Head and neck squamous cell carcinoma (HNSCC) is the 7th most common cancer globally with a 40-50 % survival rate. Although macrophage migration inhibitory factor (MIF) is overexpressed in most solid tumors and promotes tumor growth and invasion, the therapeutic potential of MIF inhibition in HNSCC is yet to be explored. In this study, we investigated the efficacy of CPSI-1306, a small-molecule MIF inhibitor, on HNSCC cell growth and cancer associated signaling pathways in vitro, as well as its impact on T cells in the HNSCC tumor microenvironment in vivo. CPSI-1306 did not reduce HNSCC cell proliferation in vitro, and mildly decreased VEGF and EGFR expression. However, CPSI-1306 significantly reduced tumor development in two orthotopic mouse oral cancer (MOC-2 and MOC-1) HNSCC models. Interestingly, CPSI-1306 treatment increased T cell infiltration to the tumor microenvironment and completely abrogated immunosuppressive checkpoint markers TIGIT, TIM3, and CTLA-4, but not PD-1 on tumor infiltrating CD8+ T cells. This was accompanied by increased CD8+ T cell expression of antitumoral cytokines IFN-γ and TNF-α in the draining lymph nodes and Granzyme B in the tumor microenvironment of CPSI-1306 treated tumor bearing mice. Our studies demonstrate that the small molecule MIF inhibitor CPSI-1306 potently inhibits T cell immunosuppression in the tumor microenvironment and reduces tumor growth in HNSCC. These studies open a novel therapeutic option for modulating anti-tumoral T cell immunity to improve HNSCC outcomes by targeting MIF.

Head and neck squamous cell carcinoma (HNSCC) is a significant public health concern worldwide. Immunomodulatory targets in the HNSCC tumor microenvironment are crucial to enhance the efficacy of HNSCC immunotherapy. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that has been linked to poor prognosis in many cancers, but the mechanistic role of MIF in HNSCC remains unclear. Using a murine orthotopic oral cancer model in Mif + / + or Mif- /- mice, we determined the function of host derived MIF in HNSCC tumor development, metastasis as well as localized and systemic tumor immune responses. We observed that Mif- /- mice have decreased tumor growth and tumor burden compared to their wild-type counterparts. Flow cytometric analysis of immune populations within the primary tumor site revealed increased Th1 and cytotoxic T cell recruitment to the HNSCC tumor microenvironment. Within the tumors of Mif- /- mice, MIF deletion also enhanced the effector function of anti-tumoral effector CD8 + T cells as well as Th1 cells and decreased the accumulation of granulocytic myeloid derived suppressor cells (g-MDSCs) in the tumor microenvironment. Furthermore, MDSCs isolated from tumor bearing mice chemotactically respond to MIF in a dose dependent manner. Taken together, our results demonstrate a chemotactic and immunomodulatory role for host derived MIF in promoting HNSCC and suggest that MIF targeted immunomodulation is a promising approach for HNSCC treatment.

Head and neck squamous cell carcinoma (HNSCC) is common and deadly, and there is a need for improved strategies to predict treatment responses. Ionizing radiation (IR) has been demonstrated to improve HNSCC outcomes, but its effects on immune responses are not well characterized. We determined the impact of IR on T cell immune responses ex vivo. Human and mouse HNSCC cells were exposed to IR ranging from 20 to 200 Gy to determine cell viability and the ability to stimulate T-cell-specific responses. Lymph node cells of LY2 and MOC2 tumor-bearing or non-tumor-bearing mice were re-stimulated with a tumor antigen derived from LY2 or MOC2 cells treated with 200 Gy IR, ultraviolet (UV) exposure, or freeze/thaw cycle treatments. T cell proliferation and cytokine production were compared to T cells restimulated with plate-bound CD3 and CD28 antibodies. Human and mouse HNSCC cells showed reduced viability in response to ionizing radiation in a dose-dependent manner, and induced expression of T cell chemotactic cytokines. Tumor antigens derived from IR-treated LY2 and MOC2 cells induced greater proliferation of lymph node cells from tumor-bearing mice and induced unique T cell cytokine expression profiles. Our results demonstrate that IR induces potent tumoral immune responses, and IR-generated tumor antigens can potentially serve as an indicator of antitumor immune responses to HNSCC in ex vivo T cell restimulation assays.

Head and neck squamous cell carcinoma (HNSCC) is a significant public health problem, with a need for novel approaches to chemoprevention and treatment. Preclinical models that recapitulate molecular alterations that occur in clinical HNSCC patients are needed to better understand molecular and immune mechanisms of HNSCC carcinogenesis, chemoprevention, and efficacy of treatment. We optimized a mouse model of tongue carcinogenesis with discrete quantifiable tumors via conditional deletion of Tgfβr1 and Pten by intralingual injection of tamoxifen. We characterized the localized immune tumor microenvironment, metastasis, systemic immune responses, associated with tongue tumor development. We further determined the efficacy of tongue cancer chemoprevention using dietary administration of black raspberries (BRB). Three Intralingual injections of 500 µg tamoxifen to transgenic K14 Cre, floxed Tgfbr1, Pten (2cKO) knockout mice resulted in tongue tumors with histological and molecular profiles, and lymph node metastasis similar to clinical HNSCC tumors. Bcl2, Bcl-xl, Egfr, Ki-67, and Mmp9, were significantly upregulated in tongue tumors compared to surrounding epithelial tissue. CD4+ and CD8 + T cells in tumor-draining lymph nodes and tumors displayed increased surface CTLA-4 expression, suggestive of impaired T-cell activation and enhanced regulatory T-cell activity. BRB administration resulted in reduced tumor growth, enhanced T-cell infiltration to the tongue tumor microenvironment and robust antitumoral CD8+ cytotoxic T-cell activity characterized by greater granzyme B and perforin expression. Our results demonstrate that intralingual injection of tamoxifen in Tgfβr1/Pten 2cKO mice results in discrete quantifiable tumors suitable for chemoprevention and therapy of experimental HNSCC.

Head and neck squamous cell carcinomas (HNSCC) are one of the most diagnosed malignancies globally, with a 5-year survival rate of approximately 40% to 50%. Current therapies are limited to highly invasive surgery, aggressive radiation, and chemotherapies. Recent reports have demonstrated the potential phytochemical properties of black raspberries in inhibiting the progression of various cancers including HNSCCs. However, the effects of black raspberry extracts on immune cells of the tumor microenvironment, specifically regulatory T cells during HNSCC, have not been investigated. We used a mouse model of 4-nitroquinoline-1-oxide (4NQO) chemically induced HNSCC carcinogenesis to determine these effects. C57BL/6 mice were exposed to 4NQO for 16 weeks and regular water for 8 weeks. 4NQO-exposed mice were fed the AIN-76A control mouse diet or the AIN76 diet supplemented with black raspberry extract. At terminal sacrifice, tumor burdens and immune cell recruitment and activity were analyzed in the tumor microenvironment, draining lymph nodes, and spleens. Mice fed the BRB extract-supplemented diet displayed decreased tumor burden compared to mice provided the AIN-76A control diet. Black raspberry extract administration did not affect overall T-cell populations as well as Th1, Th2, or Th17 differentiation in spleens and tumor draining lymph nodes. However, dietary black raspberry extract administration inhibited regulatory T-cell recruitment to HNSCC tumor sites. This was associated with an increased cytotoxic immune response in the tumor microenvironment characterized by increased CD8+ T cells and enhanced Granzyme B production during BRB extract-mediated HNSCC chemoprevention. Interestingly, this enhanced CD8+ T-cell antitumoral response was localized at the tumor sites but not at spleens and draining lymph nodes. Furthermore, we found decreased levels of PD-L1 expression by myeloid populations in draining lymph nodes of black raspberry-administered carcinogen-induced mice. Taken together, our findings demonstrate that black raspberry extract inhibits regulatory T-cell recruitment and promotes cytotoxic CD8 T-cell activity at tumor sites during HNSCC chemoprevention. These results demonstrate the immunomodulatory potential of black raspberry extracts and support the use of black raspberry-derived phytochemicals as a complementary approach to HNSCC chemoprevention and treatment.