Nano-icilin–driven TRPM8 activation elicits immunogenic exosomes with antitumor effects

Transient receptor potential melastatin 8 (TRPM8) is a cold-sensing cation channel that regulates calcium (Ca2+) levels in cells. Its overexpression is linked to tumor development and progression. TRPM8 activation by specific agonists leads to increased Ca2+ influx, causing stress and apoptosis. This stress can enhance the production and release of exosomes, which have antitumor immunity properties. We hypothesize that activating TRPM8 with nano-icilin can stimulate immune responses when administered peritumorally. 4T1 cancer cells were treated with icilin nanoparticles and hypothermia to evaluate cytotoxicity, apoptosis, calcium flux, and exosome extraction. Isolated exosomes were characterized and tested in vivo for antitumor immune response in a mouse model. Tumor growth, cytokines (IL-2, IL-12, IL-10, and IL-1β), and immunohistochemistry (IHC) were assessed. Data were analyzed using ANOVA and Duncan’s test (P ≤ 0.05). TRPM8 activation by icilin nanoparticles triggers apoptosis and calcium influx in 4T1 cells. Exosomes from treated cells exhibited altered size, charge, and increased levels of DAMPs (HMGB1, HSP70). Administering these exosomes significantly inhibited tumor growth, increased CD4+/CD8+ T cells, and elevated IL-2 and IL-12, while reducing IL-10 and PD-L1, thus preventing lung metastasis. Activation of TRPM8 by icilin or cold can induce immunogenic exosomes, enhancing T cell infiltration, proinflammatory cytokines, and tumor suppression, offering a new strategy to boost immune responses against cancer.

瞬时受体电位阳离子通道蛋白亚家族M成员8（Transient receptor potential melastatin 8, TRPM8）是一类冷敏阳离子通道，可调控细胞内钙（Ca²+）水平。该通道的过表达与肿瘤发生及进展密切相关。特定激动剂（agonists）激活TRPM8后会引发Ca²+内流增加，诱导细胞应激与凋亡。此类细胞应激可促进外泌体（exosomes）的生成与释放，而外泌体具备抗肿瘤免疫特性。我们提出如下假说：采用纳米冰素（nano-icilin）激活TRPM8并实施瘤周给药，可有效激发机体免疫应答。本研究使用冰素纳米粒联合低温处理4T1肿瘤细胞，以评估其细胞毒性、细胞凋亡、钙流变化及外泌体提取效果。对分离得到的外泌体进行表征，并通过小鼠模型开展体内抗肿瘤免疫应答实验。检测指标涵盖肿瘤生长情况、细胞因子（白细胞介素-2、白细胞介素-12、白细胞介素-10及白细胞介素-1β）水平与免疫组织化学（immunohistochemistry, IHC）结果。数据分析采用方差分析（analysis of variance, ANOVA）与邓肯检验（Duncan’s test），检验水准设定为P≤0.05。实验结果显示，冰素纳米粒激活TRPM8可诱导4T1肿瘤细胞发生凋亡并增加Ca²+内流。经处理细胞释放的外泌体其粒径、电荷发生改变，且损伤相关分子模式（damage-associated molecular patterns, DAMPs）高迁移率族蛋白B1（high mobility group box 1, HMGB1）、热休克蛋白70（heat shock protein 70, HSP70）的表达水平显著升高。将此类外泌体给药后，可显著抑制肿瘤生长，增加CD4+/CD8+ T细胞浸润，提升白细胞介素-2与白细胞介素-12的表达水平，同时降低白细胞介素-10与程序性死亡受体配体1（PD-L1）的表达，进而有效抑制肺转移。综上，冰素或低温激活TRPM8可诱导产生免疫原性外泌体，增强T细胞浸润与促炎细胞因子分泌，实现肿瘤抑制，为增强抗肿瘤免疫应答提供了全新的治疗策略。