Deoxyribonuclease type 4

Tumors were mechanically digested with accutase (PAA), collagenase IV (Worthington), hyaluronidase (Sigma), and deoxy-ribonuclease type IV (Sigma). Single-cell suspensions were then prepared and stained for flow cytometry analysis (FACS) or CD45-positive cell enrichment.

Primary tumor samples were collected during surgery. Sample collections were approved by the local ethical committee [Ethikkom-mission Nordwestschweiz (EKNZ) 2018-01990]. For the preparation of single-cell suspensions from both human and mouse tumors, tumors were collected, and surgical specimens were mechanically dissociated and subsequently digested using Accutase (PAA Laboratories), collagenase IV (Worthington), hyaluronidase (Sigma-Aldrich), and deoxyribonuclease type IV (Sigma-Aldrich) for 1 hour at 37°C under constant agitation. Cell suspensions were filtered through a 70-μm mesh, and for the analysis of tumor-infiltrating immune cells, CD45⁺ cells were further enriched by Histopaque-1119 density gradient centrifugation (Sigma-Aldrich). Splenocytes were isolated by mechanical disruption using the end of a 1-ml syringe, filtration through a 70-μm mesh, and lysis of red blood cells using red blood cell lysis buffer (eBioscience). Samples were frozen in 90% FBS and 10% dimethyl sulfoxide and stored in liquid nitrogen until the time of analysis.

The part of the lung tissue intended for flow cytometry analysis was placed into a Petri dish with Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco) without fetal bovine serum, weighed, minced into small chunks, and enzymatically digested in digestion medium (2 ml IMDM, 2 mg/ml collagenase type IV, and 50 U/ml deoxyribonuclease type IV; all obtained from Sigma-Aldrich) at 37°C for 45 min. After digestion, cells were filtered through a strainer (100 μm; Roche Diagnostics GmbH) to remove tissue debris. The single cell suspensions thus obtained were subsequently centrifuged, counted in a hemocytometer chamber after dilution with Turk’s solution (2% acetic acid), and resolved in phosphate-buffered saline for the next analysis. Cell suspension was first processed using Fcγ receptor blocking solution with purified CD16/32 (eBioscience, San Diego, CA). Subsequently, cell suspension (5 × 10⁵ cells/100 µl) was marked with the same two panels of monoclonal antibodies stated above. For better analysis of leukocytes, a common leukocytes marker, CD45, was added to both panels and CD11c was added to the second panel for the detection of alveolar macrophages.