Identification of AOC3 and LRRC17 as Colonic Fibroblast Activation Markers and Their Potential Roles in Colorectal Cancer Progression

Ahmad Zawawi, Sahira Syamimi; Mohd Azram, Nur Azlien Shahira; Sulong, Sarina; Zakaria, Andee Dzulkarnaen; Lee, Yeong Yeh; Che Jalil, Nur Asyilla; Musa, Marahaini

doi:10.31557/APJCP.2023.24.9.3099

Identification of AOC3 and LRRC17 as Colonic Fibroblast Activation Markers and Their Potential Roles in Colorectal Cancer Progression

Document Type : Research Articles

Authors

¹ Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia.

² Faculty of Applied Science, Universiti Teknologi MARA, Jengka 26400, Malaysia.

³ Department of Surgery, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia.

⁴ Department of Internal Medicine, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia.

⁵ Department of Pathology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia.

10.31557/APJCP.2023.24.9.3099

Abstract

Background: Accumulation of cancer-associated fibroblasts (CAFs) in the tumor stroma is linked to poor prognosis in colorectal cancer (CRC). CAF-cancer cell interplay, facilitated by secretomes including transforming growth factor-beta 1 (TGF-β1), supports fibroblast activation, drives colorectal carcinogenesis, and contributes to CRC aggressive phenotypes. Although widely used, traditional CAF biomarkers are found to have heterogeneous and non-specific expression. Amine oxidase copper containing 3 (AOC3) and leucine-rich repeat-containing 17 (LRRC17) have been reported to be emerging markers of myofibroblasts. Aim: Our objective was to investigate the potential of AOC3 and LRRC17 as biomarkers for fibroblast activation thus predicting their roles in CRC progression. Methods: Immunofluorescence (IF) staining of AOC3 and LRRC17 was performed on myofibroblast line (CCD-112CoN), primary fibroblasts from colorectal tumor (CAFs), and adjacent normal tissue (normal fibroblasts-NFs). SW620 (epithelial CRC cell line) was used as a control. Conventional CAF biomarker (alpha-smooth muscle actin - α-SMA) was included in the IF analysis. Fluorescence intensity was compared between groups using ImageJ software. Proliferation and contractility of treated cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and collagen gel contraction assays, respectively. Fibroblast contraction under TGF-β1 treatment was compared to those treated with complete medium (addition of 10% serum) and serum free (SF) medium. Results: Positive AOC3, LRRC17, and α-SMA expression were observed in colonic fibroblasts, more prominent in CAFs, whereas negative staining was found in SW620. Significant downregulation of AOC3, and upregulations in LRRC17 and α-SMA expression was found in TGF-β1-treated fibroblasts compared to SF medium treatment (p-value<0.05). All fibroblasts exhibited higher proliferation in complete medium and under treatment with conditioned medium from SW620 than SF medium. Significant contraction of NFs was recorded in complete medium and TGF-β1 (p-value<0.01). Conclusion: Our results demonstrate AOC3 and LRRC17 as the potential markers of CAF activation which promote CRC progression.

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