Genetic Modification of Tumor-Infiltrating Lymphocytes, Peripheral T Cells, and T-Cell Model Cell Lines.

Genetic modification of tumor-infiltrating lymphocytes (TILs) or circulating T cells has become an important avenue in cancer therapy. Here we describe a comprehensive method for establishing and expanding TIL cultures and genetically modifying them with a gene of interest (GOI) via retroviral transduction or mRNA transfection. The method includes all the important steps starting with TIL extraction from tumors through to the maintenance of the genetically modified TILs. The protocol includes instructions for retroviral transduction and mRNA transfection of circulating T cells or T-cell lines. The GOIs most commonly introduced into the target cells are chimeric antigen receptors (CARs); genetic adjuvants, such as membrane-bound interleukins; and antitumor T-cell receptors (TCRs).

Genomic Characterization of Cisplatin Response Uncovers Priming of Cisplatin-Induced Genes in a Resistant Cell Line.

Cisplatin is a chemotherapy drug that kills cancer cells by damaging their DNA. In human cells, this damage is repaired primarily by nucleotide excision repair. While cisplatin is generally effective, many cancers exhibit initial or acquired resistance to it. Here, we studied cisplatin resistance in a defined cell line system. We conducted a comprehensive genomic characterization of the cisplatin-sensitive A2780 ovarian cancer cell line compared to A2780cis, its resistant derivative. The resistant cells acquired less damage, but had similar repair kinetics. Genome-wide mapping of nucleotide excision repair showed a shift in the resistant cells from global genome towards transcription-coupled repair. By mapping gene expression changes following cisplatin treatment, we identified 56 upregulated genes that have higher basal expression in the resistant cell line, suggesting they are primed for a cisplatin response. More than half of these genes are novel to cisplatin- or damage-response. Six out of seven primed genes tested were upregulated in response to cisplatin in additional cell lines, making them attractive candidates for future investigation. These novel candidates for cisplatin resistance could prove to be important prognostic markers or targets for tailored combined therapy in the future.