Abstract 1268: A matter of TIME: Advancing a humanized mouse model to decode tumor immune microenvironment and uncover mechanisms of resistance in soft tissue sarcoma

Background: Humanized mouse models are invaluable tools in cancer research. These immunodeficient mice, engrafted with human hematopoietic stem cells (HSCs) allow the exploration of immune dynamics and therapeutic responses in cancer. However, traditional models underrepresent key immune cells, particularly natural killer (NK) cells, which are crucial for anti-tumor immunity. We developed an improved humanized mouse model with optimized NK cell reconstitution to better reflect human immune responses. Using this model, we studied soft tissue sarcoma (STS), a malignancy with an immunosuppressive tumor immune microenvironment (TIME) that contributes to therapy resistance. Our goal was to better understand immune dynamics and resistance mechanisms within the TIME and explore novel immunotherapy strategies. Methods: Humanized mice were generated by engrafting immunodeficient mice with HSCs, partially HLA-A matched to the SK-UT1 cell line for better immune representation of STS. NK cell reconstitution was improved with human IL-15 (hIL-15). Mice were engrafted with SK-UT1 STS cells to simulate the TIME. Tumor-infiltrating lymphocytes (TILs) and peripheral immune cells were analyzed by flow cytometry and single-cell sequencing to assess infiltration, activation, and exhaustion. Key immune checkpoints (PD-1, TIM-3, LAG-3, CTLA-4) and cytokines were evaluated to identify differences between TILs and peripheral immune cells. Results: Diverse immune cell populations were observed within the TIME, including T cells, NK cells, and macrophages. Tumor-infiltrating CD4+ and CD8+ T cells showed higher expression of exhaustion markers compared to circulating T cells, indicating immune suppression and impaired cytotoxic function. The model demonstrated increased infiltration of CD56+ NK cells, offering a more accurate representation of NK cell function in the TIME. The gradient of exhaustion markers across immune cells suggested a highly immunosuppressive environment, contributing to tumor progression and resistance. Conclusions: Our improved humanized mouse model offers a robust platform for studying immune-tumor interactions, particularly TILs and their role in the TIME. This model holds significant potential for testing novel immunotherapy combinations, including immune checkpoint inhibitors and targeted therapies aimed at reversing immune exhaustion. Ongoing analyses are focused on identifying key pathways involved in immune evasion through single-cell and TCR sequencing, and uncovering distinct resistance mechanisms within the tumor, potentially revealing new strategies to overcome immunotherapy resistance. These insights will reshape our understanding of the TIME and pave the way for innovative treatments to improve outcomes in STS. Citation Format: Isha Karnik, Wai Nam Liu, Min Liu, Wilson Tan, Sue Yee Tan, Shin Yie Fong, Valerie Yang, Qingfeng Chen. A matter of TIME: Advancing a humanized mouse model to decode tumor immune microenvironment and uncover mechanisms of resistance in soft tissue sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1268.