250 A specific neurotrophin network characterizes cSCC subpopulations and correlates with their behavior by patient-derived spheroids and zebrafish avatar

Cutaneous squamous cell carcinoma (cSCC) is the second most prevalent form of skin cancer. Neurotrophins (NTs) and their receptors (NTRs; CD271 and Trk) form a complex network regulating skin homeostasis. While Trk receptors are involved in stemness, CD271 is implicated in the switch between stem and early progenitors. However, their roles in cSCC need to be clarified. Rapidly Adhering (RAD) cSCC keratinocytes have been characterized as stem-like cells as compared to those that do not adhere to collagen IV within 5’ named NRAD (Not RAD). Starting from biopsies, we generated patient-derived spheroids from different tumor stages, reflecting the behavior of cSCC and its subpopulationsin vitro. Moderately/Poorly Differentiated (MD-PD) bulk cSCC cells give rise to bigger and more growing spheroids and MD-PD RAD-derived spheroids possess the highest viability and aggressiveness features. In vivo, CD271 is detectable in Well-Differentiated (WD) cSCC, while MD-PD tumors express the highest TrkA levels. Similarly, CD271 was higher in WD spheroids, while MD-PD spheroids mostly express TrkA and TrkB. By profiling in situ, WD and MD-PD RAD vs NRAD spheroids, we identified important differences in terms of NTs and NTRs expression. By CD271 overexpression in patient-derived cSCC spheroids, we found a reduced proliferation and malignancy marker expression and an increase in neoplastic cell differentiation, with a major effect on RAD spheroids. Finally, primary cSCC RAD cell injection in zebrafish produced a higher number of metastasis than NRAD. Furthermore, CD271 overexpression in cSCC cells reduced metastasis, especially for RAD cells, and promoted a higher inflammatory microenvironment. Our results indicate a significant involvement of the neurotrophin network within cSCC development and progression and it could be a path for novel therapeutic strategies.