CRC specimens were obtained from 20 patients (see Table S1 ) during therapeutic surgery at the Surgical Oncology Unit, IRCCS Ospedale Policlinico San Martino, Genoa, upon signed informed consent (Institutional and Regional Ethic Committee approval, PR163REG2014, renewed in 2017). This cohort of CRC patients was composed of newly-diagnosed patients without any kind of therapeutic treatment before surgery. Samples were anonymized as reported and referred in the paper with the year and serial number for brevity [33 (link)]; all these CRC-TAF have been used within the 8–9th culture passage.
Primary TAF lines were obtained by mincing CRC mucosa, enzyme digestion and debris removal by soft centrifugation as reported [35 (link)]; cell suspensions were then purified by density gradient centrifugation (Lympholyte, Cederlane, Burlington, ON, Canada) and seeded in culture wells for 36 h. Non-adherent cells were removed and adherent cells showing a fibroblast-like morphology were cultured for an additional 7 d in MEMalpha (Euroclone, Milan, Italy) supplemented with 10% foetal calf serum (FCS, Sigma Chemicals Co., St. Louis, MO, USA). At confluence, cell cultures were split and expanded as described [19 (link),31 (link)]. Phenotype for Intercellular Adhesion Molecule 1 (ICAM1), Fibroblast Activation Protein (FAP), Thy-1 Cell Surface Antigen (CD90), Endoglin (CD105), Epidermal Growth Factor Receptor (EGFR) and Epithelial Cell Adhesion Molecule (EPCAM) markers were analyzed by indirect immunofluorescence at different time points, along a culture period of two months as described inSection 2.5 . TAF were also tested for the expression of BTN3A1 and BTN2A1.
Primary TAF lines were obtained by mincing CRC mucosa, enzyme digestion and debris removal by soft centrifugation as reported [35 (link)]; cell suspensions were then purified by density gradient centrifugation (Lympholyte, Cederlane, Burlington, ON, Canada) and seeded in culture wells for 36 h. Non-adherent cells were removed and adherent cells showing a fibroblast-like morphology were cultured for an additional 7 d in MEMalpha (Euroclone, Milan, Italy) supplemented with 10% foetal calf serum (FCS, Sigma Chemicals Co., St. Louis, MO, USA). At confluence, cell cultures were split and expanded as described [19 (link),31 (link)]. Phenotype for Intercellular Adhesion Molecule 1 (ICAM1), Fibroblast Activation Protein (FAP), Thy-1 Cell Surface Antigen (CD90), Endoglin (CD105), Epidermal Growth Factor Receptor (EGFR) and Epithelial Cell Adhesion Molecule (EPCAM) markers were analyzed by indirect immunofluorescence at different time points, along a culture period of two months as described in
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