Single-cell RNA sequencing reveals a strong connection between Gadd45g upregulation and oncolytic HSV infection in tumor tissue

The oncolytic effect of virotherapy derives from the intrinsic capability of the applied virus in selectively infecting and killing tumor cells. Although oncolytic viruses of various constructions have been shown to efficiently infect and kill tumor cells in vitro, the efficiency of these viruses to exert the same effect on tumor cells within tumor tissues in vivo has not been extensively investigated. Here we report our studies using single-cell RNA sequencing to comprehensively analyze the gene expression profile of tumor tissues following herpes simplex virus 2-based oncolytic virotherapy. Our data revealed the extent and cell types within the tumor microenvironment that could be infected by the virus. Moreover, we observed changes in the expression of cellular genes, including antiviral genes, in response to viral infection. One notable gene found to be upregulated significantly in oncolytic virus-infected tumor cells was Gadd45g, which is desirable for optimal virus replication. These results not only help reveal the precise infection status of the oncolytic virus in vivo, but also provide insight that may lead to the development of new strategies to further enhance the therapeutic efficacy of oncolytic virotherapy. Overall design: FusON-H3, HSV-2 based oncolytic virus at 5X10^6 pfu was injected intra-tumorally into 8-10 mm subcutaneous H7, H7-Her2, MC38gp100 and MC38gp100-Her2 tumors in C57BL/6 mice (n=3). The tumors were harvested at 48 hours post-virotherapy, pooled, digested dissaggregated to single cell suspensions. The single cell preparations were then sorted into CD45+ and CD45- cells and mixed at the ratio of 3:1 and processed for scRNA-seq.