RNAseq transcriptome of draining lymph node (LN) and tumor of MC38 murine tumors treated with cryoablation and chitosan/IL-12

Focal ablation technologies are routinely used in the clinical management of inoperable solid tumors but often result in incomplete ablations leading to high recurrence rates. Adjuvant therapies capable of safely eliminating residual tumor cells are therefore of great clinical interest. Interleukin 12 (IL-12) is a potent antitumor cytokine that can be localized intratumorally through coformulation with viscous biopolymers including chitosan (CS) solutions. The objective of this research was to determine if localized immunotherapy with CS/IL-12 could prevent tumor recurrence after cryoablation (CA). Tumor recurrence, overall survival, and protective immunity were assessed. Systemic immunity was evaluated in spontaneously metastatic and bilateral tumor models. Temporal bulk RNA sequencing was performed on tumor and draining lymph node samples. In multiple murine tumor models, the addition of CS/IL-12 to CA reduced recurrence rates by 30–55%. Altogether, this cryo-immunotherapy induced com..., MC38 tumors were implanted subcutaneously in the right flanks of female C57BL/6 mice as described above. When tumor volumes measured between 200–500 mm3, CA was performed and CS/IL-12 was intratumorally injected within an hour after CA. Mice were euthanized 3 days and 7 days post-ablation to harvest tumor tissues and tumor-draining inguinal lymph nodes (dLNs). To obtain cell suspensions, dLNs were homogenized through 40 µm mesh strainers with complete T cell media. Tumor tissues were first minced into approximately 0.5 cm squares before treatment with 5X Triple Enzyme Solution (collagenase type IV 10 mg/ml, hyaluronidase 1 mg/ml, and DNase 20,000 units final concentration in Hank’s Buffered Saline Solution) for 30 minutes at 37°C with magnetic bar stirring. The resulting homogenate was filtered through a 70 µm mesh strainer into a clean tube to achieve the final cell suspension. Total RNA was extracted from the cell suspension using the GeneJET RNA Purification Kit (ThermoFisher cat #K0..., Excel file format, or .csv reader局部消融技术常规应用于不可手术实体瘤的临床诊疗，但常导致消融不完全，进而引发较高的肿瘤复发率。因此，可安全清除残留肿瘤细胞的辅助治疗手段具有极高的临床研究价值。白细胞介素12（Interleukin 12，IL-12）是一种强效抗肿瘤细胞因子，可通过与包括壳聚糖（chitosan，CS）溶液在内的粘性生物聚合物共配制，实现瘤内靶向递送。本研究旨在探究CS/IL-12局部免疫疗法是否能够预防冷冻消融（cryoablation，CA）后的肿瘤复发。研究团队对肿瘤复发情况、总生存期以及保护性免疫能力进行了评估；在自发性转移瘤模型与双侧肿瘤模型中评估了系统性免疫功能，并对肿瘤组织及引流淋巴结样本开展了时序性批量RNA测序。在多种小鼠肿瘤模型中，联合应用CS/IL-12与冷冻消融可将肿瘤复发率降低30%~55%。综上，该冷冻免疫疗法可诱导……，按照前述方法将MC38肿瘤细胞接种于雌性C57BL/6小鼠右侧背部皮下。当肿瘤体积达到200~500 mm³时，实施冷冻消融，并于术后1小时内完成CS/IL-12的瘤内注射。分别于消融后3天和7天处死小鼠，采集肿瘤组织及肿瘤引流腹股沟淋巴结（dLNs）。为制备细胞悬液，将肿瘤引流腹股沟淋巴结（dLNs）通过40μm细胞筛网进行匀浆，采用完全T细胞培养基重悬。肿瘤组织先被剪切成约0.5 cm见方的小块，随后置于5×三联酶溶液（Triple Enzyme Solution，含IV型胶原酶10 mg/ml、透明质酸酶1 mg/ml、脱氧核糖核酸酶终浓度20000 U的汉克斯缓冲盐溶液）中，于37℃环境下辅以磁力搅拌孵育30分钟。将所得匀浆通过70μm细胞筛网过滤至洁净离心管中，最终获得合格的细胞悬液。使用GeneJET RNA纯化试剂盒（GeneJET RNA Purification Kit，赛默飞世尔ThermoFisher，货号K0……）从细胞悬液中提取总RNA，文件格式支持Excel或.csv可读格式