TGF-β-independent CTGF induction regulates cell adhesion mediated drug resistance by increasing collagen I in HCC

Abstract

Hepatocellular carcinoma (HCC) is resistant to conventional chemotherapeutic agents and remains an unmet medical need. Here, we demonstrate a mechanism of cell adhesion-mediated drug resistance using a variety of HCC spheroid models to overcome environment-mediated drug resistance in HCC. We classified spheroids into two groups, tightly compacted and loosely compacted aggregates, based on investigation of dynamics of spheroid formation. Our results show that compactness of HCC spheroids correlated with fibroblast-like characteristics, collagen 1A1 (COL1A1) content, and capacity for chemoresistance. We also showed that ablation of COL1A1 attenuated not only the capacity for compact-spheroid formation, but also chemoresistance. Generally, connective tissue growth factor (CTGF) acts downstream of transforming growth factor (TGF)-β and promotes collagen I fiber deposition in the tumor microenvironment. Importantly, we found that TGF-β-independent CTGF is upregulated and regulates cell adhesion-mediated drug resistance by inducing COL1A1 in tightly compacted HCC spheroids. Furthermore, losartan, which inhibits collagen I synthesis, impaired the compactness of spheroids via disruption of cell-cell contacts and increased the efficacy of anticancer therapeutics in HCC cell line- and HCC patient-derived tumor spheroids. These results strongly suggest functional roles for CTGF-induced collagen I expression in formation of compact spheroids and in evading anticancer therapies in HCC, and suggest that losartan, administered in combination with conventional chemotherapy, might be an effective treatment for liver cancer.

Keywords: cell adhesion-mediated drug resistance (CAM-DR); collagen 1A1; connective tissue growth factor (CTGF); hepatocellular carcinoma; tumor spheroids.

Figure 1. HCC cell lines differ in spheroid-forming capacity

A. Spheroid was formed for 3 days using HCC cell lines. B. HCC cell lines were cultured in 96-well ultra-low attachment (ULA) plate for 96hr. Images of spheroids were obtained at indicated times. C. Kinetic of spheroids formation was measured in bright-field images using Operetta analysis software. The Values were normalized to 3hr of each cell lines. Data represent the mean values ± SD from three independent experiments. All bright-field images of spheroids were obtained using the Operetta® High Content Screening System and a 10× objective. *P<0.05, **P<0.005. Scale bar = 200μm.

Figure 2. Tightly compacted spheroids show strong resistance to sorafenib compared to loosely compacted aggregates

A. HCC spheroids were formed for 3 days and treated with 10 μM of sorafenib for another 4 days. B. Cell viability was measured with resazurin assay. After 5 hr of treatment with 50 μM resazurin, absorbance was measured using an Enspire plate reader. The values were normalized to control (0.01% DMSO). Data represent the mean values ± SD from three independent experiments. All bright-field images of spheroids were obtained at 7 days using the same method. *P<0.05, **P<0.005. Scale bar = 200μm.

Figure 3. HCC cell lines, which form tightly compacted spheroids, show fibroblast-like morphology and express high levels of ECM-related molecules

A. Morphology of HCC cell lines in monolayer culture. Bright-field images were obtained using the same method with a 100× objective. B, C. Expression of mRNA encoding ECM-related molecules in seven HCC cell lines was evaluated by RT-PCR (B) and real-time PCR (C). GAPDH served as a loading control, and values were normalized to GAPDH. Values were normalized to GAPDH. In real-time PCR, values of PLC/PRF/5 were normalized to 1, and other experiment groups were normalized to PLC/PRF/5. Data are shown as means ± SD from two independent experiments with duplicates. Scale bar = 50μm.

Figure 4. Collagen I expression and fibroblast-like morphology are correlated with compactness of spheroids generated by patient-derived primary HCCs

A. Spheroid formation and 2D morphology of patient-derived primary HCCs. Spheroids were formed for 3 days. Bright-field images were obtained using the same method with 10× objective for spheroids and 100× objective for monolayer cells. B. Expression of mRNA encoding ECM-related molecules in patient-derived primary HCCs was evaluated by RT-PCR. GAPDH served as a loading control. Data represent the mean values ± SD from two independent experiments. Scale bar = 200μm (spheroid), 50μm (monolayer).

Figure 5. Drug sensitivity is increased in COL1A1 knockdown HCC cell lines

A. Collagen 1A1 mRNA expression in control (siCont) and COL1A1 knockdown HCC cell lines (siCOL1A1). Expression of mRNA was evaluated by real-time PCR, and the values were normalized to GAPDH. The values of siCOL1A1 were normalized to siCont. B. After transfection of HCC cell lines with siCont and siCOL1A1, the capacity of spheroid formation was evaluated for 5 days. C, D. After 3 days of spheroid formation, Huh7 spheroids were treated with sorafenib (C) or cisplatin (D) for another 4 days. On the final day of treatment (at 7days from cell seeding), spheroids were stained with ethidium homodimer-1 (EthD-1) to evaluate the extent of cell death. After image acquisition, EthD-1 intensity was measured using the same method. The values were normalized to control (0μM) of each group. Data represent the mean values ± SD from two independent experiments. *p<0.05, **p<0.005. Scale bar = 200μm.

Figure 6. CTGF is overexpressed and regulates COL1A1 level in tightly compacted HCC spheroids

A. Lysates of HCC cell lines cultured under 2D and 3D conditions were analyzed by western blotting with anti-CTGF, anti–TGFΔ-1, and anti–Δ-actin (control) antibodies. B, C. mRNA expression in HCC cell lines cultured under 2D and 3D conditions was evaluated by RT-PCR (B) and real-time PCR (C). CTGF and TGFΔ-1 mRNA levels were examined, and GAPDH was served as a loading control. The values of Hep3B were normalized to 1, and other experiment groups were normalized to Hep3B. D. COL1A1 and CTGF mRNA levels in control siRNA transfected (siCont) and CTGF knockdown HCC cell lines (siCTGF) were examined by real-time PCR. In real-time PCR, all values were normalized to GAPDH, and the values of siCTGF were normalized to siCont. E. Spheroid formation capacity was observed in SNU475-siCont and SNU475-siCTGF for 6 days. Bright-field images were obtained using the same method. F. Drug sensitivity was measured by EthD-1 (dead cell) intensity induced by sorafenib and cisplatin at indicated concentration in Huh7-siCont and Huh7-siCTGF after 4 days from drug treatment. The values were normalized to control (0μM) of each group. Data represent the mean values ± SD from three independent experiments. *P<0.05, **p<0.005. Scale bar = 200μm.

Figure 7. Losartan inhibits COL1A1 synthesis and attenuates the compactness of spheroids without cytotoxicity

A. HCC spheroids were treated with 100 μM losartan for 3 days, and COL1A1 expression was evaluated by RT-PCR. GAPDH served as a loading control. B. Huh7 and Hep3B cells were treated with losartan at the indicated concentrations for 48hr. After treatment, nuclei were stained with Hoechst 33342 and counted. C. HCC spheroids were treated with losartan at the indicated concentrations for 3 days. Losartan-induced cell death was measured by staining with EthD-1. D. Huh7 and Hep3B spheroids were treated with 100 μM losartan for 3 days, and spheroid morphology was examined. E. Huh7 cells in monolayer culture were treated with 100 μM losartan for 72hr and nuclei were stained with Hoechst 33342. After image acquisition (left panel), nuclear volume was measured (right panel). Bright-field images were obtained using the same method with 10× objective for spheroids and 200× objective for monolayer cells. Data represent the mean values ± SD from three independent experiments relative to the value for control. Scale bar = 200μm.

Figure 8. Losartan attenuates resistance to anticancer drugs in HCC cell line- and patient-derived primary HCC spheroids

HCC spheroids were treated with 100 μM losartan for 3 days and then with sorafenib A. or cisplatin B. at the indicated concentrations for another 4 days. Then spheroids were stained with EthD-1 to detect cell death. HCCs in monolayer culture were treated with sorafenib C. or cisplatin D. at the indicated concentrations for 48 h, and nuclei were stained with Hoechst 33342 and counted. E. Patient-derived compacted spheroids (upper panel) and loosely compacted aggregates (lower panel) were treated with 100 μM losartan for 3 days and then with 10 μM sorafenib for another 4 days, and then death was measured by staining with EthD-1. All bright-field and fluorescence images were obtained using the same method. Data represent the mean values ± SD from three independent experiments relative to the value for control. *P<0.05. Scale bar = 200μm.