Celastrol-loaded ginsenoside Rg3 liposomes enhance anti-PD-L1 immunotherapy by inducing immunogenic cell death in triple-negative breast cancer

Triple-negative breast cancer (TNBC), characterized by high heterogeneity and invasiveness. The efficacy of immune checkpoint inhibitors (ICIs), particularly anti-PD1/PD-L1 antibodies (aPD-1/PD-L1), is relatively limited when treating TNBC with monotherapy. Currently, inducing immunogenic cell death (ICD) of tumor cells through approaches such as radiotherapy and chemotherapy is an effective strategy to enhance the response to aPD-L1 therapy in TNBC. However, radiotherapy and chemotherapy treatments often upregulate PD-L1 expression in tumor cells, thereby weakening the tumor cells' response to aPD-L1. Considering that TNBC cells already exhibit high baseline levels of PD-L1, additional upregulation of PD-L1 induced by external factors may deplete some aPD-L1 and potentially impair its efficacy. Therefore, there is an urgent need to identify more effective agents that can induce ICD in TNBC cells without increasing PD-L1 expression in tumor cells. Nowadays, extensive research has demonstrated that celastrol (Cel) effectively induces ICD and reduces PD-L1 expression on the tumor cell membrane, thereby enhancing the efficacy of immunotherapy. Herein, a multifunctional liposomal system which encapsulated Cel using ginsenoside Rg3 (Rg3) as the lipid membrane (Cel-Rg3-Lp) was developed. Cel-Rg3-Lp enhances the solubility and decrease the toxicity of Cel. Additionally, substituting ginsenoside Rg3 (Rg3) for cholesterol (Cho) not only mitigates the potential issue of Cho which would elevate PD-L1 levels, and also promote the accumulation of the drug in tumor tissue through the interaction between Rg3 and glucose transporter 1 (Glut1), which is overexpressed on TNBC tumor cells. Overall, although there are reports that Cel exerts anti-cancer effects by inducing ICD, its mechanism through reducing glucose regulatory protein 78 (GRP78) -mediated ER stress has not been explored. This mechanism may open unique prospects for the use of Cel in treating TNBC and enhancing the efficacy of immunotherapy.

三阴性乳腺癌（Triple-negative breast cancer, TNBC）以高度异质性与侵袭性为显著特征。免疫检查点抑制剂（immune checkpoint inhibitors, ICIs），尤其是抗PD-1/PD-L1单克隆抗体（aPD-1/PD-L1），单药治疗TNBC的临床疗效相对有限。当前，通过放疗、化疗等途径诱导肿瘤细胞发生免疫原性细胞死亡（immunogenic cell death, ICD），是提升TNBC对抗PD-L1治疗应答水平的有效策略。然而，放疗与化疗常会上调肿瘤细胞的PD-L1表达，进而削弱肿瘤细胞对aPD-L1的应答敏感性。鉴于TNBC细胞本身已存在较高基线水平的PD-L1，外源性因素额外诱导的PD-L1上调可能会消耗部分aPD-L1，甚至损害其治疗效果。因此，亟需发掘既能诱导TNBC细胞发生ICD，又不会上调肿瘤细胞PD-L1表达的更高效治疗制剂。

现有大量研究证实，雷公藤红素（celastrol, Cel）可有效诱导ICD并降低肿瘤细胞膜表面的PD-L1表达，从而增强免疫治疗的临床疗效。本研究构建了一种多功能脂质体递送系统：以人参皂苷Rg3（ginsenoside Rg3, Rg3）作为脂质膜组分包裹雷公藤红素，制备得到Cel-Rg3-Lp脂质体。该脂质体可提升雷公藤红素的水溶性并降低其全身毒副作用。此外，用人参皂苷Rg3替代胆固醇（cholesterol, Cho），不仅可规避胆固醇可能上调PD-L1水平的潜在风险，还可通过Rg3与TNBC肿瘤细胞高表达的葡萄糖转运蛋白1（glucose transporter 1, Glut1）的特异性相互作用，促进药物在肿瘤组织中的富集。

总体而言，尽管已有报道指出雷公藤红素可通过诱导ICD发挥抗癌活性，但其通过下调葡萄糖调节蛋白78（glucose regulatory protein 78, GRP78）介导的内质网应激（ER stress）发挥作用的具体分子机制尚未被阐明。该机制或可为雷公藤红素用于TNBC治疗以及增强免疫治疗疗效开辟全新的研究思路与临床转化前景。