Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian 16150, Malaysia.
Faculty of Applied Science, Universiti Teknologi MARA, Jengka 26400, Malaysia.
Asian Pac J Cancer Prev. 2023 Sep 1;24(9):3099-3107. doi: 10.31557/APJCP.2023.24.9.3099.
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.
Our objective was to investigate the potential of AOC3 and LRRC17 as biomarkers for fibroblast activation thus predicting their roles in CRC progression.
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.
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).
Our results demonstrate AOC3 and LRRC17 as the potential markers of CAF activation which promote CRC progression.
癌症相关成纤维细胞（CAFs）在肿瘤基质中的积累与结直肠癌（CRC）的预后不良有关。包括转化生长因子-β1（TGF-β1）在内的分泌组促进了 CAF-癌细胞相互作用，支持成纤维细胞激活，驱动结直肠癌变，并导致 CRC 侵袭性表型。尽管传统的 CAF 生物标志物被广泛使用，但发现它们的表达具有异质性和非特异性。含铜胺氧化酶 3（AOC3）和富含亮氨酸重复序列 17（LRRC17）已被报道为肌成纤维细胞的新兴标志物。
我们的目的是研究 AOC3 和 LRRC17 作为成纤维细胞激活标志物的潜力，从而预测它们在 CRC 进展中的作用。
对肌成纤维细胞系（CCD-112CoN）、结直肠肿瘤（CAFs）和相邻正常组织（正常成纤维细胞-NFs）中的 AOC3 和 LRRC17 进行免疫荧光（IF）染色。SW620（上皮 CRC 细胞系）用作对照。IF 分析中包括传统的 CAF 生物标志物（α-平滑肌肌动蛋白-α-SMA）。使用 ImageJ 软件比较组间荧光强度。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基-2H-四唑溴盐（MTT）和胶原凝胶收缩测定分别评估处理细胞的增殖和收缩性。比较 TGF-β1 处理下的成纤维细胞收缩与 SF 培养基处理（添加 10%血清）和无血清（SF）培养基处理下的成纤维细胞收缩。
在结肠成纤维细胞中观察到 AOC3、LRRC17 和 α-SMA 的阳性表达，在 CAFs 中更为明显，而在 SW620 中则为阴性染色。与 SF 培养基处理相比，TGF-β1 处理的成纤维细胞中 AOC3 的表达显著下调，LRRC17 和 α-SMA 的表达上调（p 值<0.05）。所有成纤维细胞在完全培养基中的增殖和条件培养基处理的 SW620 中的增殖均高于 SF 培养基。在完全培养基和 TGF-β1 处理下，NFs 的收缩显著（p 值<0.01）。
我们的研究结果表明 AOC3 和 LRRC17 是 CAF 激活的潜在标志物，可促进 CRC 的进展。
