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.

Intravesical bacillus Calmette-Guerin (BCG) G immunotherapy is the gold standard treatment for patients with non-muscle invasive bladder cancer (NMIBC). Unfortunately, more than 50% patients experience early recurrence or progression. Increased intra-tumoral immune infiltration in pre-treatment tumors has been shown to associate with poor outcomes post BCG therapy. Specifically, we recently showed that high intra-tumoral B cell density was associated with early recurrence and progression of NMIBC. Here, we address a major knowledge gap in the role of BCG induced expansion of a B cell population, called atypical B cells (ABCs), in the context of age and sex, the two major variables in the pathophysiology of NMIBC. We first investigated whether pre-BCG TLSs associate with response to BCG. Using multiplex immunofluorescence and NanoString GeoMx digital spatial profiling-based characterization, we found higher density of tumor adjacent (TA) TLSs in patients categorized as BCG non-responders. Spatial proteomic profiling of 49 immune function and phenotype associated proteins revealed increased expression of immune exhaustion associated proteins in pre-BCG TLSs in tumors from both BCG responders and non-responders. It is established that patient age is a risk factor associated with poor outcomes in NMIBC and that biological aging associates with exhausted populations of immune cells. The expansion of ABCs is known to accompany biological aging and repetitive immunizations. We hypothesized that ABCs are recruited to the bladder mucosa due to repeated BCG instillations in the induction phase and dampen local anti-tumor immunity in patients deemed as BCG non-responders. Using the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen induced murine model, we investigated the role of B cells in BCG response in aging mice. Bladder local and systemic immune alterations were characterized at post BCG therapy with or without B cell depletion. Since B cells exhibit significant shifts with biological aging in a sex associated manner, we used the four-core genotype mouse model to determine such differences following repeated treatment with BCG. BCG treatment in combination with B-cell depletion led to distinct bladder immune microenvironment states and systemic immune profiles. Overall, this study demonstrates the significant role of ABCs in disease progression and that the B cell infiltrated pre-BCG therapy TA-TLSs reflect a systemically exhausted B cell functional state in patients deemed as BCG non-responders. Findings from this study provide the first evidence suggesting the role of B cells/ABCs in BCG response and will inform future translation to development of therapies targeting the B cell exhaustion axis.

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 sexual dimorphism in the tumor immune microenvironment of non-muscle invasive bladder cancer and associated clinical outcomes. However, a significant gap in knowledge remains with respect to the current pre-clinical modeling approaches and more precisely recapitulating these differences towards improved therapeutic design. Based on 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 associated responses. Spatial profiling using markers specific to macrophages, T lymphocytes, B lymphocytes, 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 showed a higher number 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 differences in latency of tumor induction. These findings support the incorporation of sex and age as factors in pre-clinical modeling of NMIBC and will potentially advance the field of immunotherapeutic drug development to improve clinical outcomes in NMIBC.