Abstract 3987: Direct cancer cell/cancer-associated fibroblast interaction elicits resistance to oncolytic virus therapy in squamous cell carcinoma
Authors: Rullan Antonio , Milford Emma , Arwert Esther , Melcher Alan , Harrington Kevin , Sahai Erik 
Published: September 2020

Abstract
The interaction between different cells in the tumor microenvironment leads to changes in a plethora of cytokines secreted both by malignant and non-malignant cells. Invading epithelial tumors are able to break the basal membrane, which allows for direct cell-to-cell contact between cancer cells and fibroblasts present in the sub-epithelial tissues. To imitate this event, we performed co-culture experiments using human squamous cancer cells (SCC) and patient-derived cancer-associated fibroblasts (CAFs) of gynecological origin. This direct co-culture led to production of IFNβ and interferon-stimulated genes (ISGs), whereas co-culture using a transwell did not. Using siRNA, confocal microscopy and DNA damage compounds, we showed that CAFs can sense genotoxic stress present in the cancer cells (CC).Transfer of cytoplasmic material containing cGAMP from CC to CAFs triggers the STING-TBK-IRF3 pathway that induces an IFN type I response. Oncolytic viruses are approved in melanoma and are being tested in multiple clinical trials to treat human malignancies. There is very scarce information on how the interaction among cells in the tumor microenvironment affects the efficacy of viral therapy. We hypothesized that ISGs produced in the direct co-culture would impair the efficacy of oncolytic virus infection. Indeed, using a modified herpes virus (HSV1716), we showed that direct co-culture of CC and CAFs elicits resistance to viral infection. We were able to replicate this phenomenon using cancer cells and CAFs from gynecological and head and neck origin. Moreover, direct co-culture also decreased infectivity of other oncolytic viruses, such as vaccinia and reovirus. This resistance was dependent on IRF3 expression in CAFs, as silencing IRF3 in these cells abrogated this protection. We were able to rescue this effect by transfecting a siRNA-resistant form of IRF3 into the CAFs. Overexpression of IRF3 in CAFs further increased resistance to HSV infection in the cancer cells. Using a xenograft in vivo model of human gynecological SCC, we observed that co-injection of cancer cells and CAFs promoted resistance to HSV treatment. Transfection of siRNA targeting IRF3 into CAFs dramatically decreased tumor growth after HSV infection. This decrease was not seen when using CAFs expressing the siRNA-resistant form of IRF3. An additional model of human Head and Neck SCC demonstrated a gain-of-function effect of IRF3 overexpression on tumor growth after oncolytic virus treatment. Finally, through analysis of a single-cell RNA sequencing dataset and histological staining we were able to show IRF3 expression in CAFs present in the tumor microenvironment. Our data highlight the relevance of cancer cell/cancer-associated fibroblast crosstalk in the TME. Changes in cytokine and chemokines present in the tumor may result in changes of susceptibility to treatments like oncolytic viruses. Additionally, this could have a broader impact in the interaction between the tumor and the immune infiltrate.
Citation Format: Antonio Rullan, Emma Milford, Esther Arwert, Alan Melcher, Kevin Harrington, Erik Sahai. Direct cancer cell/cancer-associated fibroblast interaction elicits resistance to oncolytic virus therapy in squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3987.