Cd146

Flow cytometric analysis was performed on P3 naïve and primed IFP (n = 3) and BM (n = 3) MSC. 2.0 × 10⁵ cells were labelled with monoclonal antibodies specific for: CD10, CD44, CD56, CD73, CD90, CD105 (Biolegend, San Diego, CA), CD146, LepR (Miltenyi Biotec, Auburn, CA), CD166, CD271, NG2 (BD Biosciences, San Jose, CA), CD200, CXCR4 (Invitrogen) and the corresponding isotype controls (Supplementary Table S2). All samples included a Ghost Red Viability Dye (Tonbo Biosciences, San Diego, CA). Data were acquired using a Cytoflex S (Beckman Coulter, Brea, CA) and analysed using Kaluza analysis software (Beckman Coulter).

The following reagents were used: collagenase type IV (Invitrogen, Carlsbad, CA), trypsin inhibitor (Amresco, Cochran Solon, OH), DNase I (AppliChem, Gatersleben, Saxony-Anhalt, Germany), bovine serum albumin (BSA, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), DMEM (Dulbecco's modified Eagle's medium, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), fetal bovine serum (FBS, Hyclone, South Logan, UT), Optiprep^TM density gradient liquid (Axis-shield, Rodelokka, N-0504 Oslo, Norway), ASGPR1 (Santa Cruz Biotechnology, Dallas, TX), goat anti-mouse lgG-PE (Santa Cruz Biotechnology), F4/80 (eBioscience, Santa Clara, California), CD146 (Miltenyi Biotec, Bergisch Gladbach, Germany), CD45 (Miltenyi Biotec, Bergisch Gladbach, Germany), APC Rat IgG2b k isotype (BD Pharmingen, San Jose, CA), fluorescein isothiocyanate (FITC) Rat IgG2b k isotype (BD Pharmingen), phycoerythrin (PE) Rat IgG2b k isotype (eBioscience), IC fixation buffer (eBioscience), and permeabilization buffer (eBioscience).

Human or mouse CRC liver metastatic tissues were harvested aseptically from CRCLM patients or tumor-bearing mice and then digested with a tumor dissociation kit (Miltenyi Biotec). A single-cell suspension of tissues was obtained using a GentleMAC tissue processor (Miltenyi Biotec) according to the manufacturer’s instructions. Tumor cells were isolated using anti-EpCAM beads (Miltenyi Biotec) and characterized as EpCAM⁺ (Biolegend) with purity greater than 95% by flow cytometry. LSECs were isolated using anti-CD146 beads (Miltenyi Biotec) and characterized as CD146⁺ (Miltenyi Biotec) with purity greater than 95% by flow cytometry as described previously (64 (link)). MDSCs were characterized as negative for HLA-DR (Biolegend) and Lin (Biolegend) but positive for CD33 (Biolegend) and CD11b (Biolegend), which were sorted by flow cytometry as described previously (65 (link)).

All antibodies (Human CD31, CD45, CD146, CD166, CD90, CD74, CD105) were purchased from Miltenyi Biotech (Auburn, CA, USA). All dyes (MitoTracker Green FM (MTG) and Tetramethylrhodamine (TMRM) stain) were bought from ThermoFisher (Waltham, MA, USA). All other reagents were purchased from Sigma-Aldrich (St Louis MO, USA), unless stated explicitly below.

Passage 2 to 3 MSCs were lifted by means of trypsin, counted and put in sterile Falcon tubes at aliquots of 1 × 10⁶ cells/tube at least. The following antibody panel was used for characterization of porcine cells: CD14 (AbD Serotec), CD29 (BD Pharmingen), CD31 (BD Biosciences), CD34 (Abcam + Goat anti-rabbit IgG (PE-cy5.5), Life Technologies), CD44 (Biolegend), CD45 (Genway Biotech), CD73 (Biolegend), CD90 (BD Biosciences), CD105 (Bioss Antibodies), CD146 (Genetex). For human cells, following antibodies were used CD45, CD90, CD105, CD73, CD235a, CD34 (BD Biosciences), CD31 (BioLegend), CD33, CD14 (Beckman Coulter), and CD146 (Miltenyi Biotec) (antibodies summarized in Tables 2, 3). The cells were stained with the antibodies singularly and according to manufacturer's protocol and were assessed using a BD LSRII flow cytometer, analyzing at least 20,000 events (Becton Dickinson, Franklin Lakes, NJ, USA). The obtained data was analyzed with FlowJo software (TreeStar Inc., Ashland, OR, USA).

Flow cytometric analysis was performed on P3 eMSC (n = 3). Then, 2.0 × 10⁵ cells were labeled with antibodies specific for SUSD2 (BioLegend, San Diego, CA, USA) and CD146 (Miltenyi Biotec, Auburn, CA, USA), in addition to the corresponding isotype controls. All cells were stained with eFluor 780 fixable viability dye (Invitrogen). The fluorescent signal was acquired using a CytoFLEX S (20,000 events) and analyzed with Kaluza analysis software (Beckman Coulter, Miami, FL, USA).