Perturbational sequencing of patient tumors reveals midkine as an innate immune checkpoint in neuroendocrine tumors
Humor tumor samples were collected from patients. Resected human tumors were minced and digested using Native Bacillus licheniformis protease (Creative Enzymes, NATE-0633) on ice for 30 minutes to dissociate into single cell suspension. Tumor single cell suspensions with low viability were further processed using dead cell removal kit (Miltenyi, 130-090-101). Cultured cells were collected stained with TotalSeq anti-human hashtags C0251- C0258 (Biolegend), viability dye (zombie red, Biolegend 423109) and anti-human CD45-FITC (clone HI30, Biolegend 304038) and enriched for live cells and up to 50% immune cells using BD FACS Aria II.  Cells were plated and stimulated with 1000U/ml human IFNb (pbl assay science 11415), 500ng/ml poly:IC HMW (invivogen, tlrl-pic) transfected with lipofectamine 2000, 10ug/ml non-transfected poly(I:C) HMW, 2ug/ml SLR14 (acquired from Iwasaki lab), 1ug/ml ADU-S100 (invivogen, tlrl-nacda2r), 10ug/ml anti-human CD279 antibody (Biolegend, 329902), and cultured for up to 48 hours.  Sorted cells were immediately loaded into a 10x Chromium reaction.   Chromium Next GEM Single-Cell 5′ kits v.2 were used to generate GEX, VDJ and Cite-seq libraries according to the manufacturer's protocol.