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. 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.

实体瘤中细胞毒性T细胞（cytotoxic T cells）浸润不足是癌症免疫治疗的核心挑战之一，其主要诱因是复杂的肿瘤微环境（tumor microenvironment）。为解决这一难题，我们在体外将小鼠癌细胞系（B16-WT或B16-OVA）与癌症特异性细胞毒性T细胞（活化的OT-1）共培养，以揭示癌细胞-T细胞互作对T细胞运动能力的影响——而T细胞运动能力是实现有效肿瘤浸润的关键环节。为探究两种共培养体系中T细胞运动模式背后的潜在分子机制，我们对特定时间点从共培养系统中分选得到的癌细胞与T细胞，分别开展了批量RNA测序（bulk RNA sequencing）与单细胞RNA测序（single-cell RNA sequencing）。为评估经抗CD3/抗CD28 Dynabeads活化的OT-1 CD8+ T细胞的体内抗肿瘤特性，我们使用NOD-SCID小鼠构建黑色素瘤模型，分别向其注射转染了GCaMP6s的B16-F10-OVA细胞或B16-F10细胞。为对比体外共培养体系与体内肿瘤中的T细胞基因特征，我们在T细胞输注两天后，对携带B16-F10-OVA与B16-F10黑色素瘤的小鼠实施安乐死。使用无菌手术刀将新鲜肿瘤组织剪碎至约1 mm³的组织碎片，于37℃下在含小鼠胶原酶（1 mg/mL）与透明质酸酶（1000 U/mL）的消化液中酶解45分钟。将所得细胞悬液通过Falcon 100 µm细胞筛过滤以去除未消化的组织块，反复洗涤以清除死细胞，随后重悬于培养基中：该培养基为添加了10%胎牛血清（FBS）、1%青霉素-链霉素、1%丙酮酸钠、1% HEPES、500 µL胰岛素以及10 nM雌激素的DMEM培养基。我们使用结合CD8+抗体的Dynabeads从细胞悬液中阳性分选OT-1 CD8+ T细胞。磁分离完成后，将结合磁珠的细胞置于释放缓冲液中孵育，收集脱离磁珠的CD8+ T细胞用于批量RNA测序。与此同时，通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分离表达GCaMP6s的B16-F10-OVA与B16-F10肿瘤细胞，并对其进行批量RNA测序。