Gibco dmem medium

We utilized three well-characterized HNSCC cell lines: UDSCC1, UDSCC5, and UDSCC6 for our experiments. Cells were cultured in both 2D and 3D models. For 2D culture, cells were seeded in Gibco DMEM medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Bio&SELL GmbH, Feucht, Germany), 1% cell shield (Minerva Biolabs, Berlin, Germany), and 1% NEAA (100×, ThermoFisher Scientific, Waltham, MA, USA) in regular culture flasks. For 3D culturing, the floating sphere formation approach using Ultra-Low Attachment (ULA) surface plates (Corning, New York, NY, USA) was applied: cells were resuspended in medium containing DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) with B27 Supplement (50×, Sigma-Aldrich), supplemented with 0.4% BSA (Sigma Aldrich), 20 ng/mL EGF (Sigma Aldrich), 10 ng/mL bFGF (Thermo Scientific), and 5 µg/mL human Insulin (Roche, Basel, Switzerland) at a density of 20,000 cells per well on 6-well ULA surface plates and incubated for 72 h at 36 °C.

All the cell lines were purchased from the Stem Cell Bank, Chinese Academy of Sciences in Shanghai, China, and verified by the China Centre for Type Culture Collection in Wuhan, China. PCa cell lines (PC-3, DU-145, LNCap, and 22RV1) were cultured in Gibco RPMI-1640 medium (Thermo Fisher Scientific, USA). HEK293T cell line was cultured in Gibco DMEM medium (Thermo Fisher Scientific, USA). All the cell cultures were supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, USA) and cultured at 37 °C in a humidified incubator containing 5% CO₂. Gefitinib (S1025, Selleck), Cycloheximide (S7418, Selleck), Dactinomycin (S8964, Selleck), and EG01377 (HY-112151, MCE) were purchased from commercial sources indicated.

Deionized water, methanol (99.9%, LC/MS Grade), Gibco F12-K medium, Gibco DMEM medium, Gibco 0.25% Trypsin-EDTA, Gibco penicillin-streptomycin (P/S), phosphate-buffered saline (PBS), CellMask™ plasma membrane stain (green), LIVE/DEAD™ fixable green dead cell stain kit, and lipopolysaccharide (LPS) were obtained from Thermo Fisher Scientific (Waltham, MA). 1 N NaOH, 1 N HCl, 5 N ammonium hydroxide, glacial acetic acid (99.99%), ultra-high purity ammonium acetate (99.999%), trypan blue, and total human serum IgM and human serum IgG isolates (purity ≥95%, based on non-reduced SDS-PAGE and verified by nanoLC-MS/MS of tryptic digests) were purchased from Sigma-Aldrich (St. Louis, MO). PNGase F enzyme was from New England Biolabs (Ipswich, MA). Fetal bovine serum (FBS) was purchased from R&D Systems (Minneapolis, MN). Platelet-free anticoagulated with EDTA pooled total human plasma (from blood donated by self-declared healthy male donors of 23–67 years old) was kindly provided by Prof. Ghiran’s laboratory (BIDMC, Boston, MA). No protein depletion or enrichment was done prior to the analysis of total plasma samples in this study. All bare fused silica (BFS) capillaries (91 cm × 30 µm i.d. × 150 µm o.d.) with sheathless CESI-MS emitters in OptiMS™ cartridges were from SCIEX (Redwood City, CA). Aquapel® was purchased at Pittsburgh Glass Works (Pittsburgh, PA).

The embryonic rat heart–derived myogenic cell line H9c2 (2‐1) and mouse embryonic fibroblast (MEF) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The H9c2 (2‐1) cells and MEFs were grown in Gibco DMEM medium supplemented with 10% fetal bovine serum (Gibco, Invitrogen, Carlsbad, CA, USA), streptomycin 100 μg mL⁻¹, and penicillin 100 U mL⁻¹ (all obtained from Sigma, St Louis, MO, USA). All cells were grown at 37 °C in an atmosphere of 5% CO₂.

Three EC cell lines were used, BE3 and OE33 both derived from esophageal adenocarcinoma and OE21 derived from esophageal squamous cell carcinoma. BE3 was cultured in GIBCO DMEM medium (Life Technologies, Foster City, CA, USA) while OE33 and OE21 were cultured in GIBCO RPMI 1640 medium (Life Technologies). Media of all cell lines were supplemented with 10% FCS and 1% of penicillin/streptomycin cultured in an incubator of 5% CO₂ and 37 °C. All cell lines were passaged at 70% confluency.