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Introduction: Soft tissue sarcomas (STS) are rare, heterogenous malignancies with an unmet need for novel immunotherapies. Tumor infiltrating lymphocytes (TILs) have been linked with favorable outcomes in STS patients, though the contribution of natural killer (NK) cells and spatial relationships of TILs with MHC-I expressing cells lacks detailed characterization., Experimental Design: Using archived and prospectively collected specimens, we evaluated intratumoral NK cells by immunohistochemistry (IHC), flow cytometry, and immunofluorescence (IF). We assessed spatial localization of NK and T cells by multiplex IF, analyzing the effects of MHC-I expression status on NK and T cell clustering., Results: Both intratumoral NKp46 and CD56 dim expression were associated with significantly improved overall survival (P=0.05), while higher infiltrates of CD56bright NK cells predicted a worse prognosis (P=0.05). The presence of intratumoral NK cells was inversely proportional to CD3 + T cells. Spatial analyses showed NK cells preferentially clustering close to other NK cells with sparse CD3 + T and CD8 + T cells in range (P<0.0001). Additionally, CD3 + T and CD8 + T cells showed significantly greater co-localization with MHC-I + cells, compared to NK cells (P<0.0001). After neoadjuvant radiotherapy, there was greater CD8 clustering, while after neoadjuvant chemotherapy, there was overall lower TIL clustering., Conclusion: Intratumoral NK cells are prognostic in STS and localize closer to MHC-I- cells than T cells. Although both NK and T cells are associated with improved survival in STS, their differential distribution in the TME based on MHC-I expression status may serve as a biomarker for improved immunotherapy treatment selection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cruz, Sholevar, Judge, Darrow, Iranpur, Farley, Lammers, Razmara, Dunai, Gingrich, Persky, Mori, Thorpe, Monjazeb, Murphy and Canter.)

The microbiome has clearly been established as a cutting-edge field in tumor immunology and immunotherapy. Growing evidence supports the role of the microbiome in immune surveillance, self-tolerance, and response to immune checkpoint inhibitors such as anti PD-L1 and CTLA-4 blockade (1-6). Moreover, recent studies including those using fecal microbial transplantation (FMT) have demonstrated that response to checkpoint immunotherapies may be conferred or eliminated through gut microbiome modulation (7, 8). Consequently, studies evaluating microbiota-host immune and metabolic interactions remain an area of high impact research. While observations in murine models have highlighted the importance of the microbiome in response to therapy, we lack sufficient understanding of the exact mechanisms underlying these interactions. Furthermore, mouse and human gut microbiome composition may be too dissimilar for discovery of all relevant gut microbial biomarkers. Multiple cancers in dogs, including lymphoma, high grade gliomas, melanomas and osteosarcoma (OSA) closely resemble their human analogues, particularly in regard to metastasis, disease recurrence and response to treatment. Importantly, dogs with these spontaneous cancers also have intact immune systems, suggesting that microbiome analyses in these subjects may provide high yield information, especially in the setting of novel immunotherapy regimens which are currently expanding rapidly in canine comparative oncology (9, 10). Additionally, as onco-microbiotic therapies are developed to modify gut microbiomes for maximal responsiveness, large animal models with intact immune systems will be useful for trialing interventions and monitoring adverse events. Together, pre-clinical mechanistic studies and large animal trials can help fully unlock the potential of the microbiome as a diagnostic and therapeutic target in cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kleber, Iranpur, Perry, Cruz, Razmara, Culp, Kent, Eisen, Rebhun and Canter.)

Purpose: Clinical successes using current T-cell based immunotherapies have been limited in soft tissue sarcomas (STS), while pre-clinical studies have shown evidence of natural killer (NK) cell activity. Since tumor immune infiltration, especially tumor-infiltrating lymphocytes, is associated with improved survival in most solid tumors, we sought to evaluate the gene expression profile of tumor and blood NK and T cells, as well as tumor cells, with the goal of identifying potential novel immune targets in STS., Experimental Design: Using fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3 - CD56 + NK and CD3 + T cells and CD45 - viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis. To evaluate survival differences and validate primary DGE results, we also queried The Cancer Genome Atlas (TCGA) database to compare outcomes stratified by bulk gene expression., Results: Sorted intra-tumoral CD3 + T cells showed significant upregulation of established activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. In contrast, intra-tumoral NK cells did not exhibit upregulation of canonical cytotoxic genes (IFNG, GZMB), but rather significant DGE in mitogen signaling (DUSP4) and metabolic function (SMPD3, SLC7A5). Tumors with higher NK and T cell infiltration exhibited significantly increased expression of the pro-inflammatory receptor TLR4 in sorted CD45 - tumor cells. TCGA analysis revealed that tumors with high TLR4 expression ( P = 0.03) and low expression of STMN1 involved in microtubule polymerization ( P < 0.001) were associated with significantly improved survival., Conclusions: Unlike T cells, which demonstrate significant DGE consistent with upregulation of both activating and inhibiting receptors in tumor-infiltrating subsets, NK cells appear to have more stable gene expression between blood and tumor subsets, with alterations restricted primarily to metabolic pathways. Increased immune cell infiltration and improved survival were positively correlated with TLR4 expression and inversely correlated with STMN1 expression within tumors, suggesting possible novel therapeutic targets for immunotherapy in STS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Judge, Bloomstein, Sholevar, Darrow, Stoffel, Vick, Dunai, Cruz, Razmara, Monjazeb, Rebhun, Murphy and Canter.)