Sex differences in the aging murine urinary bladder and influence on the tumor immune microenvironment of a carcinogen-induced model of bladder cancer

Abstract

Sex and age associated differences in the tumor immune microenvironment of non-muscle invasive bladder (NMIBC) cancer and associated clinical outcomes are emerging indicators of treatment outcomes. The incidence of urothelial carcinoma of the bladder is four times higher in males than females; however, females tend to present with a more aggressive disease, a poorer response to immunotherapy and suffer worse clinical outcomes. Recent findings have demonstrated sex differences in the tumor immune microenvironment of non-muscle invasive and muscle invasive bladder cancer and associated clinical outcomes. However, a significant gap in knowledge remains with respect to the current pre-clinical modeling approaches to more precisely recapitulate these differences towards improved therapeutic design. Given the similarities in mucosal immune physiology between humans and mice, we evaluated the sex and age-related immune alterations in healthy murine bladders. Bulk-RNA sequencing and multiplex immunofluorescence-based spatial immune profiling of healthy murine bladders from male and female mice of age groups spanning young to old showed a highly altered immune landscape that exhibited sex and age associated differences, particularly in the context of B cell mediated responses. Spatial profiling of healthy bladders, using markers specific to macrophages, T cells, B cells, activated dendritic cells, high endothelial venules, myeloid cells and the PD-L1 immune checkpoint showed sex and age associated differences. Bladders from healthy older female mice also showed a higher presence of tertiary lymphoid structures (TLSs) compared to both young female and male equivalents. Spatial immune profiling of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen exposed male and female bladders from young and old mice revealed a similar frequency of TLS formation, sex differences in the bladder immune microenvironment and, age associated differences in latency of tumor induction. These findings support the incorporation of sex and age as factors in pre-clinical modeling of bladder cancer and will potentially advance the field of immunotherapeutic drug development to improve clinical outcomes.

D-mannose ameliorates age-associated cellular senescence in the bladder urothelium and NLRP3/Gasdermin/IL-1β -driven pyroptotic epithelial cell shedding

“…Recent work from our group and others has identified a new and potentially critical contributor to an altered bladder mucosal environment with age that is responsible for inflammaging, namely, formation of bladder tertiary lymphoid tissue (bTLT) with bone fide germinal centers that arise in an age-dependent manner coincident with loss of fertility in female mice (Ligon et al, 2020). Notably, bTLT only forms in a sex-dependent manner in female bladders (Hamade et al, 2022).…”

“…Recent work from our group and others has identified a new and potentially critical contributor to an altered bladder mucosal environment with age that is responsible for inflammaging, namely, formation of bladder tertiary lymphoid tissue (bTLT) with bone fide germinal centers that arise in an age-dependent manner coincident with loss of fertility in female mice (Ligon et al, 2020). Notably, bTLT only forms in a sex-dependent manner in female bladders (Hamade et al, 2022). bTLT occurs in postmenopausal women and is significantly associated with increased frequency of rUTIs and shorter time intervals between recurrent episodes (Ligon et al, 2020) and are associated with bacteria including E. coli (De Nisco et al, 2019).…”

“…Recent work has shown that aging alone initiates significant changes to the immune landscape of the aged female urinary tract including the formation of bladder tertiary lymphoid tissues (bTLT) (Hamade et al, 2022; Ligon et al, 2020). This raises important questions about how the urothelium is affected by aging and related immune changes, and what mechanistic role these changes might play in the development of UTIs and their recurrence.…”

A Computational Approach to Identification of Candidate Biomarkers in High-Dimensional Molecular Data

“…Third, we developed an intuitive, GUI that allows users, irrespective of computational expertise, to apply MFeaST on any dataset. Additionally, we successfully applied MFeaST to find biomarkers in many different omics datasets including messenger RNA-Seq [ 24 , 25 , 26 ], RT-qPCR [ 27 , 28 ], microRNA-Seq [ 29 , 30 , 31 , 32 ], NanoString profiles [ 33 ], and protein mass spectrometry [ 34 ]. These datasets were often characterized by a large number of features versus a relatively small number of samples, and usually with a lot of sparse data.…”

“…The algorithm applies a greedy method [ 23 ] to rank all available features based on the ensemble results of a diverse selection of methods and families of predictors. To date, we have successfully applied MFeaST to identify features of interest in many different -omics datasets [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. Here, we demonstrate the effectiveness of MFeaST in selecting a small set of features as potential biomarkers in prostate adenocarcinoma mRNA profiles using immune-function related genes.…”