Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4′,6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.
apoptosis; necrosis; high-throughput screening; drug discovery
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.
apoptosis; autophagy; C57BL/6 mice; dendritic cells; HTS; mitoxantrone
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.
cancer; calreticulin; chemotherapy; cisplatin; endoplasmic reticulum stress; reticulon 1C
The term “immunogenic cell death” (ICD) is commonly employed to indicate a peculiar instance of regulated cell death (RCD) that engages the adaptive arm of the immune system. The inoculation of cancer cells undergoing ICD into immunocompetent animals elicits a specific immune response associated with the establishment of immunological memory. Only a few agents are intrinsically endowed with the ability to trigger ICD. These include a few chemotherapeutics that are routinely employed in the clinic, like doxorubicin, mitoxantrone, oxaliplatin, and cyclophosphamide, as well as some agents that have not yet been approved for use in humans. Accumulating clinical data indicate that the activation of adaptive immune responses against dying cancer cells is associated with improved disease outcome in patients affected by various neoplasms. Thus, novel therapeutic regimens that trigger ICD are urgently awaited. Here, we discuss current combinatorial approaches to convert otherwise non-immunogenic instances of RCD into bona fide ICD.
ATP; autophagy; calreticulin; endoplasmic reticulum stress; HMGB1; type I interferon
ATP; autophagy; calreticulin; endoplasmic reticulum stress; HMGB1 protein; type I interferon
During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into “passive” and “active” based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches.
adoptive cell transfer; checkpoint blockers; dendritic cell-based interventions; DNA-based vaccines; immunostimulatory cytokines; peptide-based vaccines; oncolytic viruses; Toll-like receptor agonists
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 p38MAPK) and SRC family kinases (SFKs). If combined with the administration of ATRA or VD, the inhibition of p38MAPK 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.
BCR-ABL; PML-RARα; cancer; dasatinib; imatinib; monocyte-specific esterase
A chemical screen designed to identify novel inducers of autophagy led to the discovery that signal transducer and activator of transcription 3 (STAT3) inhibitors can potently stimulate the autophagic flux. Although STAT3 is best known as a pro-inflammatory and oncogenic transcription factor, mechanistic analyses revealed that autophagy is regulated by the cytoplasmic, not nuclear, pool of STAT3. Cytoplasmic STAT3 normally interacts with the eukaryotic translation initiation factor 2, subunit 1α, 35kDa (EIF2S1/eIF2α) kinase 2/protein kinase, RNA-activated (EIF2AK2/PKR), a sensor of double-stranded RNA. This interaction, which could be recapitulated using recombinant proteins in pull-down experiments, involves the catalytic domain of EIF2AK2 as well as the SH2 domain of STAT3, which can adopt a fold similar to that of EIF2S1. Thus, STAT3 may act as a competitive inhibitor of EIF2AK2. Indeed, pharmacological or genetic inhibition of STAT3 stimulates EIF2AK2-dependent EIF2S1 phosphorylation and autophagy. Conversely, the overexpression of wild-type STAT3 as well as of STAT3 mutants that cannot be phosphorylated by JAK2 or are excluded from the nucleus inhibits autophagy. However, STAT3 mutants that fail to interact with EIF2AK2 are unable to suppress autophagy. Both STAT3-targeting agents (i.e., Stattic, JSI-124 and WP1066) and EIF2AK2 activators (such as the double-strand RNA mimetic polyinosinic:polycytidylic acid) are capable of disrupting the inhibitory interaction between STAT3 and EIF2AK2 in cellula, yet only the latter does so in cell-free systems in vitro. A further screen designed to identify EIF2AK2-dependent autophagy inducers revealed that several fatty acids including palmitate trigger autophagy via a pathway that involves the disruption of the STAT3-EIF2AK2 complex as well as the phosphorylation of mitogen-activated protein kinase 8/c-Jun N-terminal kinase 1 (MAPK8/JNK1) and EIF2S1. These results reveal an unsuspected crosstalk between cellular metabolism (fatty acids), pro-inflammatory signaling (STAT3), innate immunity (EIF2AK2), and translational control (EIF2S1) that regulates autophagy.
EIF2S1S51A; endoplasmic reticulum; IRS1; palmitate; polyI:C; STAT3Y705F
B lymphocytes; SAHA; histone deacetylase inhibitors; immunogenic cell death; interferon γ; natural killer cells
ATP; caspases; chemotherapy; immunogenic cell death; radiotherapy; tumor-infiltrating lymphocytes
Although chemically non-reactive, inert noble gases may influence multiple physiological and pathological processes via hitherto uncharacterized physical effects. Here we report a cell-based detection system for assessing the effects of pre-defined gas mixtures on the induction of apoptotic cell death. In this setting, the conventional atmosphere for cell culture was substituted with gas combinations, including the same amount of oxygen (20%) and carbon dioxide (5%) but 75% helium, neon, argon, krypton, or xenon instead of nitrogen. The replacement of nitrogen with noble gases per se had no effects on the viability of cultured human osteosarcoma cells in vitro. Conversely, argon and xenon (but not helium, neon, and krypton) significantly limited cell loss induced by the broad-spectrum tyrosine kinase inhibitor staurosporine, the DNA-damaging agent mitoxantrone and several mitochondrial toxins. Such cytoprotective effects were coupled to the maintenance of mitochondrial integrity, as demonstrated by means of a mitochondrial transmembrane potential-sensitive dye and by assessing the release of cytochrome c into the cytosol. In line with this notion, argon and xenon inhibited the apoptotic activation of caspase-3, as determined by immunofluorescence microscopy coupled to automated image analysis. The antiapoptotic activity of argon and xenon may explain their clinically relevant cytoprotective effects.
antimycin A, menadione, mitochondrial membrane permeabilization, rotenone; U2OS cells, Z-VAD-fmk
antigenicity; cognate immune effectors; immunogenicity; immunosuppressive networks; innate immune effectors; MDSCs
The antineoplastic effects of anthracyclines have been shown to rely, at least in part, on a local immune response that involves dendritic cells (DCs) and several distinct subsets of T lymphocytes. Here, we show that the administration of anthracyclines to mice bearing established neoplasms stimulates the intratumoral secretion of tumor necrosis factor α (TNFα). However, blocking the TNFα/TNF receptor (TNFR) system by three different strategies—namely, (1) neutralizing antibodies, (2) etanercept, a recombinant protein in which TNFR is fused to the constant domain of an IgG1 molecule, and (3) gene knockout—failed to negatively affect the therapeutic efficacy of anthracyclines in three distinct tumor models. In particular, TNFα-blocking strategies did not influence the antineoplastic effects of doxorubicin (a prototypic anthracycline) against MCA205 fibrosarcomas growing in C57BL/6 mice, F244 sarcomas developing in 129/Sv hosts and H2N100 mammary carcinomas arising in BALB/c mice. These findings imply that, in contrast to other cytokines (such as interleukin-1β, interleukin-17 and interferon γ), TNFα is not required for anthracyclines to elicit therapeutic anticancer immune responses.
T cells; apoptosis; calreticulin; dendritic cell; immunogenic cell death; interferon γ
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.
apoptosis; autophagy; calreticulin; CD39; doxorubicin; immunogenic cell death
The BCL-2 homolog BCL-XL, one of the two protein products of BCL2L1, has originally been characterized for its prominent prosurvival functions. Similar to BCL-2, BCL-XL binds to its multidomain proapoptotic counterparts BAX and BAK, hence preventing the formation of lethal pores in the mitochondrial outer membrane, as well as to multiple BH3-only proteins, thus interrupting apical proapoptotic signals. In addition, BCL-XL has been suggested to exert cytoprotective functions by sequestering a cytosolic pool of the pro-apoptotic transcription factor p53 and by binding to the voltage-dependent anion channel 1 (VDAC1), thereby inhibiting the so-called mitochondrial permeability transition (MPT). Thus, BCL-XL appears to play a prominent role in the regulation of multiple distinct types of cell death, including apoptosis and regulated necrosis. More recently, great attention has been given to the cell death-unrelated functions of BCL-2-like proteins. In particular, BCL-XL has been shown to modulate a number of pathophysiological processes, including—but not limited to—mitochondrial ATP synthesis, protein acetylation, autophagy and mitosis. In this short review article, we will discuss the functions of BCL-XL at the interface between cell death and metabolism.
Cardiac glycosides (CGs) are natural compounds sharing the ability to operate as potent inhibitors of the plasma membrane Na+/K+-ATPase, hence promoting—via an indirect mechanism—the intracellular accumulation of Ca2+ ions. In cardiomyocytes, increased intracellular Ca2+ concentrations exert prominent positive inotropic effects, that is, they increase myocardial contractility. Owing to this feature, two CGs, namely digoxin and digitoxin, have extensively been used in the past for the treatment of several cardiac conditions, including distinct types of arrhythmia as well as contractility disorders. Nowadays, digoxin is approved by the FDA and indicated for the treatment of congestive heart failure, atrial fibrillation and atrial flutter with rapid ventricular response, whereas the use of digitoxin has been discontinued in several Western countries. Recently, CGs have been suggested to exert potent antineoplastic effects, notably as they appear to increase the immunogenicity of dying cancer cells. In this Trial Watch, we summarize the mechanisms that underpin the unsuspected anticancer potential of CGs and discuss the progress of clinical studies that have evaluated/are evaluating the safety and efficacy of CGs for oncological indications.
breast carcinoma; Digitalis purpurea; estrogen receptor; immunogenic cell death; ouabain; phytoestrogens
The illicit generation of tetraploid cells constitutes a prominent driver of oncogenesis, as it often precedes the development of aneuploidy and genomic instability. In addition, tetraploid (pre-)malignant cells display an elevated resistance against radio- and chemotherapy. Here, we report a strategy to preferentially kill tetraploid tumor cells based on the broad-spectrum kinase inhibitor SP600125. Live videomicroscopy revealed that SP600125 affects the execution of mitosis, impedes proper cell division and/or activates apoptosis in near-to-tetraploid, though less so in parental, cancer cells. We propose a novel graphical model to quantify the differential response of diploid and tetraploid cells to mitotic perturbators, including SP600125, which we baptized “transgenerational cell fate profiling.” We speculate that this representation constitutes a valid alternative to classical “single-cell fate” and “genealogical” profiling and, hence, may facilitate the analysis of cell fate within a heterogeneous population as well as the visual examination of cell cycle alterations.
cell death; cytokinesis failure; mitotic catastrophe; microtubules; polyploidy; time-lapse microscopy
One of the driving forces of oncogenesis is tetraploidy, a duplication of the DNA content that, upon asymmetric cell division or progressive chromosome loss, can originate aneuploidy. Recent findings from our group indicate the existence of an immunosurveillance system that eliminates tetraploid cancer cells. We surmise that tetraploidy-inducing chemotherapeutic agents may elicit potent anticancer responses by re-activating this immunosurveillance system.
breast carcinoma; calreticulin; HMGB1; hyperploidy; immunogenic cell death; mitotic catastrophe
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.
calreticulin; digitoxin; digoxin; HMGB1; immunogenic cell death; Na+/K+ ATPase
The genetic or functional inactivation of p53 is highly prevalent in human cancers. Using high-content videomicroscopy based on fluorescent TP53+/+ and TP53−/− human colon carcinoma cells, we discovered that SP600125, a broad-spectrum serine/threonine kinase inhibitor, kills p53-deficient cells more efficiently than their p53-proficient counterparts, in vitro. Similar observations were obtained in vivo, in mice carrying p53-deficient and -proficient human xenografts. Such a preferential cytotoxicity could be attributed to the failure of p53-deficient cells to undergo cell cycle arrest in response to SP600125. TP53−/− (but not TP53+/+) cells treated with SP600125 became polyploid upon mitotic abortion and progressively succumbed to mitochondrial apoptosis. The expression of an SP600125-resistant variant of the mitotic kinase MPS1 in TP53−/− cells reduced SP600125-induced polyploidization. Thus, by targeting MPS1, SP600125 triggers a polyploidization program that cannot be sustained by TP53−/− cells, resulting in the activation of mitotic catastrophe, an oncosuppressive mechanism for the eradication of mitosis-incompetent cells.
caspases; HCT 116; high-throughput screening; mitochondrial outer membrane permeabilization; MPS1
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.
IALT; calreticulin; immunogenic cell death; necrosis factor α; rs3751143; rs4986790; tumor
The acetylase inhibitor spermidine and the sirtuin-1 activator resveratrol disrupt the antagonistic network of acetylases and deacetylases that regulate autophagy.
Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide–dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.