Treatment sequence of stem cell delivered heterologous oncolytic virus impact on tumor microenvironment in immunocompetent ovarian cancer model.

INTRODUCTION

Ovarian cancer (OC) is a biologically heterogeneous malignancy associated with poor clinical outcomes. Oncolytic viruses (OVs) have emerged as a promising immunotherapeutic strategy; however, optimal approaches to maximize their antitumor efficacy-especially in combinatorial or sequential settings-remain insufficiently defined. To determine how the sequence of administration of two clinically relevant, stem cell-delivered OV platforms influences tumor control and immune remodeling in an immunocompetent OC model.

METHODS

We investigated the sequence of two clinically relevant products: neural stem cell delivered conditionally replication competent adenovirus (NSC.CRAd-S-pk7; NNV24) and a mesenchymal stem cell delivered vaccinia virus (MSC.VP001; SNV1). Female C57BL/6 mice were randomized into three groups: untreated tumor-bearing controls, NNV24 followed by SNV1 (NNV24+SNV1), or SNV1 followed by NNV24 (SNV1+NNV24). Therapeutic efficacy and immune responses were assessed by overall survival, histopathological evaluation, flow cytometric analysis of dendritic cell (DC) subsets and cytotoxic T lymphocytes (CTLs), and bulk RNA sequencing.

RESULTS

Treatment regimens initiated with NNV24 significantly prolonged survival compared with SNV1-first sequences. NNV24+SNV1 treatment resulted in enhanced intratumoral viral localization, increased infiltration of CTLs, mature DCs, and concomitant with a reduction in tolerogenic DC populations. Transcriptomic profiling corroborated these findings through revealing suppression of immunoregulatory and tolerogenic pathways signaling in comparison to SNV1+NNV24 group.

DISCUSSION: The treatment sequence of heterologous OV has a significant effect on the tumor microenvironment. NNV24+SNV1 promotes robust antitumor immunity and improved survival, whereas the reverse sequence compromises immune activation and therapeutic response. These findings establish a mechanistic framework for rational sequencing of OV-based immunotherapies.