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Marcin Cebula
02.11.2009
Hallmarks of cancer
METASTASIS
 Final stage in tumor progression → responsible for 90 % death
associated with solid tumors
 Process of metastasis is, complex but can be simplified to certain
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steps:
Local invasion
Intravasation
Survival in circulation
Extravasation
Colonisation
 Processes depends on genetic, epigenetic alterations, and tumor
microenvironment
METASTASIS
Loss of E-cadherin
MMP
MMP
HOW CANCER CELLS AFFECT
THE INFLAMMATORY MICROENVIROMENT
HOW THIS INFLUENCES ON METASTASIS
 Progression and metastasis – correlated with presence of infitratates of
myeloid and lymphoid cells
 Do the cancer cells secrete factors that directly activate myeloid cells to
produce tumor–promoting cytokines?
mRNA
Incubation of bone marrow–derived macrophages with
SF conditioned media from different cancer cell lines
Free of IL-6 & TNF
LLC – Lewis lung carcinoma
LCM – Lewis lung carcinoma conditioned medium
Metastatogenic function of LLC induced cytokines
(IL-6, TNF-)
LLC cells injected to IL-6-/- or TNF--/- knockout mice and WT
IL-6-/- little differences to WT were shown
IL-6 is not important for LLC metastasis
TNF- is responsible for metastasis
Which TLR are involved in sensing LCM components
 Bone marrow M from mice deficient in TLR2, TLR3, TLR4, TLR5 or
their adaptors (Myd88 and TRIF) were incubated with LCM.
IL-6 production was examined as BMDM activation marker.
Signal from LCM is recognized by TLR2 / Myd88
What is the co-receptor involved in TLR2 signaling
Bacterial lipoprotein
analog,to rule out
bacterial lipoprotein
contamination
TLR2 uses TLR6 or CD14 as co-receptors
In vivo role of TLR2 after inoculation LLC in WT and Tlr2-/mice on cytokine expression
Contribution of TLR2 signaling to LLC metastatogenesis
CD11b/ are
myeloid cells
markers
WT but not Tlr-/- lungs showed clusters with dsRED-LLC with
adjacent myeloid cells
Contribution of TLR2 signaling to survival
after inoculation LLC in WT and Tlr2-/- mice
TLR2-/- mice demonstrate higher survival and less
metastasis in comparison to WT
TLR2 is acting on BMD cells
WT mice reconstituded with WT BM (WT/WT) or
Tlr2-/- BM (WT/ Tlr2-/-)
(WT/Tlr2-/-) – improved survival
Clear suggestion that LCM contains
TLR2 activating factors
Additionally to LLC inoculation
in WT/WT or WT/Tlr2-/-, serum
free medium SFM or LLC
conditioned medium was
injected i.p
Identification of factors in LCM in terms of their
ability to induce IL-6
 LCM was seperated on mono-Q anion echange column
 Fractions inducing IL-6 were colected and separated on Superdex 200
sizing column
 Fractions inducing IL-6 eluted in high molecular fractions and contained
several polypeptides > 200 kDa
 Fractions were pooled deglycosylated and subjected to MS
Identified:
Peptides from extracellular
matrix proteins:
-versican V1
-laminin-1
-thrombospondin 2
-procollagen type III-1
Which of identified peptides are factors
inducing inflammatory cytokines
BMDM incubated with LCM
and respective antibodies to
identified peptides in LCM
-procollagen type III-1
-versican V1
-laminin-1
Role of indetified peptides in metastatogenesis
shRNA silencing
Silenced LLC cell lines
Silenced LLC cell lines injected i.v to mice
Not silenced versican
Silenced LLC cell lines
injected s.c to mice
What is versican V1 ???
Versican is able to regulate many cellular procesess including:
-adhesion
-proliferation
-apoptosis
-invasion via negatively-charged side chains
Versican expression is elevated in cancer cells:
-brain tumors, melanomas, osteosarcomas, lymphomas, breast,
prostate, colon, lung, pancreatic, oral, and ovarian cancers
-elevated levels of versican are associated with cancer relapse and poor prognosis
Confirmation of proinflamatory and prometastatic
function of versican
 Low metastatic variant of LLC
–
P29-LLC
 P29-LLC conditioned medium do not induce IL-6 in BMDM
 P29-LLC conditioned medium contain little versican
 Ectopic expression of human versican increased
How versican activates macrophages
 His-tagged versican V1 – purified
on Ni-chelate column
 Immunoprecipitation with versican
specific antibody
 Lysats from Raw264.7
macrophages incubated or not with
LCM
Summary
 Versican secretion is by LLC cells is necessary for metastatic spread to
lung, liver and adrenal gland
 Process depends on TLR2-mediated myeloid cell activation and TNF-,
(TNF- can suppress apoptosis of cancer cells, stimulate their proliferation through NFB activation, it increases vascular permeability enhancing intra and extravasations of
cancer cells)
 Response to versican requires co-receptor TLR6
 Interaction of TLR2 and versican can be direct or mediated by versican
ligand – hyaluronian which fragments can activate macropgagesTLR2
as well
New point for anti-metastatic intervention???
Metastases
development
Versican V1
Versican
binds to
TLR2 on
Macrophages
TNF-
Thank you for your attention