Characterization of Tumor Cells Resistant to Herpes Simplex Virus Infection in 3D Melanoma Cultures

Oncolytic viruses have become a viable cancer treatment modality for various malignancies in recent years. The main objective of this study was to gain insight into mechanisms of tumor resistance to viral oncolytic therapy mediated by herpes simplex virus type 1 (HSV-1) using an in vitro 3D model. Previous work found that multicellular spheroids formed by uveal melanoma cells in 3D cultures have increased resistance against HSV-1. The goal of this project was to characterize tumor cells forming HSV-1 resistant spheroids in 3D cultures for tumor cell growth (spheroid size, number of tumor cells per spheroid) and HSV-1 infection [HSV-1 protein and latency associated (LAT) RNA expression] to gain insight into mechanisms of tumor resistance to viral oncolytic therapy. 3D cultures were either infected with K26GFP (an HSV-1 strain expressing green fluorescent protein upon replication), were infected with K26GFP and treated with acyclovir, or were mock infected. Cultures were maintained for seventeen days and observed for fluorescence. On the seventeenth day, cultures were fixed, paraffin embedded, sectioned, analyzed for spheroid morphology, expression of HSV-1 proteins and LAT. We found that HSV-1 infection of 3D melanoma cultures caused the destruction of many tumor cells and was associated with a decrease of the number of multicellular spheroids. Surprisingly, residual HSV-1 resistant spheroids demonstrated increased size and cell numbers relative to mock-infected controls indicating that HSV-1 infection enhanced the growth of a subpopulation of tumor cells. We found that enlarged melanoma spheroids in HSV-1-infected 3D melanoma cultures were mostly negative for HSV-1 protein expression and for HSV-1 LAT expression in spite of HSV-1 infected cells with viral protein and LAT expression were present in the cultures. Future studies will need to further define the mechanisms by which some tumor cells escape HSV-1-mediated destruction in 3D cultures and to determine mechanisms by which HSV-1 induces increased growth in a subpopulation of melanoma cells in 3D cultures.