AlgiMatrix™ Based 3D Cell Culture System as an In-Vitro Tumor Model for Anticancer Studies

BackgroundThree-dimensional (3D) in-vitro cultures are recognized for recapitulating the physiological microenvironment and exhibiting high concordance with in-vivo conditions. Taking the advantages of 3D culture, we have developed the in-vitro tumor model for anticancer drug screening.
MethodsCancer cells grown in 6 and 96 well AlgiMatrix™ scaffolds resulted in the formation of multicellular spheroids in the size range of 100–300 µm. Spheroids were grown in two weeks in cultures without compromising the growth characteristics. Different marketed anticancer drugs were screened by incubating them for 24 h at 7, 9 and 11 days in 3D cultures and cytotoxicity was measured by AlamarBlue® assay. Effectiveness of anticancer drug treatments were measured based on spheroid number and size distribution. Evaluation of apoptotic and anti-apoptotic markers was done by immunohistochemistry and RT-PCR. The 3D results were compared with the conventional 2D monolayer cultures. Cellular uptake studies for drug (Doxorubicin) and nanoparticle (NLC) were done using spheroids.
ResultsIC50 values for anticancer drugs were significantly higher in AlgiMatrix™ systems compared to 2D culture models. The cleaved caspase-3 expression was significantly decreased (2.09 and 2.47 folds respectively for 5-Fluorouracil and Camptothecin) in H460 spheroid cultures compared to 2D culture system. The cytotoxicity, spheroid size distribution, immunohistochemistry, RT-PCR and nanoparticle penetration data suggested that in vitro tumor models show higher resistance to anticancer drugs and supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro.
ConclusionThe results from our studies are useful to develop a high throughput in vitro tumor model to study the effect of various anticancer agents and various molecular pathways affected by the anticancer drugs and formulations.

**背景**：三维（3D）体外培养体系因能够重现生理微环境、与体内状态高度契合而广受认可。本研究依托三维培养的优势，开发了用于抗癌药物筛选的体外肿瘤模型。

**方法**：将癌细胞接种于6孔及96孔AlgiMatrix™支架中，可形成尺寸介于100~300 µm的多细胞球状体。该球状体可在培养两周后仍维持其生长特性。本研究对多款已上市抗癌药物开展筛选：将药物加入三维培养体系，分别于第7、9、11天孵育24 h，随后采用AlamarBlue®法检测细胞毒性。抗癌药物的治疗效果通过球状体的数量及尺寸分布进行评估。采用免疫组织化学和逆转录聚合酶链式反应（RT-PCR）对凋亡及抗凋亡标志物进行检测。将三维培养体系的实验结果与传统二维单层培养体系进行对比。采用多细胞球状体完成了药物（多柔比星（Doxorubicin））与纳米颗粒（NLC）的细胞摄取实验。

**结果**：与二维培养模型相比，AlgiMatrix™体系中抗癌药物的半数抑制浓度（IC50）显著升高。与二维培养体系相比，H460多细胞球状体中剪切型半胱氨酸天冬氨酸蛋白酶-3的表达量显著下调，其中5-氟尿嘧啶（5-Fluorouracil）和喜树碱（Camptothecin）的下调倍数分别为2.09倍和2.47倍。细胞毒性、球状体尺寸分布、免疫组织化学、RT-PCR及纳米颗粒穿透实验数据均表明，体外肿瘤模型对抗癌药物具有更高的耐药性，证实三维培养体系是体外评估抗癌药物细胞毒性的更优模型。

**结论**：本研究结果可为构建高通量体外肿瘤模型提供依据，该模型可用于研究各类抗癌药物的作用效果，以及抗癌药物与制剂所调控的各类分子通路。