Gene-modified iPSC-derived MSCs convert immunosuppressive tumor microenvironments (TMEs) into distinct  Pro-inflammatory tmes

Abstract Background: Immunotherapy has transformed cancer care, inducing durable responses in both hematologic and solid tumors. Despite these advances, many patients derive limited benefit, potentially secondary to an immunosuppressive tumor microenvironment (TME), defined by defective antigen presentation, immunosuppressive cell infiltration, and stromal barriers that restrict T-cell trafficking. Our group has established the use of mesenchymal stem cells (MSCs) as modifiers of the tumor microenvironment (TME) (Studeny M…Andreeff, Cancer Res 2002; JNCI 2004). We demonstrated in solid tumors that intravenously injected MSCs have the innate capacity to migrate into the TME and contribute to the generation of tumor-associated fibroblasts at the invasive edge of the tumor and their metastases. While MSCs are believed to be immunosuppressive, we hypothesized that they could be modified into immunostimulatory cells and convert a “cold” TME into a pro-inflammatoryTME. In a clinical pilot trial, we demonstrated proof-of-concept that IFNβ-secreting MSCs homed to ovarian tumors and mediated intratumoral IFNβ delivery (NCT02530047)(Andreeff M et al. Cancer Res (2018)78:75). Results: We developed an immunotherapeutic strategy using induced pluripotent stem cell–derived mesenchymal stem cells (iMSCs) engineered to secrete IL-7 and IL-15, two potent cytokines selected for their robust immunostimulatory properties. iMSCs, clonally derived from iPSCs, offer superior expansion and scalable production compared to BM-MSCs. These iMSCs retained classical MSC morphology and proteogenomic profiles, supporting their identity and functionality. Telomere length and CyTOF-based cell cycle analysis revealed high proliferative capacity, far superior to BM-MSCs. Stable transgene expression was validated using GFP-tracked clones throughout differentiation and expansion. These iMSCs enhanced bone marrow regeneration following radiation therapy (Okeleji, O et al ASH2025). Proprietary gene editing ensured efficient and consistent integration of IL7 and IL15 transgenes, providing a robust platform for cytokine delivery. IL-7/IL-15-iMSCs produced supraphysiological cytokine levels (2,000–4,000 pg/mL), representing a nearly 20-fold increase over levels seen in previously engineered T cells. This enhanced cytokine output drove proliferation of resting and CD3/CD28-activated T cells, as confirmed via CFSE assays. CyTOF profiling revealed robust downstream STAT5 phosphorylation, pS6 activation, and BCL2 upregulation, indicative of efficient IL-7/IL-15 receptor downstream signaling. Hence, immunosuppressive iMSCs were modified to be potent immunostimulatory cells. We applied this concept to high-grade serous ovarian carcinomas (HGSOC). PBMCs conditioned with IL-7/IL-15-iMSCs significantly reduced the number of both platinum-sensitive and platinum-resistant ovarian cancer cells in vitro. To evaluate therapeutic efficacy in vivo, we administered IL-7/IL-15-iMSCs i.p. into the ID8 murine ovarian cancer model in immunocompetent mice. Multiplex immunofluorescence demonstrated that iMSCs home to tumors, secrete cytokines locally, and promote intratumoral T-cell, NK-cell, and macrophage infiltration, collectively leading to prolonged survival. This outcome was associated with a shift from an immune-excluded to an inflamed, immune-infiltrated TME. The ovarian TME was enriched in CD163⁺Arg1⁺PD-L1⁺M2-like macrophages, which exhibited a phenotypic shift toward an M1-like state, marked by Arginase-1 downregulation, in the IL-7/IL-15 treatment group. Experiments investigating activity of IL-7/IL-15-iMSCs in carboplatin-resistant ID8 models are ongoing and will be reported. Results in triple-negative breast cancer will be reported separately (Singh, S et al. ASH2025). Conclusion: We demonstrate that IL-7/IL-15-iMSCs display potent in vitro immunostimulatory activity, inducing robust T cell proliferation and activation. IL-7/IL-15-secreting iMSCs convert an immunosuppressive TME into a “hot” milieu, defined by massive T-cell and macrophage infiltration, M1 polarization, and restoration of T-cell functionality. Gene-modified iMSC-induced reprogramming of ovarian TME resulted in reduced tumor burden and improved survival in a murine ovarian cancer model. Results support the potential of IL-7/IL-15-iMSCs as a novel immunotherapeutic platform for overcoming immune resistance in cancer, including hematological malignancies.