Abstract
We fabricated a spheroid-forming unit (SFU) for efficient and economic production of cell spheroids. We optimized the protocol for generating large and homogenous liver cancer cell spheroids using Huh7 hepatocellular carcinoma (HCC) cells. The large Huh7 spheroids showed apoptotic and proliferative signals in the centre and at the surface, respectively. In particular, hypoxia-induced factor-1 alpha (HIF-1α) and ERK signal activation were detected in the cell spheroids. To diminish core necrosis and increase the oncogenic character, we co-cultured spheroids with 2% human umbilical vein endothelial cells (HUVECs). HUVECs promoted proliferation and gene expression of HCC-related genes and cancer stem cell markers in the Huh7 spheroidsby activating cytokine signalling, mimicking gene expression in liver cancer. HUVECs induced angiogenesis and vessel maturation in Huh7 spheroids in vivo by activating epithelial–mesenchymal transition and angiogenic pathways. The large Huh7 cell spheroids containing HUVECs survived at higher concentrations of anti-cancer drugs (doxorubicin and sorafenib) than did monolayer cells. Our large cell spheroid provides a useful in vitro HCC model to enable intuitive observation for anti-cancer drug testing.
Highlights
Hepatocellular carcinoma (HCC) occurs worldwide, with the highest incidence in Asian countries[7]
Using an ultra-low attachment plate with the same initial number of cells as that used in the spheroid-forming unit (SFU) protocol, the cells did not aggregate and were dispersed, in contrast the spheroid cultured with lower cell numbers (2 × 104 cells according to the manufacturer’s instructions) showed healthy and well-formed cell spheroid (Supplementary Fig. S1c)
Various types of 3D culture systems have been well established, using different devices or materials used for generating the 3D architecture[12, 13]
Summary
Hepatocellular carcinoma (HCC) occurs worldwide, with the highest incidence in Asian countries[7]. The mechanisms underlying tumourigenicity in HCC remain unknown. Current investigations on HCC focus on the development of suitable model systems that can be used to increase our understanding of the disease mechanisms and to develop therapeutic tools[10]. Huh[7] is a well-established carcinoma cell line derived from differentiated hepatocytes[11]. We developed and optimized a tool, which we termed ‘spheroid-forming unit’ (SFU), for generating large-size multicellular cell spheroids, using Huh[7] cells and human umbilical vein endothelial cells (HUVECs). We aimed to produce a large-size cell spheroid mimicking the human liver cancer and provide in vitro HCC model for anti-cancer drug test
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