Detection of Stage‐Dependent Collagen Remodeling at the Bladder Cancer Invasive Front by Label‐Free Multiphoton Imaging

ABSTRACT

Accurate T staging is critical for treatment decisions in bladder cancer (BCa), but extracellular matrix collagen remodeling at the tumor invasive front—an invasion correlate—is not routinely quantified. Here, we applied label‐free multiphoton microscopy (MPM) integrating second‐harmonic generation (SHG) and two‐photon excited fluorescence (TPEF) to 57 formalin‐fixed paraffin‐embedded specimens spanning T0–T3. Eight SHG‐derived collagen descriptors profiled architecture across four dimensions: content, fiber morphology, network topology, and spatial organization. We identified a non‐monotonic remodeling trajectory: collagen accumulates in non‐invasive stages, the network undergoes rarefaction and disorganization at T1, fibers become highly linearized with preferential alignment at T2, and the network re‐compacts at T3. Notably, fiber straightness peaked exclusively at T2 (all p  < 0.0001) with reduced isotropy, representing an optical phenotype of muscularis propria invasion. These findings demonstrate that label‐free SHG imaging enables quantitative characterization of stromal remodeling and provides complementary information for histopathological assessment of BCa invasion.