Project description:The mechanisms behind the antitumor effects of exercise training (ExTr) are not fully understood. Using mouse models of established breast cancer, we examined here the causal role of CD8<sup>+</sup> T cells in the benefit acquired from ExTr in tumor control, as well as the ability of ExTr to improve immunotherapy responses. We implanted E0771, EMT6, MMTV-PyMT, and MCa-M3C breast cancer cells orthotopically in wild-type or <i>Cxcr3<sup>-/-</sup></i> female mice and initiated intensity-controlled ExTr sessions when tumors reached approximately 100 mm<sup>3</sup> We characterized the tumor microenvironment (TME) using flow cytometry, transcriptome analysis, proteome array, ELISA, and immunohistochemistry. We used antibodies against CD8<sup>+</sup> T cells for cell depletion. Treatment with immune checkpoint blockade (ICB) consisted of anti-PD-1 alone or in combination with anti-CTLA-4. ExTr delayed tumor growth and induced vessel normalization, demonstrated by increased pericyte coverage and perfusion and by decreased hypoxia. ExTr boosted CD8<sup>+</sup> T-cell infiltration, with enhanced effector function. CD8<sup>+</sup> T-cell depletion prevented the antitumor effect of ExTr. The recruitment of CD8<sup>+</sup> T cells and the antitumor effects of ExTr were abrogated in <i>Cxcr3<sup>-/-</sup></i> mice, supporting the causal role of the CXCL9/CXCL11-CXCR3 pathway. ExTr also sensitized ICB-refractory breast cancers to treatment. Our results indicate that ExTr can normalize the tumor vasculature, reprogram the immune TME, and enhance the antitumor activity mediated by CD8<sup>+</sup> T cells via CXCR3, boosting ICB responses. Our findings and mechanistic insights provide a rationale for the clinical translation of ExTr to improve immunotherapy of breast cancer.
Project description:For many solid tumors, immune checkpoint blockade therapy has become first line treatment, yet a large proportion of patients with immunologically cold tumors do not benefit due to the paucity of tumor infiltrating lymphocytes. Here we show that the orphan G Protein-Coupled Receptor 182 (GPR182) contributes to immunotherapy resistance in cancer via scavenging chemokines that are important for lymphocyte recruitment to tumors. GPR182 is primarily upregulated in melanoma-associated lymphatic endothelial cells (LECs) during tumorigenesis, and this atypical chemokine receptor endocytoses chemokines promiscuously. In GPR182-deficient mice, T cell infiltration into transplanted melanomas increases, leading to enhanced effector T cell function and improved antitumor immunity. Ablation of GPR182 leads to increased intratumoral concentrations of multiple chemokines and thereby sensitizes poorly immunogenic tumors to immune checkpoint blockade and adoptive cellular therapies. CXCR3 blockade reverses the improved antitumor immunity and T cell infiltration characteristic of GPR182-deficient mice. Our study thus identifies GPR182 as an upstream regulator of the CXCL9/CXCL10/CXCR3 axis that limits antitumor immunity and as a potential therapeutic target in immunologically cold tumors.
Project description:Therapy-induced senescence-associated secretory phenotype (SASP) correlates with overcoming resistance to immune checkpoint blockade (ICB). Intrinsic resistance to ICB is a major clinical challenge. For example, ovarian cancer is largely resistant to ICB. Here we show that adoptive transfer of SASP-boosted <i>ex vivo</i> therapy-induced senescent cells sensitizes ovarian tumor to ICB. Topoisomerase 1 (TOP1) inhibitors such as irinotecan enhance cisplatin-induced SASP, which depends on the TOP1 cleavage complex-regulated cGAS pathway. Significantly, intraperitoneal transfer of cisplatin-induced, SASP-boosted senescent cells with irinotecan sensitizes ovarian tumor to anti-PD-1 antibody and improves the survival of tumor-bearing mice in an immunocompetent, syngeneic model. This correlates with the infiltration of transferred senescent cells in the established orthotopic tumors and an increase in the infiltration of activated CD8<sup>+</sup> T cells and dendritic cells in the tumor bed. Our findings indicate that adoptive transfer of SASP-boosted therapy-induced senescent cells represents a potential therapeutic strategy to sensitize tumors to ICB.
Project description:Immunologically-cold tumors including glioblastoma (GBM) are refractory to checkpoint blockade therapy, largely due to extensive infiltration of immunosuppressive macrophages (Mϕs). Consistent with a pro-tumor role of IL-6 in alternative Mϕs polarization, we here show that targeting IL-6 by genetic ablation or pharmacological inhibition moderately improves T-cell infiltration into GBM and enhances mouse survival; however, IL-6 inhibition does not synergize PD-1 and CTLA-4 checkpoint blockade. Interestingly, anti-IL-6 therapy reduces CD40 expression in GBM-associated Mϕs. We identify a Stat3/HIF-1α-mediated axis, through which IL-6 executes an anti-tumor role to induce CD40 expression in Mϕs. Combination of IL-6 inhibition with CD40 stimulation reverses Mϕ-mediated tumor immunosuppression, sensitizes tumors to checkpoint blockade, and extends animal survival in two syngeneic GBM models, particularly inducing complete regression of GL261 tumors after checkpoint blockade. Thus, antibody cocktail-based immunotherapy that combines checkpoint blockade with dual-targeting of IL-6 and CD40 may offer exciting opportunities for GBM and other solid tumors.
Project description:The development of immune checkpoint inhibitors represents a major breakthrough in cancer therapy. Nevertheless, a substantial number of patients fail to respond to checkpoint pathway blockade. Evidence for WNT/?-catenin signaling-mediated immune evasion is found in a subset of cancers including melanoma. Currently, there are no therapeutic strategies available for targeting WNT/?-catenin signaling. Here we show that a specific small-molecule tankyrase inhibitor, G007-LK, decreases WNT/?-catenin and YAP signaling in the syngeneic murine B16-F10 and Clone M-3 melanoma models and sensitizes the tumors to anti-PD-1 immune checkpoint therapy. Mechanistically, we demonstrate that the synergistic effect of tankyrase and checkpoint inhibitor treatment is dependent on loss of ?-catenin in the tumor cells, anti-PD-1-stimulated infiltration of T cells into the tumor and induction of an IFN?- and CD8+ T cell-mediated anti-tumor immune response. Our study uncovers a combinatorial therapeutical strategy using tankyrase inhibition to overcome ?-catenin-mediated resistance to immune checkpoint blockade in melanoma.
Project description:We investigated the role of chemokines in regulating T cell accumulation in solid tumors. CCL5 and CXCL9 overexpression was associated with CD8+ T cell infiltration in solid tumors. T cell infiltration required tumor cell-derived CCL5 and was amplified by IFN-?-inducible, myeloid cell-secreted CXCL9. CCL5 and CXCL9 coexpression revealed immunoreactive tumors with prolonged survival and response to checkpoint blockade. Loss of CCL5 expression in human tumors was associated with epigenetic silencing through DNA methylation. Reduction of CCL5 expression caused tumor-infiltrating lymphocyte (TIL) desertification, whereas forced CCL5 expression prevented Cxcl9 expression and TILs loss, and attenuated tumor growth in mice through IFN-?. The cooperation between tumor-derived CCL5 and IFN-?-inducible CXCR3 ligands secreted by myeloid cells is key for orchestrating T cell infiltration in immunoreactive and immunoresponsive tumors.
Project description:Transforming growth factor beta (TGF?) is a multipotent immunosuppressive cytokine. TGF? excludes immune cells from tumors, and TGF? inhibition improves the efficacy of cytotoxic and immune therapies. Using preclinical colorectal cancer models in cell type-conditional TGF? receptor I (ALK5) knockout mice, we interrogate this mechanism. Tumor growth delay and radiation response are unchanged in animals with Treg or macrophage-specific ALK5 deletion. However, CD8?Cre-ALK5flox/flox (ALK5?CD8) mice reject tumors in high proportions, dependent on CD8+ T cells. ALK5?CD8 mice have more tumor-infiltrating effector CD8+ T cells, with more cytotoxic capacity. ALK5-deficient CD8+ T cells exhibit increased CXCR3 expression and enhanced migration towards CXCL10. TGF? reduces CXCR3 expression, and increases binding of Smad2 to the CXCR3 promoter. In vivo CXCR3 blockade partially abrogates the survival advantage of an ALK5?CD8 host. These data demonstrate a mechanism of TGF? immunosuppression through inhibition of CXCR3 in CD8+ T cells, thereby limiting their trafficking into tumors.
Project description:We aimed to explore whether the combination of intradermal DNA vaccination, to boost immune response against melanoma antigens, and immune checkpoint blockade, to alleviate immunosuppression, improves antitumor effectiveness in a murine B16F10 melanoma tumor model. Compared to single treatments, a combination of intradermal DNA vaccination (ovalbumin or gp100 plasmid adjuvanted with IL12 plasmid) and immune checkpoint CTLA-4/PD-1 blockade resulted in a significant delay in tumor growth and prolonged survival of treated mice. Strong activation of the immune response induced by combined treatment resulted in a significant antigen-specific immune response, with elevated production of antigen-specific IgG antibodies and increased intratumoral CD8+ infiltration. These results indicate a potential application of the combined DNA vaccination and immune checkpoint blockade, specifically, to enhance the efficacy of DNA vaccines and to overcome the resistance to immune checkpoint inhibitors in certain cancer types.
Project description:<b>Background and purpose: </b>Combining inhibitors of vascular endothelial growth factor and the programmed cell death protein 1 (PD1) pathway has shown efficacy in multiple cancers, but the disease-specific and agent-specific mechanisms of benefit remain unclear. We examined the efficacy and defined the mechanisms of benefit when combining regorafenib (a multikinase antivascular endothelial growth factor receptor inhibitor) with PD1 blockade in murine hepatocellular carcinoma (HCC) models.<br><br><b>Basic procedures: </b>We used orthotopic models of HCC in mice with liver damage to test the effects of regorafenib-dosed orally at 5, 10 or 20?mg/kg daily-combined with anti-PD1 antibodies (10?mg/kg intraperitoneally thrice weekly). We evaluated the effects of therapy on tumor vasculature and immune microenvironment using immunofluorescence, flow cytometry, RNA-sequencing, ELISA and pharmacokinetic/pharmacodynamic studies in mice and in tissue and blood samples from patients with cancer.<br><br><b>Main findings: </b>Regorafenib/anti-PD1 combination therapy increased survival compared with regofarenib or anti-PD1 alone in a regorafenib dose-dependent manner. Combination therapy increased regorafenib uptake into the tumor tissues by normalizing the HCC vasculature and increasing CD8 T-cell infiltration and activation at an intermediate regorafenib dose. The efficacy of regorafenib/anti-PD1 therapy was compromised in mice lacking functional T cells (<i>Rag1</i>-deficient mice). Regorafenib treatment increased the transcription and protein expression of CXCL10-a ligand for CXCR3 expressed on tumor-infiltrating lymphocytes-in murine HCC and in blood of patients with HCC. Using <i>Cxcr3</i>-deficient mice, we demonstrate that CXCR3 mediated the increased intratumoral CD8 T-cell infiltration and the added survival benefit when regorafenib was combined with anti-PD1 therapy.<br><br><b>Principal conclusions: </b>Judicious regorafenib/anti-PD1 combination therapy can inhibit tumor growth and increase survival by normalizing tumor vasculature and increasing intratumoral CXCR3+CD8?T-cell infiltration through elevated CXCL10 expression in HCC cells.
Project description:Despite compelling rates of durable clinical responses to programmed cell death-1 (PD-1) blockade, advances are needed to extend these benefits to resistant tumors. We found that tumor-bearing mice deficient in the chemokine receptor CXCR3 responded poorly to anti-PD-1 treatment. CXCR3 and its ligand CXCL9 were critical for a productive CD8+ T cell response in tumor-bearing mice treated with anti-PD-1 but were not required for the infiltration of CD8+ T cells into tumors. The anti-PD-1-induced anti-tumor response was facilitated by CXCL9 production from intratumoral CD103+ dendritic cells, suggesting that CXCR3 facilitates dendritic cell-T cell interactions within the tumor microenvironment. CXCR3 ligands in murine tumors and in plasma of melanoma patients were an indicator of clinical response to anti-PD-1, and their induction in non-responsive murine tumors promoted responsiveness to anti-PD-1. Our data suggest that the CXCR3 chemokine system is a biomarker for sensitivity to PD-1 blockade and that augmenting the intratumoral function of this chemokine system could improve clinical outcomes.