Hyaluronidase type 5

Enzyme disaggregation (ED) of freshly resected breast TT and NT for immune cell isolation was performed as described previously [54 (link)]. Tissues were cut into pieces and then enzymatically digested in RPMI-1640 medium containing 1% penicillin/streptomycin and enzyme cocktail, consisting of 1mg/ml Collagenase (C0130), 100 μg/ml Hyaluronidase type V (H3506) and 30 IU/ml of Deoxyribonuclease I (D5025; all from Sigma-Aldrich) and incubated on a mixer at 37°C for 60 minutes under slow rotation. The cell suspension was then filtered through a 100 μm cell strainer (352360, BD Falcon) to remove any cellular debris and aggregates, followed by two washes with RPMI-1640 media and resuspending in complete media (RPMI-1640 media with 10% FBS and 1% penicillin/streptomycin). Cells isolated from TT (TILs, tumor-infiltrating leukocytes) and NT (NILs, non-tumor-infiltrating leukocytes) were used for flow cytometric staining.

Cell suspensions were obtained from TTs and paired non-tumor normal tissues (NT) by enzymatic digestion, as previously described (32 (link)). Briefly, surgically removed fresh colon TT and NT were cut into small pieces and suspended in RPMI media with 1% penicillin/streptomycin and enzyme cocktail (1 mg/ml collagenase, 100 µg/ml hyaluronidase type V and 30 IU/ml of deoxyribonuclease I, all from Sigma-Aldrich, UK) and incubated at 37°C under slow rotation for 60 min. The resulting cell suspension was then filtered through a 100-µm BD Falcon cell strainer (BD Biosciences, Oxford, UK), washed twice, and resuspended in complete media. Immune cells, isolated from TT (referred as TILs) and NT [referred as non-tumor-infiltrating lymphocytes (NILs)], were characterized by appropriate gating by flow cytometric analysis.

Rac2 antibody is from Novus Biologicals. Rac1 antibody is obtained from Santa Cruz Biotechnology. Vitronectin and collagen are from Sigma (Sigma-Aldrich, St. Louis, MO), fragments of fibronectin (H296 and CH271) are from R&D systems. Primary or fluorescent antibodies against CD31 (clone MEC13.3), CD11b (clone M1/70) are from BD Biosciences, F4/80 (clone BM8) is from eBiosciences. 4, 6 diamidino-2-phenylindole (DAPI) are obtained from Sigma. Alexa Flour 594 or Alexa Flour 488 is from Invitrogen life Technologies. Collagenase/Dispase is from Roche Biosciences, hyaluronidase type V and Dnase I is from Sigma. α-isonitrosopropiophenone for arginase activity and sulphanilamide for nitrite assay is from Sigma. Bouin's solution is from Sigma. MCSF is from Gibco Life technologies and GMCSF from Peprotech Life sciences. D-luciferin potassium salt is from Caliper Life sciences. CD11b magnetic beads are from Militenyi Biotec.RNA isolation kit from Qiagen. Iscript cDNA synthesis kit and SYBR green are from Biorad (Bio-Rad, Hercules, CA).

B16-F10 tumor material was collected for FACS analysis and single-cell suspension obtained with digestion mixture [0.5 mg/mL collagenase A, (Sigma Aldrich, MO, USA); 0.2 mg/mL hyaluronidase type V, (Sigma Aldrich, MO, USA); 0.02 mg/mL DNase I, (Roche Diagnostic GmbH, Germany); per each 0.25 g of tumor tissue). Red blood cells were lysed using 0.15 M ammonium chloride solution (Sigma Aldrich, MO, USA)]. Dead cells were removed by centrifugation on Lympholyte-M gradients (Cedarlane, Canada). Level of T lymphocytes was determined in homogenous single-cell suspension. To identify the subpopulations of T lymphocytes, the following antibodies were used: PE-Cy™7-CD3e, PE-CD4 and FITC-CD8a (BD, Franklin Lakes, NJ, USA). NK cells were identified with an anti-mouse CD49b antibody (Biosciences, CA, USA). Gate parameters dividing negative from positive cells were chosen based on isotype antibody control probes (Jarosz et al. 2013 (link)).

Enzyme disaggregation (ED) of fresh tumor and normal tissues from breast cancer patients, collected in cold RPMI-1640 media was performed on a rollover mixer at 37 °C for 60 min. Briefly, tissues were first washed with phosphate buffered saline (PBS) and then mechanically cut into small fragments (2–4 mm) using a surgical scalpel. Tissues were then suspended into RPMI-1640 media with 1% Penicillin/Streptomycin and an enzyme cocktail, consisting of 1 mg/ml Collagenase (Sigma–Aldrich, Dorset, UK), 100 µg/ml Hyaluronidase type V (Sigma–Aldrich) and 30 IU/ml of Deoxyribonuclease I (Sigma–Aldrich). Cell suspension was then passed through a 100 µm BD Falcon cell strainer (BD Biosciences, Oxford, UK) to remove debris and aggregates. Cells were then resuspended in RPMI-1640 media enriched with 10% FCS and 1% Penicillin/Streptomycin (complete medium) after washing with RPMI-1640 media.

All experimental protocols were approved by the Animal Care and Use Committee of Shanghai Jiaotong University and were in accordance with the guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health. After male, 6-week-old Sprague-Dawley rats were sacrificed by intraperitoneal administration of 10% chloral hydrate (3.5 mL/kg, Sigma, Missouri, USA), the AF tissues were immediately excised from L1 to L6 intervertebral discs under aseptic condition. Primary AF cell cultures were prepared as our previously described [16 (link), 17 (link)]. Briefly, AF tissues harvested were cut into small pieces (< 1 mm³) and firstly digested with 0.4% pronase (Calbiochem, San Diego, CA) for 90 min at 37 °C, followed by second-digestion with 0.025% collagenase type II (Sigma) and 0.01% hyaluronidase type V (Sigma) overnight. The suspension was filtered through a 70-μm nylon mesh filter to remove the tissue debris. Then, AF cells were seeded into culture plates and maintained at 37 °C under a humidified atmosphere of 95% air and 5% CO₂. The medium was changed every 2 days. First-passage cells in a monolayer were used throughout the experiments.