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

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. Overall design: To evaluate the antitumor properties of OT-1 CD8+ T cells activated with anti-CD3/anti-CD28 Dynabeads in vivo, melanoma mouse models were established using NOD-SCID mice injected with either B16-F10-OVA (GCaMP6s-transfected) or B16-F10 (GCaMP6s-transfected) cells. To compare T cell gene signatures between in vitro co-culture systems and in vivo tumors, B16-F10-OVA and B16-F10 melanoma-bearing mice were euthanized two days post-T cell administration. Fresh tumor tissues were minced into ~1 mm fragments using sterile scalpels and enzymatically dissociated in digestion medium containing mouse collagenase (1 mg/mL) and hyaluronidase (1000 U/mL) at 37°C for 45 minutes. The resulting cell suspension was filtered through Falcon 100 µm cell strainers to remove undigested tissue, washed repeatedly to eliminate dead cells, and resuspended in culture medium (DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, 1% sodium pyruvate, 1% HEPES, 500 µL insulin, and 10 nM estrogen). OT-1 CD8+ T cells were positively selected from the suspension using CD8+ antibody-conjugated Dynabeads. After magnetic separation, bead-bound cells were incubated with release buffer, and bead-free CD8+ T cells were collected for bulk RNA sequencing. Concurrently, GCaMP6s-expressing B16-F10-OVA and B16-F10 tumor cells were isolated via fluorescence-activated cell sorting (FACS) and processed for bulk RNA sequencing.