In Situ Analysis of UVB-Induced Keratinocyte-Melanoma Cell Communication Integrated with Scanning Electrochemical Microscopy and Microfluidic Devices

Ultraviolet B (UVB) radiation is a key environmental risk factor for remodeling the melanoma microenvironment, yet the bidirectional communication mechanisms between keratinocytes and melanoma cells remain incompletely understood. This study developed an in situ monitoring platform integrated with scanning electrochemical microscopy (SECM) and a microfluidic chip. With a fabricated Ca2+ ion-selective electrode as a SECM tip (Ca2+-ISE SECM tip), together with a lactate oxidase-modified Pt microelectrode tip (LOx-μPt tip) and a multi-potential step waveform (MPSW) method, the dynamic monitoring of Ca2+, lactate, and ROS in keratinocyte-melanoma cell communication has been achieved. UVB radiation dose-dependently enhanced A-375 melanoma cell migration in both monoculture and co-culture systems. Mechanistically, UVB induced ROS production and stimulated glycolysis in A-375 cells, resulting in elevated lactate release and autocrine-driven migration enhancement. Notably, in the co-culture system, this pro-migratory effect was significantly amplified, while inhibition by the glycolysis inhibitor 2-deoxy-d-glucose (2-DG) was minimal, suggesting a dominant paracrine signaling mechanism. In situ SECM monitoring further demonstrated that UVB also induced ROS generation, transient Ca2+ release, and significantly increased TGF-β1 secretion in HaCaT keratinocytes. N-acetylcysteine (NAC) treatment blocked Ca2+ release and TGF-β1 secretion in HaCaT cells, thereby inhibiting co-cultured A-375 cell migration. Further studies confirmed that HaCaT-derived TGF-β1 serves as a key paracrine factor, promoting migration by upregulating YB-1 expression in A-375 cells, a process dependent on extracellular Ca2+. Consequently, this work reveals an intercellular signaling axis in the UVB-exposed skin tumor microenvironment: UVB → ROS → Ca2+ release → TGF-β1 secretion → YB-1 activation, providing new mechanistic insights and potential therapeutic targets for microenvironment-dependent melanoma progression.