the positive effect of HT on melanoma cell death induction
primary triggered by RT, it should be considered to change the order of RT and
HT also in the clinics. One possibility to manipulate the cell death in an
additional way is to combine the treatments with the pan caspase inhibitor
zVAD-fmk. Pan caspase inhibitors like zVAD-fmk or IDN-6556 were proven in
preclinical studies and currently also in phase I/II trials for inflammatory
diseases and show manageable side effects [5, 120].
76
4.4.2 Induction of melanoma necroptosis by zVAD-fmk
It has been shown before for the mouse fibrosarcoma L929 cells that this pan
caspase inhibitor can induce necroptosis by blocking apoptosis [121]. Since the
characteristics of programmed necrosis like rounding of the cell, cytoplasmic
swelling, lack of DNA fragmentation and plasma membrane rupture are the
same as of accidental necrosis, necroptosis must be also considered as
immunogenic cell death form. How zVAD-fmk modulates cell death of malignant
melanoma and how it impacts on the immunogenicity of these cells is hardly
investigated. We revealed that necroptosis is inducible in B16-F10 melanoma
cells, since necrosis could be inhibited with the RIP-1 kinase inhibitor
necrostatin-1 (nec1). Necroptosis was inducible even in the natural dying cells
being present in cell culture (Figure 11A). Furthermore necrotic melanoma cell
death was fostered by blocking apoptosis with zVAD-fmk (Figure 11B). In
organisms necroptosis is seen as a backup mechanism for conditions when
apoptosis is not possible like after infection with certain viruses [84]. Many
viruses encode Bcl-2 homologs or caspase inhibitors to block apoptosis [122].
zVAD-fmk induced necrosis could be blocked by adding nec-1 (Figure 12). This
gave us a further hind that necroptosis is inducible in melanoma cells (Figure
11B).
4.5 Effects of zVAD-fmk on the immunogenic potential of B16
melanoma cells in vitro
4.5.1 Effects of zVAD-fmk on the cell cycle
Of note is that zVAD-fmk did not only modulate melanoma cell death, but also
the cell cycle. 48h after every treatment (even in mock treated cells), higher
amounts of cells were in G2 phase of the cell cycle when zVAD-fmk was
present in treatment (Figure 13B). The G2 phase of the cell cycle is more
chemo- and also radiosensitive. Cells stop in this phase of cell cycle after stress
factors like RT. This fact is used for planning fractionation schemes. In the
second or later fractions more and more cells are in the G2 phase and therefore
more sensitive for the following RT [83].
77
This suggests that a time window of 48h should be considered between the
treatments with RT, DTIC, HT and zVAD-fmk. We therefore applied the
following treatment schemes and time schedule in the in vivo experiments: RT
and injection of zVAD-fmk was performed every 24h but the second treatment
with HT and injection of DTIC was performed after 48h.
4.5.2 Effects of zVAD-fmk on the secretion of DAMPs
Normally higher amounts of necrosis correlates with an enhanced passively
release of DAMPs like ATP, HMGB1 and HSP70 (summarized in [123]). We
saw that additional incubation with zVAD-fmk results in higher amounts of
HMGB1 but not of HSP70 in the supernatants (SN) of the treated tumor cells
(Figure 14). This indicates that necroptosis induced by zVAD-fmk does not
increase the release of DAMPs in general. Only the release of HMGB1
correlated with the necrosis induction. Danger signals like HMGB1 enhance
maturation and activation of immune cells like macrophages and DCs [85].
4.5.3 Effects of zVAD-fmk on macrophages and DCs
To analyze the impact of zVAD-fmk as cell death modulator on the
immunogenicity of B16 melanoma cells in detail, we explored the impact of
tumor cell SN after the respective treatments on cells of the innate and adaptive
immune system.
Macrophages and DCs are the first cells which are recruited to the tumor and
therefore essential for the regulation and activation of the adaptive immune
system. The activation of these cells is crucial for success of an immune based
tumor therapy [85, 86]. After incubation with SN of tumor cells macrophages
increased the expression of MHCII and the activation marker CD86 on their
surface when zVAD-fmk was used for cell death modulation of B16 cells. This
was not a direct effect of the inhibitor on the immune cells, since the half- life of
zVAD-fmk is short (< 40 minutes) [124] and an incubation with the inhibitor
alone did not have any effects on macrophages and DCs (Figure 16 and 21).
The enhanced amounts of HMGB1 and ATP in the SN were in part responsible
for the increased expression of the activation markers on macrophages, since
the increased expression of these markers was not observed after incubation of
the SN with Apyrase or using peritoneal macrophages which were isolated from
MyD88 KO mice. Apyrase is a calcium-activated plasma membrane-bound
78
enzyme that catalyzes the hydrolysis of ATP to AMP and inorganic phosphate.
But Apyrase does not only degrade ATP but all purine and pyrimidine
nucleoside 5'-di- and 5'-triphosphates [125]. Therefore the experiments with
Apyrase only show that nucleotides and not specifically ATP influence the
activation of macrophages.
MyD88 is an adaptor protein for TLR signaling and is used by all TLRs except
TLR3, therefore MyD88 KO mice have defects in TLR signaling [126] and as a
result also in signaling of HMGB1 which acts through TLR2 and TLR4 [127].
Because of the existence of other ligands for TLRs than HMGB1 it is only a hind
that HMGB1 is responsible for the activation of macrophages. Other possible
ligands are for example fibrinogen, fibronectin, heparan sulfate and hyaluronan
which are all components of the tumor extracellular matrix [128].
An increased expression of MHCII and CD86 was also seen on DCs, but here
only when zVAD-fmk had been combined with RT, DTIC and HT. Again, these
effects are dependent on TLR signaling and nucleotides since the increased
expression was not detectable when using MyD88 KO mice or incubation of the
melanoma cell SN with Apyrase (Figure 24). MyD88 KO mice have defects in
TLR signaling. HMGB1 but also other molecules like fibrinogen or fibronectin
act through this pathway as mentioned above. Because of this we performed
additional experiments with a HMGB1 depleting antibody to prove that the
effects are especially dependent on HMGB1. We observed that the increased
expression of these activation markers was not present when incubating the SN
of treated tumor cells with this antibody before adding to DCs (Figure 25).
Since HMGB1 was released in the SN of melanoma cells in every treatment
when zVAD-fmk was present this indicates that not only the amount but also the
redox state of it and the combination with other molecules [12] are important for
the activation of DCs. A reductive environment is important for HMGB1 to
maintain the bioactivity [11]. We could not reconstitute the activation of DCs and
macrophages by addition of recombinant HMGB1 (data not shown). But by
addition of recombinant HMGB1 we did not adapt the milieu of the tumor cells,
therefore we could not define the redox state of HMGB1.
Danger signals enhance maturation and antigen presentation of APC [129].
Those innate immune cells are the first ones which are recruited to the tumor by
different soluble factors of the tumor microenvironment. They regulate the
79
activation or suppression of cells of the adaptive immune system and are
therefore jointly responsible for the success of an immune-based tumor therapy
(summarized in [130]). Beside the activation we analyzed the functionality of
DCs and macrophages. There are reports that soluble factors of tumors impair
the functionality of immune cells especially of DCs. By releasing factors like IL10, M-CSF, vascular endothelial growth factor (VEGF), gangliosides or
prostanoids tumors can prevent the differentiation and function of DCs
(summarized in [131]). We observed that both APCs were still functional after
contact with the treated the tumor cells. They phagocytosed and migrated
properly. Only the migration of DCs to SN of tumor cells which were treated with
RT, DTIC and HT and whose cell death was modulated with zVAD-fmk was
slightly increased (Figure 28). This suggests that this special treatment might
result in an enhanced migration into the tumor of DCs also in vivo. After
migration into the tumor, DCs have to be activated since activated DCs are then
able to activate naïve T cells [132].
4.5.4 Effects of zVAD-fmk on T cells
Activation of especially CD8+ T cells is essential for an effective anti-tumor
immune response [14]. Therefore we investigated the ability of the activated
DCs, characterized by upregulation of the activation markers MHCII and CD86
and increased release of the inflammatory cytokines TNFα and IL-1β, to
activate T cells. A slightly enhanced proliferation after specific activation of OT-1
CD8+ T cells with OVA presenting DCs which had been activated with SN of
B16-OVA cells after treatment with RT, DTIC, HT and zVAD-fmk (Figure 33C)
was observed. However, we could not detect an enhanced proliferation by MLR,
but we detected an enhanced secretion of IL-6 and INFγ after coincubation with
DCs (Figure 34A,B). It was increased by the combinatory treatment of RT, CT
and HT and additional modulation with zVAD-fmk. IL-6 has two contrary roles in
anti-tumor immunity. On the one side IL-6 can act intrinsically on tumor cells. In
this way it supports the proliferation and survival of tumor cells. On the other
side IL-6 can induce an anti-tumor immune response by leading to activation,
proliferation and survival of lymphocytes and by supporting the T cells immune
response (summarized in [114]). INFγ is in important cytokine in the initiation
and execution of the anti-tumor immunity. The efficacy of tumor infiltrating
lymphocytes is associated with their ability to secrete INFγ [115]. It leads to
80
upregulation of MHCI and therefore improves the presentation of tumor
antigens by APCs [133]. Additionally it regulates the differentiation and
functionality some immune cells. In this way it promotes a Th1 mediated
immune response and recruit CD8+ T cells [15, 17]. Furthermore it activates
macrophages to produce chemokines which results in recruitment of more
immune cells [15] (summarized in [134]).
Cell death modulation with zVAD-fmk did not only activate cells of the innate
immune system but also of the adaptive immune system in vitro.
4.5.5 Effects of zVAD-fmk on NK cells
Other effective immune cells against tumor cells are NK cells. These innate
immune cells can kill tumor cells by different ways. In contrast to T cells NK
cells can kill tumor cells independent of MHC molecules (summarized in [122]).
One possibility is the kill by binding on death receptors like Fas (CD95). They
can increase the expression of these receptors on the surface of tumor cells by
secretion of INFγ [135]. Only mature NK cell can kill tumor cells efficiently.
Treatment of tumor cells did not impact on the maturation status of NK cells
(Figure 30) or the kill of tumor cells by these cells directly (Figure 31).
However, tumor cells, which were treated with DTIC, decreased the release of
INFγ by NK cells after coincubation. So this CT agent acts immune suppressive
in this view. In contrast, cell death modulation with zVAD-fmk restored the
release of INFγ when combined with RT and HT (Figure 32). Therefore the
expression of the death receptor Fas on the surface of the tumor cells might be
increased after this multimodal treatment since release of INFγ by NK cells can
enhance Fas expression. Future experiments should examine this in more
detail.
4.6 zVAD-fmk induced B16-F10 tumor growth retardation in vivo
In our preclinical in vitro systems, the combinatory treatment of RT, DTIC and
HT in the presence of zVAD-fmk resulted in cell death of B16-F10 melanoma
cells with high immunogenic potential. To prove the immunogenic potential of
zVAD-fmk in multimodal tumor treatments, we performed mouse in vivo studies
with treatments closely resembling the human situation. For this, we established
both a local irradiation and a local hyperthermia application for mice with ectopic
81
B16-F10 tumors (Figure 7 and 8). The in vivo studies revealed that fractionated
irradiation with a clinically relevant single dose of 2Gy alone did not affect the
tumor growth. Similar results were obtained with the in vitro experiments.
Together this proves that the melanoma cells are highly radioresistant, as most
of the melanoma cells in patients [108]. Only the triple combination treatment
with fractionated RT, DTIC and HT reduced the tumor growth significantly.
Addition of zVAD-fmk further retarded the tumor growth significantly (Figure
35). The reduced tumor growth induced by zVAD-fmk is not a direct cytotoxic
effect of the inhibitor but rather dependent on the immune system (see below).
4.6.1 Immune stimulatory effects of zVAD-fmk in vivo
The combination treatment consisting of fractionated RT, DTIC and HT had
direct effects on the tumor cells but additionally, this treatment scheme changes
the tumor micromilieu. It influenced the infiltration of various immune cells into
the tumor and therefore potentially also the efficiency of the treatment. zVADfmk modulated the therapy-induced infiltration of distinct immune cells. Different
studies showed that an increased infiltration of lymphocytes is associated with
an improved survival [136-138].
4.6.2 Infiltration of immune cells into the tumor
The infiltration of innate and adaptive immune cells into the tumor is affected by
the examined treatment schemes. Fractionated irradiation alone led to an
enhanced infiltration of NK cells into the tumor, but in the presence of zVAD-fmk
only as few NK cells as in untreated tumors were observed. In contrast, the
combination treatment with RT, DTIC and HT also increased the infiltration of
NK cells and addition of zVAD-fmk further slightly enhanced this (Figure 36C).
Future work should focus on whether this is also dependent on activation of
DCs after the multimodal treatment in the presence of zVAD-fmk [84]. The
infiltration of DCs into the tumor was only increased in the presence of RT and
zVAD-fmk (Figure 36B). This highlights again that RT in multimodal settings
exerts its anti-tumor effects via activation of DCs. NK cells and cytotoxic CD8+
T cells are the key immune cell population that mediates the cytotoxic effects
induced by RT in combination with immunotherapeutic agents [83].
Regarding immune suppressive cells such as Treg and MDSCs, combination
treatment with fractionated RT, DTIC and HT significantly reduced the
82
percentage of these cells in the tumor, as also zVAD-fmk did alone (Figures 36
and 37).
To summarize these effects of zVAD-fmk on the infiltration of innate immune
cells into the tumor, one can assert that more inflammatory or effector cells like
DCs and NK cells migrate into the tumor concomitantly with less immune
suppressive cells like MDSCs, Tregs and macrophages. Further, zVAD-fmk
also modulated the infiltration of adaptive immune cells into the tumor. All
treatments reduced the infiltration of immune suppressive Tregs (Figure 37C).
We revealed that already fractionated RT alone in combination with zVAD-fmk
modulates the tumor milieu and therefore the infiltration of immune cells into the
tumor. More inflammatory or effector cells and less immune suppressive cells
were present. Nevertheless, RT alone or in combination with zVAD-fmk could
not inhibit the tumor growth significantly. We conclude that immune modulation
alone cannot be an option for a solo therapy in this case. There must be a
combination with a more locally tumor damaging therapy. Only when enough
dying cells are generated, cell death modulation by zVAD-fmk modulates the
cell death form and might cause an immune reaction via activation of DCs and
CTLs also against still viable tumor cells [139].
Since the activation of cytotoxic T cells takes place in the draining lymph nodes,
we analyzed the infiltration of NK cells, DCs and T cells into them. An enhanced
infiltration of NK cells into the lymph nodes after a combination treatment with
fractionated RT, DTIC, HT and zVAD-fmk was observed. But there were not
only more NK cells detectable, but also more DCs which are capable of
activating naïve T cells (Figure 39A). Of special note is that most of the
infiltrating DCs were positive for CD8 (Figure 39B). This DC phenotype is
especially known to efficiently cross-present antigens [112, 113]. The
percentage of CD8+ T cells in the LN was similar after all treatments, but most
likely these T cells become more activated, when more CD8+ DCs are present.
Especially after treatment of the tumor with fractionated RT, DTIC, HT and
zVAD-fmk, ex vivo re-stimulated T cells expressed more INFγ (Figure 41).
4.6.3 Tumor growth retardation in immune deficient mice
This indicates that T cells are main effector cells of this multimodal tumor
treatment scheme, which was further proven by performing similar experiments
with RAG KO mice. These mice have no functional B and T cells [140]. Here,
83
zVAD-fmk did not have any additional effects in combination treatments in these
mice (Figure 42). Nevertheless, T cells were not directly activated by the
combinatory treatment but by DCs. Treatment of tumors with RT, DTIC, HT and
zVAD-fmk results in higher amounts of necrotic cells and therefore in an
enhanced release of the danger signals ATP and HMGB1. These danger
signals activate DCs and thereby T cells. To prove the effects of the danger
signals we additionally injected Apyrase [141] or used MyD88 KO mice [142] for
the experiments. Apyrase degradates extracellular ATP and nucleotides and
MyD88 KO mice have defects in TLR signaling. There are no additional effects
of zVAD-fmk detectable when using MyD88 KO mice. Apyrase also reduced the
effects of zVAD-fmk. This is a strong evidence that HMGB1 and ATP are
responsible for the activation of DCs also in vivo (Figure 43 and 44).
4.7 Conclusion
Figure 45: Combination of RT, DTIC, HT with zVAD-fmk is capable of activating both
innate and adaptive immune cells against the tumor cells.
The combinatory treatment consisting of fractioned RT, DTIC, HT and zVAD-fmk results in an
immunogenic B16 tumor cell death: the tumor cells die more necrotic and release the danger
signals HMGB1 and most likely ATP as prominent nucleotide. This results in an activation of NK
cells and DCs. The latter in turn activate CD8+ T cells against the tumor. In contrast, the
combination treatment without zVAD-fmk results in dying and dead tumor cells with low
immunogenicity and thereby in fact acts locally but less systemically.
The detailed mechanism how zVAD-fmk in combination with RT with a single
dose of 2Gy, DTIC and HT induce an anti-tumor immune response is still not
clear. But we revealed that zVAD-fmk as cell death modulator of therapyinduced dying cells by RT, DTIC and HT fosters melanoma cell necroptosis as
an immunogenic cell death form which then activates APCs such as DCs by the
84
release or secretion of danger signals (HMGB1 and nucleotides such as ATP)
DCs and macrophages enhanced the expression of the activation markers
MHCII and CD86 and secreted higher amounts of the proinflammatory
cytokines TNFα and IL-1β. While the maturation and activation status of NK
cells is not influenced by this treatment, the secretion of INFγ by these innate
immune cells was enhanced.
In vivo, the tumor growth was already significantly decreased by the
combination treatment of fractionated RT, DTIC and HT, but zVAD-fmk
decreased the tumor growth further by enhancing the immunogenicity of the
treated tumor cells. This results in an increased infiltration of DCs and T cells
into the tumor and lymph nodes and also in an increased activation of CD8+ T
cells. These effects are dependent on the danger signals HMGB1 and ATP and
most likely also on the redox status of HMGB1. We conclude that HMGB1 in a
distinct tumor microenvironment is the main effector molecule for induction of
anti-tumor immunity by zVAD-fmk in multimodal therapies. But ATP and/or other
nucleotides further enhance these anti-tumor immune responses (Figure 43).
zVAD-fmk should be considered in the future as an additional drug for antimelanoma therapy. Moretti and colleagues already demonstrated that zVADfmk acts as radiosensitizer for tumor cells, significantly slows xenograft tumor
growth retardation induced by RT and increases the expression of HMGB1 [55].
Melanoma is a tumor with a per se “immunogenic potential” because there are
cases of spontaneous regression [143] and case reports about abscopal effects
[144-146]. The latter are a phenomenon in which local RT induces a regression
of (metastatic) cancer at a distance from the irradiated site (summarized in
[147]). This is especially observed in patients with melanoma. Postow et al
postulated a case report of an abscopal effect in a patient with melanoma
treated with ipilimumab and RT [148]. The patient showed a systemic response
to localized radiotherapy after receiving Ipilimumab. Additionally, there was a
systemic increase of activated CD4+ T cells and a decrease of MDSCs in the
serum of the patient detectable.
This raises hope that multimodal treatment settings that induce activation of
immune cells will increase survival of melanoma patients in the future. Our data
suggest including zVAD-fmk in multimodal melanoma treatments to foster the
induction of immunogenic tumor cell death and therefore anti-melanoma
85
immunity. As zVAD-fmk showed some side effects in phase I clinical studies as
liver toxicities [120], other caspase inhibitors with lower toxicities were
developed such as IDN-6556 (PF-03491390) and should be tested also in
combination with RT, DTIC and HT in the future. In general, caspase inhibitors
have been proven in preclinical and current phase I/II clinical trials to be suitable
drugs for inflammatory diseases and tissue damages with manageable side
effects [5, 120]. Therefore, the way into the clinics of caspase inhibitors for the
treatment of melanoma should be feasible.
4.8 Outlook
zVAD-fmk should be tested in long time experiment to even better simulate the
clinical situation. Different fractionation schemes of RT should be analyzed and
the most beneficial chronology of combining RT with zVAD-fmk and further
agents should be uncovered [63]. To prove that zVAD-fmk in multimodal
settings leads to a long lasting immune memory against the tumor, challenge
experiments have to be performed. After that zVAD-fmk or new less toxic
caspase inhibitors such as IDN-6556 could be tested in clinical phase I/II
studies for treatment of melanoma. Additionally, zVAD-fmk should be tested as
immune-stimulating molecule in combination with distinct fractionation schemes
of RT and chemotherapeutic agents for other tumor entities than melanoma.
86
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Jendrossek, V., The intrinsic apoptosis pathways as a target in
anticancer therapy. Curr Pharm Biotechnol, 2012. 13(8): p. 1426-38.
Giblin, A.V. and J.M. Thomas, Incidence, mortality and survival in
cutaneous melanoma. J Plast Reconstr Aesthet Surg, 2007. 60(1): p. 3240.
Friedlander, R.M., Apoptosis and caspases in neurodegenerative
diseases. N Engl J Med, 2003. 348(14): p. 1365-75.
Marek Los, S.B.G., Apoptotic Pathways as Targets for Novel Therapies
in Cancer and Other Diseases. 2005: Springer.
Iwata, A., et al., Extracellular BCL2 proteins are danger-associated
molecular patterns that reduce tissue damage in murine models of
ischemia-reperfusion injury. PLoS One, 2010. 5(2): p. e9103.
Umansky, V., et al., Extracellular adenosine metabolism in immune cells
in melanoma. Cancer Immunol Immunother, 2014. 63(10): p. 1073-80.
Belka, C., et al., Impact of localized radiotherapy on blood immune cells
counts and function in humans. Radiother Oncol, 1999. 50(2): p. 199204.
Heylmann, D., et al., Human CD4+CD25+ regulatory T cells are sensitive
to low dose cyclophosphamide: implications for the immune response.
PLoS One, 2013. 8(12): p. e83384.
Khan, N., et al., The evolving role of radiation therapy in the
management of malignant melanoma. Int J Radiat Oncol Biol Phys,
2011. 80(3): p. 645-54.
Richter, K., M. Haslbeck, and J. Buchner, The heat shock response: life
on the verge of death. Mol Cell, 2010. 40(2): p. 253-66.
Vezzoli, M., et al., High-mobility group box 1 release and redox
regulation accompany regeneration and remodeling of skeletal muscle.
Antioxid Redox Signal, 2011. 15(8): p. 2161-74.
Pisetsky, D.S., The translocation of nuclear molecules during
inflammation and cell death. Antioxid Redox Signal, 2014. 20(7): p. 111725.
Lauber, K., et al., Apoptotic cells induce migration of phagocytes via
caspase-3-mediated release of a lipid attraction signal. Cell, 2003.
113(6): p. 717-30.
Melief, C.J., Tumor eradication by adoptive transfer of cytotoxic T
lymphocytes. Adv Cancer Res, 1992. 58: p. 143-75.
Hu, X., S.D. Chakravarty, and L.B. Ivashkiv, Regulation of interferon and
Toll-like receptor signaling during macrophage activation by opposing
feedforward and feedback inhibition mechanisms. Immunol Rev, 2008.
226: p. 41-56.
Kunz, M., et al., Strong expression of the lymphoattractant C-X-C
chemokine Mig is associated with heavy infiltration of T cells in human
malignant melanoma. J Pathol, 1999. 189(4): p. 552-8.
Mukai, S., et al., Infiltration of tumors by systemically transferred tumorreactive T lymphocytes is required for antitumor efficacy. Cancer Res,
1999. 59(20): p. 5245-9.
87
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
Zitvogel, L., A. Tesniere, and G. Kroemer, Cancer despite
immunosurveillance: immunoselection and immunosubversion. Nat Rev
Immunol, 2006. 6(10): p. 715-27.
Kim, R., M. Emi, and K. Tanabe, Cancer immunoediting from immune
surveillance to immune escape. Immunology, 2007. 121(1): p. 1-14.
Vesely, M.D., et al., Natural innate and adaptive immunity to cancer.
Annu Rev Immunol, 2011. 29: p. 235-71.
Dunn, G.P., et al., Cancer immunoediting: from immunosurveillance to
tumor escape. Nat Immunol, 2002. 3(11): p. 991-8.
Schreiber, R.D., L.J. Old, and M.J. Smyth, Cancer immunoediting:
integrating immunity's roles in cancer suppression and promotion.
Science, 2011. 331(6024): p. 1565-70.
Bennaceur, K., et al., Immunosuppressive networks in the tumour
environment and their effect in dendritic cells. Biochim Biophys Acta,
2009. 1795(1): p. 16-24.
Wilczynski, J.R. and M. Duechler, How do tumors actively escape from
host immunosurveillance? Arch Immunol Ther Exp (Warsz), 2010. 58(6):
p. 435-48.
Chang, C.C., S.P. Murphy, and S. Ferrone, Differential in vivo and in vitro
HLA-G expression in melanoma cells: potential mechanisms. Hum
Immunol, 2003. 64(11): p. 1057-63.
Garcia-Lora, A., I. Algarra, and F. Garrido, MHC class I antigens,
immune surveillance, and tumor immune escape. J Cell Physiol, 2003.
195(3): p. 346-55.
Valenti, R., et al., Tumor-released microvesicles as vehicles of
immunosuppression. Cancer Res, 2007. 67(7): p. 2912-5.
Ostrand-Rosenberg, S., Immune surveillance: a balance between
protumor and antitumor immunity. Curr Opin Genet Dev, 2008. 18(1): p.
11-8.
Mills, C.D., et al., M-1/M-2 macrophages and the Th1/Th2 paradigm. J
Immunol, 2000. 164(12): p. 6166-73.
Zhang, J.G., et al., The role of adenosine A2A and A2B receptors in the
regulation of TNF-alpha production by human monocytes. Biochem
Pharmacol, 2005. 69(6): p. 883-9.
Link, A.A., et al., Ligand-activation of the adenosine A2a receptors
inhibits IL-12 production by human monocytes. J Immunol, 2000. 164(1):
p. 436-42.
Balkwill, F. and A. Mantovani, Inflammation and cancer: back to
Virchow? Lancet, 2001. 357(9255): p. 539-45.
Coussens, L.M. and Z. Werb, Inflammation and cancer. Nature, 2002.
420(6917): p. 860-7.
Kerr, J.F., A.H. Wyllie, and A.R. Currie, Apoptosis: a basic biological
phenomenon with wide-ranging implications in tissue kinetics. Br J
Cancer, 1972. 26(4): p. 239-57.
Elmore, S., Apoptosis: a review of programmed cell death. Toxicol
Pathol, 2007. 35(4): p. 495-516.
Kurosaka, K., et al., Silent cleanup of very early apoptotic cells by
macrophages. J Immunol, 2003. 171(9): p. 4672-9.
Savill, J. and V. Fadok, Corpse clearance defines the meaning of cell
death. Nature, 2000. 407(6805): p. 784-8.
88
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
Tesniere, A., et al., Immunogenic death of colon cancer cells treated with
oxaliplatin. Oncogene, 2010. 29(4): p. 482-91.
Zitvogel, L., et al., The anticancer immune response: indispensable for
therapeutic success? J Clin Invest, 2008. 118(6): p. 1991-2001.
Yuan, J. and G. Kroemer, Alternative cell death mechanisms in
development and beyond. Genes Dev, 2010. 24(23): p. 2592-602.
Hu, X. and Y. Xuan, Bypassing cancer drug resistance by activating
multiple death pathways--a proposal from the study of circumventing
cancer drug resistance by induction of necroptosis. Cancer Lett, 2008.
259(2): p. 127-37.
Kono, H. and K.L. Rock, How dying cells alert the immune system to
danger. Nat Rev Immunol, 2008. 8(4): p. 279-89.
Leist, M. and M. Jaattela, Four deaths and a funeral: from caspases to
alternative mechanisms. Nat Rev Mol Cell Biol, 2001. 2(8): p. 589-98.
Nicotera, P., M. Leist, and E. Ferrando-May, Intracellular ATP, a switch
in the decision between apoptosis and necrosis. Toxicol Lett, 1998. 102103: p. 139-42.
Clarke, P.G., Developmental cell death: morphological diversity and
multiple mechanisms. Anat Embryol (Berl), 1990. 181(3): p. 195-213.
Hitomi, J., et al., Identification of a molecular signaling network that
regulates a cellular necrotic cell death pathway. Cell, 2008. 135(7): p.
1311-23.
Gilbert, J.B., et al., Determination of lithium-ion distributions in
nanostructured block polymer electrolyte thin films by X-ray
photoelectron spectroscopy depth profiling. ACS Nano, 2015. 9(1): p.
512-20.
Anselmo, A.C., et al., Monocyte-mediated delivery of polymeric
backpacks to inflamed tissues: a generalized strategy to deliver drugs to
treat inflammation. J Control Release, 2015. 199: p. 29-36.
de Moraes, M.A., et al., Factors controlling the deposition of silk fibroin
nanofibrils during layer-by-layer assembly. Biomacromolecules, 2015.
16(1): p. 97-104.
Vercammen, D., et al., Inhibition of caspases increases the sensitivity of
L929 cells to necrosis mediated by tumor necrosis factor. J Exp Med,
1998. 187(9): p. 1477-85.
Mocarski, E.S., J.W. Upton, and W.J. Kaiser, Viral infection and the
evolution of caspase 8-regulated apoptotic and necrotic death pathways.
Nat Rev Immunol, 2012. 12(2): p. 79-88.
Vandenabeele, P., et al., Molecular mechanisms of necroptosis: an
ordered cellular explosion. Nat Rev Mol Cell Biol., 2010. 11(10): p. 70014. doi: 10.1038/nrm2970. Epub 2010 Sep 8.
Heuer, A. and O. Rubner, Optimizing the prediction process: from
statistical concepts to the case study of soccer. PLoS One, 2014. 9(9): p.
e104647.
Wu, Y.T., et al., zVAD-induced necroptosis in L929 cells depends on
autocrine production of TNFalpha mediated by the PKC-MAPKs-AP-1
pathway. Cell Death Differ, 2011. 18(1): p. 26-37.
Moretti, L., et al., Radiosensitization of solid tumors by Z-VAD, a pancaspase inhibitor. Mol Cancer Ther., 2009. 8(5): p. 1270-9. Epub 2009
May 5.
89
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
Voll, R.E., et al., Immunosuppressive effects of apoptotic cells. Nature,
1997. 390(6658): p. 350-1.
Vierling, F., et al., Surprising prenatal toxicity of epidermal lipoxygenase3. Placenta, 2014. 35(9): p. 776-9.
Matzinger, P., The danger model: a renewed sense of self. Science,
2002. 296(5566): p. 301-5.
Polak, R., et al., Sugar-mediated disassembly of mucin/lectin multilayers
and their use as pH-Tolerant, on-demand sacrificial layers.
Biomacromolecules, 2014. 15(8): p. 3093-8.
Srinivasan, S., et al., Quantification of feather structure, wettability and
resistance to liquid penetration. J R Soc Interface, 2014. 11(96): p.
20140287.
Rubner, Y., et al., Fractionated radiotherapy is the main stimulus for the
induction of cell death and of Hsp70 release of p53 mutated glioblastoma
cell lines. Radiat Oncol, 2014. 9(1): p. 89.
Kulzer, L., et al., Norm- and hypo-fractionated radiotherapy is capable of
activating human dendritic cells. J Immunotoxicol, 2014. 11(4): p. 328-36.
Frey, B., et al., Induction of abscopal anti-tumor immunity and
immunogenic tumor cell death by ionizing irradiation - implications for
cancer therapies. Curr Med Chem, 2012. 19(12): p. 1751-64.
Dong Xda, E., et al., High mobility group box I (HMGB1) release from
tumor cells after treatment: implications for development of targeted
chemoimmunotherapy. J Immunother, 2007. 30(6): p. 596-606.
Park, J.S., et al., Involvement of toll-like receptors 2 and 4 in cellular
activation by high mobility group box 1 protein. J Biol Chem, 2004.
279(9): p. 7370-7.
Apetoh, L., et al., Toll-like receptor 4-dependent contribution of the
immune system to anticancer chemotherapy and radiotherapy. Nat Med,
2007. 13(9): p. 1050-9.
Downing, A., J.A. Newton-Bishop, and D. Forman, Recent trends in
cutaneous malignant melanoma in the Yorkshire region of England;
incidence, mortality and survival in relation to stage of disease, 19932003. Br J Cancer, 2006. 95(1): p. 91-5.
Arnold-Schild, D., et al., Cutting edge: receptor-mediated endocytosis of
heat shock proteins by professional antigen-presenting cells. J Immunol,
1999. 162(7): p. 3757-60.
Multhoff, G., Activation of natural killer cells by heat shock protein 70. Int
J Hyperthermia, 2002. 18(6): p. 576-85.
Mariathasan, S., et al., Cryopyrin activates the inflammasome in
response to toxins and ATP. Nature, 2006. 440(7081): p. 228-32.
Coade, S.B. and J.D. Pearson, Metabolism of adenine nucleotides in
human blood. Circ Res, 1989. 65(3): p. 531-7.
Di Virgilio, F., Liaisons dangereuses: P2X(7) and the inflammasome.
Trends Pharmacol Sci, 2007. 28(9): p. 465-72.
Aymeric, L., et al., Tumor cell death and ATP release prime dendritic
cells and efficient anticancer immunity. Cancer Res, 2010. 70(3): p. 8558.
Ghiringhelli, F., et al., Activation of the NLRP3 inflammasome in dendritic
cells induces IL-1beta-dependent adaptive immunity against tumors. Nat
Med, 2009. 15(10): p. 1170-8.
90
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
Sabirov, R.Z., A.K. Dutta, and Y. Okada, Volume-dependent ATPconductive large-conductance anion channel as a pathway for swellinginduced ATP release. J Gen Physiol, 2001. 118(3): p. 251-66.
Hisadome, K., et al., Volume-regulated anion channels serve as an
auto/paracrine nucleotide release pathway in aortic endothelial cells. J
Gen Physiol, 2002. 119(6): p. 511-20.
Pellegatti, P., et al., A novel recombinant plasma membrane-targeted
luciferase reveals a new pathway for ATP secretion. Mol Biol Cell, 2005.
16(8): p. 3659-65.
Ohshima, Y., et al., gamma-Irradiation induces P2X(7) receptordependent ATP release from B16 melanoma cells. Biochim Biophys
Acta, 2010. 1800(1): p. 40-6.
Salomaa, S.I., et al., Editorial. Non-DNA targeted effects. Mutat Res,
2010. 687(1-2): p. 1-2.
Goodhead, D.T., Initial events in the cellular effects of ionizing radiations:
clustered damage in DNA. Int J Radiat Biol, 1994. 65(1): p. 7-17.
Ward, J.F., Radiation mutagenesis: the initial DNA lesions responsible.
Radiat Res, 1995. 142(3): p. 362-8.
Sinclair, W.K., Cyclic X-ray responses in mammalian cells in vitro. 1968.
Radiat Res, 2012. 178(2): p. Av112-24.
Scholch, S., et al., Radiotherapy combined with TLR7/8 activation
induces strong immune responses against gastrointestinal tumors.
Oncotarget, 2015. 6(7): p. 4663-76.
Vitale, M., et al., Effect of tumor cells and tumor microenvironment on
NK-cell function. Eur J Immunol, 2014. 44(6): p. 1582-92.
M, B.F., et al., Caco-2 cells permeability evaluation of nifuroxazide
derivatives with potential activity against methicillin-resistant
Staphylococcus aureus (MRSA). Drug Dev Ind Pharm, 2014: p. 1-7.
Ramirez, K., et al., Imipramine attenuates neuroinflammatory signaling
and reverses stress-induced social avoidance. Brain Behav Immun,
2015.
Deep, G. and R. Agarwal, New combination therapies with cell-cycle
agents. Curr Opin Investig Drugs, 2008. 9(6): p. 591-604.
Hill, G.J., 2nd, E.T. Krementz, and H.Z. Hill, Dimethyl triazeno imidazole
carboxamide and combination therapy for melanoma. IV. Late results
after complete response to chemotherapy (Central Oncology Group
protocols 7130, 7131, and 7131A). Cancer, 1984. 53(6): p. 1299-305.
Loo, T.L., et al., Mechanism of action and pharmacology studies with
DTIC (NSC-45388). Cancer Treat Rep, 1976. 60(2): p. 149-52.
Sanada, M., et al., Modes of actions of two types of anti-neoplastic
drugs, dacarbazine and ACNU, to induce apoptosis. Carcinogenesis,
2007. 28(12): p. 2657-63.
Zolzer, F., Radiation and/or hyperthermia sensitivity of human melanoma
cells after several days of incubation in media lacking serum or certain
serum components. Int J Radiat Oncol Biol Phys, 2000. 46(2): p. 491-7.
Bickels, J., et al., Coley's toxin: historical perspective. Isr Med Assoc J,
2002. 4(6): p. 471-2.
Nauts, H.C. and J.R. McLaren, Coley toxins--the first century. Adv Exp
Med Biol, 1990. 267: p. 483-500.
Hanson, D.F., Fever, temperature, and the immune response. Ann N Y
Acad Sci, 1997. 813: p. 453-64.
91
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
Takahashi, I., et al., Clinical application of hyperthermia combined with
anticancer drugs for the treatment of solid tumors. Surgery, 2002. 131(1
Suppl): p. S78-84.
Matsumoto, Y., et al., A possible mechanism for hyperthermic
radiosensitization mediated through hyperthermic lability of Ku subunits
in DNA-dependent protein kinase. Biochem Biophys Res Commun,
1997. 234(3): p. 568-72.
Franckena, M., et al., Long-term improvement in treatment outcome after
radiotherapy and hyperthermia in locoregionally advanced cervix cancer:
an update of the Dutch Deep Hyperthermia Trial. Int J Radiat Oncol Biol
Phys, 2008. 70(4): p. 1176-82.
Colombo, R., et al., Combination of intravesical chemotherapy and
hyperthermia for the treatment of superficial bladder cancer: preliminary
clinical experience. Crit Rev Oncol Hematol, 2003. 47(2): p. 127-39.
Overgaard, J., et al., Randomised trial of hyperthermia as adjuvant to
radiotherapy for recurrent or metastatic malignant melanoma. European
Society for Hyperthermic Oncology. Lancet, 1995. 345(8949): p. 540-3.
Bichay, T.J., M.M. Feeley, and G.P. Raaphorst, A comparison of heat
sensitivity, radiosensitivity and PLDR in four human melanoma cell lines.
Melanoma Res, 1992. 2(1): p. 63-9.
Schildkopf, P., et al., Radiation combined with hyperthermia induces
HSP70-dependent maturation of dendritic cells and release of proinflammatory cytokines by dendritic cells and macrophages. Radiother
Oncol., 2011. 101(1): p. 109-15. doi: 10.1016/j.radonc.2011.05.056.
Epub 2011 Jun 23.
Riccardi, C. and I. Nicoletti, Analysis of apoptosis by propidium iodide
staining and flow cytometry. Nat Protoc, 2006. 1(3): p. 1458-61.
Schleicher, U. and C. Bogdan, Generation, culture and flow-cytometric
characterization of primary mouse macrophages. Methods Mol Biol,
2009. 531: p. 203-24.
Lutz, M.B., et al., An advanced culture method for generating large
quantities of highly pure dendritic cells from mouse bone marrow. J
Immunol Methods, 1999. 223(1): p. 77-92.
Grossmayer, G.E., et al., IgG autoantibodies bound to surfaces of
necrotic cells and complement C4 comprise the phagocytosis promoting
activity for necrotic cells of systemic lupus erythaematosus sera. Ann
Rheum Dis., 2008. 67(11): p. 1626-32. doi: 10.1136/ard.2007.081828.
Epub 2007 Dec 28.
Geran, R.I., et al., Modified protocol for the testing of new synthetics in
the L1210 lymphoid leukemia murine model in the DR&D program, DCT,
NCI. Natl Cancer Inst Monogr, 1977(45): p. 151-3.
Kepp, O., et al., The immunogenicity of tumor cell death. Curr Opin
Oncol, 2009. 21(1): p. 71-6.
Anvekar, R.A., et al., Sensitization to the mitochondrial pathway of
apoptosis augments melanoma tumor cell responses to conventional
chemotherapeutic regimens. Cell Death Dis., 2012. 3:e420.(doi): p.
10.1038/cddis.2012.161.
Ko, S.H., et al., Optimizing a novel regional chemotherapeutic agent
against melanoma: hyperthermia-induced enhancement of temozolomide
cytotoxicity. Clin Cancer Res., 2006. 12(1): p. 289-97.
92
110.
111.
112.
113.
114.
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
Triantopoulou, S., et al., Radiotherapy in conjunction with superficial and
intracavitary hyperthermia for the treatment of solid tumors: survival and
thermal parameters. Clin Transl Oncol, 2012. 21: p. 21.
Kim, S., et al., In vivo developmental stages in murine natural killer cell
maturation. Nat Immunol, 2002. 3(6): p. 523-8.
Schnorrer, P., et al., The dominant role of CD8+ dendritic cells in crosspresentation is not dictated by antigen capture. Proc Natl Acad Sci U S
A, 2006. 103(28): p. 10729-34.
den Haan, J.M., S.M. Lehar, and M.J. Bevan, CD8(+) but not CD8(-)
dendritic cells cross-prime cytotoxic T cells in vivo. J Exp Med, 2000.
192(12): p. 1685-96.
Fisher, D.T., M.M. Appenheimer, and S.S. Evans, The two faces of IL-6
in the tumor microenvironment. Semin Immunol, 2014. 26(1): p. 38-47.
Sadanaga, N., et al., Local secretion of IFN-gamma induces an antitumor
response: comparison between T cells plus IL-2 and IFN-gamma
transfected tumor cells. J Immunother, 1999. 22(4): p. 315-23.
Khan, N., et al., The evolving role of radiation therapy in the
management of malignant melanoma. Int J Radiat Oncol Biol Phys.,
2011. 80(3): p. 645-54. doi: 10.1016/j.ijrobp.2010.12.071. Epub 2011 Apr
12.
Overgaard, J., et al., Hyperthermia as an adjuvant to radiation therapy of
recurrent or metastatic malignant melanoma. A multicentre randomized
trial by the European Society for Hyperthermic Oncology. Int J
Hyperthermia., 1996. 12(1): p. 3-20.
Wolter, K.G., et al., Therapeutic window for melanoma treatment
provided by selective effects of the proteasome on Bcl-2 proteins. Cell
Death Differ., 2007. 14(9): p. 1605-16. Epub 2007 Jun 1.
Kroemer, G., et al., Immunogenic Cell Death in Cancer Therapy. Annu
Rev Immunol, 2012. 12: p. 12.
Shiffman, M.L., et al., Clinical trial: the efficacy and safety of oral PF03491390, a pancaspase inhibitor - a randomized placebo-controlled
study in patients with chronic hepatitis C. Aliment Pharmacol Ther, 2010.
31(9): p. 969-78.
Christofferson, D.E. and J. Yuan, Necroptosis as an alternative form of
programmed cell death. Curr Opin Cell Biol., 2010. 22(2): p. 263-8. doi:
10.1016/j.ceb.2009.12.003. Epub 2010 Jan 4.
Zamai, L., et al., NK cells and cancer. J Immunol, 2007. 178(7): p. 40116.
Krysko, D.V., et al., Immunogenic cell death and DAMPs in cancer
therapy. Nat Rev Cancer, 2012. 12(12): p. 860-75.
Garcia-Calvo, M., et al., Inhibition of human caspases by peptide-based
and macromolecular inhibitors. J Biol Chem, 1998. 273(49): p. 32608-13.
Hirayama, H., et al., Contrasting effects of ghrelin and des-acyl ghrelin
on the lumbo-sacral defecation center and regulation of colorectal motility
in rats. Neurogastroenterol Motil, 2010. 22(10): p. 1124-31.
Takeuchi, O., K. Hoshino, and S. Akira, Cutting edge: TLR2-deficient and
MyD88-deficient mice are highly susceptible to Staphylococcus aureus
infection. J Immunol, 2000. 165(10): p. 5392-6.
Yu, M., et al., HMGB1 signals through toll-like receptor (TLR) 4 and
TLR2. Shock, 2006. 26(2): p. 174-9.
93
128.
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
Huang, B., et al., TLR signaling by tumor and immune cells: a doubleedged sword. Oncogene, 0000. 27(2): p. 218-224.
Srikrishna, G. and H.H. Freeze, Endogenous Damage-Associated
Molecular Pattern Molecules at the Crossroads of Inflammation and
Cancer. Neoplasia (New York, N.Y.), 2009. 11(7): p. 615-628.
Weiss, E.M., et al., Selected anti-tumor vaccines merit a place in
multimodal tumor therapies. Front Oncol., 2012. 2:132.(doi): p.
10.3389/fonc.2012.00132. Epub 2012 Oct 9.
Pinzon-Charry, A., T. Maxwell, and J.A. Lopez, Dendritic cell dysfunction
in cancer: A mechanism for immunosuppression. Immunol Cell Biol,
2005. 83(5): p. 451-461.
Tan, J.K. and H.C. O'Neill, Maturation requirements for dendritic cells in
T cell stimulation leading to tolerance versus immunity. J Leukoc Biol,
2005. 78(2): p. 319-24.
Seliger, B., F. Ruiz-Cabello, and F. Garrido, IFN inducibility of major
histocompatibility antigens in tumors. Adv Cancer Res, 2008. 101: p.
249-76.
Zaidi, M.R. and G. Merlino, The two faces of interferon-gamma in cancer.
Clin Cancer Res, 2011. 17(19): p. 6118-24.
Selleck, W.A., et al., IFN-gamma sensitization of prostate cancer cells to
Fas-mediated death: a gene therapy approach. Mol Ther, 2003. 7(2): p.
185-92.
Loi, S., et al., Prognostic and predictive value of tumor-infiltrating
lymphocytes in a phase III randomized adjuvant breast cancer trial in
node-positive breast cancer comparing the addition of docetaxel to
doxorubicin with doxorubicin-based chemotherapy: BIG 02-98. J Clin
Oncol, 2013. 31(7): p. 860-7.
Gooden, M.J., et al., The prognostic influence of tumour-infiltrating
lymphocytes in cancer: a systematic review with meta-analysis. Br J
Cancer, 2011. 105(1): p. 93-103.
Adams, S., et al., Prognostic value of tumor-infiltrating lymphocytes in
triple-negative breast cancers from two phase III randomized adjuvant
breast cancer trials: ECOG 2197 and ECOG 1199. J Clin Oncol, 2014.
32(27): p. 2959-66.
Frey, B., et al., Antitumor immune responses induced by ionizing
irradiation and further immune stimulation. Cancer Immunol Immunother,
2014. 63(1): p. 29-36.
Shinkai, Y., et al., RAG-2-deficient mice lack mature lymphocytes owing
to inability to initiate V(D)J rearrangement. Cell, 1992. 68(5): p. 855-67.
Cauwels, A., et al., Extracellular ATP drives systemic inflammation,
tissue damage and mortality. Cell Death Dis, 2014. 5: p. e1102.
Kawai, T., et al., Unresponsiveness of MyD88-deficient mice to
endotoxin. Immunity, 1999. 11(1): p. 115-22.
Kalialis, L.V., K.T. Drzewiecki, and H. Klyver, Spontaneous regression of
metastases from melanoma: review of the literature. Melanoma Res,
2009. 19(5): p. 275-82.
Postow, M.A., et al., Immunologic correlates of the abscopal effect in a
patient with melanoma. N Engl J Med, 2012. 366(10): p. 925-31.
Hiniker, S.M., D.S. Chen, and S.J. Knox, Abscopal effect in a patient with
melanoma. N Engl J Med, 2012. 366(21): p. 2035; author reply 2035-6.
94
146.
147.
148.
Grimaldi, A.M., et al., Abscopal effects of radiotherapy on advanced
melanoma patients who progressed after ipilimumab immunotherapy.
Oncoimmunology, 2014. 3: p. e28780.
Rubner, Y., et al., How does ionizing irradiation contribute to the
induction of anti-tumor immunity? Front Oncol., 2012. 2:75.(doi): p.
10.3389/fonc.2012.00075. Epub 2012 Jul 25.
Postow, M.A., et al., Immunologic correlates of the abscopal effect in a
patient with melanoma. N Engl J Med., 2012. 366(10): p. 925-31. doi:
10.1056/NEJMoa1112824.
95
Publications
Original articles:
- Werthmöller N, Frey B, Wunderlich R, Fietkau R, Gaipl US. Modulation of
radiochemoimmunotherapy-induced B16 melanoma cell death by the pan-caspase
inhibitor zVAD-fmk induces anti-tumor immunity in a HMGB1-, nucleotide, and T celldependent manner. Cell Death Dis. 2015, accepted
- Frey B, Stache C, Rubner Y, Werthmöller N, Schulz K, Sieber R, Semrau S, Rödel
F, Fietkau R, Gaipl US. Combined treatment of human colorectal tumor cell lines with
chemotherapeutic agents and ionizing irradiation can in vitro induce tumor cell death
forms with immunogenic potential. J Immunotoxicol. 2012 Jul;9(3):301-13
Reviews:
- Frey B, Rubner Y, Kulzer L, Werthmöller N, Weiss EM, Fietkau R, Gaipl US.
Antitumor immune responses induced by ionizing irradiation and further immune
stimulation. Cancer Immunol Immunother. 2014 Jan;63(1):29-36
- Rubner Y, Wunderlich R, Rühle PF, Kulzer L, Werthmöller N, Frey B, Weiss
EM, Keilholz L, Fietkau R, Gaipl US. How does ionizing irradiation contribute to
the induction of anti-tumor immunity? Front Oncol. 2012 Jul 25;2:75. doi:
10.3389/fonc.2012.00075. eCollection 2012
Short Publications:
- Finkel P, Meyer F, Bosl K , Werthmöller N, Mackensen A, Frey B , Gaipl U
Ullrich E. Bivalent role of NK cells in antitumor immunization strategies
combining X-ray and hyperthermia. ONCOLOGY RESEARCH AND
TREATMENT Volume: 37 Supplement: 5 Pages: 255-256 Meeting Abstract:
P780 Published: OCT 2014
- Werthmöller N, Frey B, Fietkau R, Gaipl US. Modulation of
radiochemoimmunotherapy-induced B16 melanoma cell death by zVAD-fmk
has immunostimulant anti-tumor effects. STRAHLENTHERAPIE UND
ONKOLOGIE Volume: 190 Supplement: 1 Pages: 76-76 Published: JUL 2014
- Frey B, Weber J, Kohn K, Werthmöller N, Brandt T, Lotter M, Rodel F,
Fietkau R, Gaipl US. Norm-and hypofractionated local irradiated of CT26 colon
cancer tumors of BALB/c mice induce a timely restricted infiltration of antigen
presenting cells and CD8+T cells. STRAHLENTHERAPIE UND ONKOLOGIE
Volume: 190 Supplement: 1 Pages: 85-85 Published: JUL 2014
- Werthmöller N, Frey B; Fietkau R; Gaipl US. The influence of standard
combinatory tumor treatments and the apoptosis inhibitor zVAD-fmk on B16
melanoma cell death forms and consecutively on macrophages and dendritic
cells. STRAHLENTHERAPIE UND ONKOLOGIE Volume: 189 Supplement: 1
Pages: 120-120 Published: MAY 2013
96
- Gabriel S; Werthmöller N, Weber J, Fietkau R, Gaipl US, Frey B. Time
kinetics of cell death of irradiated CT26 colorectal tumor cells after single and
fractionated irradiation - implications for combining radiotherapy with the
immune stimulator AnnexinA5. STRAHLENTHERAPIE UND ONKOLOGIE
Volume: 188 Supplement: 1 Pages: 128-128 Published: JUN 2012
Manuscript in preparation:
Patrick Finkel, Benjamin Frey, Friederike Mayer, Karina Bösl, Nina
Werthmöller, Andreas Mackensen, Udo Gaipl*, Evelyn Ullrich*. Bivalent role of
NK cells in antitumor reactions triggered by ionizing radiation in combination
with hyperthermia. Submission to Oncoimmunology
97