Cd45 cells

The patients with lung cancer, specimen acquisition and processing and scRNA-seq and analysis of human samples are described elsewhere^{6 (link)}. In brief, tumour and non-involved lung resection specimens were obtained from patients undergoing surgery at the Mount Sinai Medical Center in collaboration with the Biorepository and Department of Pathology and under the provisions of a protocol reviewed and approved by Mount Sinai’s Institutional Review Board (IRB HS 10–00472 and HS 00135). Informed consent was obtained from the participants. Tissues were rinsed in PBS, minced and incubated for 40 min at 37 °C in collagenase IV 0.25 mg/ml, collagenase D 200 U/ml and DNase I 0.1mg/ml (all Sigma). Cell suspensions were aspirated through a 18G needle 10 times and strained through 70-μm mesh before RBC lysis. Suspensions were enriched for immune cells by magnetic-bead positive selection of CD45⁺ cells (Miltenyi) before processing for scRNA-seq. A detailed unsupervised analysis of the human scRNA-seq dataset has been published elsewhere^{6 (link)}.

Primary murine lung EC isolation was performed as we previously did^{12 (link)–14 (link)}. Briefly, 14–18wk old mice were euthanized using 100% CO₂ inhalation followed by cervical dislocation. The chest was immediately opened through a midline sternotomy. The left ventricle was identified and the ventricular cavity was entered through the apex with a 27-gauge needle. The right atrium was identified and an incision was made to exsanguinate the animal and to allow the excess perfusate to exit the vascular space. The animal was perfused with 20 ml of cold PBS. The lung tissue was collected and minced finely with scissors. The tissue fragments were digested in DMEM medium containing 1 mg/mL Collagenase D/Dispase (Roche, Switzerland) and 25 U/mL DNase (Sigma, St. Louis, MO) at 37C for 2hr with shaking, after which the suspension was homogenized by triturating. The homogenate was filtered through a 70μm nylon mesh (BD Biosciences, San Jose, CA) and pelleted by centrifugation (400g for 5 min). Cells were first depleted for CD45⁺ cells (MiltenyiBiotec) and then positively selected for CD31⁺ cells (Miltenyi Biotec) using magnetically labeled microbeads according to the manufacturer’s protocol. Isolated ECs (CD45^-CD31⁺) were cultured in EC culture medium with no medium change for the first 72hrs to allow EC attachment followed by medium change about every 3 days.