J Liver Cancer.  2023 Sep;23(2):341-349. 10.17998/jlc.2023.04.30.

Isolation and characterization of cancer-associated fibroblasts in the tumor microenvironment of hepatocellular carcinoma

• Mun K
• Han J
• Roh P
• Park J
• Kim G
• Hur W
• Jang J
• Choi J
• Yoon S
• You Y
• Choi H
• Sung P

• KMID: 2546414
• DOI: http://doi.org/10.17998/jlc.2023.04.30

Abstract

Background
/Aim: Cancer-associated fibroblasts (CAFs) play an immunosuppressive role in the tumor microenvironment (TME) of human cancers; however, their characteristics and role in hepatocellular carcinoma (HCC) remain to be elucidated.
Methods
Nine tumor and surrounding liver tissue samples from patients with HCC who underwent surgery were used to isolate patient-derived CAFs. Cell morphology was observed using an optical microscope after culture, and cell phenotypes were evaluated using flow cytometry and immunoblotting. Cytokines secreted by CAFs into culture medium were quantified using a multiplex cytokine assay.
Results
CAFs were abundant in the TME of HCC and were adjacent to immune cells. After culture, the CAFs and non-tumor fibroblasts exhibited spindle shapes. We observed a robust expression of alpha-smooth muscle actin and fibroblast activation protein in CAFs, whereas alpha-fetoprotein, epithelial cell adhesion molecule, platelet/endothelial cell adhesion molecule-1, and E-cadherin were not expressed in CAFs. Furthermore, CAFs showed high secretion of various cytokines, namely C-X-C motif chemokine ligand 12, interleukin (IL)-6, IL-8, and C-C motif chemokine ligand 2.
Conclusions
CAFs are abundant in the TME of HCC and play a crucial role in tumor progression. These fibroblasts secrete cytokines that promote tumor growth and metastasis.

Keyword

Carcinoma, hepatocellular; Cancer-associated fibroblasts; Tumor microenvironment; Cytokines

Figure

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  Figure 1. Morphology of CAFs isolated from tumor tissues of patients with HCC and inhibition of immune cells infiltrating the tumor. (A) Schematic depicting the isolation procedure of CAFs from tumor tissues of patients with HCC. (B) Phase contrast microscope images of isolated CAFs at 1, 3, and 5 days after culture. Images were captured at 20× magnification. (C) Immunohistochemical staining of tumor tissues of patients with HCC using glypican3, Ki-67, vimentin, CD38, CD68, CD15, CD3, CD20, and PD-L1 antibodies as well as H&E and Masson's trichrome stains. Vimentin and Masson's trichrome staining were positive in CAFs in the peri-tumor and some clusters within the intra-tumor tissues (black arrowheads). Immune cells positive for CD38+, CD68+, CD15+, CD3+, CD20+, and PD-L1+ were located in close proximity to fibroblast clusters (red arrowheads). (D) Quantification of vimentin+ cells correlated with CD68+ or CD3+ cells. All means were statistically analyzed. CAF, cancer-associated fibroblast; HCC, hepatocellular carcinoma; H&E, hematoxylin and eosin; NT, non-tumor; T, tumor.

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  Figure 2. CAFs robustly express FAP and α-SMA, but do not express EpCAM, E-cadherin, and PECAM-1. (A) Single-stain flow cytometry was performed for SMA, FAP, AFP, and EpCAM in CAFs and non-tumor fibroblasts isolated from patients with HCC. (B) Similarly, single-stain flow cytometry was performed for the same markers in HCC cells. Graphs show the expression of each marker. (C) Expression of α-SMA, vimentin, and E-cadherin in CAFs, non-tumor fibroblasts, and hepatoma cell lines was analyzed using immunoblotting. (D) Expression of E-cadherin, PECAM-1, vimentin, and GAPDH in CAFs, non-tumor fibroblasts, and HUVECs was analyzed using immunoblotting. All means were statistically analyzed; *P<0.05, **P<0.01, ***P<0.001. CAF, cancer-associated fibroblast; FAP, fibroblast activation protein-α; SMA, alpha-smooth muscle actin; EpCAM, epithelial cell adhesion molecule; MFI, mean fluorescence intensity; NF, non-tumor fibroblast; PECAM-1, platelet and endothelial cell adhesion molecule 1; HCC, hepatocellular carcinoma.

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  Figure 3. CAFs secrete various cytokines. CAF-cultured medium was analyzed using multiplex analysis to detect the level of cytokines secreted by CAFs. The graph shows the level of each cytokine. Statistical analysis was performed; *P<0.01, **P<0.001. CAF, cancer-associated fibroblast; CCL, C-C motif chemokine ligand; CXCL, C-X-C motif ligand; IL, interleukin; TNF-α, tumor necrosis factor-alpha.

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Reference

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