Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate.

Abstract

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-β2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.

• Authors: Sang-Yun Lee, Yvonne Teng, Miseol Son, Bosung Ku, Hyun Ju Hwang, Vinay Tergaonkar, Pierce Kah-Hoe Chow, Dong Woo Lee, Do-Hyun Nam
• Journal: Molecules (Basel, Switzerland) (Molecules) Vol. 26 Issue 16 (Aug 16 2021) ISSN: 1420-3049 [Electronic] Switzerland
• PMID: 34443536 (Publication Type: Journal Article)

Chemical References

• Antineoplastic Agents
• Biomarkers, Tumor
• Cytokines
• Tight Junction Proteins

Topics

• Antineoplastic Agents (analysis, pharmacology)
• Biomarkers, Tumor (metabolism)
• Carcinoma, Hepatocellular (pathology)
• Cell Aggregation (drug effects)
• Cell Line, Tumor
• Cell Proliferation (drug effects)
• Cytokines (metabolism)
• Drug Resistance, Neoplasm (drug effects)
• Drug Screening Assays, Antitumor
• Epithelial Cells (drug effects, metabolism)
• Extracellular Matrix (drug effects, metabolism)
• Fluorescence
• High-Throughput Screening Assays
• Humans
• Imaging, Three-Dimensional
• Liver Neoplasms (pathology)
• Models, Biological
• Reproducibility of Results
• Spheroids, Cellular (drug effects, pathology)
• Tight Junction Proteins (metabolism)