NextSeq 500 paired end sequencing; DNA barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and in response to immunotherapy
DNA barcoding reveals ongoing immunoediting of clonal cancer populations during metastatic progression and in response to immunotherapy
4T1 cells were obtained from ATCC. The ClonTracer library was a gift from Dr Frank Stegmeier (Addgene #67267). Lentiviral particles containing the high-complexity barcode library were produced by transfecting 293T cells. 4T1 and EMT6 cancer cell lines were barcoded by lentiviral infection using 0.8 Âµg/ml polybrene. Cells from each line, were infected with a target MOI of 0.1, corresponding to 10% infectivity to ensure single lentiviral integration. A starting population of 4T1 cells (highly metastatic) was transfected with a lentivirus vector carrying the ClonTracer Barcoding Library. 50 000 cells were inoculated into the mammary fat pad of wild-type (WT) Balb/c mice. Primary tumours were resected 15 days following inoculation to allow metastases to develop and mice were subsequently treated with either combination immunotherapy (anti-PD1 + anti-CTLA4) or control antibodies. Lung metastases were then checked for clonal composition. IE1 and IE2 were two clones enriched following immunotherapy treatment in vivo, each with unique transcriptional profiles, while NT1 and NT2 were two independent control clones with barcodes that were not enriched following immunotherapy. These clonal cell lines were then isolated and propagated in vitro from the starting population of 4T1 cells, and clonal cell lines confirmed with Sanger sequencing. RNA was extracted from established subclonal cell lines (P2) using the QIAGEN RNeasy Mini Kit.  Libraries were prepared using the KAPA RNA HyperPrep Kit with RiboErase.