Document resource
Background The immunosuppressive tumor microenvironment (TME) remains a significant barrier to solid tumor immunotherapy. Tumor immunosuppression is primarily driven by myeloid cells, including both tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC). We previously demonstrated that interleukin 12-secreting genetically engineered myeloid cells (IL-12 GEMys) prolong survival in a syngeneic mouse model of rhabdomyosarcoma and become curative when combined with cyclophosphamide and fludarabine (Cy/Flu) preconditioning. IL-12 GEMy therapy was dependent upon an anti-tumor CD8 T cell response. However, the mechanisms of why Cy/Flu is required for cures and how this therapy overcomes tumor myeloid immunosuppression remains unexplored. Further, we have only applied IL-12 GEMys in a limited number of tumor models, therefore testing in additional malignancies is needed to support future clinical translation. Osteosarcoma is a difficult to treat malignant bone tumor with high rates of metastasis, making it an important disease for continued studies of IL-12 GEMy therapy.Methods F42010 murine syngeneic osteosarcoma cells were orthotopically injected into the tibia of C57BL/6 mice and tissues were analyzed by flow cytometry and multiplex immunofluorescence. For treatment experiments, tumors were palpable prior to treatment (typically 10 days). Cy/Flu was given two days prior to GEMy injection by tail vein. GEMys were generated from bone marrow hematopoietic progenitor and stem cells harvested by magnetic bead isolation (StemCell) and transduced with lentivirus during a five day culture in Stemspan SFEM media (StemCell) supplemented with growth factors.Results Orthotopic F42010 osteosarcoma tumors were dominated by an immunosuppressive myeloid infiltrate. Treatment with Cy/Flu depleted most circulating immune cells and TAMs. Cy/Flu plus IL-12 GEMy therapy activated intratumoral CD8+ T cells and was curative. Six days after IL-12 GEMy treatment there was pronounced systemic reprogramming of monocytes and macrophages to a major histocompatibility complex class II (MHCII) positive phenotype. In vitro skewing of bone marrow derived macrophages (BMDM) revealed that pretreatment with tumor conditioned media impaired IFNg-mediated MHCII expression, whereas pretreatment with IFNg prior to TCM exposure overcame suppression of MHCII.Conclusions These results provide preclinical support for IL-12 GEMys in osteosarcoma and shed light on how Cy/Flu plus IL-12 GEMy therapy overcomes tumor immunosuppression by depleting TAMs in the tumor and reprogramming myeloid cells to an MHCII-high phenotype. Our results suggest that exposure to IFNg prior to tumor factors facilitates robust and complete anti-tumor skewing, which may be a key function of Cy/Flu preconditioning by eliminating tumor-skewed myeloid cells and inducing production of naive monocytes.Ethics Approval Mouse experiments were reviewed and approved by the Animal Care and Use Committee (ACUC) at the National Institutes of Health.