Increase in CD4+FOXP3+ regulatory T cell number and upregulation of the HGF/c-Met signaling pathway during the liver metastasis of colorectal cancer

Xiaoming Huang; Zexian Chen; Nanrong Zhang; Caiyan Zhu; Xutao Lin; Jiandong Yu; Zhiping Chen; Ping Lan; Yunle Wan

doi:10.3892/ol.2020.11785

Increase in CD4⁺FOXP3⁺ regulatory T cell number and upregulation of the HGF/c-Met signaling pathway during the liver metastasis of colorectal cancer

Oncol Lett. 2020 Sep;20(3):2113-2118. doi: 10.3892/ol.2020.11785. Epub 2020 Jun 26.

Authors

Xiaoming Huang^{1

2}, Zexian Chen^{2

3}, Nanrong Zhang⁴, Caiyan Zhu⁵, Xutao Lin⁶, Jiandong Yu¹, Zhiping Chen¹, Ping Lan^{2

3}, Yunle Wan¹

Affiliations

¹ Department of Hepatobiliary Surgery, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510655, P.R. China.
² Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Diseases, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510655, P.R. China.
³ Department of Colorectal Surgery, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510655, P.R. China.
⁴ Department of Anesthesiology, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510655, P.R. China.
⁵ Department of Pharmacy, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510655, P.R. China.
⁶ Department of Endoscopy Center, The Sixth Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510655, P.R. China.

Abstract

Colorectal cancer (CRC) is the third and second most common type of cancer diagnosed in males and females, respectively, and is the fourth leading cause of cancer-associated mortality worldwide. Liver metastasis is the primary cause of mortality in patients with CRC, and therefore requires therapeutic focus. Regulatory T cells (Tregs) and hepatic stellate cells (HSCs) are potentially involved in regulating the immune response during liver metastasis. The aim of the present study was to evaluate the influence of CD4⁺ forkhead box p3 (Foxp3)⁺ Tregs and the HGF/c-Met signaling pathway in the liver metastasis of CRC. A model of the latter was established using Balb/c mice via splenic injection of human CRC cells (CT-26 line). The mice were monitored for 3 weeks after being injected, and the spleens and livers were removed on day 22 for further analysis. Moreover, the single-cell suspensions were labeled with CD4 and Foxp3 antibodies, and were analyzed using flow cytometry. Expression levels of α-smooth muscle actin (SMA), hepatocyte growth factor (HGF) and hepatocyte growth factor receptor (c-Met) were analyzed using immunohistochemistry. Mice injected with CT-26 cells exhibited signs of illness and significant weight loss, compared with the control mice (P=0.013), and they also developed liver metastases, at an average of 20.5 tumors per mouse. Pathological evaluation using hematoxylin and eosin staining confirmed the tumors as liver metastases of CRC. The numbers of CD4⁺ T cells were significantly decreased in the spleen (P<0.001) and liver (P=0.003) of tumor-bearing mice, while the proportions of CD4⁺FOXP3⁺ Tregs increased significantly in the spleen (P<0.001) and liver (P=0.026) compared with that in the controls. Additionally, α-SMA, HGF and c-Met levels increased significantly during metastatic growth in the liver. In conclusion, CD4⁺FOXP3⁺ Treg levels increased and the HGF/c-Met pathway was upregulated during the liver metastasis of CRC in mice, indicating the presence of potential therapeutic targets.

Keywords: CD4+ T cells; CD4+FOXP3+ Treg; colorectal cancer; hepatic stellate cells; liver metastasis.