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. 2019 Feb;10(2):277-288.
doi: 10.1111/1759-7714.12947. Epub 2019 Jan 3.

Clinical significance and biological role of cancer-derived Type I collagen in lung and esophageal cancers

Shuo Fang  1   2 , Yongdong Dai  3 , Yan Mei  4 , Mingming Yang  5 , Liang Hu  6 , Hong Yang  7 , Xininyuan Guan  2   4 , Jiangchao Li  4   5
Affiliations

Clinical significance and biological role of cancer-derived Type I collagen in lung and esophageal cancers

Shuo Fang et al. Thorac Cancer. 2019 Feb.

Abstract

Background: Extracellular matrix (ECM) is remodeled during carcinogenesis. An abundant constituent of ECM is collagen. Type I collagen is secreted by fibroblasts, is important for tumor growth and epithelial-mesenchymal transition, and may also be secreted by cancer cells. However, the role and function of cancer-derived Type I collagen in the tumor microenvironment remains unclear.

Methods: We used immunohistochemistry and Western blot to detect Type I collagen expression in non-small cell lung cancer (NSCLC) and esophageal squamous cell carcinoma (ESCC) cell lines, respectively. We assessed the migration and adhesion capability of these cells in vivo by inhibiting Type I collagen in tumors. Relevant data were extracted from a large cohort study of The Cancer Genome Atlas to analyze messenger RNA levels. Protein expression of Type I collagen was further determined in tumor tissues of patients using tissue microarray.

Results: Cancer cell lines secreted Type I collagen. The molecular weight of cancer-derived Type I collagen was different from that secreted by cancer-associated fibroblasts and normal fibroblasts. Expression levels of COL1A1 and COL1A2 (subtypes of Type I collagen) messenger RNA in NSCLC and ESCC tumors were higher than in normal tissues, but were not associated with tumor node metastasis stages. Low expression of Type I collagen was significantly associated with poor overall survival and cancer cell differentiation.

Conclusion: NSCLC and ESCC cells could produce Type I collagen endogenously, revealing the potential functions of Type I collagen in cancer development. Cancer-derived Type I collagen was associated with overall survival and cancer cell differentiation.

Keywords: Type I collagen; esophagus cancer; extracellular matrix; non-small cell lung cancer; tumor microenvironment.

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Figures

Figure 1
Figure 1
Masson's staining revealed partial expression of Type I collagen in lung cancer nests. (a) Type I collagen expression was detected in non‐small cell lung cancer tissue using immunohistochemistry staining (brown) and was mainly located in the extracellular matrix (left), with little located in the tumor, as shown by high power microscope field (right, 400×, bar = 25 μm). (b) After Type I collagen was stained blue with Masson's staining, we observed that the bronchus was encircled by Type I collagen (arrows), indicating that Type I collagen was expressed in normal lung tissue. (c) We also observed several blue lines in the lung cancer nest. (d,e) Similar findings were observed in lung squamous and lung adenocarcinoma.
Figure 2
Figure 2
Type I collagen, but not Type III or IV, was identified in lung cancer nests. (a) To explore which type of collagen was expressed in the lung cancer nests, we conducted immunohistochemistry on the tumor tissue. Type I collagen expression was found in human lung tumor tissues. (b) We further assessed xenograft tissue sections from mice transplanted with lung cancer cells using antibodies recognizing Type I, III, and IV collagen. Under 200× magnification, Type I collagen was observed in the cancer nest, while the expression of Type III and IV was barely observed.
Figure 3
Figure 3
Lung cancer cell lines produce Type I collagen. (a) Western blotting results show that non‐small cell cancer cell lines could produce Type I collagen, which could be detected in the culture medium. Several cell lines expressed two types of Type I collagen, but their identities are unknown. The second protein band might represent a small degraded fraction derived from Type I collagen. (b) To confirm the specificity of the antibody, we examined extracts of mouse tail, which mainly comprise Type I collagen. The ladder band of Type I collagen displayed different molecular weights. The molecular weight of Type I collagen was approximately 128 kDa and other ladder bands were degradation products. (c) Primary cancer cell culture with immunohistochemistry (IHC) was used to analyze cancer cells and fibroblasts derived from tumor tissue. Type I collagen was expressed in both cancer cells and fibroblasts (400×). (d,e) Data were extracted from the protein atlas database to analyze the protein expression of Type I collagen in various cancer types using antibodies recognizing COL1A1 and COL1A2. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase. formula image H, formula image M, formula image L, formula image Not, formula image H, formula image M, formula image L, formula image Not.
Figure 4
Figure 4
Inhibition of cancer‐derived Type I collagen suppressed tumor clone formation while increasing the invasive ability of cancer cells. (a,b) Wound‐healing assays show that cell motility and invasion were markedly increased in an A549 cell line treated with a Type I collagen inhibitor. Representative images were taken at 0, 24, and 48 hours after scratching. (c, top) After Type I collagen was inhibited, a significant reduction in the number of tumor clones was observed on the culture dishes, indicating that the Type I collagen inhibitor significantly suppressed tumor clone formation. (c, bottom) Cancer cell invasion was also promoted when Type I collagen was inhibited. The results are presented as the mean ± standard deviation. (d) Column charts of the statistical results (P, *< 0.05, **< 0.01). formula image Control, formula image C inhibitor.
Figure 5
Figure 5
The expression of Type I collagen messenger RNA (mRNA) was higher in tumor tissue than in normal tissue. (a) Clinical data of 501 patients with esophageal squamous cell carcinoma (ESCC) and 535 patients with lung cancer (LUAD and LUSC) from The Cancer Genome Atlas were collected, and the mRNA expression level of Type I collagen (COL1A1 and COL1A2) in these patients was analyzed. The level of mRNA expression of Type I collagen was significantly higher in tumor tissue than in normal tissue (P < 0.0001). (b) mRNA expression of Type I collagen was not associated with cancer stage (P > 0.05).
Figure 6
Figure 6
Type I collagen is secreted by esophageal squamous cell carcinoma (ESSC) and has clinical significance. (a) Type I collagen was determined in ESCC cells lines using Western blotting. (b) Comparison between cancer‐derived and fibroblast‐derived Type I collagen revealed that they have different molecular weights. Cancer‐derived Type I collagen was represented by 72 kDa and 95 kDa bands, while the tumor fibroblast (TF) and normal fibroblast (NF)‐derived samples only show the 95 kDa band. They both expressed a 120 kDa band (data not shown). (c) A tissue microarray assay using immunohistochemistry shows that Type I collagen was significantly overexpressed in the tumor tissues of 258 patients with ESCC from Lin County (Henan province, China) compared to non‐tumor tissues. Kaplan–Meier analysis showed that low expression levels of Type I collagen were significantly associated with poor overall survival and cell differentiation. (d) Considering that stem cells are related to cell differentiation, we used a teratoma tumor model made from human embryonic stem cell lines in vivo to verify that Type I collagen was present in situ in cancer stem‐like cells, which maintain a differentiation state. All values were expressed as the average of three biological replicates. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; IgG, immunoglobulin.
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