3D生物打印胶质瘤体外肿瘤模型及初步应用研究

Glioma is still difficult to treat because of its high malignancy, high recurrence rate, and high resistance to anticancer drugs. Glioma stem cells (GSCs) are considered to be critical for glioma development. Lack of in vitro tumor models that mimic the in vivo microenvironment of glioma limits the research of glioma development, progression, recurrence, and resistance mechanisms. In this application, using the applicants’ established advantages, 3D bioprinting techniques will be combined with optimized bioprintable material mimicking the neural tissue extracellular and biofabricated in vitro to create a pre-vascularized tumor model with spatial structural microenvironment. Similarities and differences between 3D bioprinted glioma model and in vivo glioma tissue microenvironment will be compared by histological, pathological analyses and gene expression levels in vitro and in animal model. Furthermore, sensitivity to chemotherapeutic drugs (temozolomide) will be compared with that of 3D bioprinted glioma cell line, glioma stem cell line, primary cultured glioma cells, and primary cultured sorted glioma stem cells compared to corresponding 2D culture models. It is a new in vitro tumor model that can mimic the tumor microenvironment in vivo, and provide a new model for in vitro study of the mechanism of gliomagenesis and clinical drug screening.

脑胶质瘤因高度恶性、高复发性和高耐药性而难以攻克，胶质瘤干细胞更被认为是罪魁祸首。能够模拟胶质瘤体内微环境的体外肿瘤模型的缺乏限制了对胶质瘤发生、进展、复发、耐药机制的研究。本申请使用3D生物打印技术结合优选的能够模拟神经组织细胞外基质的可生物打印材料和申请人课题组在胶质瘤干细胞研究的传统优势，体外生物制造具有空间结构微环境的预血管化肿瘤模型；通过体外、体内组织形态、病理和基因表达水平对比3D生物打印胶质瘤模型与胶质瘤组织微环境的异同性；进一步，使用3D生物打印胶质瘤细胞系、胶质瘤干细胞系、原代培养胶质瘤细胞和原代培养分选的胶质瘤干细胞与各自2D培养模型比较对化疗药物（替莫唑胺）的敏感性。为体外研究胶质瘤发生机制、临床药物筛选等研究提供更加能够模拟体内肿瘤微环境的新的体外肿瘤模型。

肿瘤生物模型是研究肿瘤发生机制、检验肿瘤放化疗效果的有效工具。脑恶性胶质瘤正因缺乏合适的肿瘤模型而研究进展受限，建立能够模拟体内微环境的3D胶质瘤模型，有望为研究胶质瘤发生机制，提高胶质瘤放化疗敏感性和治疗预后提供可能。本文通过3D生物打印技术体外建立胶质瘤干细胞的3D肿瘤模型，首先体外评估胶质瘤微环境中干细胞特征；其次通过改进的生物打印方法建立壳-芯结构肿瘤细胞线模型，用转染LOXP-STOP-LOXP-RFP基因的胶质瘤干细胞（GSCs）和转染CRE酶的人骨髓间充质干细胞（hMSCs）的肿瘤模型研究胶质瘤干细胞与微环境中骨髓间充质干细胞的作用关系；再同轴生物打印构建的壳-GSC23/芯-U118模型模拟胶质瘤微环境，对比替莫唑胺化疗药物敏感性；最后在此模型基础上拓展研究了胶质瘤干细胞3D肿瘤模型放射敏感性差异及探讨了初步分子机制。结果发现1）3D生物打印胶质瘤干细胞模型不仅能够高效维持干细胞比例，而且具有潜在血管内皮转分化潜能，同时发现体外3D胶质瘤模型具有富集胶质瘤干细胞的效果；2）双色荧光开关基因载体系统示踪研究发现同轴3D生物打印多细胞自组装异质肿瘤组织样细胞线为体外研究肿瘤微环境特别是肿瘤与间质相互作用关系提供了更优越的3D模型，胶质瘤干细胞和骨髓间充质干细胞在体外3D空间结构微环境中发生细胞融合现象；3）同轴生物打印构建的壳-GSC23/芯-U118模型模拟胶质瘤微环境，发现肿瘤侵袭和耐药相关基因的表达显著增强；4）拓展研究发现3D生物打印胶质瘤模型较2D模型更加耐受放射治疗，转录组测序和实验验证提示ITGA2过表达通过AKT分子通路参与到3D胶质瘤模型的放疗耐受。本研究利用3D生物打印技术建立胶质瘤体外3D模型，发现了胶质瘤干细胞富集效应和转分化血管内皮潜能，以及胶质瘤干细胞与骨髓间充质干细胞融合的现象，证实3D胶质瘤模型具有更强的侵袭和替莫唑胺耐药特征，拓展发现3D生物打印胶质瘤通过ITGA2/AKT分子通路耐受放射治疗。