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  • The following are the supplementary data related to


    The following are the supplementary data related to this article.
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    Introduction Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death worldwide [1]. The majority of patients with early-stage CRC can be cured by surgery [2]. Nevertheless, late-stage CRC patients suffer from a poor 5-year survival, especially, metastatic CRC patients, with a 5-year survival rate lower than 10% [3]. Angiogenesis is a comprehensive progression linking cells, soluble factors, and extracellular matrix (ECM) components [4], which is vital to tumorigenesis, tumor development and metastasis [5], [6]. By decreasing blood supply to inhibit tumor progression, anti-angiogenesis therapy has become a key therapeutic strategy in CRC treatment [7]. Chemokines are a superfamily of small-molecular weight proteins that exert diverse functions by binding to their receptors [8]. Chemokine receptors, consisting of seven-transmembrane domain, G protein-coupled receptors, are defined by their ability to signal on binding chemokines [9]. Chemokines and their receptors not only contribute to inducing immune responses [10], but also shape the tumor microenvironment either by promoting or suppressing tumorigenesis [6]. For example, CXCR1 is involved in the progression of gastric cancer [11], hemangioendothelioma [12], and non-small cell lung cancer [13]. Previously, we revealed that CXCL5 and CCR4 can promote CRC metastasis, and the Dorsomorphin of CCR4 and CXCL5 predicted poor prognosis in CRC patients [14], [15]. CCR6 belongs to a subclass of CCR chemokine receptors and functions via binding to CCL20, which regulates immune function by mediating memory T cell responsiveness and immune cell homing [9]. High expression of CCR6 promotes metastasis in colorectal cancer [16] and hepatocellular carcinoma [17] and predicts poor clinical outcomes, whereas the role of CCR6 in angiogenesis in CRC remains poorly understood. Herein, we investigated the relationship between CCR6 high expression and CRC patient prognosis. Moreover, we also studied the function of CCR6 on angiogenesis in vitro and in vivo and found that CCR6 promoted angiogenesis in CRC via activating AKT/NF-κB/VEGF pathway. These findings indicate that CCR6 may act as a potential therapeutic target in CRC anti-angiogenesis treatment.
    Materials and methods
    Discussion The dynamic interaction between tumor and host cells leads to the formation of the tumor microenvironment (TME), which elicits multiple effects on tumor biological processes, including growth, metastasis and angiogenesis [5]. Chemokines and their receptors can link the tumor to the TME and affect the progression of CRC [23]. The expression of several chemokines and chemokine receptors have been detected in CRC and the up-regulation of CCR6 has been preliminarily shown. CCL20, also known as liver activation-regulated chemokine, the sole ligand for CCR6, was also overexpressed in CRC [24]. We found that CCL20 was expressed in CRC cell lines and overexpressed in CRC tissues. We confirmed that CCL20 was expressed in culture media of CRC cell lines by ELISA. CCL20-CCR6 interaction promoted epithelial-to-mesenchymal (EMT) in colon cancer cells [25]. Bisweswar Nandi et al. [26] found that stromal CCR6 drove tumor growth in a murine transplantable colon cancer through recruitment of tumor-promoting macrophages. Ulrich Keilholz et al. [27] found that CCR6 staining was significantly stronger in tumor cells, and CCR6 staining in the primary tumor was independently associated with the presence of liver metastases. Therefore, the researchers concluded that CCR6 and its ligand may be involved in the metastatic spread to the liver. In immunohistology, we used IRS scores to evaluate the expression level of CCR6 in CRC tissues, and the cutoff value was generated by X-tile software. Most importantly, our study was focused on the role of CCR6 in angiogenesis in CRC and found that CCR6 promoted tumor angiogenesis via the AKT/NF-κB/VEGF pathway in colorectal cancer. Jinlin Liu et al. demonstrated that up-regulated CCR6 indicated poor prognosis for CRC patients and CCR6 expression should be recognized as an independent prognostic factor in CRC [16]. In our study, multivariate analysis suggested that CCR6 was not an independent prognostic risk factor in CRC. This may be because their sample size (191 samples) is larger than ours (78 samples).