Transcriptional effects of co-culture on the spatiotemporal dynamics of T cell motility and cancer-T cell interactions [scRNA-seq]

Insufficient infiltration of cytotoxic T cells into solid tumors poses a major challenge in cancer immunotherapy, largely due to the intricate tumor microenvironment. To address this, we co-cultured mouse cancer cell lines (B16-WT or B16-OVA) with cancer-specific cytotoxic T cells (activated OT-1) in vitro to uncover the impact of cancer-T cell interactions on T cell motility, pivotal for effective tumor infiltration. To investigate the potential molecular mechanisms underlying T cell motility patterns in the two coculture contexts, we performed both bulk and single-cell RNA sequencing of cancer and T cells sorted from the co-culture systems at specific time points. In this experiment, we aimed to explore the quantitative effects on spatiotemporal dynamics of T cell motility within a co-culture system of murine melanoma cells and cancer-specific OT-1 CD8+ T cells. B16-F10 cells overexpressing ovalbumin and the Ca2+ sensor GCaMp6s (referred to as B16-OVA) were generated via lentivirus infection. CD8+ T cells isolated from C57BL/6J-OT-I spleens were activated using Dynabeads® Mouse T-Activator CD3/CD28. Co-cultures were established with B16-F10-WT and B16-F10-OVA cells seeded onto Poly-D-lysine-coated µ-Slide 8 well plates, followed by the addition of activated OT-1 CD8+ T cells. Fluorescence-activated cell sorting (FACS) was employed to sort cells using EpCam antibody for cancer cells and CD8 antibody for T cells. Bulk RNA sequencing was performed on cells from co-culture at 0h, 6h, and 12h, while single-cell RNA sequencing was specifically conducted on cells obtained from the 12h co-culture.