Molecular determinants of immunogenic cell death elicited by radiation therapy.

Cancer cells undergoing immunogenic cell death (ICD) can initiate adaptive immune responses against dead cell-associated antigens, provided that (1) said antigens are not perfectly covered by central tolerance (antigenicity), (2) cell death occurs along with the emission of immunostimulatory cytokines and damage-associated molecular patterns (DAMPs) that actively engage immune effector mechanisms (adjuvanticity), and (3) the microenvironment of dying cells is permissive for the initiation of adaptive immunity. Finally, ICD-driven immune responses can only operate and exert cytotoxic effector functions if the microenvironment of target cancer cells enables immune cell infiltration and activity. Multiple forms of radiation, including non-ionizing (ultraviolet) and ionizing radiation, elicit bona fide ICD as they increase both the antigenicity and adjuvanticity of dying cancer cells. Here, we review the molecular determinants of ICD as elicited by radiation as we critically discuss strategies to reinforce the immunogenicity of cancer cells succumbing to clinically available radiation strategies.

Type I interferon and cancer.

Type I interferon (IFN) is a class of proinflammatory cytokines with a dual role on malignant transformation, tumor progression, and response to therapy. On the one hand, robust, acute, and resolving type I IFN responses have been shown to mediate prominent anticancer effects, reflecting not only their direct cytostatic/cytotoxic activity on (at least some) malignant cells, but also their pronounced immunostimulatory functions. In line with this notion, type I IFN signaling has been implicated in the antineoplastic effects of various immunogenic therapeutics, including (but not limited to) immunogenic cell death (ICD)-inducing agents and immune checkpoint inhibitors (ICIs). On the other hand, weak, indolent, and non-resolving type I IFN responses have been demonstrated to support tumor progression and resistance to therapy, reflecting the ability of suboptimal type I IFN signaling to mediate cytoprotective activity, promote stemness, favor tolerance to chromosomal instability, and facilitate the establishment of an immunologically exhausted tumor microenvironment. Here, we review fundamental aspects of type I IFN signaling and their context-dependent impact on malignant transformation, tumor progression, and response to therapy.

Inhibition of MYC translation through targeting of the newly identified PHB-eIF4F complex as a therapeutic strategy in CLL.

Dysregulation of messenger RNA (mRNA) translation, including preferential translation of mRNA with complex 5' untranslated regions such as the MYC oncogene, is recognized as an important mechanism in cancer. Here, we show that both human and murine chronic lymphocytic leukemia (CLL) cells display a high translation rate, which is inhibited by the synthetic flavagline FL3, a prohibitin (PHB)-binding drug. A multiomics analysis performed in samples from patients with CLL and cell lines treated with FL3 revealed the decreased translation of the MYC oncogene and of proteins involved in cell cycle and metabolism. Furthermore, inhibiting translation induced a proliferation arrest and a rewiring of MYC-driven metabolism. Interestingly, contrary to other models, the RAS-RAF-(PHBs)-MAPK pathway is neither impaired by FL3 nor implicated in translation regulation in CLL cells. Here, we rather show that PHBs are directly associated with the eukaryotic initiation factor (eIF)4F translation complex and are targeted by FL3. Knockdown of PHBs resembled FL3 treatment. Importantly, inhibition of translation controlled CLL development in vivo, either alone or combined with immunotherapy. Finally, high expression of translation initiation-related genes and PHBs genes correlated with poor survival and unfavorable clinical parameters in patients with CLL. Overall, we demonstrated that translation inhibition is a valuable strategy to control CLL development by blocking the translation of several oncogenic pathways including MYC. We also unraveled a new and direct role of PHBs in translation initiation, thus creating new therapeutic opportunities for patients with CLL.

Cellular senescence in the response of HR+ breast cancer to radiotherapy and CDK4/6 inhibitors.

BACKGROUND: Preclinical evidence from us and others demonstrates that the anticancer effects of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors can be enhanced with focal radiation therapy (RT), but only when RT is delivered prior to (rather than after) CDK4/6 inhibition. Depending on tumor model, cellular senescence (an irreversible proliferative arrest that is associated with the secretion of numerous bioactive factors) has been attributed beneficial or detrimental effects on response to treatment. As both RT and CDK4/6 inhibitors elicit cellular senescence, we hypothesized that a differential accumulation of senescent cells in the tumor microenvironment could explain such an observation, i.e., the inferiority of CDK4/6 inhibition with palbociclib (P) followed by RT (P→RT) as compared to RT followed by palbociclib (RT→P). METHODS: The impact of cellular senescence on the interaction between RT and P was assessed by harnessing female INK-ATTAC mice, which express a dimerizable form of caspase 8 (CASP8) under the promoter of cyclin dependent kinase inhibitor 2A (Cdkn2a, coding for p16Ink4), as host for endogenous mammary tumors induced by the subcutaneous implantation of medroxyprogesterone acetate (MPA, M) pellets combined with the subsequent oral administration of 7,12-dimethylbenz[a]anthracene (DMBA, D). This endogenous mouse model of HR+ mammary carcinogenesis recapitulates key immunobiological aspects of human HR+ breast cancer. Mice bearing M/D-driven tumors were allocated to RT, P or their combination in the optional presence of the CASP8 dimerizer AP20187, and monitored for tumor growth, progression-free survival and overall survival. In parallel, induction of senescence in vitro, in cultured human mammary hormone receptor (HR)+ adenocarcinoma MCF7 cells, triple negative breast carcinoma MDA-MB-231 cells and mouse HR+ mammary carcinoma TS/A cells treated with RT, P or their combination, was determined by colorimetric assessment of senescence-associated ß-galactosidase activity after 3 or 7 days of treatment. RESULTS: In vivo depletion of p16Ink4-expressing (senescent) cells ameliorated the efficacy of P→RT (but not that of RT→P) in the M/D-driven model of HR+ mammary carcinogenesis. Accordingly, P→RT induced higher levels of cellular senescence than R→TP in cultured human and mouse breast cancer cell lines. CONCLUSIONS: Pending validation in other experimental systems, these findings suggest that a program of cellular senescence in malignant cells may explain (at least partially) the inferiority of P→RT versus RT→P in preclinical models of HR+ breast cancer.

Eµ-TCL1 adoptive transfer mouse model of chronic lymphocytic leukemia.

Despite being the most common adult leukemia in the western world, Chronic Lymphocytic Leukemia (CLL) remains a life-threatening and incurable disease. Efforts to develop new treatments are highly dependent on the availability of appropriate mouse models for pre-clinical testing. The Eµ-TCL1 mouse model is the most established pre-clinical approach to study CLL pathobiology and response to treatment, backed by numerous studies highlighting its resemblance to the most aggressive form of this malignancy. In contrast to the transgenic Eµ-TCL1 model, employing the adoptive transfer of Eµ-TCL1-derived splenocytes in immunocompetent C57BL/6 mice results in a comparably rapid (e.g., leukemic development within weeks compared to months in the transgenic model) and reliable model mimicking CLL. In this chapter, we would like to provide readers with a thoroughly optimized, detailed, and comprehensive protocol to use the adoptive transfer Eµ-TCL1 model in their research.

Assessment of translation rate in leukemic cells and immune cells of the microenvironment by OPP protein synthesis assay.

Despite being tightly regulated, messenger RNA (mRNA) translation, a manner in which cells control expression of genes and rapidly respond to stimuli, is highly dysfunctional and plastic in pathologies including cancer. Conversely, the investigation of molecular mechanisms whereby mRNA translation becomes aberrant in cancer, as well as inhibition thereof, become critical in developing novel therapeutic approaches. More specifically, in malignancies such as chronic lymphocytic leukemia in which aberrant global and transcript specific translation has been linked with poorer patient outcomes, targeting translation is a relevant approach, with various translation inhibitors under development. Here we elaborate on a protein synthesis assay by flow cytometry, O-propargyl-puromycin, demonstrating global mRNA translation rate with a variety of different applications including cell lines, primary cells or co-culture systems in vitro. This method provides a comprehensive tool in quantifying the rate of global mRNA translation in cancer cells, as well as that of the tumor microenvironment cells, or in response to inhibitory therapeutic agents while offering the possibility to simultaneously assess other cellular markers.

Flow cytometry-assisted quantification of cell cycle arrest in cancer cells treated with CDK4/6 inhibitors.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (i.e., palbociclib, abemaciclib, and ribociclib) are well known for their capacity to mediate cytostatic effects by promoting cell cycle arrest in the G1 phase, thus inhibiting cancer cell proliferation. Cytostatic effects induced by CDK4/6 inhibitors can be transient or lead to a permanent state of cell cycle arrest, commonly defined as cellular senescence. Induction of senescence is often associated to metabolic modifications and to the acquisition of a senescence-associated secretory phenotype (SASP) by cancer cells, which in turn can promote or limit antitumor immunity (and thus the efficacy of CDK4/6 inhibitors) depending on SASP components. Thus, although accumulating evidence suggests that anti-cancer effects of CDK4/6 inhibitors also depend on the promotion of antitumor immune responses, assessing cell cycle arrest and progression in cells treated with palbociclib remains a key approach for investigating the efficacy of CDK4/6 inhibitors. Here, we describe a method to assess cell cycle distribution simultaneously with active DNA replication by flow cytometry in cultured hormone receptor-positive breast cancer MCF7 cells.

Quantification of beta-galactosidase activity as a marker of radiation-driven cellular senescence.

Cellular senescence is a permanent state of cell cycle arrest that can be triggered by different stressors, including cancer treatments (the so-called "therapy-induced senescence"), such as radiation therapy (RT). Although senescent cells do not proliferate, they remain metabolically active and play a critical role in tumor progression, metastasis, and response to therapy. Therefore, investigating the induction of cellular senescence upon RT treatment is a critical read out for investigating RT efficacy or combinatorial strategies in cancer research. Senescent cells are characterized by a plethora of markers, including an increased content and activity of lysosomes, which can be detected by the activity of the lysosomal enzyme senescence-associated ß-galactosidase. In this chapter, we present a protocol for the gold standard cytochemical method for quantification of the activity of the senescence-associated ß-galactosidase in irradiated murine breast cancer cells in vitro.

Immunologically relevant effects of radiation therapy on the tumor microenvironment.

Focal radiation therapy (RT) has been successfully employed to clinically manage multiple types of cancer for more than a century. Besides being preferentially cytotoxic for malignant cells over their nontransformed counterparts, RT elicits numerous microenvironmental alterations that appear to factor into its therapeutic efficacy. Here, we briefly discuss immunostimulatory and immunosuppressive microenvironmental changes elicited by RT and their impact on tumor recognition by the host immune system.

The DNA Damage Response and Inflammation in Cancer.

Genomic stability in normal cells is crucial to avoid oncogenesis. Accordingly, multiple components of the DNA damage response (DDR) operate as bona fide tumor suppressor proteins by preserving genomic stability, eliciting the demise of cells with unrepairable DNA lesions, and engaging cell-extrinsic oncosuppression via immunosurveillance. That said, DDR sig-naling can also favor tumor progression and resistance to therapy. Indeed, DDR signaling in cancer cells has been consistently linked to the inhibition of tumor-targeting immune responses. Here, we discuss the complex interactions between the DDR and inflammation in the context of oncogenesis, tumor progression, and response to therapy. SIGNIFICANCE: Accumulating preclinical and clinical evidence indicates that DDR is intimately connected to the emission of immunomodulatory signals by normal and malignant cells, as part of a cell-extrinsic program to preserve organismal homeostasis. DDR-driven inflammation, however, can have diametrically opposed effects on tumor-targeting immunity. Understanding the links between the DDR and inflammation in normal and malignant cells may unlock novel immunotherapeutic paradigms to treat cancer.

Multimodal immunostimulation to control BRCA1-defective ovarian carcinoma.

Depending on intensity and duration, STING1 (stimulator of interferon response cGAMP interactor 1) signaling can restrain or promote tumor progression via both cancer cell-intrinsic and -extrinsic pathways. Bruand et al. recently identified a novel STING1-driven immunosuppressive pathway that can be targeted toward superior disease control in preclinical models of homologous recombination deficient (HRD) ovarian carcinoma.

Myeloid-Derived Suppressor Cells and Radiotherapy.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of pathologically activated, mostly immature, myeloid cells that exert robust immunosuppressive functions. MDSCs expand during oncogenesis and have been linked to accelerated disease progression and resistance to treatment in both preclinical tumor models and patients with cancer. Thus, MDSCs stand out as promising targets for the development of novel immunotherapeutic regimens with superior efficacy. Here, we summarize accumulating preclinical and clinical evidence indicating that MDSCs also hamper the efficacy of radiotherapy (RT), as we critically discuss the potential of MDSC-targeting strategies as tools to achieve superior immunotherapeutic tumor control by RT in the clinic.

Cytofluorometric assessment of cell cycle progression in irradiated cells.

Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects upon the accumulation of unrepaired damage to macromolecules, notably DNA. The ability of ionizing radiation to prevent malignant cells from replicating and to cause their demise is indeed an integral component of the anticancer activity of RT. Neoplastic cells are generally more sensitive to the cytostatic and cytotoxic effects of RT than their healthy counterparts as they exhibit increased proliferative rate and limited capacity for DNA repair. This provides a rather comfortable therapeutic window for clinical RT usage, especially with the development of novel, technologically superior RT modalities that minimize the exposure of normal tissues. Thus, while accumulating evidence indicates that cancer control by RT also involves the activation of tumor-targeting immune responses, assessing cell cycle progression in irradiated cells remains a central approach for investigating radiosensitivity in preclinical tumor models. Here, we detail a simple, flow cytometry-assisted method to simultaneously assess cell cycle distribution and active DNA replication in cultured estrogen receptor (ER)+ breast cancer MCF7 cells. With minimal variations, the same technique can be straightforwardly implemented to a large panel of human and mouse cancer cell lines.

Quantification of cytosolic DNA species by immunofluorescence microscopy and automated image analysis.

When employed according to specific doses and fractionation schedules, radiation therapy (RT) elicits potent tumor-targeting immune responses that rely on the secretion of type I interferon (IFN) by irradiated cancer cells. Most often, this is initiated by the ability of RT to promote the cytosolic accumulation of double-stranded DNA (dsDNA) molecules, which are detected by cyclic GMP-AMP synthase (CGAS) to engage the stimulator of interferon response cGAMP interactor 1 (STING1)-dependent transactivation of type I IFN-coding genes via interferon regulatory factor 3 (IRF3). Here, we describe a simple protocol for the quantification of cytosolic dsDNA species by immunofluorescence microscopy coupled to automated image analysis, as enabled by precise sample processing conditions that permeabilize plasma-but not nuclear or inner mitochondrial-membranes. As compared to subcellular fractionation-based techniques, this approach is compatible with assessments in individual cells aimed at gauging inter-cellular heterogeneity, as well as subcellular tests including co-localization studies.

RT-PCR-assisted quantification of type I IFN responses in irradiated cancer cells.

It is now clear that radiation therapy (RT) can be delivered in doses and according to fractionation schedules that actively elicit immunostimulatory effects. While such effects are often sufficient to drive potent anticancer immunity culminating with systemic disease eradication, the immunostimulatory activity of RT stands out as a promising combinatorial partner for bona fide immunotherapeutics including immune checkpoint inhibitors (ICIs). Accumulating preclinical and clinical evidence indicates that the secretion of type I interferon (IFN) by irradiated cancer cells is a sine qua non for RT to initiate ICI-actionable tumor-targeting immune responses. Here, we detail a simple protocol to quantitatively assess type I IFN responses in irradiated mouse hormone receptor (HR)+ TS/A cells by RT-PCR. With minimal variations, the same technique can be straightforwardly adapted to quantify type I IFN-associated transcriptional responses in a variety of human and mouse cancer cells maintained in vitro.

Cytofluorometric assessment of acute cell death responses driven by radiation therapy.

Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects on malignant cells, largely reflecting the ability of ionizing radiation to cause direct and indirect damage to macromolecules including DNA and lipids. While low-dose RT generally causes limited cytotoxicity in an acute manner (as it imposes insufficient cellular damage to compromise homeostasis, or instead induces the delayed demise of cells that fail to complete mitosis successfully), high RT doses can mediate an acute wave of cell death that begins to manifest shortly (24-72h) after irradiation. Here, we provide two straightforward techniques to assess the acute cytotoxic effects of RT by the flow cytometry-assisted quantification of plasma membrane permeabilization (PMP, a late-stage manifestation of cell death) and either mitochondrial outer membrane permeabilization (MOMP) or phosphatidylserine (PS) externalization (two early-stage signs of cell death) in mouse mammary adenocarcinoma TS/A cells. With minor variations, the same protocols can be straightforwardly adapted to measure acute cell death responses as elicited by RT in a large panel of human and mouse cancer cells lines of different histological derivation.

Immunological control of ovarian carcinoma by chemotherapy and targeted anticancer agents.

At odds with other solid tumors, epithelial ovarian cancer (EOC) is poorly sensitive to immune checkpoint inhibitors (ICIs), largely reflecting active immunosuppression despite CD8+ T cell infiltration at baseline. Accumulating evidence indicates that both conventional chemotherapeutics and targeted anticancer agents commonly used in the clinical management of EOC not only mediate a cytostatic and cytotoxic activity against malignant cells, but also drive therapeutically relevant immunostimulatory or immunosuppressive effects. Here, we discuss such an immunomodulatory activity, with a specific focus on molecular and cellular pathways that can be harnessed to develop superior combinatorial regimens for clinical EOC care.