Pronase e solution

PGN was isolated essentially following a published procedure [29 ]. Briefly, biomass was harvested from 1 l of culture grown to the stationary phase by centrifugation (5000 g, 30 min, 4 °C), resuspended in 60 ml distilled water and transferred drop-wise into 65 ml of boiling 8% sodium dodecyl sulfate (SDS; Sigma) under constant stirring to lyse cells. The suspension was further boiled for 1 h, reduced to the former volume using a rotary evaporator, and stirred overnight. SDS was removed by several washing steps with distilled water, 60 ml, each, using an Optima L-100XP ultracentrifuge from Beckman Coulter (rotor Ti70, 35,000 rpm, 30 min, 40 °C) followed by dialysis against distilled water for 4 days at room temperature. For the total volume of 12 ml of that PGN solution, 200 μl of an α-amylase solution (24 mg ml^− 1; Sigma) were added and the mixture was incubated at 37 °C for 2 h under constant shaking. Further, 320 μl of pre-incubated Pronase E solution (10 mg ml^− 1 in 10 mM Tris-HCl, pH 7.5; Sigma) was added and incubated at 60 °C for 1.5 h. Preparations were washed, boiled for 1 h, washed again, and dried in a Speed Vac vacuum centrifuge (Thermo Fisher Scientific, Vienna, Austria).

Primary tumor cells were obtained from 38 NSCLC fresh samples. Freshly resected tumors were cut into small pieces, then digested overnight at +4°C in a 0.1% Pronase E solution (Sigma-Aldrich, Saint-Louis, MO) and seeded in type IV collagen-coated dishes with CnT-17 medium (CELLnTEc, Bern, Switzerland). After the proliferation phase, cells were cultured in bronchial epithelial cell growth medium (BEGM) (Lonza, Walkersville, MD). The sensitivity to the HER2 tyrosine kinase inhibitor (TKI) tucatinib (irbinitinib, ARRY-380, ONT-380) (HY-16069, MedChemtronica, Sollentuna, Sweden) was assessed by MTT assay as previously described (7 (link)). Further culture samples were also frozen for protein extraction and RNA extraction. FHIT and pHER2 status were assessed by western blotting as previously described (Supplementary Figure S1) (7 (link)). Six FHIT^low/pHER2^high tumors and 6 other tumors were selected for RNA-sequencing analysis (Supplementary Figure S1 and Supplementary Table S1).