5 ml polystyrene tubes

Mice carrying patient-derived tumors were sacrificed before or when tumor implants had reached the ethical size limit. The implanted tumors, spleens and whole blood were collected from the sacrificed mice. Single-cell suspensions of tumor implants were obtained using the same protocol as described above. Spleens were cut into 3 × 3 mm pieces that were passed through a 70 μm cell strainer (BD) with a syringe plunger, and the obtained cell suspensions were washed with PBS. Blood was collected into Microvette EDTA tubes (Sarstedt, Nümbrecht, Germany) from the hind leg vein (vena saphena) and centrifuged for 5 min at 2000× g. Subsequently, the plasma was removed and replaced with PBS prior to PBMC isolation with a 2 mL Ficoll gradient in 5 mL polystyrene tubes (Corning, Amsterdam, The Netherlands). Sanger sequencing for KRAS status was outsourced to Eurofins Genomics (Ebersberg, Germany).

Heparinized whole blood samples were cryopreserved using Proteomic Stabilizer (Smart Tube Inc., San Carlos, CA, USA) as indicated by the manufacturer within 30 min after phlebotomy and stored at −80 °C. On the day of processing, samples were thawed in a stirred water bath at 10 °C and incubated with 5 mL of thaw-lyse buffer (Smart Tube Inc.) for 10 min at room temperature in 15 mL centrifuge tubes. Cells were centrifuged at 700× g for 5 min and the erythrocyte lysis step was repeated with 25 mL of thaw-lyse buffer for 5 min at room temperature in 50 mL centrifuge tubes. Peripheral blood leukocytes were washed twice with cell staining medium (1 × PBS made from 10 × PBS ((Rockland Immunochemicals, Gilbertsville, PA, USA) using MilliQ water, supplemented with 0.5% (w/v) BSA (PANBiotech, Aidenbach, Germany) and 0.02% sodium azide (Sigma-Aldrich, St. Louis, MO, USA)). Cell counts were determined volumetrically using a MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch-Gladbach, Germany). Prior to further processing in 5 mL polystyrene tubes (Corning, Corning, NY, USA), cell counts were adjusted to 1.6 × 10⁶ cells per sample.

Single cell suspensions from tumor and colon tissues were resuspended in complete medium to 1 million cells/mL in 5-mL polystyrene tubes (Corning, Amsterdam, Netherlands). Stimulation of tissue APCs by polyclonally activated co-residing T cells was performed by addition of 2.5 µL microbeads loaded with anti-CD2, CD3 and CD28 antibodies (Miltenyi Biotec, Auburn, CA, USA), which represent 1 bead: 4 cell ratio. In parallel, cell suspensions were stimulated with 0.2 μg/mL recombinant IFN-γ (Biolegend, San Diego, CA, USA). After 6 h of culture, supernatants from unstimulated and microbeads-stimulated cell suspensions were collected from the top layer of the culture medium to avoid contamination of sedimented cells or microbeads. Moreover, autologous PBMCs were also cultured for 12 h in 90% complete media and 10% supernatant from microbeads-stimulated tissue suspensions. Remaining supernatants were stored at −20 °C until use. Activation of APCs was analyzed by flow cytometry.