Sandy Adjemian

Autophagy-Dependent Anticancer Immune Responses Induced by Chemotherapeutic Agents in Mice

The release of adenosine triphosphate through autophagy can promote antitumor immune responses. Antineoplastic chemotherapies are particularly efficient when they elicit immunogenic cell death, thus provoking an anticancer immune response. Here we demonstrate that autophagy, which is often disabled in cancer, is dispensable for chemotherapy-induced cell death but required for its immunogenicity. In response to chemotherapy, autophagy-competent, but not autophagy-deficient, cancers attracted dendritic cells and T lymphocytes into the tumor bed. Suppression of autophagy inhibited the release of adenosine triphosphate (ATP) from dying tumor cells. Conversely, inhibition of extracellular ATP-degrading enzymes increased pericellular ATP in autophagy-deficient tumors, reestablished the recruitment of immune cells, and restored chemotherapeutic responses but only in immunocompetent hosts. Thus, autophagy is essential for the immunogenic release of ATP from dying cells, and increased extracellular ATP concentrations improve the efficacy of antineoplastic chemotherapies when autophagy is disabled.

Consensus guidelines for the detection of immunogenic cell death

Apoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named “immunogenic cell death” (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

An immunosurveillance mechanism controls cancer cell ploidy

Keeping Cancer Cells At Bay Cancer cells are often aneuploid; that is, they have an abnormal number of chromosomes. But to what extent this contributes to the tumorigenic phenotype is not clear. Senovilla et al. (p. 1678; see the Perspective by Zanetti and Mahadevan) found that tetraploidization of cancer cells can cause them to become immunogenic and thus aid in their clearance from the body by the immune system. Cells with excess chromosomes put stress on the endoplasmic reticulum, which leads to movement of the protein calreticulin to the cell surface. Calreticulin exposure in turn caused recognition of cancer cells in mice by the host immune system. Thus, the immune system appears to serve a protective role in eliminating hyperploid cells that must be overcome to allow unrestricted growth of cancer cells. Polyploid cancer cells trigger an immune response owing to proteins aberrantly exposed on their outer surfaces. Cancer cells accommodate multiple genetic and epigenetic alterations that initially activate intrinsic (cell-autonomous) and extrinsic (immune-mediated) oncosuppressive mechanisms. Only once these barriers to oncogenesis have been overcome can malignant growth proceed unrestrained. Tetraploidization can contribute to oncogenesis because hyperploid cells are genomically unstable. We report that hyperploid cancer cells become immunogenic because of a constitutive endoplasmic reticulum stress response resulting in the aberrant cell surface exposure of calreticulin. Hyperploid, calreticulin-exposing cancer cells readily proliferated in immunodeficient mice and conserved their increased DNA content. In contrast, hyperploid cells injected into immunocompetent mice generated tumors only after a delay, and such tumors exhibited reduced DNA content, endoplasmic reticulum stress, and calreticulin exposure. Our results unveil an immunosurveillance system that imposes immunoselection against hyperploidy in carcinogen- and oncogene-induced cancers.

Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress.

In contrast to other cytotoxic agents including anthracyclins and oxaliplatin (OXP), cisplatin (CDDP) fails to induce immunogenic tumor cell death that would allow to stimulate an anticancer immune response and hence to amplify its therapeutic efficacy. This failure to induce immunogenic cell death can be attributed to CDDPs incapacity to elicit the translocation of calreticulin (CRT) from the lumen of the endoplasmic reticulum (ER) to the cell surface. Here, we show that, in contrast to OXP, CDDP is unable to activate the protein kinase-like ER kinase (PERK)-dependent phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). Accordingly, CDDP also failed to stimulate the formation of stress granules and macroautophagy, two processes that only occur after eIF2α phosphorylation. Using a screening method that monitors the voyage of CRT from the ER lumen to the cell surface, we identified thapsigargin (THAPS), an inhibitor of the sarco/ER Ca2+-ATPase as a molecule that on its own does not stimulate CRT exposure, yet endows CDDP with the capacity to do so. The combination of ER stress inducers (such as THAPS or tunicamycin) and CDDP effectively induced the translocation of CRT to the plasma membrane, as well as immunogenic cell death, although ER stress or CDDP alone was insufficient to induce CRT exposure and immunogenic cell death. Altogether, our results underscore the contribution of the ER stress response to the immunogenicity of cell death.

Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy

The success of some chemo- and radiotherapeutic regimens relies on the induction of immunogenic tumor cell death and on the induction of an anticancer immune response. Cells succumbing to immunogenic cell death undergo specific changes in their surface characteristics and release pro-immunogenic factors according to a defined spatiotemporal pattern. This stimulates antigen presenting cells such as dendritic cells to efficiently take up tumor antigens, process them, and cross-prime cytotoxic T lymphocytes, thus eliciting a tumor-specific cognate immune response. Such a response can also target therapy-resistant tumor (stem) cells, thereby leading, at least in some instances, to tumor eradication. In this review, we shed some light on the molecular identity of the factors that are required for cell death to be perceived as immunogenic. We discuss the intriguing observations that the most abundant endoplasmic reticulum protein, calreticulin, the most abundant intracellular metabolite, ATP, and the most abundant non-histone chromatin-binding protein, HMGB1, can determine whether cell death is immunogenic as they appear on the surface or in the microenvironment of dying cells.

Cardiac glycosides exert anticancer effects by inducing immunogenic cell death.

Cardiac glycosides kill cancer cells in a way that stimulates the immune response. A Cancer Double Feature—3807 A traditional chemotherapeutic drug performs a one-act play: It enters and kills a dividing cancer cell and then takes its bow. However, some chemotherapeutics have a wider range—they not only kill individual cancer cells but also do so in such a way that the dead cells function as a vaccine that primes the immune system to attack other cancer cells. Menger et al. now identify cardiac glycosides as potent inducers of this so-called immunogenic cell death. Using fluorescence microscopy to detect the hallmarks of immunogenic cell death, the authors identified cardiac glycosides, such as the heart drug digoxin, as immunogenic cell death inducers. They then verified that these drugs had anticancer effects in mice with intact immune systems but not in mice that lacked functional immunity. Cancer cells that died from digoxin exposure then effectively functioned as a vaccine—stimulating the immune system so that growth of future cancers is prevented. Indeed, human cancer patients on chemotherapy who happened to be taking the cardiac glycoside digoxin to treat other medical conditions had improved overall survival compared with patients who were not taking these drugs. Although efficacy in cancer patients remains to be formally tested, cardiac glycosides may augment chemotherapeutic response—forcing cancer to bow out. Some successful chemotherapeutics, notably anthracyclines and oxaliplatin, induce a type of cell stress and death that is immunogenic, hence converting the patient’s dying cancer cells into a vaccine that stimulates antitumor immune responses. By means of a fluorescence microscopy platform that allows for the automated detection of the biochemical hallmarks of such a peculiar cell death modality, we identified cardiac glycosides (CGs) as exceptionally efficient inducers of immunogenic cell death, an effect that was associated with the inhibition of the plasma membrane Na+- and K+-dependent adenosine triphosphatase (Na+/K+-ATPase). CGs exacerbated the antineoplastic effects of DNA-damaging agents in immunocompetent but not immunodeficient mice. Moreover, cancer cells succumbing to a combination of chemotherapy plus CGs could vaccinate syngeneic mice against a subsequent challenge with living cells of the same type. Finally, retrospective clinical analyses revealed that the administration of the CG digoxin during chemotherapy had a positive impact on overall survival in cohorts of breast, colorectal, head and neck, and hepatocellular carcinoma patients, especially when they were treated with agents other than anthracyclines and oxaliplatin.

Trial watch: Prognostic and predictive value of the immune infiltrate in cancer.

Solid tumors are constituted of a variety of cellular components, including bona fide malignant cells as well as endothelial, structural and immune cells. On one hand, the tumor stroma exerts major pro-tumorigenic and immunosuppressive functions, reflecting the capacity of cancer cells to shape the microenvironment to satisfy their own metabolic and immunological needs. On the other hand, there is a component of tumor-infiltrating leucocytes (TILs) that has been specifically recruited in the attempt to control tumor growth. Along with the recognition of the critical role played by the immune system in oncogenesis, tumor progression and response to therapy, increasing attention has been attracted by the potential prognostic and/or predictive role of the immune infiltrate in this setting. Data from large clinical studies demonstrate indeed that a robust infiltration of neoplastic lesions by specific immune cell populations, including (but not limited to) CD8+ cytotoxic T lymphocytes, Th1 and Th17 CD4+ T cells, natural killer cells, dendritic cells, and M1 macrophages constitutes an independent prognostic indicator in several types of cancer. Conversely, high levels of intratumoral CD4+CD25+FOXP3+ regulatory T cells, Th2 CD4+ T cells, myeloid-derived suppressor cells, M2 macrophages and neutrophils have frequently been associated with dismal prognosis. So far, only a few studies have addressed the true predictive potential of TILs in cancer patients, generally comforting the notion that—at least in some clinical settings—the immune infiltrate can reliably predict if a specific patient will respond to therapy or not. In this Trial Watch, we will summarize the results of clinical trials that have evaluated/are evaluating the prognostic and predictive value of the immune infiltrate in the context of solid malignancies.

Cytoplasmic STAT3 represses autophagy by inhibiting PKR activity.

In a screen designed to identify novel inducers of autophagy, we discovered that STAT3 inhibitors potently stimulate the autophagic flux. Accordingly, genetic inhibition of STAT3 stimulated autophagy in vitro and in vivo, while overexpression of STAT3 variants, encompassing wild-type, nonphosphorylatable, and extranuclear STAT3, inhibited starvation-induced autophagy. The SH2 domain of STAT3 was found to interact with the catalytic domain of the eIF2α kinase 2 EIF2AK2, best known as protein kinase R (PKR). Pharmacological and genetic inhibition of STAT3 stimulated the activating phosphorylation of PKR and consequent eIF2α hyperphosphorylation. Moreover, PKR depletion inhibited autophagy as initiated by chemical STAT3 inhibitors or free fatty acids like palmitate. STAT3-targeting chemicals and palmitate caused the disruption of inhibitory STAT3-PKR interactions, followed by PKR-dependent eIF2α phosphorylation, which facilitates autophagy induction. These results unravel an unsuspected mechanism of autophagy control that involves STAT3 and PKR as interacting partners.

Chemotherapy-induced antitumor immunity requires formyl peptide receptor 1

How dying tumor cells get noticed Besides killing tumor cells directly, some chemotherapies, such as anthracyclines, also activate the immune system to kill tumors. Vacchelli et al. discovered that in mice, anthracycline-induced antitumor immunity requires immune cells to express the protein formyl peptide receptor 1 (FPR1). Dendritic cells (DCs) near tumors expressed especially high amounts of FPR1. DCs normally capture fragments of dying tumor cells and use them to activate nearby T cells to kill tumors, but DCs lacking FPR1 failed to do this effectively. Individuals with breast or colon cancer expressing a variant of FPR1 and treated with anthracyclines showed poor metastasis-free and overall survival. Thus, FPR1 may affect anti-tumor immunity in people, too. Science, this issue p. 972 Formyl peptide receptor 1 helps the immune system sense dying tumor cells. Antitumor immunity driven by intratumoral dendritic cells contributes to the efficacy of anthracycline-based chemotherapy in cancer. We identified a loss-of-function allele of the gene coding for formyl peptide receptor 1 (FPR1) that was associated with poor metastasis-free and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy. The therapeutic effects of anthracyclines were abrogated in tumor-bearing Fpr1−/− mice due to impaired antitumor immunity. Fpr1-deficient dendritic cells failed to approach dying cancer cells and, as a result, could not elicit antitumor T cell immunity. Experiments performed in a microfluidic device confirmed that FPR1 and its ligand, annexin-1, promoted stable interactions between dying cancer cells and human or murine leukocytes. Altogether, these results highlight the importance of FPR1 in chemotherapy-induced anticancer immune responses.

Surface-exposed calreticulin in the interaction between dying cells and phagocytes

Phagocytosis is essential for pathogen elimination and for the removal of apoptotic corpses, a process that has been long considered immunologically silent. The phagocytic uptake of apoptotic/necrotic cells involves a plethora of molecules, including immunoglobulins, lectins, components of the complement system (all of which act as opsonins), as well as the phospholipid phosphatidylserine (PS) and the endoplasmic reticulum chaperone calreticulin (CRT), both of which can be exposed on the surface of dying cells. For a long time, surface‐exposed CRT was believed to participate in phagocytosis, mostly as a (co)receptor for specific opsonins. Recently, this view has been challenged by the observations that, similar to PS, CRT acts as a facultative recognition ligand on apoptotic cells, and that cytotoxic agents such as anthracyclines induce the exposure of CRT on the surface of dying tumor cells, thereby generating an engulfment signal that stimulates the uptake of apoptotic corpses and the presentation of the corresponding antigens by dendritic cells. Here, we summarize the current knowledge on the role of CRT and CRT‐interacting proteins during corpse removal.