Bioengineered three‐dimensional co‐culture of cancer cells and endothelial cells: A model system for dual analysis of tumor growth and angiogenesis

Abstract

Angiogenesis marks the transformation of a benign local tumor into a life-threatening disease. Many in vitro assays are available on two-dimensional (2D) platforms, however, limited research has been conducted to investigate the behavior of tumors and endothelial cells (ECs) grown on three-dimensional (3D) platforms. This study provides a 3D co-culture spheroid of tumor cells with ECs to study the interplay between ECs and tumor cells. In a 3D co-culture with HepG2 hepatocellular carcinoma (HCC) cells, ECs differentiate to form tubule networks when in co-culture. Addition of angiogenic factors or angiogenesis inhibitors to the model system enhanced or inhibited endothelial differentiation in the 3D model, enabling investigations of the cellular signaling pathways utilized in HCC development. The 3D model demonstrated similar protein expression levels as a HCC xenograft, as well as exhibited upregulation of essential signaling proteins such as Akt/mTor in the 3D model, which is not reflected in the 2D model. The effects of several anti-angiogenic agents, such as sorafenib, sunitinib, and axitinib were analyzed in the 3D co-culture model by utilizing fluorescent proteins and a fluorescence resonance energy transfer (FRET)-based caspase-3 sensor in the ECs, which can detect apoptosis in real time. The apoptotic capability of a drug to inhibit angiogenesis in the 3D model can be easily distinguished via the FRET sensor, and dual screening of anti-angiogenesis and anti-tumor drugs can be achieved in a single step via the 3D co-culture model. In summary, a 3D co-culture model is constructed, where a HCC tumor microenvironment with a hypoxic core and true gradient penetration of drugs is achieved for drug screening purposes and in vitro studies utilizing a small HCC tumor. Biotechnol. Bioeng. 2017;114: 1865-1877. © 2017 Wiley Periodicals, Inc.

Innovation for hepatotoxicity in vitro research models: A review

“…Chiew et al developed a 3D spheroid including HepG2 cells and LSECs. The 3D models demonstrated similar protein expression levels as a liver cancer xenograft, as well as upregulation of essential signaling proteins such as Akt/mTor, which was not reflected in the 2D models (Chiew, Wei, Sultania, Lim, & Luo, ).…”

Rapid identification of antimicrometastases drugs using integrated model systems with two dimensional monolayer, three dimensional spheroids, and zebrafish xenotransplantation tumors

“…A nonadhesive coating method was used to generate 3D multicellular tumor spheroids as reported in our previous study (Anand et al, ; G. G. Y. Chiew et al, ). Briefly, low adherent round‐bottom 96‐well plates were coated with 1% pluronic F‐127 (Sigma‐Aldrich, Steinheim, Germany) to generate a nonadhesive surface.…”

“…A nonadhesive coating method was used to generate 3D multicellular tumor spheroids as reported in our previous study (Anand et al, 2015;G. G. Y. Chiew et al, 2017).…”

“…Therefore, 3D cell culture models have been widely established to improve the successful rate of anticancer drug discovery (Muranen et al, ). Recently, we developed caspase‐3 sensor‐based 3D spheroid models for anticancer drug screening (Anand, Fu, Teoh, & Luo, ; G. G. Y. Chiew, Wei, Sultania, Lim, & Luo, ). In this study, to further mimic the in vivo microenvironment of metastasis in a noninvasive manner, we developed a biosensor‐based zebrafish xenotransplantation model for testing antimetastasis drugs.…”

Implementation of a dynamic culture condition to the heterotypic 3D breast cancer model

“…Nowadays, for cancer studies, the most common 3D models are: (a) multicellular tumor spheroids generated by various methods including the static suspension cultures and dynamic device‐assisted cultures, (b) matrix‐embedded cultures that provide some elements of ECM microenvironment, and (c) scaffold‐based cultures that provide the physical support for cells allowing them to form an appropriate cell–cell or cell–ECM interactions (Lv, Hu, Lu, Lu, & Xu, ). Some of the examined 3D cancer models were composed of cells of various origin what provided the cells heterogeneity in the model (Chiew, Wei, Sultania, Lim, & Luo, ; Heneweer et al, ; Jaganathan et al, ). However, the other significant aspect of in vitro cancer modeling is the incorporation of stress forces (Chim & Mikos, ; Mina, Huang, Murray, & Mahler, ).…”