Engineering Mesenchymal Stromal Cells as Antigen-Presenting Cells to Enhance Anti-Tumor Immunity and Cabozantinib Efficacy in Osteosarcoma

Osteosarcoma (OS) is the most common primary bone malignancy of children and adolescents and disproportionately affects underserved populations, including rural residents, patients with public insurance, and racial/ethnic minorities. These patients more frequently present with metastatic disease, experience higher relapse rates, and suffer worse survival compared to non-underserved groups. Despite advances in multimodal therapy, survival rates for OS have stagnated for decades, and immune checkpoint blockade (ICB), transformative in other cancers, has shown minimal benefit in OS. This reflects profound biological barriers: low tumor mutational burden, a myeloid-skewed immunosuppressive tumor microenvironment (TME), and strikingly low expression of MHC class II (MHC-II) molecules, which impairs CD4⁺ T-cell priming and undermines adaptive immunity. The goal of this project is to overcome these barriers by engineering induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs), abundant in the OS TME and naturally tumor-tropic, into functional antigen-presenting cells. Using a doxycycline-inducible PiggyBac system, iMSCs will be engineered to co-express CIITA, CD80, and CD86. This innovation directly addresses the two critical deficits in OS: MHC-II-restricted antigen presentation and co-stimulation for CD4⁺ T-cell activation. We hypothesize that reprogrammed iMSCs will restore CD4⁺ T-cell priming, amplify antigen presentation, and convert the TME from ICB-refractory to ICB-permissive. Aim 1 will determine how CIITA/CD80/ CD86 engineering enhances iMSC-mediated antigen presentation and CD4⁺ T-cell activation, using MOS-J-OVA lysates and OT-II CD4⁺ T- cells. Aim 2 will test whether engineered iMSCs synergize with a sequential three-step regimen. Step 1: Establish the K7M2 tumor model by intratibial inoculation of K7M2 cell. Step 2: Activate doxycycline-inducible, tumor-tropic iPS-MSC-APCs to prime MHC-II antigen presentation. Step 3: tumoricidal effect, antigen generation and neutralization of suppressive TAMs/ MDSCs via Cabozantinib. Efficacy will be evaluated by survival, and mechanistic profiling of tumor-infiltrating lymphocytes, as well as Th1 polarization and antigen-presentation programs, using flow cytometry, immunohistochemistry, and spatial transcriptomics. This project is significant because it directly addresses the biological mechanisms driving poor outcomes in OS and the inequities that disproportionately affect underserved patients. This approach is innovative because it converts iMSCs into inducible antigen-presenting cells that can home to tumors naturally, stay within the tumor microenvironment, and overcome critical barriers to an effective immune response. If successful, this strategy could improve treatment of osteosarcoma and provide the foundation for treatment of other solid tumors that are not effectively treated with current therapies.