{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T16:02:29Z","timestamp":1791475349117,"version":"4.3.4"},"reference-count":51,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,11,4]],"date-time":"2024-11-04T00:00:00Z","timestamp":1730678400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>Nadofaragene firadenovec (Ad-IFN\u03b1\/Syn3) is now approved for BCG-unresponsive bladder cancer (BLCA). IFN\u03b1 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 IFN\u03b1 (muAd-Ifn\u03b1) gene therapy on cancer cells and the tumor microenvironment using a novel murine equivalent of Nadofaragene firadenovec (muAd-Ifn\u03b1).<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>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-Ifn\u03b1 (3x10<jats:sup>11<\/jats:sup> 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\u2019 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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>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-Ifn\u03b1 and Ad-Ctrl were identified. Within the specific cell clusters, IPA analysis revealed significant differences between muAd-Ifn\u03b1 and control. IFN\u03b1 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-Ifn\u03b1 treated tumors compared to controls. PD1\/PD-L1 expression was reduced at 72h.<\/jats:p><\/jats:sec><jats:sec><jats:title>Discussion<\/jats:title><jats:p>This single-cell analysis builds upon our understanding of the impact of Ad-IFN\u03b1 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.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fimmu.2024.1387229","type":"journal-article","created":{"date-parts":[[2024,11,4]],"date-time":"2024-11-04T04:30:27Z","timestamp":1730694627000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["Single-cell RNA sequencing analysis identifies acute changes in the tumor microenvironment induced by interferon \u03b1 gene therapy in a murine bladder cancer model"],"prefix":"10.3389","volume":"15","author":[{"given":"Alexis R.","family":"Steinmetz","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Morgan","family":"Pierce","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alberto","family":"Martini","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Come","family":"Tholomier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ganiraju","family":"Manyam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akshay","family":"Sood","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan J.","family":"Duplisea","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"3rd","given":"Burles A.","family":"Johnson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bogdan A.","family":"Czerniak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Byron H.","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chinnaswamy","family":"Jagannath","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seppo","family":"Yla-Herttuala","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nigel R.","family":"Parker","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David J.","family":"McConkey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Colin P.","family":"Dinney","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sharada","family":"Mokkapati","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2024,11,4]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1016\/j.urolonc.2007.11.033","article-title":"Failure to achieve a complete response to induction BCG therapy is associated with increased risk of disease worsening and death in patients with high risk non-muscle invasive bladder cancer","volume":"27","author":"Lerner","year":"2009","journal-title":"Urol Oncol"},{"key":"B2","unstructured":"D.I.S.C.O. 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