Mopc 21

PBMC, 1x10⁶ cells/ml of RPMI 1640 + 5% heat inactivated fetal bovine serum were incubated without PHA or BTP2 (control), with BTP2 (1000 nM), with PHA (2 μg/ml), or with PHA (2 μg/ml) + BTP2 (1000 nM) for 16 hours at 37°C in 95% air and 5% CO₂ atmosphere. At the end of incubation, the cells were washed and labeled with FITC mouse IgG2a,k isotype control mAb (G155-178, BD Pharmingen) and PE Mouse IgG1, k isotype control mAb (MOPC-21, BD Pharmingen) or FITC mouse IgG2a, k anti-human CD3E mAb (HIT3a, BD Pharmingen) and PE mouse IgG1, k anti-human CD25 mAb (M-A251, BD Pharmingen). Each isotype mAb (10 μl) and each antibody marker (10 μl) for CD3E and CD25 was added to 100 μl of cell suspension in 5ml falcon tube. The sample tubes were vortexed and incubated in dark at 4 C for 30 min in refrigerator. After incubation, cells were washed by adding 3 ml of cold working wash buffer (PBS containing 1% FBS and 0.1% FBS and 0.1% wt/vol sodium azide). Cells were then resuspended in 400 μL of working fixative reagent (PBS + 0.1% sodium azide +1% fetal bovine serum + 37% formalin) and flow cytometry analysis was performed. Flow cytometry data was acquired on a FACSCanto II (BD Biosciences) using FACSDiva software (8.0.1) and the FCS files were analyzed using FlowJo 10.8.1 software (Beckton Dickinson and Company).

Cells were collected, stained, and analyzed by flow cytometry (BD Cytoflex S flow cytometer) as follows. Staining for membrane surface molecules included use of the following antibodies: EpCAM-PE (phycoerythrin): [VU-1D9] (Abcam) and isotype control [MOPC-21] (BD Pharmingen); E-cadherin-PE: CD324 [DECMA-1] BioLegend) and isotype control [A95-1] (BD Pharmingen); and N-cadherin-APC (Allophycocyanin): CD325 [8C11] (BioLegend) and isotype control [27 (link)–35 (link)] (BD Pharmingen). Staining was performed following standard methods. Cells were stained for 30 min (EpCAM) and 20 min (E-cadherin and N-cadherin). For intracellular staining of CCT2 we used the antibody CCT2-PE: [NP_006422] (LSBio) and the isotype control [MOPC-21] (LSBio), following the method in ThermoFisher Scientific’s “Protocol A: two-step protocol: intracellular (cytoplasmic) proteins” and incubating the antibody for 70 min. When optimizing the CCT2 intracellular stain for CSS Autoprep conditions, we adjusted the antibody staining protocol for CCT2-PE to match the 20 min in the CSS Autoprep automated conditions. All data was generated using FCS Express 6 software (De Novo).

Cell surface antigens were analysed by FACS on a BD FACSCalibur after 20 min staining at 4°C with antibodies to HLA class I–FITC (W6/32; BD), HLA-DR–FITC (L243; BD), IgG1–APC (MOPC-21; BD), IgG2a–FITC (G155-178; BD), IgG2a–Phycoerythrin (G155-178; BD), anti-CD46–APC (MEM-258; ImmunoTools, Friesoythe, Germany), anti-CD55–Phycoerythrin (IA10; BD) and anti-CD59–APC (OV9A2, eBioscience).

The VLA-4-specific ligand 4-((N’-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L-aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine (LDV) and its FITC-conjugated analog (LDV-FITC probe) [30 (link),33 (link)] were synthesized at Commonwealth Biotechnologies. Mouse anti-human CD29/integrin β₁ chain, clone MAR4 (PE), mouse anti-human CD49d/ integrin α₄ chain, clone 9F10 (PE), and isotype control (mouse IgG1 κ, PE) clone MOPC-21 were purchased from BD Biosciences and used according to the instructions of the manufacturer. Human recombinant CXCL12/SDF-1α and recombinant human CXCL8/IL-8 were obtained from R&D Systems, Inc. All other reagents were from Sigma-Aldrich. Stock solutions were prepared in DMSO, at concentrations ~1000-fold higher than the final concentration. Usually, 1 μl of stock solution was added to 1 ml of cell suspension, yielding a final 0.1% DMSO concentration. Control samples were treated with an equal amount of pure DMSO (vehicle).

To determine HER2 and PD-L1 surface expression, organoids were dissociated into single cells using TryplE supplemented with 1:1000 Rho kinase inhibitor. 25,000 single tumor cells were stained with Live/Dead^® Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, Cat. No. L34957) for 30 min on ice. Tumor cells were washed with PBS/EDTA buffer (PBS/2 mM EDTA/0.5% HSA) and stained with mouse anti-human HER2-APC (Neu24.7, BD Biosciences, Cat. No. 340554), anti-PD-L1-PE (29E.2A3, BD, Cat. No. 568079), or matched isotype controls IgG1-APC (MOPC21, BD Biosciences, Cat. No. 550854) and IgG2b,k-PE (27-35, BD, Cat. No. 555058) for 30 min at 4°C. All acquisitions were performed on a BD FACS Canto II. Data were analyzed with FlowJo v10.6.1 (TreeStar).