Fig. 4

Bladder immune microenvironment following BBN carcinogen exposure in male and female mice. Whole bladder image captured using Phenochart Software image viewer 1.0.9 (Akoya Biosciences) showing low magnification image of multiplex immunofluorescence stained whole bladders from 12- and 15-month old male (A, M) and female (E, I), respectively, at 12 weeks post BBN exposure. Corresponding H&E stained section of a male (B, N) and female (F, J) bladder showing high-grade dysplasia, carcinoma in situ and early invasion. Multiplex immunofluorescence stained image showing F4/80+ macrophages, CD163+ M2-like macrophages, PD-L1 immune checkpoint, EpCAM+ epithelial cells (C, G, K and O) and CD11b+ myeloid cells, Pax5+ B cells, CD8+ cytotoxic T cells and CD3+ T cells (D, H, L, P). High magnification (×20) mages representative of n = 3–11 regions of interest from bladders of 3 to 5 mice in each sex