In Vitro and In Vivo Characterization of Porcine Urothelial Cells as an Intravesical Cellular Xenotransplantation Immunotherapeutic Agent for Bladder Cancer.

BACKGROUND

Cellular xenotransplantation offers a unique immunotherapeutic strategy for cancer by utilizing xenogeneic cells to elicit robust host immune responses. Innovative treatments are required for non-muscle invasive bladder cancer (NMIBC) due to the limited effectiveness of current standard therapies. Intravesical xenogeneic porcine urothelial cell (PUC) immunotherapy provides a novel mechanism-of-action option.

OBJECTIVE

This study evaluates cellular xenotransplantation utilizing primary PUCs in vitro and in vivo to characterize their potential as an intravesical immunotherapeutic agent for NMIBC.

METHODS

Primary PUC phenotypic and transcriptomic identities were first characterized using flow cytometry and bulk RNA sequencing. Subsequently, the effects of basic fibroblast growth factor (bFGF) and the ROCK inhibitor Y-27632 on PUC expansion capacity, gene expression, and senescence were investigated. Immune-stimulatory potency was measured through cell-based co-culture bioassays, and in vivo biodistribution and immune recruitment were assessed in mice following intravesical instillation.

RESULTS

Expanded PUCs demonstrated high epithelial purity (92.16% ± 5.38% CD49f+). RNA-seq profiles confirmed a distinct urothelial identity (e.g., UPK3A, TP63, KRT7), with unsupervised clustering clearly separating PUCs from skeletal muscle comparators. Combined treatment with bFGF and Y-27632 synergistically promoted growth and upregulated differentiation markers while significantly suppressing p16-mediated senescence. PUCs significantly stimulated immune cell proliferation and IFN-γ/IL-2 secretion through direct, F-actin-polarized interactions. in vivo, PUCs remained localized and were cleared within 28 days, inducing a peak recruitment of CD4+ and CD8+ T cells at Day 14 and enhancing systemic cytotoxicity.

CONCLUSION

PUCs serve as a potent, localized, and transient cellular medicine that primes systemic antitumor-like immunity. The optimized expansion protocol facilitates the scalable production of non-senescent porcine cells for clinical applications in bladder cancer.