Figure 1.
Characterization and distribution of H-CAFs in vivo and in vitro.
(A) The expression of α-SMA, FAP, FSP, vimentin, fibronectin and cytokeratin in the four types of purified fibroblasts cultured for 3–10 passages was determined by immunofluorescent staining. All four fibroblast types showed high expression of the mesenchymal markers FSP, vimentin and fibronectin. NLFs, H-CAFs, PTFs and NLFs displayed high expression of α-SMA, which suggests that these fibroblasts exist in an activated state under cell culture conditions. However, another activation hallmark FAP was highly expressed in H-CAFs and PTFs but not in NSFs and NLFs. (B) Western blotting showed differences in α-SMA and FAP expression among the four fibroblasts. Consistent with the results of the immunofluorescent staining, H-CAFs, PTFs and NLFs displayed high expression of α-SMA. FAP was highly expressed in H-CAFs and PTFs but not in NSFs and NLFs. (C) The distribution of H-CAFs identified by α-SMA (+) CD31 (−) expression in each specimen from malignant, peri-tumor and normal liver regions was detected through immunohistochemistry in serial pathological sections. α-SMA expression was detected to confirm the presence of H-CAFs. In addition, CD31 expression was evaluated to exclude the presence of vascular endothelial cells, which co-express α-SMA and CD31. H-CAFs were more abundant in tumor tissue, compared with peri-tumor and normal liver tissue.
Figure 2.
H-CAFs promoted the proliferation of HCC cells both in vivo and vitro.
(A and B) 97L cells (A) and Hep3B cells (B) were cultured in conditioned medium from different fibroblasts, and the proliferation of malignant cells was assayed by CCK-8 analysis. NLFs, PTFs and H-CAFs significantly increased HCC cell proliferation relative to NSFs and the control group. (C and D) A BALB/c nude mouse xenograft model based on the co-injection of HCC cells with or without two fibroblast types (NSFs or H-CAFs) was used to investigate the in vivo interaction between H-CAFs and HCC cells. Tumor volumes of tumor nodes generated by the co-injection of HCC cells and H-CAFs were consistently significantly larger than those formed by HCC cells without co-injection of H-CAFs. NSFs did not significantly increase tumor growth relative to the control. In addition, fibroblasts did not generate tumors when injected alone. (C) Gross tumor specimens at the end of the experiment are shown. Larger HCC tumors are formed when HCC cells are co-injected with H-CAFs (n = 5 per group) (D). (* P<0.05; ** P<0.01).
Figure 3.
H-CAFs promoted malignant cell proliferation and protected malignant cells from necrosis in tumor xenograft, as revealed by immunohistochemical tests and H&E staining.
(A) Ki-67 expression in HCC cells was substantially higher in tumor nodes formed by the co-injection of 97L cells and H-CAFs, which was in contrast with the results of tumors formed by the co-injection of 97L and NSFs or 97L cells alone. (B) Ki-67-positive cells were abundant near α-SMA (+) CD31 (−) fibroblasts within serial tumor sections. In samples that were double immunostained with α-SMA (red color, black row) and CD31 (brown color, red row), CD31 was not expressed in α-SMA-positive cells (Upper panel). When double immunostained with α-SMA (red color) and Ki-67 (brown color), a high incidence of tumor cells with Ki-67 expression (brown color, red row) was observed found in the vicinity of H-CAFs with α-SMA-positive staining (red color, black row) (Lower panel). (C) Necrosis was massively reduced in tumor nodes generated by the co-injection of fibroblasts (+NSFs or +H−CAFs), compared with tumors formed by 97L cell injection alone (control). (D) α-SMA expression was observed in all tumor xenografts, including the tumor nodes formed without fibroblast co-injection (control group).
Figure 4.
The proliferative enhancement of HCC cells by H-CAFs was partially mediated by HGF in a paracrine model.
(A) The level of SDF-1, HGF, TGF-β and EGF in the conditioned medium derived from H-CAFs was determined by ELISA. H-CAFs and PTFs secreted HGF at significantly higher levels than NLFs, whereas NSFs secreted almost no detectable HGF. However, NSFs secreted a higher level of SDF1 than the other three fibroblast types. TGF-β and EGF secretion were similar for the four fibroblast types. (B) Cell proliferation increased upon exposure to different concentrations of HGF, as demonstrated by CCK-8 tests. HGF promoted HCC cell proliferation at all concentration levels. (C) The increase in proliferation of HCC cells as mediated by H-CAF conditioned medium was significantly antagonized by HGF-neutralizing antibody. Moreover, anti-HGF did not interfere with the proliferation of 97L cells. (* P<0.05; ** P<0.01).
Figure 5.
H-CAFs are pathologically activated in tumors, whereas NSFs and PTFs remain inactivated.
(A) Microscope images of H-CAFs, PTFs and NSFs incubated for 24 hours. When originally isolated from the tumor specimens, PTFs and NLFs presented with a static, pericyte-like phenotype, whereas H-CAFs harbored an activated phenotype resembling myofibroblasts. (B) The expression of α-SMA, FAP, FSP and fibronectin in H-CAFs, PTFs and NSFs incubated for 24 hours was determined by immunofluorescence staining. α-SMA expression was remarkably higher in H-CAFs and relatively non-existent in the other fibroblast types when analyzed immediately following harvest from tumor specimens. This difference suggests the pathologically activated nature of H-CAFs and the relatively static nature of the other fibroblasts within the original tissues.
Figure 6.
The abundance of H-CAFs positively correlated with tumor volume in human HCC samples.
(A) The abundance of H-CAFs was dichotomized into two levels, high density and low density. (B) Tumor volumes of the high-density group (n = 27) were significantly higher than tumor volumes of the low-density group (n = 16, P = 0.006).