Panoply™ Human CTGF Over-expressing Stable Cell Line

Connective tissue growth factor (CTGF) is a matricellular protein associated with liver fibrosis. Here, researchers investigated the role of CTGF in hepatocellular carcinoma (HCC), given that HCC frequently develops from fibrotic liver. CTGF was highly expressed in 93 human HCC samples compared to non-tumor tissues, with expression localized primarily to tumor cells. Elevated CTGF expression correlated with malignant clinicopathological features of HCC. Hepatic stellate cells (HSCs) were present in both human and murine liver tumors, and the expression of α-SMA-a marker of HSC activation-positively correlated with CTGF expression. Forced expression of CTGF did not affect the growth of PLC/PRF/5 cells (an HCC cell line with low endogenous CTGF expression) in isolation; however, in the presence of LX-2 cells (an HSC cell line), CTGF promoted the growth of the HCC cells. Co-culture with LX-2 cells promoted the growth of HepG2 cells (an HCC cell line with high endogenous CTGF expression) compared to monoculture. Anti-CTGF antibodies blocked the growth-promoting effect of LX-2 cells on tumors in both in vitro cultures and xenograft models. Co-culture of LX-2 cells with HepG2 cells induced IL-6 production in LX-2 cells, subsequently leading to STAT-3 activation and HepG2 cell proliferation. Anti-CTGF antibodies reduced IL-6 production in LX-2 cells and inhibited STAT-3 activation in HepG2 cells. Collectively, these data indicate that tumor-derived CTGF is a key factor in the HCC microenvironment, capable of activating neighboring HSCs to transmit growth-promoting signals to HCC cells.

To investigate the effect of CTGF on hepatocellular carcinoma (HCC) cell proliferation, researchers generated a CTGF-overexpressing PLC/PRF/5 cell line; among various HCC cell lines (including Huh7, HLF, HepG2, PLC/PRF/5, and Hep3B), the PLC/PRF/5 line exhibited the lowest levels of endogenous CTGF expression and secretion. In vitro experiments showed no significant difference in growth between CTGF-overexpressing PLC/PRF/5 cells and either parental or empty-vector-transfected PLC/PRF/5 cells (Figure 1A). Following xenograft transplantation into NOG mice, tumors derived from CTGF-overexpressing cells showed no growth differences compared to those derived from empty-vector-transfected cells, consistent with the in vitro findings (Figure 1B). Subsequently, the researchers examined the effect of CTGF on HCC cell proliferation in the presence of hepatic stellate cells (HSCs). In a Transwell co-culture system, CTGF-overexpressing PLC/PRF/5 cells proliferated more rapidly than empty-vector-transfected PLC/PRF/5 cells when co-cultured with the human HSC cell line LX-2 (Figure 1C). In the xenograft model, when co-injected with LX-2 cells, tumors formed by CTGF-overexpressing PLC/PRF/5 cells were significantly larger than those formed by empty-vector-transfected PLC/PRF/5 cells (Figure 1D). Although α-SMA-positive cells and fibrous bands were observed in the xenograft tumors formed by co-injection with LX-2 cells (Figure 1E), the predominant cell population within the tumors remained the PLC/PRF/5 cells. These results indicate that CTGF promotes HCC cell growth in the presence of HSCs, both in vitro and in xenograft models.

Figure 1. Forced expression of CTGF does not affect the growth of PLC/PRF/5 cells alone but increases their growth in the presence of LX-2 cells. (Makino Y, et al., 2018)