Identification of oncolytic vaccinia restriction factors in canine high-grade mammary tumor cells using single-cell transcriptomics.

Béatrice Cambien,Marine Gautier-Isola,Jérôme Henriques,Olivier Ferraris,Nicolas Nottet,Roger Rezzonico,Virginie Magnone,Christophe N Peyrefitte,Catherine Compin,Georges Vassaux,Robert Barthel,Sophie Giorgetti-Peraldi,Audrey Lamit,Pascal Barbry,Bernard Mari,Alberto Baeri,Frédéric Bost,Laure-Emmanuelle Zaragosi,Kevin Lebrigand

doi:10.1371/journal.ppat.1008660

Abstract

Mammary carcinoma, including triple-negative breast carcinomas (TNBC) are tumor-types for which human and canine pathologies are closely related at the molecular level. The efficacy of an oncolytic vaccinia virus (VV) was compared in low-passage primary carcinoma cells from TNBC versus non-TNBC. Non-TNBC cells were 28 fold more sensitive to VV than TNBC cells in which VV replication is impaired. Single-cell RNA-seq performed on two different TNBC cell samples, infected or not with VV, highlighted three distinct populations: naïve cells, bystander cells, defined as cells exposed to the virus but not infected and infected cells. The transcriptomes of these three populations showed striking variations in the modulation of pathways regulated by cytokines and growth factors. We hypothesized that the pool of genes expressed in the bystander populations was enriched in antiviral genes. Bioinformatic analysis suggested that the reduced activity of the virus was associated with a higher mesenchymal status of the cells. In addition, we demonstrated experimentally that high expression of one gene, DDIT4, is detrimental to VV production. Considering that DDIT4 is associated with a poor prognosis in various cancers including TNBC, our data highlight DDIT4 as a candidate resistance marker for oncolytic poxvirus therapy. This information could be used to design new generations of oncolytic poxviruses. Beyond the field of gene therapy, this study demonstrates that single-cell transcriptomics can be used to identify cellular factors influencing viral replication.

Highlights

Oncolytic vaccinia virus (VACV) represents a new class of anticancer agent with multiple mechanisms of action
In the context of oncolytic virotherapy, the susceptibility of different grades of primary low-passage mammary carcinoma cells of canine origin to an oncolytic vaccinia virus (VV)
Single-cell RNAseq performed on triple-negative breast carcinomas (TNBC) cell samples infected with VV suggested that the reduced activity of the virus was associated with a higher mesenchymal status of the cells

Summary

Introduction

Oncolytic vaccinia virus (VACV) represents a new class of anticancer agent with multiple mechanisms of action. VACV has been shown to act at three distinct levels [1]. VACV infects and selectively replicates in cancer cells, leading to primary oncolysis and resulting in cancer cell destruction [2]. It disrupts the tumor vasculature [3] and reduces tumor perfusion. The release of tumor antigens from dead tumor cells participates in the initiation of an immune response that may be effective against tumor cells [1, 4,5,6,7]. VACV, administered intratumorally or systemically has been well tolerated in various clinical trials [4]

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