A non-invasive assessment of TILs would be valuable as obtaining bladder cancer tissue often requires an invasive procedure under anesthesia. Also, tissue-based biomarkers are subject to potential spatial heterogeneity. A non-invasive biomarker of TILs might generate a potential opportunity for medical therapy in the absence of surgical biopsy.
Previous work suggested that flow cytometry, RNA-Sequencing, and single-cell RNA-Sequencing of urinary immune cells corresponded to various populations of bladder cancer TILs.3,4 To better understand the relevance of immune cells in bladder cancer, we applied high-dimensional multispectral flow cytometry (Cytek Aurora 5L platform) to study urine-derived lymphocytes (UDLs), TILs, and peripheral blood mononuclear cells (PBMC) in a prospectively accruing cohort of patients with treatment-naïve high-grade urothelial bladder cancers undergoing surgery at The Ohio State University. In a subset of our 21-patient cohort, we also performed multiplexed immunofluorescence of tumor tissues to provide spatial insights into TILs.
Our main findings were that, unlike PMBCs, CD4+ and CD8+ UDLs phenotypically mirror TILs from the same patient. Our profiling methods revealed a wide range of T cell developmental and functional states present among UDLs and TILs, ranging from naïve to activated to exhausted T cell clusters. Because our prior pre-clinical work demonstrated that CD8+ T cells among bladder cancer TILs were more often exhausted in male subjects as compared to females,5 we analyzed our patient cohort’s UDLs and TILs by sex. CD4+ and CD8+ T cell clusters in males were more often exhausted, and T cell clusters in females were more often activated, lending important clinical validation to our prior findings. Further, the quantification of male vs female exhausted TILs cells via multiplexed immunofluorescence was consistent with these results.
Our UDL profiling workflow is compatible with both fresh and frozen biospecimens, thus presenting an opportunity to investigate UDLs as a correlative biomarker in bladder cancer clinical trials. To this end, we have profiled UDLs and are analyzing results from NCT02891161 (durvalumab + radiation for locally advanced bladder cancer). Additionally, we are collecting biospecimens from NCT06263153 (durvalumab + futibatinib for muscle-invasive bladder cancer) and planning to analyze UDLs from NCT05521698 (AR inhibitor for non-muscle-invasive bladder cancer) and NCT07000084 (BCG ± gemcitabine for BCG-exposed non-muscle-invasive bladder cancer). The goal of these efforts is to develop and validate the use of UDLs as a robust non-invasive predictive biomarker that corresponds with treatment response for patients with bladder cancer.
Written by: Johanna Schafer, PhD1,2,3 No Joon Song, PhD1,2 Debasish Sundi, MD1,2,4
- Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA
- Pelotonia Institute for Immuno-Oncology, The Ohio State University, Columbus, Ohio, USA
- Roche Diagnostics Solutions, Oro Valley, Arizona, USA
- Department of Urology, The Ohio State University Wexner Medical Center, Columbus, Ohio, USA
- Robertson et al., Nature Communications (2023): Expression-based subtypes define pathologic response to neoadjuvant immune-checkpoint inhibitors in muscle-invasive bladder cancer
- Boll et al., Nature Communications (2025): Predicting immunotherapy response of advanced bladder cancer through a meta-analysis of six independent cohorts
- Wong et al., Journal of Experimental Medicine (2018): Urine-derived lymphocytes as a non-invasive measure of the bladder tumor immune microenvironment
- Tran et al., Journal of Experimental Medicine (2024): Urine scRNAseq reveals new insights into the bladder tumor immune microenvironment
- Kwon et al., Science Immunology (2022): Androgen conspires with the CD8+ T cell exhaustion program and contributes to sex bias in cancer