INTRODUCTION AND OBJECTIVE: Prune Belly Syndrome (PBS) is characterized by bladder dysmyogenesis, yielding a dysfunctional compliant thick wall with excess collagen deposition. To dissect the cellular heterogeneity and gene expression networks altered in PBS, we report the cell type composition and transcriptional activity of PBS human bladder by using single cell RNA sequencing (scRNA-seq).METHODS: Using IRB-approved methods, bladder dome from 2 PBS and 6 non-PBS control (CO) males underwent fresh single-cell digestion. scRNA-seq was performed and 5277 and 31828 bladder cells from PBS and CO patients was detected, respectively. Cell type clusters were graphically displayed by Uniform Manifold Approximation and Projection (UMap) plot and differentially expressed genes (DEGs) were generated to assign each cluster identity. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis was performed for PBS affected genes.RESULTS: We identified 17 distinct bladder cell clusters, including 6 fibroblast (1, 2, 3, and 4, immunofibroblast, myofibroblast), 1 smooth muscle (SM), and 2 urothelial (umbrella and basalþintermediate) clusters (Fig 1A-B). Counts of individual cell types were expressed as relative proportions, identifying significant PBS fibroblast enrichment, (67% PBS vs 40% CO). Five of 6 PBS fibroblast sub-types are proportionately fewer in number than in CO. The exception is a dominant fibroblast sub-type we label as fibroblast 4, (61% of all PBS fibroblasts vs <10% CO fibroblast subtypes). SM and urothelial cell populations are dramatically reduced in PBS (SM: 5% PBS vs 11% CO and urothelial: <1% PBS vs 7% CO) (Fig 1C-E). PBS fibroblast DEGs, but not SM cells, are enriched in collagen genes. Fibroblast markers (DCN and PLA2G2A) and SM genes (DES, TPM2 and TAGLN) are reduced (by 4, 13, 2, 4, and 2x respectively) in PBS cells (Fig 1G). KEGG pathways analysis for fibroblasts and SM showed a highly similar enrichment for neurodegenerative disease pathways (Fig 1H-I).CONCLUSIONS: Using scRNA-seq, we identified and characterized the disarrayed cell type populations in PBS bladders, generating their unbiased transcriptomic signatures which highlight commonality with neurodegenerative diseases. This PBS transcriptomic map is a step toward potential markers for diagnosis and therapeutic intervention.

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Checkpoint inhibitor (CPI) therapy represents an exciting new treatment option for patients with advanced malignancies. However, several adverse events (AEs) that affect multiple body systems are associated with CPI treatment, including colitis. The mechanisms mediating these AEs remain to be fully elucidated. That mice treated with CPI fail to develop the spectrum of AEs observed in patients hampers the field's ability to develop supportive care treatment options. Here we sought to examine colitis in tumor bearing mice undergoing CPI therapy. Briefly, naïve TH cells and Treg cells were harvested from C57Bl/6 donor mice and were adoptively transferred into RAG2−/- recipients. Animals were inoculated with B16F10 cells into the left flank seven days later. T cell engraftment was assessed after 13 days, and animals were randomized into treatment groups by rate of T cell engraftment; animals were treated Q3D with either vehicle or anti-CTLA4 for an additional four weeks. Animals were monitored for body weight loss and systemic AEs (gastrointestinal, pulmonary, neurological) daily and tumor volume was measured by digital caliper three times per week. Extent of colitis was evaluated by video endoscopy at several timepoints and colitis severity was scored using a 0-4 scale. Animals developed body weight loss approximately two weeks after T cell transfer and CPI treatment increased the severity of body weight loss observed. Colitis was seen by video endoscopy within a similar timeframe. Mean tumor volume was decreased in animals treated with CPI as compared to those treated with vehicle. Diarrhea, pulmonary symptoms, and neurological symptoms were observed in a subset of animals over the study period; anti-CTLA4 treatment increased the overall incidence of pulmonary symptoms. These data demonstrate the utility of this system for assessment of colitis or other AE supportive care treatment options in the context of CPI-treated, tumor-bearing mice. Citation Format: Caitlin S. Parello, Benjamin G. Cuiffo, Kasey Reardon, Alexandria Fusco, Brett Van Dam, Gregory D. Lyng, Stephen T. Sonis. Colitis in tumor-bearing mice undergoing checkpoint inhibitor therapy [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 585.