Wang Mei, Wu Chunping, Guo Yu, Cao Xiaojuan, Zheng Wenwei, Fan Guo-Kang
1 Department of Otolaryngology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
2 Department of Otolaryngology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Tumour Biol. 2017 May;39(5):1010428317705512. doi: 10.1177/1010428317705512.
Most primarily cultured laryngeal squamous cell carcinoma cells are difficult to propagate in vitro and have a low survival rate. However, in our previous work to establish a laryngeal squamous cell carcinoma cell line, we found that laryngeal cancer-associated fibroblasts appeared to strongly inhibit the apoptosis of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In this study, we investigated whether paired laryngeal cancer-associated fibroblasts alone can effectively support the growth of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In all, 29 laryngeal squamous cell carcinoma specimens were collected and primarily cultured. The laryngeal squamous cell carcinoma cells were separated from cancer-associated fibroblasts by differential trypsinization and continuously subcultured. Morphological changes of the cultured laryngeal squamous cell carcinoma cells were observed. Immunocytofluorescence was used to authenticate the identity of the cancer-associated fibroblasts and laryngeal squamous cell carcinoma cells. Flow cytometry was used to quantify the proportion of apoptotic cells. Western blot was used to detect the protein levels of caspase-3. Enzyme-linked immunosorbent assay was used to detect the levels of chemokine (C-X-C motif) ligand 12, chemokine (C-X-C motif) ligand 7, hepatocyte growth factor, and fibroblast growth factor 1 in the supernatants of the laryngeal squamous cell carcinoma and control cells. AMD3100 (a chemokine (C-X-C motif) receptor 4 antagonist) and an anti-chemokine (C-X-C motif) ligand 7 antibody were used to block the tumor-supporting capacity of cancer-associated fibroblasts. Significant apoptotic changes were detected in the morphology of laryngeal squamous cell carcinoma cells detached from cancer-associated fibroblasts. The percentage of apoptotic laryngeal squamous cell carcinoma cells and the protein levels of caspase-3 increased gradually in subsequent subcultures. In contrast, no significant differences in the proliferation capacity of laryngeal squamous cell carcinoma cells cocultured with cancer-associated fibroblasts were detected during subculturing. High level of chemokine (C-X-C motif) ligand 12 was detected in the culture supernatant of cancer-associated fibroblasts. The tumor-supporting effect of cancer-associated fibroblasts was significantly inhibited by AMD3100. Our findings demonstrate that the paired laryngeal cancer-associated fibroblasts alone are sufficient to support the primary growth of laryngeal squamous cell carcinoma cells in vitro and that the chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 axis is one of the major contributors.
大多数原代培养的喉鳞状细胞癌细胞在体外难以增殖，存活率较低。然而，在我们之前建立喉鳞状细胞癌细胞系的工作中，我们发现喉癌相关成纤维细胞似乎在体外能强烈抑制原代培养的喉鳞状细胞癌细胞的凋亡。在本研究中，我们调查了单独的配对喉癌相关成纤维细胞是否能在体外有效支持原代培养的喉鳞状细胞癌细胞的生长。总共收集了29个喉鳞状细胞癌标本并进行原代培养。通过差异胰蛋白酶消化将喉鳞状细胞癌细胞与癌相关成纤维细胞分离，并进行连续传代培养。观察培养的喉鳞状细胞癌细胞的形态变化。采用免疫细胞荧光法鉴定癌相关成纤维细胞和喉鳞状细胞癌细胞的身份。采用流式细胞术定量凋亡细胞的比例。采用蛋白质免疫印迹法检测半胱天冬酶 - 3的蛋白水平。采用酶联免疫吸附测定法检测喉鳞状细胞癌和对照细胞上清液中趋化因子（C - X - C基序）配体12、趋化因子（C - X - C基序）配体7、肝细胞生长因子和成纤维细胞生长因子1的水平。使用AMD3100（一种趋化因子（C - X - C基序）受体4拮抗剂）和抗趋化因子（C - X - C基序）配体7抗体来阻断癌相关成纤维细胞的肿瘤支持能力。在从癌相关成纤维细胞分离的喉鳞状细胞癌细胞形态中检测到明显的凋亡变化。在随后的传代培养中，凋亡的喉鳞状细胞癌细胞百分比和半胱天冬酶 - 3的蛋白水平逐渐增加。相比之下，在传代培养期间，与癌相关成纤维细胞共培养的喉鳞状细胞癌细胞的增殖能力未检测到显著差异。在癌相关成纤维细胞的培养上清液中检测到高水平的趋化因子（C - X - C基序）配体12。AMD3100显著抑制了癌相关成纤维细胞的肿瘤支持作用。我们的研究结果表明，单独的配对喉癌相关成纤维细胞足以在体外支持喉鳞状细胞癌细胞的原代生长，并且趋化因子（C - X - C基序）配体12/趋化因子（C - X - C基序）受体4轴是主要贡献者之一。
