Sofia R. Gameiro

Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death

Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy‐induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from ICD. Docetaxel treatment of tumor cells did not induce ATP or high‐mobility group box 1 (HMGB1) secretion, or cell death. However, calreticulin (CRT) exposure was observed in all cell lines examined after chemotherapy treatment. Killing by carcinoembryonic antigen (CEA), MUC‐1, or PSA‐specific CD8+ CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen‐processing machinery, and mediated largely by CRT membrane translocation, as determined by functional knockdown of CRT, PERK, or CRT‐blocking peptide. A docetaxel‐resistant cell line was selected (MDR‐1+, CD133+) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. Here, we provide an operational definition of “immunogenic modulation,” where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to ICD and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy.

The Tipping Point for Combination Therapy: Cancer Vaccines With Radiation, Chemotherapy, or Targeted Small Molecule Inhibitors

Therapeutic cancer vaccines are a unique treatment modality in that they initiate a dynamic process of activating the host immune system, which can then be exploited by concurrent or subsequent therapies. The addition of immunotherapy to standard-of-care cancer therapies has shown evidence of efficacy in preclinical models and in the clinical setting. This review examines the preclinical and clinical interactions between vaccine-mediated tumor-specific immune responses and local radiation, systemic chemotherapy, or select small molecule inhibitors, as well as the potential synergy between these modalities.

Abscopal Regression of Antigen Disparate Tumors by Antigen Cascade After Systemic Tumor Vaccination in Combination with Local Tumor Radiation

Radiation is a primary modality in cancer treatment. Radiation can also reduce tumor growth outside the treatment field, often referred to as the abscopal effect. The mechanisms and therapeutic potential of the abscopal effect have not been fully elucidated. We evaluated the role of vaccination directed against a tumor-associated antigen (TAA) in the induction and amplification of radiation induced abscopal effects. Active-specific immunotherapy with a TAA-specific vaccine regimen was used to induce and potentiate T-cell responses against carcinoembryonic antigen (CEA) in combination with local irradiation of subcutaneous tumors. We examined the potential synergy of a poxvirus-based CEA vaccine regimen in CEA-transgenic (Tg) mice in combination with either external beam radiation or brachytherapy of local tumors. The induction of CD8(+) T cells specific for multiple TAAs not encoded by the vaccine was observed after the combination therapy. In two tumor models, the antigen cascade responses induced by vaccine and local irradiation mediated the regression of antigen negative metastases at distal subcutaneous or pulmonary sites. Clinically, local control of the primary tumor is necessary and can sometimes prevent metastases; however, irradiation generally fails to control preexisting metastases. These studies suggest that by coupling tumor irradiation with immunotherapy, the abscopal effect can transcend from anecdotal observation to a defined mechanism that can be exploited for the treatment of systemic disease.

Exploitation of differential homeostatic proliferation of T-cell subsets following chemotherapy to enhance the efficacy of vaccine-mediated antitumor responses.

The 5-year survival rate for stage IB-III non-small-cell lung cancer (NSCLC) remains 15%. Surgical resection followed by adjuvant chemotherapy with cisplatin and vinorelbine is one standard-of-care. We sought to determine in a preclinical model whether (a) the combination of cisplatin and vinorelbine could positively modulate components of the immune system independent of antitumor activity, and (b) there were synergistic effects of this drug combination and vaccine immunotherapy. We examined the effect of cisplatin/vinorelbine on gene expression, cell-surface phenotype, and CTL-mediated cytolysis of murine lung carcinoma cells in vitro; we also assessed the effects of cisplatin/vinorelbine on immune subsets and function of Tregs in vivo. Finally, we evaluated the potential synergy between chemotherapy and a recombinant yeast-CEA vaccine in a murine model transgenic for CEA with mice bearing lung tumors. These studies demonstrate that exposure of lung tumor cells to the platinum doublet cisplatin/vinorelbine modulates tumor cell phenotype and increases sensitivity to CTL-mediated cytolysis. These studies also demonstrate that cisplatin/vinorelbine (a) induces sub-myeloablative leucopenia that differentially modulates reconstitution of Treg versus effector T-cell subsets and (b) can be employed synergistically with vaccine, exploiting homeostatic peripheral expansion of T cells. Antitumor studies show for the first time that cisplatin/vinorelbine combined with vaccine increases the survival of mice with established NSCLC. These findings provide the rationale for the potential clinical benefit of the combined use of vaccine with cisplatin/vinorelbine chemotherapy regimens.

Radiation-induced modulation of costimulatory and coinhibitory T-cell signaling molecules on human prostate carcinoma cells promotes productive antitumor immune interactions

We sought to determine if single-dose external beam radiation therapy (EBRT) could modulate the expression signature of T-cell costimulatory and coinhibitory molecules in human prostate cancer (PCa) cell lines in vitro. We investigated the functional impact of irradiated PCa cells with a modulated costimulatory profile on responder T-cell activity. We used three PCa cell lines (DU145, PC3, and LNCaP) and two epithelial cell lines from noncancerous prostate and lung tissue. After 72 hours of EBRT, surface expression of four immunostimulatory molecules (CD70, CD275/ICOSL, CD134L/OX40L, and CD137L/41BBL) and two immunosuppressive markers (CTLA-4/CD152 and PD-L1/CD274) were evaluated by flow cytometry. We evaluated the impact of several radiation doses and the longevity of modulated expression. We examined the functional impact of radiation-induced modulation of cancer cells by cytotoxic T cells (CTL) cytotoxicity and ELISPOT assay for interferon-gamma (IFN-γ) production. Last, we evaluated whether IFN-γ-induced PD-L1 expression could be reversed by EBRT. After 10 Gy EBRT, expression of OX40L and 41BBL increased in all three PCa cell lines; expression of CD70 and ICOSL increased in PC3 cells. Conversely, a decrease in PD-L1 expression in DU145 and PC3 cells was detectable up to 144 hours after EBRT. No PD-L1 was detected in LNCaP. Epithelial cells from normal prostate were not modulated by radiation. CTL cytolytic activity and IFN-γ production were enhanced by interaction with irradiated PCa cells. Finally, EBRT failed to prevent IFN-γ-induced upregulation of PD-L1. We demonstrate that a single dose of EBRT increased surface expression of costimulatory molecules and decreased the expression of coinhibitory molecules in human PCa cell lines. Changes in irradiated tumor cells led to functional enhancement of T-cell activity, despite EBRT failing to reduce IFN-γ-induced expression of PD-L1. These data suggest that combining radiotherapy with T-cell stimulating immunotherapy may be an attractive strategy for cancer treatment.

Combination Therapy with Local Radiofrequency Ablation and Systemic Vaccine Enhances Antitumor Immunity and Mediates Local and Distal Tumor Regression

Purpose Radiofrequency ablation (RFA) is a minimally invasive energy delivery technique increasingly used for focal therapy to eradicate localized disease. RFA-induced tumor-cell necrosis generates an immunogenic source of tumor antigens known to induce antitumor immune responses. However, RFA-induced antitumor immunity is insufficient to control metastatic progression. We sought to characterize (a) the role of RFA dose on immunogenic modulation of tumor and generation of immune responses and (b) the potential synergy between vaccine immunotherapy and RFA aimed at local tumor control and decreased systemic progression. Experimental Design Murine colon carcinoma cells expressing the tumor-associated (TAA) carcinoembryonic antigen (CEA) (MC38-CEA+) were studied to examine the effect of sublethal hyperthermia in vitro on the cells’ phenotype and sensitivity to CTL-mediated killing. The effect of RFA dose was investigated in vivo impacting (a) the phenotype and growth of MC38-CEA+ tumors and (b) the induction of tumor-specific immune responses. Finally, the molecular signature was evaluated as well as the potential synergy between RFA and poxviral vaccines expressing CEA and a TRIad of COstimulatory Molecules (CEA/TRICOM). Results In vitro, sublethal hyperthermia of MC38-CEA+ cells (a) increased cell-surface expression of CEA, Fas, and MHC class I molecules and (b) rendered tumor cells more susceptible to CTL-mediated lysis. In vivo, RFA induced (a) immunogenic modulation on the surface of tumor cells and (b) increased T-cell responses to CEA and additional TAAs. Combination therapy with RFA and vaccine in CEA-transgenic mice induced a synergistic increase in CD4+ T-cell immune responses to CEA and eradicated both primary CEA+ and distal CEA– s.c. tumors. Sequential administration of low-dose and high-dose RFA with vaccine decreased tumor recurrence compared to RFA alone. These studies suggest a potential clinical benefit in combining RFA with vaccine in cancer patients, and augment support for this novel translational paradigm.

Tumor Cells Surviving Exposure to Proton or Photon Radiation Share a Common Immunogenic Modulation Signature, Rendering Them More Sensitive to T Cell-Mediated Killing.

PURPOSE To provide the foundation for combining immunotherapy to induce tumor antigen-specific T cells with proton radiation therapy to exploit the activity of those T cells. METHODS AND MATERIALS Using cell lines of tumors frequently treated with proton radiation, such as prostate, breast, lung, and chordoma, we examined the effect of proton radiation on the viability and induction of immunogenic modulation in tumor cells by flow cytometric and immunofluorescent analysis of surface phenotype and the functional immune consequences. RESULTS These studies show for the first time that (1) proton and photon radiation induced comparable up-regulation of surface molecules involved in immune recognition (histocompatibility leukocyte antigen, intercellular adhesion molecule 1, and the tumor-associated antigens carcinoembryonic antigen and mucin 1); (2) proton radiation mediated calreticulin cell-surface expression, increasing sensitivity to cytotoxic T-lymphocyte killing of tumor cells; and (3) cancer stem cells, which are resistant to the direct cytolytic activity of proton radiation, nonetheless up-regulated calreticulin after radiation in a manner similar to non-cancer stem cells. CONCLUSIONS These findings offer a rationale for the use of proton radiation in combination with immunotherapy, including for patients who have failed radiation therapy alone or have limited treatment options.

Attacking malignant cells that survive therapy: Exploiting immunogenic modulation

We have recently defined “immunogenic modulation,” a mechanism whereby malignant cells that survive anticancer therapy, due to sublethal delivery or development of treatment resistance, become nonetheless more sensitive to killing by cytotoxic T lymphocytes. This mechanism can be exploited to identify which therapies will best synergize with immunotherapy, potentially maximizing patient clinical benefit.

Inhibitors of histone deacetylase 1 reverse the immune evasion phenotype to enhance T-cell mediated lysis of prostate and breast carcinoma cells

The clinical promise of cancer immunotherapy relies on the premise that the immune system can recognize and eliminate tumor cells identified as non-self. However, tumors can evade host immune surveillance through multiple mechanisms, including epigenetic silencing of genes involved in antigen processing and immune recognition. Hence, there is an unmet clinical need to develop effective therapeutic strategies that can restore tumor immune recognition when combined with immunotherapy, such as immune checkpoint blockade and therapeutic cancer vaccines. We sought to examine the potential of clinically relevant exposure of prostate and breast human carcinoma cells to histone deacetylase (HDAC) inhibitors to reverse tumor immune escape to T-cell mediated lysis. Here we demonstrate that prostate (LNCAP) and breast (MDA-MB-231) carcinoma cells are more sensitive to T-cell mediated lysis in vitro after clinically relevant exposure to epigenetic therapy with either the pan-HDAC inhibitor vorinostat or the class I HDAC inhibitor entinostat. This pattern of immunogenic modulation was observed against a broad range of tumor-associated antigens, such as CEA, MUC1, PSA, and brachyury, and associated with augmented expression of multiple proteins involved in antigen processing and tumor immune recognition. Genetic and pharmacological inhibition studies identified HDAC1 as a key determinant in the reversal of carcinoma immune escape. Further, our findings suggest that the observed reversal of tumor immune evasion is driven by a response to cellular stress through activation of the unfolded protein response. This offers the rationale for combining HDAC inhibitors with immunotherapy, including therapeutic cancer vaccines.

Cancer vaccines targeting carcinoembryonic antigen: state-of-the-art and future promise

Concurrent with the US FDA’s approval of the first therapeutic cancer vaccine, and supported by mounting clinical evidence indicating that targeting carcinoembryonic antigen (CEA) can safely overcome pre-existing tolerance, a multitude of novel CEA cancer vaccines are now in various stages of development. Since cancer-driven immune suppression often limits the efficacy of vaccines, numerous strategies are being examined in both preclinical and clinical settings to overcome immunosuppressive elements, including the combined use of vaccines with certain chemotherapies, immune checkpoint inhibitors, small-molecule targeted therapies and radiation. This review discusses the current state and future direction of therapeutic cancer vaccines targeting CEA, based on advances achieved over the last 5 years.