Macs dissociator

Cells were isolated and cultured as previously reported [24 (link)]. In brief, washed pieces of 1 mm³ were dissociated in digestion medium (10 mg/mL collagenase IV (Worthington, USA), 0.25% Trypsin-EDTA (Sigma-Aldrich, Switzerland), and 0.2 mg/mL DNase (Roche, Switzerland) in advanced DMEM-F12, Hepes 10 mM, 1% L-glutamine, 1% penicillin-streptomycin-amphotericin B) using a gentle MACSTM dissociator (Miltenyi Biotec, Switzerland). Cells were filtered through a 70 µm strainer to remove collagen debris, and red blood cells were lysed with ACK lysis buffer (Thermo Fisher, Scientific, USA). After mechanical fibroblast removal and single-cell dissociation, cells were resuspended and maintained in AdvDMEM + GF medium. After 2 days of recovery phase, cells were counted and resuspended in fresh AdvDMEM + GF medium supplemented with growth-factor-reduced Matrigel and plated in 96-well ULA plates (3–4 × 10³ cells/well).

For isolation of total RNA, lysis buffer from the RNeasy^® kit (Qiagen, Hilden, Germany) was added to snap frozen resections. Samples were shredded in M tubes (# 130-093-236, Miltenyi Biotec) in a gentle MACS^TM dissociator (Miltenyi Biotec). Total RNA was prepared according to the manufacturer’s protocol and stored at −80 °C. Quality of total RNA was controlled with the Agilent 2100 expert EukaryoteTotal RNA Nano Assay. Only RNA with a RNA integrity number > 6 was used for analysis. The isolated RNA was reverse-transcribed using a Reverse Transcription System (Applied Biosystems). Quantitative mRNA analysis by real-time PCR was performed using Taqman^® gene Expression Assays for mouse collagen type I alpha 1 (Col1a1) Mm00801666_g1, TGF-β1 Mm01178820_m1, MMP-9 Mm00442991_m1, tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) Mm00441818_m1, hypoxia inducible factor 1 alpha subunit (HIF-1α) Mm01283760_m1 and GAPDH # 4352339E TaqMan® Gene Expression Assays. The relative cDNA concentration for the gene of interest was calculated using the ddCt-method.

Lungs were perfused with PBS. Bronchial lymph nodes were dissected from the lung tissue under a microscope and prepared separately for flow cytometric analysis by meshing through a 70-μm sieve. Single-cell suspensions from lung tissue were prepared using the gentle MACS dissociator (Miltenyi Biotec) and digestion with 0.5 mg ml⁻¹ collagenase D and 20 μg ml⁻¹ DNase I (Roche) for 25 min at 37 °C. Spleens were meshed through a sieve, and bone marrow was flushed out of femur and tibia of hind legs. All cells were counted using a Guava EasyCyte capillary flow cytometer and ViaCount solution (Millipore).

Fresh OSCC patient-derived tumor tissues were harvested and manually cut into 2 × 1 mm pieces. Subsequently, the tissue pieces were dissociated into homogenates with a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany; 130-093-235). Then, the samples were digested in RPMI medium containing Collagenase D (Roche, Basel, Switzerland; 11088858001) at 1 mg/mL, hyaluronidase at 0.1 mg/mL (Sigma–Aldrich, St. Louis, MO, USA; H1136), and DNases at 0.2 mg/mL (Sigma–Aldrich, St. Louis, MO, USA; AMPD1) for 2 h at 37 °C. After filtering the cells through 70 μm cell strainers, the obtained cells were subsequently separated using Lymphoprep^TM (STEMCELL Technologies, Vancouver, BC, Canada; 07801). The TILs were collected and then subjected to flow cytometry analysis.