Abstract 3143: Leveraging chemo-immunotherapy to remodel the tumor microenvironment and enhance treatment efficacy in osteosarcoma lung metastases

Osteosarcoma (OS), the most common primary bone cancer across all ages, predominantly metastasizing to lungs. At diagnosis, 20% of OS patients present gross OS lung metastases (OSLM), while most others harbor undetectable micrometastases. Despite current treatments, including systemic chemotherapy and experimental therapeutics like immune checkpoint inhibitors, the 5-year survival rate for OSLM continues to be low (ca. 20-30%), making it the leading cause of death in OS patients. Thus, there is an urgent need for innovative therapies to shift OSLM tumor microenvironment (TME) towards a more immunogenic state to overcome tumor tolerance. Gemcitabine (GMT), has shown limited efficacy in relapsed OSLM when administered systemically. Pulmonary administration (PA) of GMT demonstrated promise in reducing tumor burden but does not eradicate it. To address this gap, we propose a novel strategy combining inhaled GMT with colony-stimulating factor-1 receptor inhibitors (CSF-1Ris); pexidartinib (PLX-3397), that target tumor-associated macrophages (TAMs), both delivered locally to lungs via PA. GMT's pulmonary efficacy is partially linked to its ability to upregulate Fas, a death ligand on tumor cells, promoting Fas-mediated apoptosis, and CSF-1Ris are expected to shift TAMs towards a more inflammatory, M1-like phenotype. Our studies began with evaluating GMT's immunomodulatory effects on Fas expression in vitro using OSLM cells. Moving to 3D multicellular tumor spheroid (MCT) model composed of OSLM cells, macrophages, and fibroblasts, we confirmed GMT's ability to increase Fas expression, simulating the results we obtained in OSLM in vivo model. Concurrently, we tested the ability of PLX to shift macrophages from tumor-promoting M2-like phenotype to tumor-suppressing M1-like phenotype. RAW 264.7 macrophages polarized to M2-like state with IL-4 were treated with PLX, resulting in a significant reduction of M2-like macrophages. Similar effects were observed in the 3D MCT model, which recapitulates again our in vivo studies. In vivo, Balb/c mice with OSLM were treated via PA with GMT (5 mg/kg weekly for 2 weeks) and PLX (2 mg/kg every other day for 3 weeks). Tumor burden, tracked with bioluminescence imaging (BLI), has significantly reduced for combination therapy. GMT increased Fas expression in tumor cells, while PLX shifted TAM toward an anti-tumorigenic state, reducing M2 TAMs and enhancing M1/M2 ratio. The treatment was well-tolerated without affecting body weight or overall health. In conclusion, we showed that chemo-immunotherapy combination via PA can effectively remodel the TME in an immunocompetent OSLM mouse model. We also developed a 3D spheroid model with the relevant immune-cells that mimic key aspects for the in vivo model. These studies help us identify clinically translatable combination therapies for OSLM as both GMT and PLX are approved clinically. Citation Format: Raneem R. Aldaqqa,Fatemah S. Sunbul,Victoria A. Garcia,Asma M. Al-terawi,Joshua J. Reineke,Sandro R. da Rocha. Leveraging chemo-immunotherapy to remodel the tumor microenvironment and enhance treatment efficacy in osteosarcoma lung metastases [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 3143.