Lionel Apetoh

Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy

Conventional cancer treatments rely on radiotherapy and chemotherapy. Such treatments supposedly mediate their effects via the direct elimination of tumor cells. Here we show that the success of some protocols for anticancer therapy depends on innate and adaptive antitumor immune responses. We describe in both mice and humans a previously unrecognized pathway for the activation of tumor antigen–specific T-cell immunity that involves secretion of the high-mobility-group box 1 (HMGB1) alarmin protein by dying tumor cells and the action of HMGB1 on Toll-like receptor 4 (TLR4) expressed by dendritic cells (DCs). During chemotherapy or radiotherapy, DCs require signaling through TLR4 and its adaptor MyD88 for efficient processing and cross-presentation of antigen from dying tumor cells. Patients with breast cancer who carry a TLR4 loss-of-function allele relapse more quickly after radiotherapy and chemotherapy than those carrying the normal TLR4 allele. These results delineate a clinically relevant immunoadjuvant pathway triggered by tumor cell death.

Calreticulin exposure dictates the immunogenicity of cancer cell death.

Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.

Immunological aspects of cancer chemotherapy.

Accumulating evidence indicates that the innate and adaptive immune systems make a crucial contribution to the antitumour effects of conventional chemotherapy-based and radiotherapy-based cancer treatments. Moreover, the molecular and cellular bases of the immunogenicity of cell death that is induced by cytotoxic agents are being progressively unravelled, challenging the guidelines that currently govern the development of anticancer drugs. Here, we review the immunological aspects of conventional cancer treatments and propose that future successes in the fight against cancer will rely on the development and clinical application of combined chemo- and immunotherapies.

5-Fluorouracil Selectively Kills Tumor-Associated Myeloid-Derived Suppressor Cells Resulting in Enhanced T Cell–Dependent Antitumor Immunity

Myeloid-derived suppressor cells (MDSC) accumulate in the spleen and tumor bed during tumor growth. They contribute to the immune tolerance of cancer notably by inhibiting the function of CD8(+) T cells. Thus, their elimination may hamper tumor growth by enhancing antitumor T-cell functions. We have previously reported that some anticancer agents relied on T cell-dependent anticancer responses to achieve maximal efficacy. However, the effect of anticancer agents on MDSC has remained largely unexplored. In this study, we observed that gemcitabine and 5-fluorouracil (5FU) were selectively cytotoxic on MDSC. In vivo, the treatment of tumor-bearing mice with 5FU led to a major decrease in the number of MDSC in the spleens and tumor beds of animals whereas no significant effect on T cells, natural killer cells, dendritic cells, or B cells was noted. Interestingly, 5FU showed a stronger efficacy over gemcitabine to deplete MDSC and selectively induced MDSC apoptotic cell death in vitro and in vivo. The elimination of MDSC by 5FU increased IFN-gamma production by tumor-specific CD8(+) T cells infiltrating the tumor and promoted T cell-dependent antitumor responses in vivo. Altogether, these findings suggest that the antitumor effect of 5FU is mediated, at least in part, by its selective cytotoxic action on MDSC.

The Intestinal Microbiota Modulates the Anticancer Immune Effects of Cyclophosphamide

The Microbiota Makes for Good Therapy The gut microbiota has been implicated in the development of some cancers, such as colorectal cancer, but—given the important role our intestinal habitants play in metabolism—they may also modulate the efficacy of certain cancer therapeutics. Iida et al. (p. 967) evaluated the impact of the microbiota on the efficacy of an immunotherapy [CpG (the cytosine, guanosine, phosphodiester link) oligonucleotides] and oxaliplatin, a platinum compound used as a chemotherapeutic. Both therapies were reduced in efficacy in tumor-bearing mice that lacked microbiota, with the microbiota important for activating the innate immune response against the tumors. Viaud et al. (p. 971) found a similar effect of the microbiota on tumor-bearing mice treated with cyclophosphamide, but in this case it appeared that the microbiota promoted an adaptive immune response against the tumors. The gut microbiota promote the efficacy of several antineoplastic agents in mice. Cyclophosphamide is one of several clinically important cancer drugs whose therapeutic efficacy is due in part to their ability to stimulate antitumor immune responses. Studying mouse models, we demonstrate that cyclophosphamide alters the composition of microbiota in the small intestine and induces the translocation of selected species of Gram-positive bacteria into secondary lymphoid organs. There, these bacteria stimulate the generation of a specific subset of “pathogenic” T helper 17 (pTH17) cells and memory TH1 immune responses. Tumor-bearing mice that were germ-free or that had been treated with antibiotics to kill Gram-positive bacteria showed a reduction in pTH17 responses, and their tumors were resistant to cyclophosphamide. Adoptive transfer of pTH17 cells partially restored the antitumor efficacy of cyclophosphamide. These results suggest that the gut microbiota help shape the anticancer immune response.

Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells

Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.

The anticancer immune response: indispensable for therapeutic success?

Although the impact of tumor immunology on the clinical management of most cancers is still negligible, there is increasing evidence that anticancer immune responses may contribute to the control of cancer after conventional chemotherapy. Thus, radiotherapy and some chemotherapeutic agents, in particular anthracyclines, can induce specific immune responses that result either in immunogenic cancer cell death or in immunostimulatory side effects. This anticancer immune response then helps to eliminate residual cancer cells (those that fail to be killed by chemotherapy) or maintains micrometastases in a stage of dormancy. Based on these premises, in this Review we address the question, How may it be possible to ameliorate conventional therapies by stimulating the anticancer immune response? Moreover, we discuss the rationale of clinical trials to evaluate and eventually increase the contribution of antitumor immune responses to the therapeutic management of neoplasia.

Immunogenic death of colon cancer cells treated with oxaliplatin

Both the pre-apoptotic exposure of calreticulin (CRT) and the post-apoptotic release of high-mobility group box 1 protein (HMGB1) are required for immunogenic cell death elicited by anthracyclins. Here, we show that both oxaliplatin (OXP) and cisplatin (CDDP) were equally efficient in triggering HMGB1 release. However, OXP, but not CDDP, stimulates pre-apoptotic CRT exposure in a series of murine and human colon cancer cell lines. Subcutaneous injection of OXP-treated colorectal cancer (CRC), CT26, cells induced an anticancer immune response that was reduced by short interfering RNA-mediated depletion of CRT or HMGB1. In contrast, CDDP-treated CT26 cells failed to induce anticancer immunity, unless recombinant CRT protein was absorbed into the cells. CT26 tumors implanted in immunocompetent mice responded to OXP treatment in vivo, and this therapeutic response was lost when CRT exposure by CT26 cells was inhibited or when CT26 cells were implanted in immunodeficient mice. The knockout of toll-like receptor 4 (TLR4), the receptor for HMGB1, also resulted in a deficient immune response against OXP-treated CT26 cells. In patients with advanced (stage IV, Duke D) CRC, who received an OXP-based chemotherapeutic regimen, the loss-of-function allele of TLR4 (Asp299Gly in linkage disequilibrium with Thr399Ile, reducing its affinity for HMGB1) was as prevalent as in the general population. However, patients carrying the TLR4 loss-of-function allele exhibited reduced progression-free and overall survival, as compared with patients carrying the normal TLR4 allele. In conclusion, OXP induces immunogenic death of CRC cells, and this effect determines its therapeutic efficacy in CRC patients.

A novel dendritic cell subset involved in tumor immunosurveillance

The interferon (IFN)-γ–induced TRAIL effector mechanism is a vital component of cancer immunosurveillance by natural killer (NK) cells in mice. Here we show that the main source of IFN-γ is not the conventional NK cell but a subset of B220+Ly6C− dendritic cells, which are atypical insofar as they express NK cell-surface molecules. Upon contact with a variety of tumor cells that are poorly recognized by NK cells, B220+NK1.1+ dendritic cells secrete high levels of IFN-γ and mediate TRAIL-dependent lysis of tumor cells. Adoptive transfer of these IFN-producing killer dendritic cells (IKDCs) into tumor-bearing Rag2−/−Il2rg−/− mice prevented tumor outgrowth, whereas transfer of conventional NK cells did not. In conclusion, we identified IKDCs as pivotal sensors and effectors of the innate antitumor immune response.

The interaction between HMGB1 and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy.

Summary: For the last four decades, the treatment of cancer has relied on four treatment modalities, namely surgery, radiotherapy, cytotoxic chemotherapy, and hormonotherapy. Most of these therapies are believed to directly attack and eradicate tumor cells. The emerging concept that cancer is not just a disease of a tissue or an organ but also a host disease relies on evidence of tumor‐induced immunosuppression and polymorphisms in genes involved in host protection against tumors. This theory is now gaining new impetus, based on our recent data showing that optimal therapeutic effects require the immunoadjuvant effect of tumor cell death induced by cytotoxic anticancer agents. Here, we show that the release of the high mobility group box 1 protein (HMGB1) by dying tumor cells is mandatory to license host dendritic cells (DCs) to process and present tumor antigens. HMGB1 interacts with Toll‐like receptor 4 (TLR4) on DCs, which are selectively involved in the cross‐priming of anti‐tumor T lymphocytes in vivo. A TLR4 polymorphism that affects the binding of HMGB1 to TLR4 predicts early relapse after anthracycline‐based chemotherapy in breast cancer patients. This knowledge may be clinically exploited to predict the immunogenicity and hence the efficacy of chemotherapeutic regimens.