Download Capitalizing on the Immunogenicity of Dying Tumor Cells

Molecular Pathways
Capitalizing on the Immunogenicity of Dying Tumor Cells
Catia Fonseca and Glenn Dranoff
Abstract
Cancer cell death occurs continually during tumor development and progression, whereas the
selective killing of surviving cancer cells remains the primary objective of antineoplastic treatments. Recent insights into the immunologic consequences of cancer cell death have begun to
elucidate the ways in which host antitumor immunity is shaped during cancer pathogenesis and
then modulated by therapeutic intervention. Dying tumor cells evoke a range of host responses,
dependent in part upon the mode of cell death, which may either impede or foster additional
immune-mediated cancer destruction. Within the tumor microenvironment, the capture of
apoptotic tumor cells by macrophages and dendritic cells may trigger tolerance networks that
contribute to immune suppression, whereas the uptake of necrotic cancer cells may engender
inflammatory pathways that fuel antitumor cytotoxicity. Milk fat globule epidermal growth factor
8, a phosphatidylserine-binding protein, and MHC class I chain ^ related protein A, an NKG2D
ligand, play key roles in these competing outcomes. A deeper understanding of the mechanisms
underlying the immunogenicity of dying cells informs the crafting of strategies that exploit endogenous or treatment-induced cancer cell death as the basis for stimulating sustained host antitumor
cytotoxic reactions.
Background
Cancer cells devise a myriad of strategies to antagonize cell
death, but these countermeasures prove to be imperfect (1).
Although the economies of cell proliferation and accumulation
dominate cell demise during disease progression, pathologic
examination of established cancers typically reveals evidence
for both single cell and zonal areas of tumor death. Mixtures
of apoptosis, necrosis, and autophagy frequently may be
discerned histologically. These varying forms of cellular
destruction reflect the failure of tumors to cope adequately
with the challenging burdens of cellular stress (2). Such
ongoing insults include DNA damage and genomic instability;
perturbed signal transduction and transcriptional networks;
the unfolded protein response, hypoxia, nutrient deprivation,
immunologic attack; and likely many others (3). Notwithstanding the intense effort directed toward unraveling the
mechanisms underlying cancer cell death, much less attention
have been devoted to understanding the host response to
tumor cell demise.
A spectrum of host responses to cell death empowers the
immune system with the dual abilities to monitor the integrity
of healthy tissues and to respond rapidly to tissue injury (4).
Authors’Affiliation: Department of Medical Oncology and CancerVaccine Center,
Dana-Farber Cancer Institute and Department of Medicine, Brigham and Women’s
Hospital and Harvard Medical School, Boston, Massachusetts
Received 11/29/07; revised 1/3/08; accepted 1/4/08.
Requests for reprints: Glenn Dranoff, Dana-Farber Cancer Institute, Dana 520C,
44 Binney Street, Boston, MA 02115. Phone: 617-632-5051; Fax: 617-632-5167;
E-mail: glenn_dranoff@ dfci.harvard.edu.
F 2008 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-07-2245
www.aacrjournals.org
Under steady-state conditions, the capture of apoptotic cells
serves as an immunoregulatory event that helps maintain
tolerance, whereas the detection of necrotic cells evokes an
inflammatory cascade that mobilizes effector responses. However, the development and progression of cancer typically
involves both apoptotic and necrotic forms of cell death. What
are the implications of this mixed pattern of cellular demise for
the induction of antitumor immunity?.
The crafting of genetic and biochemical strategies to
characterize cancer antigens has yielded the insight that most
patients mount tumor-specific T cell and antibody reactions
(5). Because cancer cells typically fail to express the repertoire
of costimulatory molecules required for stimulating T cells
directly, adaptive immune recognition is likely accomplished
through an indirect mechanism that exploits the capture of
dying tumor cells by host mononuclear phagocytes (6). The
concurrent acquisition of apoptotic and necrotic cells by
dendritic cells and macrophages in the tumor microenvironment, however, might result in a set of conflicting signals that
drive tolerance and effector responses in parallel. Under these
conditions, a form of immunologic ‘‘gridlock’’ may ensue, in
which neither the regulatory nor activation pathways achieve
full control.
Histopathologic analysis typically reveals the presence of
various immune cells within or circumscribing tumors,
indicative of a nascent host response, whereas specific
morphologic patterns of reactivity have been linked with
varying clinical outcomes. Although dense intratumor T-cell
infiltrates are rare, they are tightly associated with a decreased
incidence of recurrent disease and reduced mortality in
multiple cancer types, including malignant melanoma, follicular lymphoma, and carcinomas of the colon, ovary, liver, and+
kidney (7 – 13). The combination
of brisk intratumoral CD8
+
T cells and rare FoxP3 Tregs connotes a particularly favorable
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Clin Cancer Res 2008;14(6) March 15, 2008
Molecular Pathways
prognostic index, suggesting that the balance of effector and
regulatory cells might modulate disease course in some
patients.
In murine models of methlycholanthrene-induced carcinogenesis, endogenous host responses contribute to tumor
dormancy (14). In this state of immune equilibrium, the
coordinated activities of CD4+ and CD8+ T cells, IFN-g, and
interleukin (IL)-12 mediate disease control. The attainment
of immune equilibrium in humans is highlighted through
multiple case reports of the inadvertent transplantation of
metastatic melanoma to recipients of renal allografts who were
maintained on immunosuppression; tumors arose in these
patients notwithstanding intervals as long as 16 years from
the putative surgical cures of early stage melanomas in the
donors (15).
This dynamic of host reactivity raises the possibility that
antitumor immunity might contribute to the therapeutic
efficacy of some conventional cancer treatments. Consistent
with this idea, the durability of clinical responses to
oncologic therapy is closely linked to the presence of brisk
intratumoral T-cell infiltrates in diverse cancers (6). Perhaps
antineoplastic treatments modulate the mixture of apoptotic
and necrotic cell death within tumors, thereby altering the
balance of effector and regulatory T cells in favor of more
intense and sustained protection. How might this be
achieved?
The principles governing the host reaction to the turnover of
normal cells provide a useful framework for considering the
consequences of cancer cell destruction (4). The programmed
death of normal cells (apoptosis) involves a well-orchestrated
sequence of events that culminates in the formation of surface
blebs and the sequestration of intracellular components with
the potential to effectuate lysis (16). Apoptosis is a critical
feature of tissue homeostasis, and the orderly detection and
clearance of cellular corpses is essential for proper tissue
remodeling and renewal. Defects in the management of early
apoptotic cells may allow the resurrection of some cells that
otherwise were committed to die, thereby increasing the risk
of transformation (17, 18). Various parenchymal cells seem
capable of ingesting their dying neighbors, but the host
immune system is especially well-adapted for scavenging
cellular remnants and debris (19).
The principal immune components charged with this task
are macrophages and dendritic cells, the key mononuclear
phagocytes. These antigen-presenting cells use a multiplicity
of receptor/ligand pairs to recognize and engulf apoptotic
cells (20). Whereas scavenger receptors, complement components, and collectins all play important roles in clearance,
a primary ‘‘eat me’’ signal seems to be phosphatidylserine.
The centrality of this detection scheme is underscored by
its conservation throughout metazoans, including the paradigmatic model system, Caenorhabditis elegans (21). Phosphatidylserine is normally retained within the inner leaflet
of the surface membrane through the tonic activities of a
transporter/flippase, but upon execution of programmed
death, this lipid is translocated to the exterior envelope of
the cell (22).
Mononuclear phagocytes accomplish phosphatidlyserinebased uptake of apoptotic cells through several secreted and
surface proteins (Fig. 1). Milk fat globule epidermal growth
factor 8 (MFG-E8) and the closely related molecule Del-1 are
Clin Cancer Res 2008;14(6) March 15, 2008
released into the tissue microenvironment, wherein their
discoidin domains bind phosphatidylserine on cellular corpses. These are then internalized through the binding of RGD
sequences, encoded in the epidermal growth factor domains
of MFG-E8 and Del-1, to the avh3 and avh5 integrins
expressed on the phagocyte surface (23, 24). A second set of
opsonins that similarly bind phosphatidylserine are growth
arrest – specific gene 6 and protein S, although these secreted
proteins promote phagocyte engulfment through interactions
with the tyro family of receptor tyrosine kinases (Mer, Axl,
and Tyro-3; refs. 25, 26). The membrane proteins TIM-1 and
TIM-4 additionally detect phosphatidlyserine and contribute
to the uptake of apoptotic cells by phagocytes (27). The
essential role of these pathways in tissue homeostasis is
underscored by the development of persistent inflammation
in mice harboring mutations in these gene products (28).
Indeed, the efficient clearance of cellular corpses by mononuclear phagocytes results in the elaboration of immunosuppressive mediators, particularly transforming growth factor
h and IL-10, which empower dendritic cells and macrophages
to stimulate FoxP3+ expressing and other regulatory T-cell
subsets that support the maintenance of immune tolerance
(29, 30).
In contrast to the orderly clearance of apoptotic cells, necrotic
cell death triggers a vigorous inflammatory response that is
aimed at restraining the cause of tissue damage (31).
Phagocytes that encounter necrotic debris produce a broad
range of proinflammatory cytokines such as IL-1h, IL-6, IL-12,
and IL-23. These in turn engage a network of soluble and
membrane factors that orchestrate the recruitment and activation of multiple innate and adaptive immune elements that
control invading pathogens and noxious agents (Fig. 1).
Concurrently, the elaboration of regulatory molecules such as
transforming growth factor h and IL-10 is attenuated, thereby
temporarily dampening the immunosuppressive circuits, which
facilitates the mobilization and evolution of the effector
cascade.
The triggering of this inflammatory response by mononuclear phagocytes requires the MyD88 adapter protein, which
functions in both IL-1 and Toll-like receptor signaling (32). The
latter pattern-recognition molecules detect not only microbial
signatures but also the biochemical sequelae of necrotic cell
death, which include the spillage of cytoplasmic heat shock
proteins and the release of nuclear HMGB-1, a normal
component of chromatin (33). The phagocyte ingestion of
damaged nucleic acids may further provoke a cytosolic
recognition system for DNA that engenders the production of
inflammatory cytokines, particularly type I IFNs (34). The
engulfment of uric acid generated in foci of necrosis may also
engage the inflammasome, a cytoplasmic protein complex that
stimulates IL-1h secretion, which in turn triggers the nuclear
factor-nB – and Jun kinase – signaling cascades to elicit the
transcription of multiple inflammatory cytokine genes (35).
The surface exposure of calreticulin, a protein usually resident
in the endoplasmic reticulum, may additionally serve as a key
signal that activates macrophages and dendritic cells to
propagate the effector response (36).
How do these insights into the host response to apoptotic
and necrotic death inform our understanding of the immune
consequences of oncologic treatments? Recent work indicates
that some forms of cytotoxic cancer therapy, particularly
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Capitalizing on the Immunogenicity of DyingTumor Cells
Fig. 1. The uptake of dying tumor cells by
macrophages and dendritic cells may evoke
either tolerance or protective immunity (top).
The recognition of phosphatidylserine on apoptotic
cells promotes the production of immunosuppressive
mediators, such as transforming growth factor h
and IL-10, and the stimulation of regulatoryTcells.
MFG-E8, Del-1, growth arrest ^ specific gene 6,
protein S,TIM-1, and TIM-4 bind phosphatidylserine
that is mobilized to the exterior of the cell membrane
during programmed cell death. Phagocyte
engulfment is accomplished through avh3 and avh5
integrins and Tyro kinase family members (Tyro-3,
Axl, and Mer; bottom). Immunogenic cell death
evokes the production of inflammatory cytokines
and enhances antitumor cytotoxicity. HMGB-1, uric
acid, heat shock proteins, and calreticulin trigger
dendritic cell maturation, cross-presentation of
captured tumor antigens, and CD8+ T-cell expansion.
DNA damage up-regulates MICA, which triggers
NKG2D-dependent cytotoxicity in innate and
adaptive lymphocytes.
radiation, anthracyclines, and platinum-based compounds,
induce an immunogenic form of cell death characterized by
the surface mobilization of calreticulin and the extracellular
release of the nuclear protein HMGB1 (33, 36). Together,
these modifications activate dendritic cells, through a Toll-like
receptor – 4 and MyD88-dependent pathway, to capture and
cross-present efficiently tumor cell debris to evoke cytotoxic
T-cells responses. Variants of Toll-like receptor – 4 with high
avidity for HMGB-1 are associated with superior clinical
outcomes after anthracycline-based adjuvant chemotherapy
in women with early stage breast carcinoma. Tumor cell
immunogenicity may be further augmented through the
induction of DNA damage that generates novel protein
epitopes that provoke T-cell recognition (37). Cytotoxic
therapies may additionally compromise regulatory T-cell
circuits through a reduction in antigen load and the modification of lymphocyte subset dynamics during homeostatic
expansion (38).
Oncologic treatments may also affect the expression of
NKG2D ligands, which are induced on the surface of tumor
cells in response to DNA damage (39). The human NKG2D
www.aacrjournals.org
ligands include MICA, a MHC class I – related molecule,
the closely related MICB, and five UL16-binding proteins
(40). These gene products manifest limited expression in
healthy tissues but may be up-regulated in response to
double-stranded DNA breaks through a pathway that involves
ATM, ATR, CHK-1, and CHK-2 (39). Ligand-triggered NKG2D
activation in natural killer cells, gy-T cells, and CD8+ ah-T
cells culminates in potent cytotoxicity (41). In murine models,
the engineered expression of NKG2D ligands on tumors provokes rejection in wild-type hosts through the coordinated
activities of natural killer cells, CD8+ T lymphocytes, and
perforin, whereas the administration of anti-NKG2D blocking
antibodies increases susceptibility to chemical carcinogens
(42 – 44). Notwithstanding the ability of NKG2D signaling to
engender innate and adaptive antitumor responses, immune
escape is frequently accomplished in patients through the
shedding of MICA from tumor cells, which is mediated in part
through the protein disulfide isomerase ERp5 (45). Shedding
of MICA elicits the down-regulation of surface NKG2D,
resulting in impaired cytotoxic function and tumor progression (46).
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Molecular Pathways
Fig. 2. Antibodies to MICA and MFG-E8 may enhance the immunogenicity of dying tumor cells (top). MICA is shed from the surface of tumor cells through a
mechanism that involves the protein disulfide isomerase ERp5, whereupon soluble MICA evokes down-regulation of NKG2D on innate and adaptive cytotoxic lymphocytes.
Anti-MICA antibodies may complex shed ligand, restoring NKG2D levels and engendering protective immunity (bottom). Anti ^ MFG-E8 antibodies block
phosphatidlyserine-based uptake of apoptotic cells by antigen-presenting cells, resulting in the enhanced induction of antitumor CD8+ cytotoxic T cells and the inhibition
of FoxP3+ regulatory T cells.
Clinical/Translational Advances
If host immunity participates in the response to conventional
oncologic treatments, can induced tumor cell death be rendered
more immunogenic? In this context, we found that some
patients who achieved clinical responses to immunotherapy with
granulocyte macrophage colony-stimulating factor – secreting
tumor cell vaccines or blockade of CTL-associated antigen
4 – generated high titer antibodies against MICA and ERp5
(ref. 47).1 These humoral reactions antagonized the immunosuppressive effects of shedding of MICA and restored innate
1
Fonseca et. al, submitted.
Clin Cancer Res 2008;14(6) March 15, 2008
and adaptive cytotoxicity. The antibodies also promoted the
cross-presentation of ingested tumor antigens by dendritic cells
and thereby augmented antitumor cellular immunity. Together, these findings raise the possibility that therapeutic
monoclonal antibodies against MICA and ERp5 might be
useful for cancer treatment, especially in combination with
DNA-damaging agents that enhance NKG2D ligand expression
(Fig. 2).
Will the concurrent uptake of apoptotic cells limit the ability
of these strategies to enhance tumor cell immunogenicity?
MFG-E8 and perhaps other phosphatidylserine-binding proteins are produced in the tumor microenvironment, where they
may promote Treg expansion and local immunosuppression
(30). Inhibiting the capture of dying cells through these
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Capitalizing on the Immunogenicity of DyingTumor Cells
pathways might therefore attenuate maintenance of the local
tolerance networks (Fig. 2). In a therapeutic model of
established melanoma in mice, the combination of granulocyte
macrophage colony-stimulating factor – secreting tumor cell
vaccines with a dominant-negative MFG-E8 mutant, which
blocks phosphatidylserine-based phagocytosis (48), effectuated
the complete regressions of established tumors in the absence
of significant toxicities. The therapeutic immunity involved the
inhibition of Tregs and the concurrent expansion of CD8+
cytotoxic T cells. Thus, systemic administration of anti – MFGE8 antibodies or other approaches that mask phosphatidylserine
might complement strategies that trigger immunogenic cell
death. Furthermore, the gene products involved in the clearance
of apoptotic cells play decisive roles in tumor progression
through fostering angiogenesis, matrix metalloproteinase
production, epithelial-to-mesenchymal transformation, and
epithelial cell migration (49, 50). Thus, targeting these pathways might not only stimulate tumor immunity but also
antagonize pathogenic tumor mechanisms.
Conclusions
Accumulating evidence indicates that the clinical activity
of many cancer therapies involves the interplay of tumor
cell autonomous effects and host immunity. An improved
understanding of the host response to dying tumor cells
provides compelling opportunities to render existing oncologic treatments more efficacious and to develop novel
schemes to intensify endogenous antitumor reactivity.
Analogous to the way in which an intact host response is
required for antimicrobial agents to achieve pathogen
control in infectious diseases, protective antitumor immunity will likely synergize with multiple forms of cancer
therapy. Paradoxically, the critical role of genomic instability in driving tumor pathogenesis and the evolution of
drug-resistant variants renders cancer cells highly amenable
to immune recognition. Perhaps this vulnerability may now
be actualized through capitalizing on the host response to
death.
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