Identification of a novel biomarker-CCL5 using antibody microarray for colorectal cancer

https://doi.org/10.1016/j.prp.2019.02.011Get rights and content

Abstract

Purpose

To screen novel candidate biomarkers in primary colorectal cancer (CRC), and indentify their clinical valuation in progress of colorectal cancer.

Methods

By using antibody microarray, 274 target proteins in tissue samples from primary colorectal cancer patients were detected. Among differently expressed proteins in CRC tissues, As promising candidate biomarker, RANTES/CCL5 was validated by enzyme-linked immunosorbentassay and immunohistochemistry (IHC), and the clinical significance of CCL5 was analyzed.

Results

Totally, 25 differentially expressed proteins were indentified between colorectal cancers and matched normal mucosa. CCL5 expression was significantly associated with adverse pathological progress, apt to lymph node metastasis and higher T stage.

Conclusions

CCL5 may contribute to promoting tumor growth, and CCL5 is a promising target that may help in understanding the pathogenesis of CRC.

Introduction

Colorectal cancer is the most common gastrointestinal cancer and the fourth leading cause of cancer death worldwide [1]. In China, the increasing colorectal cancer incidence especially among young adults, reflect an increased prevalence of risk factors such as smoking, drinking, unhealthy diet, etc. To the present, the efficacy of cytotoxic drugs and targeted therapies have been proved in the treatment of advanced colorectal cancer. However, the targeted agents, including anti-VEGF and anti-EGFR antibodies, benefit only certain patients who harbor relevant variation. Discovering new therapeutic targets for personalized treatment is still an urgent need in colorectal cancer research.

Currently, more and more studies indicate that chemokine plays a important role in tumor growth and metastasis. Different members of chemokine family eitherpromote or inhibit tumor growth by promoting or inhibiting the tumor angiogenesis. Besides leukocytes and vascular endothelial cells, the epithelial cells and colorectal cancer cells could also secrete chemokines to recruit effector cells or regulating cells with concentration dependent manners to the source of chemokines. Researches provide evidence that chemokines produced by the tumor microenvironment can induce tumor proliferation, infiltration, promote angiogenesis, and stimulate tumor cell metastasis CC chemokine motif ligand 5 (CCL5), also known as RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted), belongs to CC chemokine family whose members include monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, I-309, macrophage inhibitory protein-1a and macrophage inhibitory protein-1β [2]. The combination of CCL5 and its receptor C-C chemokine receptor type 5 (CCR5) is involved in malignant transformation, invasion and metastasis of tumors [3]. Expressions of CCL5 have been reported in breast cancer and cervical carcinoma specimens, and high plasma CCL5 level correlated with advanced progression [[4], [5], [6]]. The plasma CCL5 levels in patients with advanced ovarian cancer or advanced gastric cancer were apparently higher than those with early ovarian cancers or gastric cancers [7]. Using real time RT-PCR, Cambien et al. [8] found that the expression of CCL5 in colorectal cancer cells was higher than that in normal mucosa, systemic treatment with anti−CCL5 antibodies could delaying the progression of colon cancer. However, Szczepanik et al. [9] detected the level of CCL5 in the serum of colorectal cancer patients by Cytometric Bead Array and observed no correlation between CCL5 levels and the prognosis of colorectal cancer. Although many studies have focused on the roles CCL5 played in CRC growth and metastasis, its mechanism remains uncertain.

In this study, we detect the expression of cytokines and chenokinesin colorectal cancer using antibody microarray. Our result demonstrated that RANTES/CCL5 was higher in CRC tissue than matched normal colorectal mucosa. To validate the result of the antibody microarray expression analysis, we examined CCL5 levels in flesh colorectal cancer specimens by enzyme-linked immunosorbentassay (ELISA), and measured CCL5 expression in colorectal cancer paraffin specimens by IHC. Then the associations of CCL5 expression with clinical pathologic parameters were analyzed and the influence of CCL5 expression on colorectal cancer progression was evaluated.

Section snippets

Materials and methods

Paired tissue samples of CRC and paired normal colorectal mucosa were taken from 4 patients with primary CRC. The clinicopathological data for CRC patients are documented in Table 1. The tissue samples obtained intraoperatively were immediately frozen in liquid nitrogen and stored at −70 °C until protein extraction. The study was approned by the ethical committees of Qian-Fo-Shan Hospital Affiliated Shandong University.

The antibody microarray was used according to manufacturer's protocol with

Elisa

Paired samples of colorectal cancer and mucosa were obtained from 38 patients during surgery in Qian-Fo-Shan Hospital Affiliated Shandong University. Tumor samples were taken from vital areas of histopathologically confirmed adenocarcinomas. Mucosa samples were taken from unaffected mucosa, 5 cm distal to the tumor margin. The tissue was harvested immediately after resection of the colon and rectal, washed in ice cold phosphate-buffered saline (PBS). Tissue samples were minced and homogenized

Immunohistochemistry

Immunohistochemical studies were performed on tissue specimens from 60 patients who had been clinically diagnosed with primary CRC between 2014 and 2015 at Yantaishan Hospital. Tissues (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections) underwent heat mediated antigen retrieval in sodium citrate buffer (pH 6.0). Sections were reacted with Polyclonal mouse Anti-RANTES antibody (dilution 1:150; abcam) and incubated with sample at 4 °C overnight. A HRP-labeled polymer detection system was

Statistical analysis

All statistical analyses were performed using the IBM SPSS Statistics 20.The differences of target proteins levels detected by antibody microarray between groups were assessed by paired t-tests. CCL5 protein concentration determined by an ELISA assay were shown as MEAN ± SEM and analyzed by Wilcoxon matched-pair signed-rank (two samples). Spearman Rank Correlation Test was used to evaluate the association between CCL5 expression and clinicopathological characteristics.

Results

Among 274 human proteins detected in 4 CRC tissues, 25 differently expressed proteins were found in tissues of colorectal cancer compared to their matched normal colorectal mucosa, including 14 up-regulated and 11 down –regulated proteins (Fig. 1), based on the significant difference (P < 0.05) of light signal intensities between two samples. The up-regulated proteins were shown in red and down –regulated proteins in green (Fig. 1a). Bar graph shows the relative expression levels of target

Discussion

In 2013, Japanese scholars Hisaaki Miyoshi et al. detected the expression of 506 target proteins of colorectal cancer with biotin-labeled antibody microarray and found that, in CRC tissues, IL-1α, GRO, Glut5, MIG, ICAM-5, VE-cadherin, uPA and Leptin R were increased when comparing to normal colon tissues, MPIF-1/CCL23, FGF R5, MIP2, SAA and IL-18 Rβ were strongly up-regulated in rectal cancer when comparing to the levels in non-rectal cancer [10].

In this study, we used a novel antibody

Acknowledgements

Many thanks to Shengjie Dong pH.D for consultations on statistical analyses. The study was approned by the ethical committees of Qian-Fo-Shan Hospital Affiliated Shandong University. The study was supported by the National Natural Science Foundation of China (No. 81272420), the Scientific and Technological Development Projects in Shandong Province of China (No. 2011GSF11838), Shandong Province Natural Science Foundation (No. ZR2012HM085), and the Scientific and Technological Development

References (19)

  • H.K. Kim et al.

    Elevated levels of circulating platelet microparticles, VEGF, IL-6 and RANTES in patients with gastric cancer: possible role of a metastasis predictor

    Eur. J. Cancer

    (2003)
  • G. Soria et al.

    The inflammatory chemokines CCL2 and CCL5 in breast cancer

    Cancer Lett.

    (2008)
  • D.M. Parkin et al.

    Estimating the world cancer burden: Globocan 2000

    Int. J. Cancer

    (2001)
  • D. Lv et al.

    CCL5 as a potential immunotherapeutic target in triple-negative breast cancer

    Cell. Mol. Immunol.

    (2013)
  • D. Aldinucci et al.

    The inflammatory chemokine CCL5 and cancer progression

    Mediators Inflamm.

    (2014)
  • E. Azenshtein et al.

    The CC chemokine RANTES in breast carcinoma progression: regulation of expression and potential mechanisms of promalignant activity

    Cancer Res.

    (2002)
  • Y. Niwa et al.

    Correlation of tissue and plasma RANTES levels with disease course in patients with breast or cervical cancer

    Clin. Cancer Res.

    (2001)
  • S.A. Eissa et al.

    Importance of serum IL-18 and RANTES as markers for breast carcinoma progression

    J. Egypt. Cancer Inst.

    (2005)
  • B. Cambien et al.

    CCL5 neutralization restricts cancer growth and potentiates the targeting of PDGFRbeta in colorectal carcinoma

    PLoS One

    (2011)
There are more references available in the full text version of this article.

Cited by (12)

View all citing articles on Scopus
View full text
© 2019 Elsevier GmbH. All rights reserved.