Abstract A major challenge in osteosarcoma (OS) immunotherapy is overcoming the immunosuppressive tumor microenvironment (TME), to restore immune recognition and destruction of tumor cells. One of the key immune cell subsets known to contribute to the immunosuppressive TME are M2-polarized tumor-associated macrophages (TAMs). Our work aims to identify novel oncolytic virus (OV) combination immunotherapies to induce direct lysis of OS tumor cells, activate immune-mediated destruction of OS, and remodel the immunosuppressive TME. The direct lytic activity of oncolytic herpes simplex virus (oHSV; HSV1716) treatment was assessed using LIVE/DEAD flow cytometry and viral replication was measured using standard plaque assay. NK cell degranulation and killing assays were performed by co-culture of OS cell lines with peripheral blood mononuclear cells (PBMC) treated ± oHSV; NK cell degranulation and target cell death was assessed by flow cytometry. TAMs were generated in vitro by co-culture of PBMC with OS cell lines or primary bone marrow-derived mesenchymal stem cells (MSCs) for 7 days. Phagocytosis assays were performed by co-culture of TAMs with OS cells using fluorescent cell tracker stains and flow cytometry. Multicellular spheroids were generated by co-culture of firefly luciferase-expressing OS cell lines with CD14+ monocytes and bone marrow derived MSCs for 7 days in low adhesion plates, viability of tumor cells after treatment was assessed by addition of d-luciferin and quantification of luminescence. Treatment of four OS cell lines with oHSV for 72 hours resulted in <20% cell death by direct lysis. However, after treatment with oHSV-GFP, >95% of OS cells were GFP+ and viral titer increased up to 105-fold when compared to viral input. This highlighted high levels of infection and replication of oHSV within OS cell lines, despite limited oncolysis. Moreover, oHSV treatment of PBMC significantly enhanced the immune-mediated killing of OS cell lines, and this was NK cell dependent. Monolayer co-culture of PBMC with OS cell lines or MSC generated TAMs with significantly increased pro-tumor M2-like phenotype (CD14+CD206+CD163+), when compared with PBMC cultured alone, and TAMs suppressed NK cell degranulation against OS cell lines. Treatment of TAMS with oHSV encoding GM-CSF reduced the proportion of M2 TAMs. Moreover, treatment of OS target cells with anti-EGFR monoclonal antibody (mAb) and co-culture with in vitro generated TAMs significantly increased their phagocytic activity. Multicellular spheroids incorporating MSCs and CD14+ monocytes displayed a high degree of resistance against immune-mediated killing, when compared with OS cell line alone as monolayer or spheroid cultures. However, combination of OV with anti-EGFR mAb significantly increased immune-mediated killing against multicellular spheroids. oHSV combination strategies with mAb may be a promising treatment strategy against OS, to exert direct oncolysis, stimulate immune-mediated destruction, and remodel the immunosuppressive TME. Citation Format: Tyler K. Barr, Victoria A. Jennings, Alison Taylor, Jessica Murby, Natasha J. Caplen, Javed Khan, Richard Baugh, Heather E. Owston, Dennis McGonagle, Fiona Errington-Mais, Graham P. Cook. Oncolytic HSV1716-GMCSF combination strategies to remodel the immunosuppressive osteosarcoma tumor-microenvironment and promote anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B049.
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