Gene expression profile at single cell level of mouse lung cancer cell-line tumors grown subcutaneously in adult male C57BL/6 mice with and without high-fat diet-induced obesity
Obesity affects the progress of lung cancer. To understand this more, we performed single-cell RNA sequencing of dissociated tumors of four mouse lung cancer cell-lines grown subcutaneously in adult male C57BL/6 mice with and without high-fat diet-induced obesity. Overall design: Groups (n = 6-8) of male C57BL/6 mice of about 4 months age had been kept on ad libitum for about 3 months on high-fat or control diet (Bioserv products S3282 and F4031, with 60% and 10% fat calories respectively), when they were injected subcutaneously in body flank with 100k cells of one of four mouse lung cancer cell-lines (four separate experiments, one per cell-line). All tumors were dissected after euthanizing mice when the largest tumor reached a maximum dimension of 2 cm. Tumor ulcerations occurred in case of the firefly luciferase-expressing Lewis lung carcinoma (LLC-Luc) cell-line. KPN1.1 cells were from Joshi lab of Yale University. CMT167 cell-line clones engineered with a CRISPR-Cas system for Stk11 knockout (ko) and its negative control were from Yau lab of Roswell Park Cancer Center. Tumors were dissociated with enzymatic and mechanical means in GentleMACS device with Miltenyi Bio reagents. Dissociated tumors were enriched for live cells using magnetic cell separation of dead cells, fixed using Parse Biosciences Cell Fixation Kit, and stored at -80 ºC until all experiments were completed. In all, 24 dissociated tumors were subjected to single-cell RNA sequencing with Parse Biosciences Evercode Whole Transcriptome v3 Kit – the samples underwent three rounds of combinatorial barcoding together (5k cells per sample) resulting in a single library that was sequenced and demultiplexed. For barcoding, 2 neighboring wells (total 5213 projected barcoded cells) were used for each sample, with wells A1 to D12 of the barcoding plate used in order of sample listing in the samples.tsv file.