Leucosep tube

Human PBMCs were obtained from anonymous, healthy donors from the NIH Blood Bank. Cells were separated with LeucoSep tube filled with Ficoll-Paque-plus (GE Healthcare, Pittsburgh, PA) according to the manufacturer’s instruction. CD8+ cells were isolated from PBMC using Dynabeads™ CD8 Positive Isolation Kit (TermoFisher Scientific, Waltham, MA). Three other primary cells including Human Lung Microvascular Endothelial Cells (HMVEC-L), Human Bone Marrow Stromal Cells (BMSC) and mouse spleen cells were kindly provided by our collaborators. Culture cell lines, including KLM1, 293T and MEF were harvested with trypsin–EDTA and single-cell suspension was prepared following 10× Genomics Single cell protocols: Cell preparation guide (CG00053, Rev C). Both PBMC and cell lines were washed twice to remove ambient RNA and finally resuspended in 1× PBS (calcium and magnesium free) containing 0.04% BSA. Cell concentration and viability were determined twice on a Guava^® easyCyte Single Sample Flow Cytometer (MilliporeSigma, Burlington, MA) using Guava^® ViaCount^® Assay. Cells with viability of greater than 90% were used and kept on ice for fixation and single cell RNA-Seq analysis.

20 ml citrated peripheral venous blood were drawn from HNSCC patients directly before and immediately after curative tumor resection and processed within 24 h after collection. Blood sample preparation was done as described previously [17] (link), [19] . Briefly, 20 ml of blood were diluted with 10 ml PBS and carefully layered into a Leucosep tube containing 16 ml Ficoll-Paque (GE-Healthcare) below a porous barrier. After buoyant density gradient centrifugation (1600×g, 20°C, 20 min) the interphase consisting of peripheral blood mononuclear cells (PBMNC) and CTC was removed and washed. The cell suspension was spun onto 2–4 glass slides per sample containing a maximum of 6×10⁶ cells per slide using the Cell Spin II centrifuge (Tharmac, Waldsolms, Germany), air-dried and subsequently fixated with 96% Ethanol. Slides were stored at 4°C until subjected to immunocytochemical staining (Fig. 1). The presence of CTC was verified by multi-immunofluorescence staining against pan-CK (epithelial), N-cadherin (mesenchymal), CD133 (stem-cell-like), counterstaining with CD45 (hematopoietic) and DAPI (nucleus). CTC and hematopoietic cells were enumerated, normalized and expressed as number of CTC per 1000 PBMNC. Individual cell type profiles were analyzed and correlated to therapeutic outcome in 10 patients.