Dnase type 4

Hippocampi were isolated from C57BL6/J mice (0–1 day old), and tissues were digested with trypsin type XI (Sigma, Rehovot, Israel, Cat # T1005), and DNase type IV (Sigma, cat #D5025). Cells were suspended in a plating medium including MEM supplemented with 10% FBS, transferrin (0.089 mg/m), GlutaMAX (0.75 U/mL) (Sigma, Cat # 35050-038), insulin (16 μM) (Roche, Basel, Switzerland, Cat # 45865100), and SM1 neuronal supplement (STEMCELL, Seattle, WA, USA, Cat # 05711), and plated on glass coverslips coated with Matrigel (Corning, NY, USA, Cat# 356234) in a 24-well plate. The day after plating and twice a week thereafter half of the medium was removed and was replaced with by fresh feeding medium (plating medium lacking insulin and containing Ara-C (3 μM)) (Sigma, Cat# C6645).

For the preparation of single‐cell suspensions from mouse tumors and tissues, specimens were mechanically dissociated and subsequently digested using, collagenase IV (Sigma‐Aldrich, 9001‐12‐1), Trypsin (Sigma‐Aldrich, T‐3924), and DNase type IV (Sigma‐Aldrich, 9003‐98‐9) for 30 min at 37 °C under constant agitation. Cell suspensions were filtered through 70‐μm mesh and lysed for red blood cells using BD Pharmlyse™ lysing buffer (BD Biosciences, 555899).

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, Germany), collagenase IV (Worthington, United States), hyaluronidase (Sigma, United States), and DNase type IV (Sigma, United States) for 1 h at 37°C under constant agitation. Cell suspensions were filtered through 70-µm mesh twice and lysed for red blood cells using RBC lysis buffer (eBioscience, United States). PBMCs were isolated by density gradient centrifugation using Histopaque-1077 (Sigma, United States) from buffy coats. Mice splenocytes were isolated by mechanical disruption using the end of a 1-ml syringe, lysed for red blood cells using RBC lysis buffer, then digested with Collagenase D (Roche, Switzerland) and DNase I (Roche, Switzerland). Samples were either used directly or frozen (in 90% FBS, 10% DMSO) and stored in liquid nitrogen until the time of analysis.

To obtain single-cell suspensions, human and mouse tumors were mechanically dissociated and subsequently enzymatically digested using accutase (PAA Laboratories), collagenase IV (Worthington), hyaluronidase (Sigma) and DNase type IV (Sigma) for 1 h at 37 °C under constant agitation. Afterward, the samples were filtered using a 70 µM cell strainer and washed. Precision counting beads (BioLegend) were added to all mouse tumors to calculate the number of cells per gram of tumor.
PBMCs were isolated from buffy coats by density gradient centrifugation using Hisopaque-1077 (Millipore) and SepMate PBMC isolation tubes (StemCell) according to the manufacturer’s protocol, followed by red blood cell lysis using RBC lysis buffer (eBioscience) for 2 min at RT. Subsequently, the cells were washed with PBS and ready for further analysis.
For splenocyte isolation, freshly harvested murine spleens were mechanically dissociated by filtering through a 100 µM filter. After washing, the red blood cells were lysed as described above.
For murine PBMC analysis, blood was collected from the tail vein of mice on Day 14 of the experiment via tail vein puncture. After washing, the red blood cells were lysed as described above, and the samples were immediately subjected to lectin staining.
Single-cell suspensions were used immediately or frozen for later analysis in liquid nitrogen (in 90% FBS and 10% DMSO).

Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation, using Histopaque (Sigma-Aldrich, 10771), from buffy coats obtained from healthy blood donors (Blood Bank, University Hospital Basel, Switzerland). Fresh tumor samples were obtained from two ovarian cancer patients undergoing tumor resections at University Hospital Basel, Switzerland. Patient characteristics are summarized in Supplementary Table 1. The study was approved by the local Ethical Review Board (Ethikkommission Nordwestschweiz) and University Hospital Basel, Switzerland. Written consent to use their tumor samples for research purposes was obtained from all patients. Fresh tumor samples were mechanically dissociated and digested using accutase (Innovative Cell Technologies, AT-104), collagenase IV (Worthington, LS004188), hyaluronidase (Sigma-Aldrich, H6254), and DNAse type IV (Sigma-Aldrich, D5025), directly after excision. Single-cell suspensions were prepared and samples were stored in liquid nitrogen until further use. In the following assays, single-cell suspensions derived from ovarian cancer samples were maintained in RPMI medium containing L-glutamine (Sigma-Aldrich, R8758) supplemented with 1 × penicillin/streptomycin (Sigma-Aldrich, P4333) and 10% FBS (Pan Biotech, P30-5500).