Isolation and Culture of Primary cSCC Cells

Primary cell lines were derived from 6 fresh MDHNcSCC tissue samples from the original cohort of 15 patients. The samples were cultured within a Matrigel (cat.#354234; Corning, Tewksbury, Maine) explant and extracted following their abundant growth.^{27 (link)} The extracted cells were grown and passaged in DMEM medium (cat.#10569010; Thermo Fisher Scientific, Waltham, Mass.) supplemented with 1% fetal calf serum (cat.#10091148; Thermo Fisher Scientific), 5% mTeSR (cat.#85850; STEMCELL Technologies, Vancouver, British Columbia, Canada), 1% penicillin–streptomycin (cat.#15140122; Thermo Fisher Scientific), and 0.2% gentamicin/amphotericin B (cat.#R01510; Thermo Fisher Scientific). All cultures were maintained in a humidified 37°C incubator with 5% CO₂. All primary cells used in the experiments were passages 6–8.