Abstract 5891: Identification of intratumor pathogens from single-cell RNA sequencing, cellular dysfunction and immune response
Authors: Bukavina Laura , Sindhani Mohit , Valentine Henkel , Ranti Daniel , Mishra Kirtishri , Kutikov Alexander , Gupta Shilpa , Sfakiano John , Ghannoum Mahmoud , Retuerto Mauricio , Correa Andres , Uzzo Robert , Abbosh Philip 
Published: April 2023

Abstract
Introduction: We aim to assess the transcriptomic features of the single cell level to identify intracellular pathogens in patients with high grade bladder cancer.
Methodology: CellRanger and Alevin were utilized to extract cell barcodes and unique molecular identifiers. After quality control, reads were aligned with human reference genome with Kraken2 for species level identification of bacteria. Contaminants (false positives) were removed based on higher frequencies and negative control (reference control) of 2,491 sterile cell experiments. Seuret Findmarkers, scIntegrate, and scDensity Plot were utilized to normalize the data, cluster the cells, and calculate markers in each cluster of differentially expressed genes between the two conditions. We focused our analysis on Pseudomonas aeruginosa due to high urogenital virulence, and immunomodulation capabilities by pattern recognition receptors and inflammasome.
Results: We identified presence of three over-represented intracellular taxa in tissue biopsies of patients with BC compared to healthy controls (after false discovery rate correction and cross validation of contaminants). Patients with history of high-grade BC demonstrated increased presence of Aeromonas (175,473 reads, 54%), Pseudomonas (75,002 reads, 26.73%), and Bacillus (15,410 reads, 5.49%) in their tissue samples, not visualized in healthy controls (counts &lt;10). Among 8,324 cells (across 7 patients), classical CD14+ monocyte subtype was most likely to be exposed to Pseudomonas species (4150/5069, 81.8%), followed by non-classical CD16+ monocytes (722/1040, 69.4%), and CD8+ Tcells (348/680, 51.1%). (Figure 1A-C). Relative gene expression level between exposed and non-exposed monocytes demonstrated total 25 upregulated genes, with increased expression GNBL21 (aka: RACK1, log10 4.1), MALAT1(log10 3.7), TCEB2(log10 2.9) (increasing proliferation, migration, and reduction of cell autophagy) compared to non-infected, and normal controls. Pseudomonas involvement of non-monocyte cells (CD8+, CD4+Th17, CD4+ naïve) was associated with disease progression (2/5 patients), as well as increased expression of IFI6/27(interferon alpha inducible protein 6 &27), ITAC (interferon inducible T cell alpha-chemoattractant).
Conclusion: Altogether, our analysis depicted the distribution of intracellular bacteria not previously recognized within tumor microenvironment utilizing scRNAseq, and revealed overexpression of RACK1 in Pseudomonas positive cells, associated with apoptosis resistance.
Citation Format: Laura Bukavina, Mohit Sindhani, Henkel Valentine, Daniel Ranti, Kirtishri Mishra, Alexander Kutikov, Shilpa Gupta, John Sfakiano, Mahmoud Ghannoum, Mauricio Retuerto, Andres Correa, Robert Uzzo, Philip Abbosh. Identification of intratumor pathogens from single-cell RNA sequencing, cellular dysfunction and immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5891.