Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells.

The therapeutic efficacy of anthracyclines relies on antitumor immune responses elicited by dying cancer cells. How chemotherapy-induced cell death leads to efficient antigen presentation to T cells, however, remains a conundrum. We found that intratumoral CD11c(+)CD11b(+)Ly6C(hi) cells, which displayed some characteristics of inflammatory dendritic cells and included granulomonocytic precursors, were crucial for anthracycline-induced anticancer immune responses. ATP released by dying cancer cells recruited myeloid cells into tumors and stimulated the local differentiation of CD11c(+)CD11b(+)Ly6C(hi) cells. Such cells efficiently engulfed tumor antigens in situ and presented them to T lymphocytes, thus vaccinating mice, upon adoptive transfer, against a challenge with cancer cells. Manipulations preventing tumor infiltration by CD11c(+)CD11b(+)Ly6C(hi) cells, such as the local overexpression of ectonucleotidases, the blockade of purinergic receptors, or the neutralization of CD11b, abolished the immune system-dependent antitumor activity of anthracyclines. Our results identify a subset of tumor-infiltrating leukocytes as therapy-relevant antigen-presenting cells.

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.

[Contributions of immunogenic cell death to the efficacy of anticancer chemotherapy]. / Contribution de la mort immunogène à l'efficacité de la chimiothérapie anticancéreuse.

Immunogenic cell death, characterized by calreticulin exposure on the surface of the dying cell, release of the nuclear protein high mobility group box 1 (HMGB1), and release of ATP, enables stimulation of the immune system. We outlined the importance of this kind of cell death for the success of some anticancer chemotherapies. However, defects in the immunogenic cell death signalling pathway can lead to therapeutic failure, apparently because anticancer immune responses must contribute to the efficacy of chemotherapeutic regimens. These defects can be related to the therapy, the tumour cell, the host or the tumour-host interface. It is necessary to characterize these defects to restore and improve the efficacy of anticancer chemotherapies.

Synthetic induction of immunogenic cell death by genetic stimulation of endoplasmic reticulum stress.

Cis-diamminedichloridoplatinum(II) (CDDP), commonly referred to as cisplatin, is a chemotherapeutic drug used for the treatment of a wide range of solid cancers. CDDP is a relatively poor inducer of immunogenic cell death (ICD), a cell death modality that converts dying cells into a tumor vaccine, stimulating an immune response against residual cancer cells that permits long-lasting immunity and a corresponding reduction in tumor growth. The incapacity of CDDP to trigger ICD is at least partially due to its failure to stimulate the premortem endoplasmic reticulum (ER)-stress response required for the externalization of the "eat-me" signal calreticulin (CRT) on the surface of dying cancer cells. Here, we developed a murine cancer cell line genetically modified to express the ER resident protein reticulon-1c (Rtn-1c) by virtue of tetracycline induction and showed that enforced Rtn-1c expression combined with CDDP treatment promoted CRT externalization to the surface of cancer cells. In contrast to single agent treatments, the tetracycline-mediated Rtn-1c induction combined with CDDP chemotherapy stimulated ICD as measured by the capacity of dying tumor cells, inoculated into syngenic immunocompetent mice, to mount an immune response to tumor re-challenge 1 week later. More importantly, established tumors, forced to constitutively express Rtn-1c in vivo by continuous treatment with tetracycline, became responsive to CDDP and exhibited a corresponding reduction in the rate of tumor growth. The combined therapeutic effects of Rtn-1c induction with CDDP treatment was only detected in the context of an intact immune system and not in nu/nu mice lacking thymus-dependent T lymphocytes. Altogether, these results indicate that the artificial or "synthetic" induction of immunogenic cell death by genetic manipulation of the ER-stress response can improve the efficacy of chemotherapy with CDDP by stimulating anticancer immunity.

Screening of novel immunogenic cell death inducers within the NCI Mechanistic Diversity Set.

Immunogenic cell death (ICD) inducers can be defined as agents that exert cytotoxic effects while stimulating an immune response against dead cell-associated antigens. When initiated by anthracyclines, ICD is accompanied by stereotyped molecular changes, including the pre-apoptotic exposure of calreticulin (CRT) on the cell surface, the lysosomal secretion of ATP during the blebbing phase of apoptosis, and the release of high mobility group box 1 (HMGB1) from dead cells. By means of genetically engineered human osteosarcoma U2OS cells, we screened the 879 anticancer compounds of the National Cancer Institute (NCI) Mechanistic Diversity Set for their ability to promote all these hallmarks of ICD in vitro. In line with previous findings from our group, several cardiac glycosides exhibit a robust propensity to elicit the major manifestations of ICD in cultured neoplastic cells. This screen pointed to septacidin, an antibiotic produced by Streptomyces fibriatus, as a novel putative inducer of ICD. In low-throughput validation experiments, septacidin promoted CRT exposure, ATP secretion and HGMB1 release from both U2OS cells and murine fibrosarcoma MCA205 cells. Moreover, septacidin-killed MCA205 cells protected immunocompetent mice against a re-challenge with living cancer cells of the same type. Finally, the antineoplastic effects of septacidin on established murine tumors were entirely dependent on T lymphocytes. Altogether, these results underscore the suitability of the high-throughput screening system described here for the identification of novel ICD inducers.

Quantification of cell cycle-arresting proteins.

Cellular senescence, which can be defined as a stress response preventing the propagation of cells that have accumulated potentially oncogenic alterations, is invariably associated with a permanent cell cycle arrest. Such an irreversible blockage is mainly mediated by the persistent upregulation of one or more cyclin-dependent kinase inhibitors (CKIs), including (though not limited to) p16( INK4A ) and p21( CIP1 ) and p27( KIP1 ). CKIs operate by binding to cyclin-dependent kinases (CDKs), de facto inhibiting their enzymatic activity. Here, we provide an immunoblotting-based method for the detection and quantification of CKIs in vitro and ex vivo, together with a set of guidelines for the interpretation of results.

Fluorescent biosensors for the detection of HMGB1 release.

During necrosis and following some instances of apoptosis (in particular in the absence of a proficient phagocytic system), the nonhistone chromatin component high-mobility group box 1 (HMGB1) is released in the extracellular space. In vivo, extracellular HMGB1 can bind Toll-like receptor 4 on the surface of dendritic cells, de facto operating as a danger-associated molecular pattern and alarming the organism to the presence of stressful conditions. Recent results indicate that the release of HMGB1 is one of the key features for cell death to be perceived as immunogenic, i.e., to be capable of triggering a cognate immune response in vivo. Thus, only anticancer agents that-among other features-allow for the release of HMGB1 as they induce cell death are expected to stimulate anticancer immune responses. To investigate the immunogenic potential of conventional anticancer agents and novel cell death inducers on a high-throughput scale, we engineered human osteosarcoma U2OS cells to express HMGB1 fused at the N-terminus of the green fluorescent protein (GFP). Coupled to fluorescence microscopy workstations for automated image acquisition and analysis, this HMGB1-GFP-based biosensor is amenable for the identification of potential inducers of immunogenic cell death among large chemical libraries.

EGFR inhibitors exacerbate differentiation and cell cycle arrest induced by retinoic acid and vitamin D3 in acute myeloid leukemia cells.

By means of an unbiased, automated fluorescence microscopy-based screen, we identified the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib as potent enhancers of the differentiation of HL-60 acute myeloid leukemia (AML) cells exposed to suboptimal concentrations of vitamin A (all-trans retinoic acid, ATRA) or vitamin D (1α,25-hydroxycholecalciferol, VD). Erlotinib and gefitinib alone did not promote differentiation, yet stimulated the acquisition of morphological and biochemical maturation markers (including the expression of CD11b and CD14 as well as increased NADPH oxidase activity) when combined with either ATRA or VD. Moreover, the combination of erlotinib and ATRA or VD synergistically induced all the processes that are normally linked to terminal hematopoietic differentiation, namely, a delayed proliferation arrest in the G0/G1 phase of the cell cycle, cellular senescence, and apoptosis. Erlotinib potently inhibited the (auto)phosphorylation of mitogen-activated protein kinase 14 (MAPK14, best known as p38(MAPK)) and SRC family kinases (SFKs). If combined with the administration of ATRA or VD, the inhibition of p38(MAPK) or SFKs with specific pharmacological agents mimicked the pro-differentiation activity of erlotinib. These data were obtained with 2 distinct AML cell lines (HL-60 and MOLM-13 cells) and could be confirmed on primary leukemic blasts isolated from the circulation of AML patients. Altogether, these findings point to a new regimen for the treatment of AML, in which naturally occurring pro-differentiation agents (ATRA or VD) may be combined with EGFR inhibitors.

Crosstalk between ER stress and immunogenic cell death.

Preclinical and clinical findings suggest that tumor-specific immune responses may be responsible--at least in part--for the clinical success of therapeutic regimens that rely on immunogenic cell death (ICD) inducers, including anthracyclines and oxaliplatin. The molecular pathways whereby some, but not all, cytotoxic agents promote bona fide ICD remain to be fully elucidated. Nevertheless, a central role for the endoplasmic reticulum (ER) stress response has been revealed in all scenarios of ICD described thus far. Hence, components of the ER stress machinery may constitute clinically relevant druggable targets for the induction of ICD. In this review, we will summarize recent findings in the field of ICD research with a special focus on ER stress mechanisms and their implication for cancer therapy.

ATP-dependent recruitment, survival and differentiation of dendritic cell precursors in the tumor bed after anticancer chemotherapy.

Tumor cells succumb to chemotherapy while releasing ATP. We have found that extracellular ATP attracts dendritic cell (DC) precursors into the tumor bed, facilitates their permanence in the proximity of dying cells and promotes their differentiation into mature DCs endowed with the capacity of presenting tumor-associated antigens.

Cisplatin resistance associated with PARP hyperactivation.

Non-small cell lung carcinoma patients are frequently treated with cisplatin (CDDP), most often yielding temporary clinical responses. Here, we show that PARP1 is highly expressed and constitutively hyperactivated in a majority of human CDDP-resistant cancer cells of distinct histologic origin. Cells manifesting elevated intracellular levels of poly(ADP-ribosyl)ated proteins (PAR(high)) responded to pharmacologic PARP inhibitors as well as to PARP1-targeting siRNAs by initiating a DNA damage response that translated into cell death following the activation of the intrinsic pathway of apoptosis. Moreover, PARP1-overexpressing tumor cells and xenografts displayed elevated levels of PAR, which predicted the response to PARP inhibitors in vitro and in vivo more accurately than PARP1 expression itself. Thus, a majority of CDDP-resistant cancer cells appear to develop a dependency to PARP1, becoming susceptible to PARP inhibitor-induced apoptosis.

Subversion of the chemotherapy-induced anticancer immune response by the ecto-ATPase CD39.

The efficacy of antineoplastic chemotherapies with anthracyclines or oxaliplatin relies on the induction of immunogenic cell death, thus provoking an anticancer immune response. Recently, we observed that overexpression of CD39, an ATP-degrading enzyme expressed on the cell surface, abolishes the immunogenicity of cell death, thus rendering cancers resistant against chemotherapy.

Premortem autophagy determines the immunogenicity of chemotherapy-induced cancer cell death.

One particular strategy to render anticancer therapies efficient consists of converting the patient's own tumor cells into therapeutic vaccines, via the induction of immunogenic cell death (ICD). One of the hallmarks of ICD dwells in the active release of ATP by cells committed to undergo, but not yet having succumbed to, apoptosis. We observed that the knockdown of essential autophagy-related genes (ATG3, ATG5, ATG7 and BECN1) abolishes the pre-apoptotic secretion of ATP by several human and murine cancer cell lines undergoing ICD. Accordingly, autophagy-competent, but not autophagy-deficient, tumor cells treated with ICD inducers in vitro could induce a tumor-specific immune response in vivo. Cancer cell lines stably depleted of ATG5 or ATG7 normally generate tumors in vivo, and such autophagy-deficient neoplasms, upon systemic treatment with ICD inducers, exhibit the same levels of apoptosis (as monitored by nuclear shrinkage and caspase-3 activation) and necrosis (as determined by following the kinetics of HMGB1 release) as their autophagy-proficient counterparts. However, autophagy-incompetent cancers fail to release ATP, to recruit immune effectors into the tumor bed and to respond to chemotherapy in conditions in which autophagy-competent tumors do so. The intratumoral administration of ecto-ATPase inhibitors increases extracellular ATP concentrations, re-establishes the therapy-induced recruitment of dendritic cells and T cells into the tumor bed, and restores the chemotherapeutic response of autophagy-deficient cancers. Altogether, these results suggest that autophagy-incompetent tumor cells escape from chemotherapy-induced (and perhaps natural?) immunosurveillance because they are unable to release ATP.

Anticancer activity of cardiac glycosides: At the frontier between cell-autonomous and immunological effects.

Retrospective clinical data indicate that cardiac glycosides (CGs), notably digoxin, prolong the survival of carcinoma patients treated with conventional chemotherapy. CGs are known to influence the immune response at multiple levels. In addition, recent results suggest that CGs trigger the immunogenic demise of cancer cells, an effect that most likely contributes to their clinical anticancer activity.

Loss-of-function alleles of P2RX7 and TLR4 fail to affect the response to chemotherapy in non-small cell lung cancer.

The success of anticancer chemotherapy relies at least in part on the induction of an immune response against tumor cells. Thus, tumors growing on mice that lack the pattern recognition receptor TLR4 or the purinergic receptor P2RX7 fail to respond to chemotherapy with anthracyclins or oxaliplatin in conditions in which the same neoplasms growing on immunocompetent mice would do so. Similarly, the therapeutic efficacy (measured as progression-free survival) of adjuvant chemotherapy with anthracyclins is reduced in breast cancer patients bearing loss-of-function alleles of TLR4 or P2RX7. TLR4 loss-of-function alleles also have a negative impact on the therapeutic outcome of oxaliplatin in colorectal cancer patients. Here, we report that loss-of-function TLR4 and P2RX7 alleles do not affect overall survival in non-small cell lung cancer (NSCLC) patients, irrespective of the administration and type of chemotherapy. The intrinsic characteristics of NSCLC (which near-to-always is chemoresistant and associated with poor prognosis) and/or the type of therapy that is employed to treat this malignancy (which near-to-always is based on cisplatin) may explain why two genes that affect the immune response to dying cells fail to influence the clinical progression of NSCLC patients.

Immunohistochemical detection of cytoplasmic LC3 puncta in human cancer specimens.

Autophagy is an evolutionarily conserved catabolic process that involves the entrapment of cytoplasmic components within characteristic vesicles for their delivery to and degradation within lysosomes. Alterations in autophagic signaling are found in several human diseases including cancer. Here, we describe a validated immunohistochemical protocol for the detection of LC3 puncta in human formalin-fixed, paraffin-embedded cancer specimens that can also be applied to mouse tissues. In response to systemic chemotherapy, autophagy-competent mouse tumors exhibited LC3 puncta, which did not appear in mouse cancers that had been rendered autophagy-deficient by the knockdown of Atg5 or Atg7. As compared with normal tissues, LC3 staining was moderately to highly elevated in the large majority of human cancers studied, albeit tumors of the same histological type tended to be highly heterogeneous in the number and intensity of LC3 puncta per cell. Moreover, tumor-infiltrating immune cells often were highly positive for LC3. Altogether, this protocol for LC3 staining appears suitable for the specific detection of LC3 puncta in human specimens, including tissue microarrays. We surmise that this technique can be employed for retrospective or prospective studies involving large series of human tumor samples.

Cardiac glycosides exert anticancer effects by inducing immunogenic cell death.

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.

Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice.

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.