Peritumoral activated hepatic stellate cells are associated with hepatic recurrence for resectable colorectal adenocarcinoma liver metastasis following resection

Abstract

The formation of the pre-metastatic niche (PMN), which precedes the establishment of tumor lesions, plays a critical role in cancer recurrence and metastasis. Hepatic stellate cells (HSCs), a critical liver stromal cell component, can be induced to facilitate metastasis by modeling liver PMN formation. In the present study, activated HSCs were observed in the peritumor non-cancerous liver tissues (PNLT) colorectal adenocarcinoma liver metastasis (CRALM), and the density of activated HSCs was higher in PNLT compared with that in normal liver tissues (NLT). High density of activated HSC in the PNLT was positively associated with the number of tumor liver metastases (P=0.036), maximum diameter of liver metastases (P=0.002), and recurrence following synchronous radical resection (P=0.003). High density of activated HSCs in the PNLT was identified as a significant and independent prognostic factor for disease-free survival (HR, 2.083; 95% CI, 1.504-2.885; P=0.016) and overall survival (HR, 2.039; 95% CI, 1.312-3.169; P=0.019). Functionally, in vitro assays revealed that activated HSCs facilitated colorectal adenocarcinoma (CRA) cells to colonize the liver. Molecularly, it was demonstrated that the pro-recurrence of activated HSCs depended on paracrine hepatic growth factor. Taken together, the present results showed that high density of activated HSCs in the PNLT was an independent predictor for CRALM recurrence following resection, and they exerted their roles via their effect on CRA cell recruitment and proliferation by paracrine HGF.

Keywords: colorectal cancer; hepatic recurrence; hepatic stellate cells; liver metastasis; pre-metastatic niche.

Figure 1.

A flowchart revealing the clinical experimental design.

Figure 2.

Density of activated HSCs is significantly higher in PNLT with colorectal adenocarcinoma liver metastasis. (A) Immunohistochemistry was used to determine the number of activated HSCs with α-SMA antibody. The typical images of PNLT and NLT are shown. (B) The density of activated HSCs was significantly higher in PNLT compared with that in NLT. The scatter graph was used to show the differential densities and a Student's t-test was used to analyze the difference between percentage of activated HSCs in PNLTs and NLTs. ***P<0.001. PNLT, peritumor non-cancerous liver tissue; NLT, normal liver tissue; HSC, hepatic stellate cells.

Figure 3.

High-density of activated HSCs is associated with poor prognosis. (A) ROC curve. The sensitivity and 1-specificity of the density of activated HSCs in colorectal adenocarcinoma tumor tissues was plotted (black line). The AUC and the P-value was calculated using ROC curve analysis: AUC, 0.712 and P=0.001. The highest Youden index used to determine the cut-off value (10%) for density, <10% was used for low-density of activated HSCs and >10% for high-density. Then the sensitivity and 1-specificity of low-density of activated HSCs in PNLT were plotted (red line). The AUC and the P-value was calculated using ROC curve analysis: AUC, 0.660 and P=0.012. The orange line works as the reference line. Kaplan-Meier analysis of the association between high- and low-density of activated HSCs and (B) disease-free survival and (C) overall survival. Compared with those with low-density of activated HSCs (<10%) in PNLT, patients with high-density of activated HSCs in the PNLT exhibited worse DFS and poor OS. ROC, receiver operating characteristic; AUC, area under the curve; HSCs, hepatic stellate cells.

Figure 4.

Activated HSCs promote CRA cell recruitment and proliferation in vitro. (A) HSCs were activated by CRA cells. A Transwell co-cultured module (left panel) was used to demonstrate the effect of CRA cells on HSCs activation. The results revealed that HSCs co-cultured with Lovo cells for 7 days, were activated with characterization of elevated α-SMA expression. However, HSCs co-cultured with FHC cells for 7 days, were not activated as their α-SMA expression was not elevated. (B) Activated HSCs promote CRA cell recruitment. An in vitro chemotaxis Boyden chamber module assay (left panel) was used to determine the effect of activated HSCs on CRA cell recruitment. The results showed that compared with the blank control and inactivated HSCs, a higher number of CRA cells migrated into the lower wells when co-cultured with activated HSCs. (C) Activated HSCs promoted CRA cell viability using a MTT assay. Compared with that in the blank control and inactivated HSCs, the supernatant of activated HSCs exhibited a strong effect on supporting cancer cell viability. (D) EdU assays showed activated HSCs promoted CRA cell proliferation. Compared with that in the blank control and inactivated HSCs, the supernatant of activated HSCs exhibited a strong effect on supporting cancer cell proliferation. **P<0.01. HSCs, hepatic stellate cells; CRA, colorectal adenocarcinoma; α-SMA, α-smooth muscle actin; MTT, methyl thiazolyl tetrazolium; EdU, 5-ethynyl-2′-deoxyuridine.

Figure 5.

Activated HSCs induces disseminated CRA cell recruitment and growth in vivo. (A) Representative liver image from mice injected with activated or inactivated HSCs, followed by Lovo cells or with injection of Lovo cells alone. Compared with mice injected with inactivated HSCs, followed by Lovo cells and mice injected with Lovo cells alone, the liver of mice injected with activated HSCs, followed by Lovo cells had a higher number of metastatic foci (yellow arrows). The data was quantified (lower panel) and ANOVA was used to analyze the data statistically. **P<0.01. (B) Masson's trichrome staining was used to determine the desmoplastic stromal reaction in metastatic foci. The tumors from the mice injected with activated HSCs, followed by Lovo cells exhibited a desmoplastic stromal reaction, which were mostly comprised of fibrils and collagen. (C) Immunohistochemistry was used to show the cellular components of the desmoplastic stroma. Results revealed that the cellular components of the desmoplastic stroma were primarily composed of activated HSCs, characterized by α-SMA expression. HSCs, hepatic stellate cells; CRA, colorectal adenocarcinoma; α-SMA, α-smooth muscle actin.

Figure 6.

HGF secreted by activated HSCs induces colorectal adenocarcinoma cell recruitment and growth in the liver. (A) The HGF concentration of inactivated or activated HSC supernatant was analyzed using ELISA. HGF concentration was significantly upregulated in the HSC supernatant, followed by activation with Lovo cells. (B) In vitro chemotaxis Boyden chamber model assays (left panel) showed the HGF antibody counteracted the effect of activated HSCs on Lovo cell recruitment. (C) MTT assays were used to determine the effect on viability. The results revealed that the supernatant of activated HSCs promoted the viability of Lovo cells; however, this effect was reversed with the addition of the HGF antibody. **P<0.01. HGF, hepatic growth factor.