基于肿瘤微环境重塑的PSCs靶向制剂用于晚期胰腺癌化疗增敏及其机理研究

Pancreatic cancer (PC) is one of the most aggressive tumors with a high mortality rate and short survival. The high resistance of advanced PC to conventional chemotherapy treatments is one of the principal reason for the poor prognosis. The enriched stroma of PC is believed to be an important contributing factor to the poor therapeutic response. The pancreatic stellate cells (PSCs) activated by transforming growth factor-β (TGF-β) are thought to play a crucial role in the formation of enriched stroma microenvironment. In this study, a novel sequential combination therapy strategy was put forward to increase the treatment of advanced PC, i.e., “remodeling the tumor microenvironment by a PSCs-targeting nanopreparation, followed by chemotherapy with higher sensitivity”. PSCs exhibit high expression of fibroblast activation protein α (FAPα) on the surface. Therefore, a FAPα-responsive nanopreparation for PSCs-specific targeting was developed to delivery TGF-β receptor inhibitor, galunisertib, which could efficiently inhibit the activation of PSCs and down-regulate the interstitial fibrosis in the stroma. On the base of the remodeled tumor microenvironment induced by the PSCs-targeting nanopreparation, a conventional chemotherapy was sequentially performed and an increased anti-tumor efficiency was achieved. In this study, PSCs-targeting nanopreparation was prepared and the physicochemical properties were studied in detail. Moreover, the PSCs targeting abilities and the capacity of remodeling the stroma microenvironment were studied using two-dimensional cell model, three-dimensional cell ball model with core-shell structure and orthotopic tumor models. The sequential combination of PSCs-targeting nanopreparation and conventional chemotherapy were performed to on orthotopic tumor models, and the anti-tumor efficiency and safety were observed. Moreover, the mechanism of increased sensitivity of PC to chemotherapy was explored by charactering the tumor microenvironment. This sequential combination therapy strategy will provide an efficient and promising mean for the advanced PC treatment，which also has important significance for the effective treatment of other stroma-rich solid tumors.

高纤维结缔间质是晚期胰腺癌产生化疗抵抗的重要原因之一，而其形成与TGF-β刺激胰腺星状细胞（PSCs）活化密切相关。本项目首次提出“间质重塑-化疗增敏”的联合治疗策略，即针对PSCs表面过度表达纤维细胞激活蛋白α（FAPα），设计FAPα生物响应型PSCs靶向制剂，将TGF-β受体激酶抑制剂高效靶向递送至PSCs并响应释药，从而有效抑制PSCs活化，下调间质结缔组织增生；在重塑微环境基础上序贯联用临床化疗制剂，可显著提高胰腺癌化疗效果。本项目将系统考察PSCs靶向制剂理化性质，通过二维细胞模型、壳-核结构3D细胞球模型和原位荷瘤动物模型系统评价PSCs靶向性及对微环境重塑能力，并建立该制剂和化疗制剂序贯治疗方案，考察化疗敏感性，并在微环境表征基础上阐明化疗增敏机制，为建立安全、高效的晚期胰腺癌联合治疗方案提供实验基础和理论依据，对提高富含间质实体瘤的临床治疗水平具有重要理论意义和应用价值。

晚期胰腺癌特有的高度纤维结缔增生和极度乏氧的病理微环境，极大减少药物对靶细胞的可及性及靶细胞对药物的敏感性，而造成胰腺癌患者化疗响应低下。转化生长因子-β（TGF-β）是促进其病理微环境形成的重要细胞因子，可通过激活胰腺星状细胞（PSCs），促使其分泌大量细胞外基质（ECM），在挤压血管削弱药物和氧气向肿瘤灌注的同时，亦阻碍其向瘤内深层浸润。本论文针对PSCs表面特异性表达成纤维细胞激活蛋白α（FAP-α），以FAP-α响应肽（Gly-Pro, GP）为连接臂，成功构建了具有FAP-α响应功能的葡聚糖（Dex）-脱氧胆酸（DOCA）两亲性偶联物Dex-GP-DOCA，可将TGF-β抑制药物高效靶向递送于PSCs并响应FAP-α快速释药，从而实现对TGF-β相关信号通路的高效抑制，肿瘤微环境得到显著而持久的改善，表现为ECM表达显著下调，血管形态得以恢复，血液灌注量显著增加，肿瘤深层乏氧亦显著改善，肿瘤细胞耐药蛋白P-gp表达显著下调。此外，本研究亦发现，所构建的FAP-α生物响应PSCs靶向制剂亦能显著抑制PSCs促有丝分裂生长因子Wnt16的分泌，有利于抑制肿瘤细胞增殖。基于所构建的FAP-α生物响应PSCs靶向制剂对肿瘤微环境的多方位重塑，本研究进一步序贯联用胰腺癌一线化疗药物白蛋白结合型紫杉醇纳米粒（Abraxane）以及吉西他滨，发现化疗药物在胰腺癌瘤内的分布量和浸润深度皆显著增加，且瘤内乏氧的改善显著提高了肿瘤细胞对化疗的敏感度，胰腺癌的化疗效果得到极显著的提升，抑制率分别高达87.5%和85.2%。该研究工作为建立安全、高效的晚期胰腺癌联合治疗方案提供了实验基础和理论依据，对提高富含间质实体瘤的临床治疗水平具有重要理论意义和应用价值。