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.
Recent reports suggest that glucocorticoids (GCs), which can be synthesized in the oral mucosa, play an important role in cancer development. Therefore, the objectives of this study were to characterize the role of the oral GC system in oral cancer, and determine the effect of black raspberry (BRB) administration on GC modulation during oral cancer chemoprevention. We determined the expression of GC enzymes in various oral cancer cell lines, and investigated the role of the GC inactivating enzyme HSD11B2 on CAL27 oral cancer cells using siRNA mediated knockdown approaches. Using two in vivo models of oral carcinogenesis with 4-nitroquinoline-1-oxide (4NQO) carcinogen on C57Bl/6 mice and F344 rats, we determined the effect of BRB on GC modulation during HNSCC chemoprevention. Our results demonstrate that HSD11B2, which inactivates cortisol to cortisone, is downregulated during oral carcinogenesis in clinical and experimental models. Knockdown of HSD11B2 in oral cancer cells promotes cellular proliferation, invasion and expression of angiogenic biomarkers EGFR and VEGFA. An ethanol extract of BRB increased HSD11B2 expression on oral cancer cells. Dietary administration of 5% BRB increased Hsd11b2 gene and protein expression and reduced the active GC, corticosterone, in cancer-induced mouse tongues. Our results demonstrate that the oral GC system is modulated during oral carcinogenesis, and black raspberry administration upregulates Hsd11b2 during oral cancer chemoprevention. In conclusion, our findings challenge the use of synthetic glucocorticoids in head and neck cancer, and support the use of natural product alternatives that potentially modulate GC metabolism in a manner that supports oral cancer chemoprevention.
Progress in stem cell research has revolutionized the medical field for more than two decades. More recently, the discovery of induced pluripotent stem cells (iPSCs) has allowed for the development of advanced disease modeling and tissue engineering platforms. iPSCs are generated from adult somatic cells by reprogramming them into an embryonic-like state via the expression of transcription factors required for establishing pluripotency. In the context of the central nervous system (CNS), iPSCs have the potential to differentiate into a wide variety of brain cell types including neurons, astrocytes, microglial cells, endothelial cells, and oligodendrocytes. iPSCs can be used to generate brain organoids by using a constructive approach in three-dimensional (3D) culture in vitro. Recent advances in 3D brain organoid modeling have provided access to a better understanding of cell-to-cell interactions in disease progression, particularly with neurotropic viral infections. Neurotropic viral infections have been difficult to study in two-dimensional culture systems in vitro due to the lack of a multicellular composition of CNS cell networks. In recent years, 3D brain organoids have been preferred for modeling neurotropic viral diseases and have provided invaluable information for better understanding the molecular regulation of viral infection and cellular responses. Here we provide a comprehensive review of the literature on recent advances in iPSC-derived 3D brain organoid culturing and their utilization in modeling major neurotropic viral infections including HIV-1, HSV-1, JCV, ZIKV, CMV, and SARS-CoV2.
Background Head and neck squamous cell carcinoma (HNSCC) is a significant problem and is frequently resistant to current treatments. STAT1 is important in anti-tumour immune responses against HNSCC. However, the role of STAT1 expression by tumour cells and its regulation during HNSCC is unclear. Methods We determined the effects of STAT1 inhibition on tumour development and immunity in CAL27 and UMSCC22A HNSCC cell lines in vitro and in a HNSCC carcinogen-induced model in vivo. Results STAT1 siRNA knockdown in human HNSCC cells impaired their proliferation and expression of the immunosuppressive marker PD-L1. Stat1-deficient mice displayed increased oral lesion incidence and multiplicity during tumour carcinogenesis in vivo. Immunosuppressive markers PD-1 in CD8+ T cells and PD-L1 in monocytic MDSCs and macrophages were reduced in oral tumours and draining lymph nodes of tumour-bearing Stat1-deficient mice. However, STAT1 was required for anti-tumour functions of T cells during HNSCC in vivo. Finally, we identified TRIM24 to be a negative regulator of STAT1 that plays a similar tumorigenic function to STAT1 in vitro and thus may be a potential target when treating HNSCC. Conclusion Our findings indicate that STAT1 activity plays an important role in tumorigenicity and immunosuppression during HNSCC development.
Cancers of the oral cavity and pharynx contribute 3% of yearly cancer cases in the United States, primarily as head and neck squamous cell carcinoma (HNSCC). HNSCC presents with a five‐year survival rate of 66.2%, with primary risk factors being alcohol abuse or human papillomavirus infection. Myeloid derived suppressor cells (MDSC) are a myeloid lineage cell population known to accumulate within HNSCC tumors and promote a pro‐tumor microenvironment by suppressing the host immune response against tumor cells. Macrophage migration inhibitory factor (MIF) is a cytokine released by immune cells and mediates inflammatory and chemotactic effects upon interaction with its receptor, CD74, expressed by antigen presenting cells (APC), including MDSC. Given their role in promoting tumor growth, our group sought to elucidate the function of host versus tumor cell derived MIF on MDSC. Given the chemotactic effects of MIF upon APC, we hypothesized that knockdown of Mif gene expression would result in a better outcome and reduced tumor burden as a result of reduced MDSC accumulation within the primary tumor microenvironment. Using murine HNSCC cell lines LY2 and MOC2 in conjunction with Mif+/+, Mif‐/‐ and myeloid specific MIF knockout mice (m‐Mif‐/‐), our group has revealed an anti‐immunosuppressive role for host‐derived MIF through its interaction with MDSC, with tumor derived MIF being immunosuppressive in nature. After injection of the tumor cell lines into the buccal mucosa of age matched mice, we monitored tumor development until terminal sacrifice. At this point, we extracted tumor cells for flow cytometric analysis and isolated tumor associated Gr‐1+ MDSC. Extracted MDSC were co‐cultured with naïve T cells fluorescently labeled with CFSE, to enable quantification of suppression of T cell proliferation. Furthermore, we extracted isolated MDSC RNA for analysis by RT‐qPCR. Unexpectedly, our initial finding ran contrary to our hypothesis, as we found that Mif‐/‐ mice had a mean tumor burden of 108.3 mg compared to Mif+/+ mice who presented with a mean tumor of only 64 mg (p = 0.00624). Flow cytometry revealed increased CD11b+ F4/80+ macrophages in mice expressing MIF. Interestingly, a smaller proportion of these cells were identified as MDSC in Mif+/+ and m‐Mif‐/‐ compared to Mif‐/‐, determined by expression of MDSC markers Ly6G and Ly6C. Our group found that both Mif+/+ and m‐Mif‐/‐ demonstrated reduced suppression of T cell proliferation compared to Mif‐/‐. Gene expression analysis revealed in Mif‐/‐ MDSC upregulation of immunosuppressive genes Arg1, Ptgs2, S100a8 and S100a9. Seeing that both Mif+/+ and m‐Mif‐/‐ demonstrate similar anti‐tumor phenotypic and molecular responses to HNSCC, our group has concluded that in response to host derived, but not tumor derived, MIF, MDSC at the tumor site shift towards a more anti‐tumor M1 phenotype and additionally that MIF derived from non‐myeloid host sources within the tumor microenvironment are sufficient to stimulate this response.
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