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Updated: May 10, 2026
An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment
Published on: July 28, 2012
Single-cell RNA sequencing analysis identifies acute changes in the tumor microenvironment induced by interferon α
Alexis R Steinmetz1, Morgan Pierce1, Alberto Martini1
1Department of Urology, The University of Texas MD Anderson Cancer Center, Houston, TX, 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.
Insights
This study explored the effects of adenoviral interferon-alpha (Ad-IFNα) gene therapy in a bladder cancer model. Ad-IFNα modulated immune responses and cell death pathways, offering insights for improving cancer treatments.
Area of Science:
- Oncology
- Immunology
- Gene Therapy
Background:
- Nadofaragene firadenovec (Ad-IFNα/Syn3) is approved for BCG-unresponsive bladder cancer.
- Interferon-alpha (IFNα) exhibits anti-cancer properties by inducing apoptosis, inhibiting angiogenesis, and activating the immune system.
Purpose of the Study:
- To establish and utilize an immunocompetent murine bladder cancer model to evaluate the therapeutic effects of murine adenoviral IFNα (muAd-Ifnα).
- To investigate the impact of muAd-Ifnα gene therapy on cancer cells and the tumor microenvironment at a single-cell level.
Main Methods:
- A murine bladder cancer model was established using MB49 cells.
- Mice were treated with muAd-Ifnα or an adenovirus control.
- Single-cell RNA sequencing (scRNAseq) was performed on bladder tissues 72 hours post-treatment to analyze cellular and molecular changes.
Main Results:
- scRNAseq identified 16 distinct cell clusters, including tumor, endothelial, and immune cells.
- IFNα signaling and cell death pathways were upregulated in tumor and endothelial cells.
- T-cells showed increased immunogenic cell death signaling and decreased Th2 pathway activity, while macrophages exhibited altered PD1/PD-L1 and activation pathways.
Conclusions:
- Single-cell analysis reveals Ad-IFNα's broad impact on the tumor microenvironment.
- Findings provide mechanistic insights to enhance patient selection and therapeutic efficacy for Ad-IFNα treatments in bladder cancer.
