Alexis R. Steinmetz ¹ Morgan Pierce ¹ Alberto Martini ¹ Côme Tholomier ¹ Ganiraju Manyam ² Yan Chen ¹ Jonathan J. Duplisea ¹ Burles Johnson III ³ Bogdan A. Czerniak ⁴ Byron H. Lee ¹ Chinnaswamy Jagannath ⁵ Seppo Ylä-Herttuala ⁶ Nigel R. Parker ⁶ David J. Mcconkey ⁷ Colin Dinney ¹ Sharada Mokkapati ^1*

• ¹ Department of Urology, University of Texas MD Anderson Cancer Center, Houston, United States
• ² Department of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, Texas, United States
• ³ Johns Hopkins Greenberg Bladder Cancer Institute, Brady Urological Institute, Johns Hopkins University, Baltimore, Maryland, United States
• ⁴ Department of Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas, United States
• ⁵ Department of Pathology and Genomic Medicine, Houston Methodist Research Institute, Houston, Texas, United States
• ⁶ A.I. Virtanen Institute for Molecular Sciences, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Northern Savonia, Finland
• ⁷ ohns Hopkins Greenberg Bladder Cancer Institute, Brady Urological Institute, Johns Hopkins University, Baltimore, Maryland, United States

Introduction: Nadofaragene firadenovec (Ad-IFNα/Syn3) is now approved for BCG-unresponsive bladder cancer (BLCA). IFNα is a pleiotropic cytokine that causes direct tumor cell killing via TRAIL-mediated apoptosis, angiogenesis inhibition, and activation of the innate and adaptive immune system. We established an immunocompetent murine BLCA model to study the effects of murine adenoviral IFNα (muAd-Ifnα) gene therapy on cancer cells and the tumor microenvironment using a novel murine equivalent of Nadofaragene firadenovec (muAd-Ifnα). Methods: Tumors were induced by instilling MB49 cells into the bladders of mice; luciferase imaging confirmed tumor development. Mice were treated with adenovirus control (Ad-Ctrl; empty vector), or muAd-Ifnα (3x1011 VP/mL), and survival analysis was performed. For single-cell sequencing (scRNAseq) analysis (72h), bladders were harvested and treated with collagenase/hyaluronidase and TrypLE for cell dissociation. Single cells were suspended in PBS/1% FBS buffer; viability was assessed with Vicell cell counter. scRNAseq analysis was performed using 10X genomics 3' sequencing. Raw RNAseq data were pre-processed using Cell Ranger single-cell software. Seurat (R package) was used to normalize and cluster the scRNA data. Pooled differential gene expression analysis in specific cell clusters was performed with DESeq2. Results: We identified 16 cell clusters based on marker expression which were grouped into epithelial (tumor), uroplakin-enriched, endothelial, T-cells, neutrophils, and macrophage clusters. Top differentially expressed genes between muAd-Ifnα and Ad-Ctrl were identified. Within the specific cell clusters, IPA analysis revealed significant differences between muAd-Ifnα and control. IFNα signaling and hypercytokinemia/chemokinemia were upregulated in all clusters. Cell death pathways were upregulated in tumor and endothelial clusters. T-cells demonstrated upregulation of the immunogenic cell death signaling pathway and a decrease in the Th2 pathway genes. Macrophages showed upregulation of PD1/PD-L1 pathways along with downregulation of macrophage activation pathways (alternate and classical). Multiplex immunofluorescence confirmed increased infiltration with macrophages in muAd-Ifnα treated tumors compared to controls. PD1/PD-L1 expression was reduced at 72h. Discussion: This single-cell analysis builds upon our understanding of the impact of Ad-IFNα on tumor cells and other compartments of the microenvironment. These data will help identify mechanisms to improve patient selection and therapeutic efficacy of Nadofaragene firadenovec.