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.

Poor response to Bacillus Calmette-Guérin (BCG) immunotherapy remains a major barrier in the management of patients with non–muscle invasive bladder cancer (NMIBC). Multiple factors are associated with poor outcomes, including biological aging and female sex. More recently, it has emerged that a B-cell–infiltrated pretreatment immune microenvironment of NMIBC tumors can influence the response to intravesically administered BCG. The mechanisms underlying the roles of B cells in NMIBC are poorly understood. Here, we show that B-cell–dominant tertiary lymphoid structures (TLSs), a hallmark feature of the chronic mucosal immune response, are abundant and located close to the epithelial compartment in pretreatment tumors from BCG non-responders. Digital spatial proteomic profiling of whole tumor sections from male and female patients with NMIBC who underwent treatment with intravesical BCG, revealed higher expression of immune exhaustion–associated proteins within the tumor-adjacent TLSs in both responders and non-responders. Chronic local inflammation, induced by the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen, led to TLS formation with recruitment and differentiation of the immunosuppressive atypical B-cell (ABC) subset within the bladder microenvironment, predominantly in aging female mice compared to their male counterparts. Depletion of ABCs simultaneous to BCG treatment delayed cancer progression in female mice. Our findings provide evidence indicating a role for ABCs in BCG response and will inform future development of therapies targeting the B-cell–exhaustion axis.

Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC₄), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC₄ binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC₄ binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC₄-binding site.

Multidrug resistance protein 1 (MRP1) (gene symbol ABCC1) is an ATP-binding cassette (ABC) transporter which effluxes xeno- and endobiotic organic anions including estradiol glucuronide and the pro-inflammatory leukotriene C₄. MRP1 also confers multidrug resistance by reducing intracellular drug accumulation through active efflux. MRP1 has three membrane spanning domains (MSD), and two nucleotide binding domains (NBD). MSD1 and MSD2 are linked to NBD1 and NBD2 by connecting regions (CR) 1 and CR2, respectively. Here we targeted four residues in CR1 (Ser⁶¹², Arg⁶¹⁵, His⁶²², Glu⁶²⁴) for alanine substitution and unexpectedly, found that cellular levels of three mutants (S612A, R615A, E624A) in transfected HEK cells were substantially lower than wild-type MRP1. Whereas CR1-H622A properly trafficked to the plasma membrane and exhibited organic anion transport activity comparable to wild-type MRP1, the poorly expressing R615A and E624A (and to a lesser extent S612A) mutant proteins were retained intracellularly. Analyses of cryogenic electron microscopic and atomic homology models of MRP1 indicated that Arg⁶¹⁵ and Glu⁶²⁴ might participate in bonding interactions with nearby residues to stabilize expression of the transporter. However, this was not supported by double exchange mutations E624K/K406E, R615D/D430R and R615F/F619R which failed to improve MRP1 levels. Nevertheless, these experiments revealed that the highly conserved CR1-Phe⁶¹⁹ and distal Lys⁴⁰⁶ in the first cytoplasmic loop of MSD1 are also essential for expression of MRP1 protein. This study is the first to demonstrate that CR1 contains several highly conserved residues critical for plasma membrane expression of MRP1 but thus far, currently available structures and models do not provide any insights into the underlying mechanism(s). Additional structures with rigorous biochemical validation data are needed to fully understand the bonding interactions critical to stable expression of this clinically important ABC transporter.