CITEseq analysis of non-small-cell lung cancer lesions

Immunotherapy is becoming a mainstay in the treatment of NSCLC. We profiled immune cells of 35 early stage NSCLC lesions using multiscale single cell sequencing, including scRNAseq, CITEseq, and TCRseq. Samples of tumor and non-involved lung were obtained from surgical specimens of patients undergoing resection at Mount Sinai Hospital (New York, NY) after obtaining informed consent in accordance with a protocol reviewed and approved by the Institutional Review Board at the Icahn School of Medicine at Mount Sinai (IRB Human Subjects Electronic Research Applications 10-00472 and 10-00135) and in collaboration with the Biorepository and Department of Pathology. Tissues were rinsed in PBS, minced and incubated for 40 minutes at 37C in Collagenase IV 0.25mg/ml, Collagenase D 200U/ml and DNAse I 0.1mg.ml (all Sigma). Cell suspensions were then aspirated through a 18G needle ten times and strained through a 70-micron mesh prior to RBC lysis. Cell suspensions were enriched for CD45+ cells by either bead positive selection (Miltenyi) per kit instructions or FACS sorting on a BD FACSAria flow sorter. Cells were directly loaded onto a 10X Chromium single-cell encapsulation chip according to manufacturer instructions for scRNAseq, or were stained with CITEseq and hashing antibody panels prior to loading, then subjected to downstream encapsulation and respective library prep. Alternatively, cells were subject to CD2+ bead enrichment prior to encapsulation and joint TCR/gene expression RNAseq. Raw data files are uploaded on NCBI with SRA Study SRP251372 BioProject ID PRJNA609924