MCO Molecular and Clinical Oncology 2049-9450 2049-9469 D.A. Spandidos 10.3892/mco.2015.665 MCO-0-0-665 Articles CXCL10/CXCR3 overexpression as a biomarker of poor prognosis in patients with stage II colorectal cancer BAIMING 1 * CHENXIA 2 * BAYI 1 Department of Gastrointestinal Medical Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, P.R. China Department of Pediatric Hematology Center, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300041, P.R. China Correspondence to: Dr Yi Ba, Department of Gastrointestinal Medical Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, 4th floor, Building A, Huan Hu Xi Road, Hexi, Tianjin 300060, P.R. China, E-mail: dryiba@gmail.com

Contributed equally

 01 2016 30 10 2015 4 1 23 30 19022015 28072015 Copyright: © Bai et al. 2015 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

The CXCL10/CXCR3 axis of inflammatory mediators is one of the most important groups of chemokine axes, which has been proven to be a lymphocyte-associated metastasis mediator in several tumors. The term inflammatory adhesions refers to tumors found to be attached to the surrouding tissues during surgery, although no cancer cell infiltration is later identified on pathological examination. The aim of the present study was to investigate the clinical characteristics of stage II colorectal cancer (CRC) and determine the correlation between the CXCL10/CXCR3 axis, inflammatory adhesions and prognosis. Clinicohistopathological data were collected from 401 CRC patients who had undergone R0 resection. Statistical analysis was performed with SPSS 17.0 software. Immunohistochemistry (IHC) was applied to measure the expression of CXCL10 and CXCR3 in 71 recurrent CRC patients, 72 non-recurrent CRC patients and 10 samples from normal peritumoral tissues, all retrieved from the Tianjin Medical University Cancer Institute and Hospital, Tianjin, China. Inflammatory adhesions, tumor location and size and the number of high-risk factors for reccurrence were more significantly associated with overall survival (OS) rather than disease-free survival in all the patients as determined by the log-rank and Cox's regression hazard analysis. Further analysis demonstrated that only the presence of inflammatory adhesions (P=0.025) was associated with the OS of recurrent patients. Patients with recurrence exhibited higher CXCR3 (P<0.001) and CXCL10 (P<0.001) expression compared with non-recurrent patients, as determined by IHC. The correlation between clinicopathological variables, CXCL10/CXCR3 expression and survival was also analyzed: Inflammatory adhesions and general tumor type (ulcerated vs. elevated) exhibited a significant correlation with CXCR3; however, the expression of CXCL10 was not significantly correlated with tumor location, histological type, size, gender, or preoperative carcinoembryonic antigen and hemoglobin levels. Furthermore, patients exhibiting a high expression of CXCR3 presented with a higher risk of relapse; among those, patients with inflammatory adhesions always exhibited worse survival. However, no such association was identified for CXCL10 expression. In conclusion, CXCR3 expression may be used as a prognostic marker and may contribute to the prediction of clinical outcome in stage II CRC patients.

 inflammatory adhesion CXCL10 CXCR3 colorectal cancer relapse Introduction

Colorectal cancer (CRC) is a common malignancy of the gastrointestinal tract, which is fourth in incidence and second in terms of cancer-related mortality in America (1). Several factors and genes are associated with the process of tumor angiogenesis, invasion, growth and metastasis in CRC. A number of stage III/IV patients succumb to metastasis and stage II patients to recurrence, particularly in the liver, lungs and lymph nodes (2). Despite the advances in identifying high-risk factors for recurrence in stage II CRC patients, the benefit from chemotherapy administration remains uncertain (3–5).

Over the last few decades, microsatellite instability (MSI) has been identified as a biomarker in previous clinical trials (4,5), which demonstrated that patients with MSI treated with 5-fluorouracil exhibited a significant survival benefit compared with the non-MSI and the surgery alone groups. Numerous tumor cell-derived factors and microenvironment molecules, such as chemokines, are involved in cancer cell metastasis and migration (6,7). Chemokines (8–10 kDa) are chemotactic cytokines that cause directed migration of numerous cells, including leukocytes, and are induced by inflammatory cytokines, growth factors and pathogenic stimuli. The chemokine-receptor axis allows cells to move towards high local concentrations of chemokines during inflammation, as well as the homeostatic transport of lymphocytes and dentritic cells. To date, >50 chemokines and 20 seven-transmembrane-domain receptors, which belong to G-protein coupled families, have been identified. One receptor may generally bind to more than one cytokines. A number of human cancers characterized by leukocyte infiltration possess a complex chemokine network that affects tumor cell growth, survival, infiltration, migration and angiogenesis.

Latest research demonstrated that the expression of the majority of chemokines and their receptors, such as CXCL10/CXCR3, CXCL12/CXCR4, CCL21/CCR7 and CCL25/CCR9, is associated with CRC. Dwinell et al (8) reported that CXCR3 is not present in normal colonic epithelial cells, but in mononuclear cells in the lamina propria. Kawada et al (9) observed that CXCL10 enhances CRC cell survival and gelatinase expression in culture and upregulates cell surface expression of CXCR3. Furthermore, CXCL10 has been found to be overexpressed in several cases of CRC as a Ras target gene (10), although Jiang et al (11) reported opposite findings. It appears that chemokines exert their tumor-associated activies by inducing immune-stimulating and angiostatic effects and constituting the tumor microenviroment. However, the precise role of CXCL10/CXCR3 in solid cancers remains poorly understood.

The aim of the present study was to investigate CXCL10 and CXCR3 expression in stage II CRC, in order to determine its clinicopathological significance and role in disease recurrence and optimise postoperative treatment in patients with stage II CRC.

 Patients and methods <sec> <title>Patients and materials

A series of 401 stage II CRC patients who underwent radical resection at Tianjin Medical University Cancer Institute and Hospital between 2005 and 2009 were included in this study. None of the patients had received preoperative neoadjuvant chemotherapy or radiotherapy. The patients were divided into two groups, the recurrence group (RG) and the non-recurrence group (NRG). We collected paraffin-embedded samples from 71 recurrent cases, 12 non-recurrent cases and 10 normal tissue samples. All the samples were independently reviewed by two pathologists and the histological diagnoses were classified according to the 2010 World Health Organization Classification of Digestive System Tumors (12). The recurrence risk factors of stage II CRC according to the guidelines of the National Comprehensive Cancer Network included poor differentiation, lymph node or blood vessel infiltration, intestinal obstruction, <12 lymph nodes retrieved, perineural invasion, partial perforation and positive resection margin. The term inflammatory adhesions refers to tumors found to be attached to the surrouding tissues during surgery, although no cancer cell infiltration is later identified on pathological examination.

 Immunohistochemical analysis

Tumor samples were collected from the Tianjin Medical University Cancer Institute and Hospital (Tianjin, China), fixed in formalin, embedded in paraffin and sectioned at 4 µm. A polyclonal rabbit anti-human CXCL10 antibody (cat. no. (C-20) sc-6226; dilution, 1:120; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and polyclonal rabbit anti-human CXCR3 antibody (cat. no. sc-101500; dilution, 1:200) were separately added to the sections following deparaffinization, hydration, antigen repair and endogenous peroxidase blocking. Immunoperoxidase staining was performed with the two-step EnVision™ method (DakoCytomation, Glostrup, Denmark) according to the manufacturer's instructions and visualized with 3,3′-diaminobenzidine (Sigma, St. Louis, MO, USA). The phosphate-buffered saline buffer was used to prepare negative control samples. Cell membrane and cytoplasmic staining were measured for these antibodies. Two pathologists independently counted the positive cells. The 4-tiered scoring system (−/+/++/+++), which took into account the percentage of positive cells and staining intensity, was used in our evaluation. The expression level of a certain target was determined according to the respective median values of a tumor indicator. Lower than the median was defined as ‘low expression’ and higher as ‘strong expression’.

 Follow-up

Follow-up data were collected through telephone communication and from the database of the Medical Records Department of our hospital. The time interval from the operative date to clinical relapse was defined as the disease-free survival (DFS), and to death or last follow-up as overall survival (OS).

 Statistical analysis

All the data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). The t-test and analysis of variance were used for numerical variables and the χ2 test for qualitative variables. For survival analysis, survival curves were generated by the Kaplan-Meier method. The univariate survival analysis was performed using the log-rank test and the multivariate Cox proportional hazards model was used to identify the independent prognostic factors. P<0.05 was considered to indicate statistically significant differences.

 Results <sec> <title>Clinicopathological characteristics

A total of 229 male and 172 female patients, aged 7–87 years (mean age, 60.5 years), were initially included in the study. Of these, 54.86% (220/401) had colon cancer, 45.89% (181/401) had rectal cancer. Patients with colon cancer exhibited a survival advantage compared with those with rectal cancer (P=0.026). The general classification was ulcerated type in 288/401, elevated type in 111/401, colloid type in 1/401 and infiltrative type in 1/401. The pathological results of all kinds of carcinomas were adenoma, histologically classified as tubular (n=198), papillary (n=15), mucinous (n=25) and mixed (n=54). Of the 401 cases, 22 were classified as well-differentiated, 335 as moderately differentiated and 44 as poorly differentiated. A maximum diameter of >10 cm was observed in 4.99% (20/401) of the cases, with a significantly different DFS compared with cases with smaller tumors (P<0.051). In 91 cases, inflammatory adhesions to the surrounding tissues were identified, which statistically significantly affected OS (P=0.024), but not DFS (P=0.214). A backward Cox's proportional hazards regression analysis of the clinicopathological parameters yielded a hazard ratio of 1.823 (95% confidence interval: 1.093–3.039; P=0.013) for adhesion-positive compared with adhesion-negative patients. Patients with more risk factors exhibited shorter DFS and OS (P<0.05). The multivariate Cox analysis indicated that location, tumor size and the number of high-risk factors were independent variables (P=0.013, 0.033 and 0.036), as was the presence of inflammatory adhesions, and were all significant prognostic factors for poor OS (Table IA and B). Colon cancer patients with larger tumors who had >1 high-risk factor and inflammatory adhesions were more likely to succumb to the disease.

From the follow-up data, we identified 106 patients with relapse or metastasis, with a median DFS of 21.8 months and a median OS of 39.6 months. The follow-up time ranged from 2 to 36 months. Of the 401 patients, 286 survived without evidence of cancer, 39/401 remained alive with recurrent lesions, 67/401 succumbed to CRC and 9/401 succumbed due to other causes.

 Inflammatory adhesions

In the RG, only the presence of inflammatory adhesions was associated with OS (P=0.025) (Table II). However, there was no such association with the remaining characteristics. A total of 28 (28/106) of the relapsed patients exhibited inflammatory adhesions surrounding the tumor and the majority survived for <3 years [67.86 (19/28)]. The median survival time of relapsed patients with adhesions was 26.63 months, while in the NRG it was 61.67 months. It is considered that adhesion had been reported previously to OS. It was concluded that the presence of inflammatory adhesions in a proportion of the patients was not associated with recurrence, but was closely associated with OS; however, for patients who presented with adhesions as well as recurrence, the prognosis was worse.

 Immunohistochemistry (IHC) in CRC

Immunostaining with anti-CXCL10 or -CXCR3 was considered to be antibody-specific by using the immunising peptide for each antibody as the target. In order to elucidate the association between clinical characteristics and the CXCL10/CXCR3 axis, we examined 143 CRC specimens and 10 peritumoral tissues at a distance of ≥5 cm from the resection margin (normal intestinal epithelia). CXCL10 and CXCR3 exhibited distinct characteristics in each group (Fig. 1).

As determined by IHC, CXCL10 was poorly expressed in 36 out of 72 cases (50.0%) in NRG and 19 out of 71 cases (26.76%) in RG, as well as strongly expressed in 10 out of 72 cases (13.89%) in the NRG and 27 out of 71 cases (38.03%) in the RG. We also found that 48 samples (66.67%) exhibited low CXCR3 expression in the NRG and 21 samples (29.58%) in the RG, whereas 12 cases (16.90%) exhibited high CXCR3 expression in the RG and 2 (2.78%) in the NRG. In the normal group, none of the normal tissue samples expressed CXCR3, whereas 6 samples poorly expressed CXCL10. The remaining pathological sections were not stained by the specific antibodies (Tables III and IV). There was a significant difference beween normal tissues and CRC RG or NRG in terms of CXCL10 and CXCR3 expression (P<0.05).

 Effect of clinicopathological characteristics and biomarker expression on survival

The univariate variables were discussed and analyzed at the beginning of this research. We aimed to determine the association between prognosis and the expression of CXCL10 or CXCR3 with the assistance of Kaplan-Meier plots. High expression of CXCR3 was found to be associated with shorter OS and DFS (both P-values <0.0001; Fig. 2, Table V). However, CXCL10 expression was significantly associated with DFS (P<0.0001), but not with OS (P=0.181) (Table V). Further analysis demonstrated that general classification and the presence of inflammatory adhesions were correlated with CXCR3 (χ2=7.074, P=0.029) and CXCL10 (χ2=4.863, P=0.088). The fact that ulcerated CRCs stained deeper compared with elevated CRCs may be attributed to CXCR3, as one of the common inflammatory cytokines, being involved in ulcer-related inflammation.

 Effect of inflammatory adhesions and CXCR3 expression on survival

Of the 401 stage II CRC patients, 91 had inflammatory adhesions, of whom 28 patients developed disease recurrence (Table IA). All the recurrent tumors with inflammatory adhesions were collected for IHC. In view of the results of the log-rank test, we found that the presence of inflammatory adhesions was associated with OS and CXCR3 expression; therefore, the statistical significance of the association was investigated. Under the same conditions of cancer surrounded by adhesions, patients with lower CXCR3 expression exhibited a better OS. However, patients with the same expression level of CXCR3 and different inflammatory adhesion status exhibited no differences in terms of OS (P>0.05). On multivariate analysis, only CXCR3 expression (P=0.003) was found to be an independent factor predicting a poorer prognosis (Fig. 3).

 Discussion

In this study, we first analyzed the clinicopathological characteristics of stage II CRC and found that colon cancer, a higher number of risk factors, the presence of inflammatory adhesions and tumor size were associated with OS and were independent variables; the presence of inflammatory adhesions, in particular, was found to be a significant factor for recurrent patients. Subsequently, we further investigated the association between the CXCL10/CXCR3 axis and inflammatory adhesions and observed that strong CXCL10 or CXCR3 expression in stage II CRC patients predicted short DFS and OS, particularly CXCR3 expression, which exhibited statistical significance. Furthermore, CXCR3 was found to be closely associated with inflammatory adhesions and OS. We suggest that CXCR3 is a strong indicator of relapse in stage II CRC patients; in addition, CXCR3 was a long-term prognostic biomarker for relapsed stage II patients. The chemokine axis CXCL10/CXCR3 may be the molecular mechanism underlying the development of inflammatory adhesions.

The morbidity and mortality of CRC are on the increase worldwide. Chemokines, as inflammatory cytokines, were first investigated in the context of hematological diseases (13), such as primary thrombocythemia, leukemia, multiple myeloma and von Willebrand syndrome. The association of chemokines with cancer has been attracting increasing attention. Schimanski et al (14) and Ottaiano et al (15) used IHC to detect CXCL12/CXCR4 expression in CRC and reported significantly higher expression in stage III/IV compared with stage I/II disease, suggesting that high expression was associated with lymph node and distant metastasis. Similar findings were reported for CCR7 in CRC (16,17), breast cancer (18) and pancreatic cancer (19). Chemokines in tumors mainly function in two ways: They either alter tumor cell actin aggregation, increase or decrease the formation of pseudopodia and affect tumor cell migration, or they promote the secretion of certain cytokines, such as metalloproteinases, degrade the extracellular matrix, damage the endothelial cells and alter vascular permeability, thereby affecting the process of invasion or metastasis.

CXCL10 belongs to the ELR (Glu-Leu-Arg) motif-negative subfamily and acts as an angiogenesis inhibitor; it has also been characterized as a prognostic marker predicting clinical outcome in uterine cervical cancer (20) and melanoma (21). It has been demonstrated that CXCL10 exerts its antitumor effect through its immune-stimulating and angiostatic properties. Of note, the additional roles of CXCL10 in the tumor microenvironment are also important. CXCL10 may activate RhoA and Racl and trigger migration of cancer cells (22). The CXCL10/CXCR3-mediated chemotaxis was found to promote lymph node metastasis in CRC by Kawada et al (9). As one of the first immune defense components, CXCL10 levels increase sharply in the liver, lungs and lymph nodes in CRC by combining to its specific receptor CXCR3. In order to elucidate the mechanism of action, previous researchers constructed a CRC cell metastasis model and found that cells from both primary and metastatic lesions expressed increased levels of CXCL10 and CXCR3 (23,24); they confirmed that tumor cells are able to activate and increase the level of lymphocytes in the microenviroment, upregulate the expression of interferon-γ and promote secretion of chemokine axes such as CXCL10/CXCR3, which is a cascade reaction. The underlying molecular mechanism may be CXCL10 promoting the CRC cells to secrete matrix metalloproteinases (MMPs) by inhibiting extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and repressing the ERK signaling pathway. The MMPs may initiate other metastasis-related pathways or alter the adhesive properties of cancer cells, directly promoting invasion. Metzner et al (25) previously analyzed the expression of chemokines in melanoma cells by flow cytometric measurements, ELISA and reverse transcription polymerase chain reaction; they hypothesized that constitutive chemokine expression enables an autocrine growth mechanism in epidermoid carcinoma cells. Numorous studies have demonstrated that CRC cells have the ability to secrete CXCL10 and CXCR3, whereas normal epithelial cells may not express CXCR3. In the present study, although CXCL10 as well as CXCR3 differed between RG and NRG, CXCL10 did not statistically significantly affect DFS or OS. As mentioned above, we hypothesized that the main function of CXCL10 expressed by stage II CRC cells is to promote the secretion of CXCR3 in an autocrine manner and initiate a cascade reaction, promoting invasion and distant migration of malignant carcinoma cells.

Regardless of the local or systemic inflammatory response, cancer-associated inflammation, another studying point, appears to be associated with tumor formation, progression and metastasis and may be of prognostic value in patients with CRC. Hu et al (26) found that microbiota in the gastrointestinal tract may enhance the expression of chemokine CCL5 and induce CCL5-mediated inflammation, which in turn promotes epithelial cell proliferation through local activation of the interleukin 6 (IL-6) pathway, leading to tumorigenesis. Components of the gut microbiota may be associated with individual susceptibility. Through measuring IL-6, IL-10, neutrophil-lymphocyte ratio, neutrophil count and other inflammatory factors, it was previously demonstrated that elevated circulating IL-6 concentration is associated with tumor necrosis (27). The key pathway may be Wnt signaling recognized by the transcription factor nuclear factor-κB (28). There are no studies on the latent function of peritumoral imflammatory adhesions; however, the association between CXCR3 and inflammation is clear. Dysregulation of CXCR3 expression has been found in viral infections (29), autoimmune diseases (30), allergy and asthma. To the best of our knowledge, the present study is the first to provide supportive evidence for the hypothesis that CRC prognosis is associated with the presence of inflammatory adhesions and CXCR3 expression. In turn, the CXCL10/CXCR3 axis may participate in the peritumoral inflammatory response and promote tumor progression.

In conclusion, we observed that CXCL10 and CXCR3 are upregulated in recurrent CRC tissues. CXCR3 expression may be used as one of the predictors of prognosis in postoperative stage II CRC patients. The cause of adhesion formation remains unclear, but may pertain to the upregulated expression of CXCR3. Therefore, postoperative stage II CRC patients exhibiting strong expression of CXCR3 should be closely followed up.

 References JemalABrayFCenterMMFerlayJWardEFormanDGlobal cancer statisticsCA Cancer J Clin616990201110.3322/caac.2010721296855 ChambersAFGroomACMacDonaldICDissemination and growth of cancer cells in metastatic sitesNat Rev Cancer2563572200210.1038/nrc86512154349 BensonABIIISchragDSomerfieldMRCohenAMFigueredoATFlynnPJKrzyzanowskaMKMarounJMcAllisterPVan CutsemEAmerican Society of Clinical Oncology recommendations on adjuvant chemotherapy for stage II colon cancerJ Clin Oncol2234083419200410.1200/JCO.2004.05.06315199089 RibicCMSargentDJMooreMJTumor microsatellite-instability status as a predictor of benefit from fluorouracil-based adjuvant chemotherapy for colon cancerN Engl J Med349247257200310.1056/NEJMoa02228912867608 SargentDJMarsoniSMongesGThibodeauSNLabiancaRHamiltonSRFrenchAJKabatBFosterNRTorriVDefective mismatch repair as a predictive marker for lack of efficacy of fluorouracil-based adjuvant therapy in colon cancerJ Clin Oncol2832193226201010.1200/JCO.2009.27.182520498393 ZlotnikAChemokines in neoplastic progressionSemin Cancer Biol14181185200410.1016/j.semcancer.2003.10.00415246053 BalkwillFCancer and the chemokine networkNat Rev Cancer4540550200410.1038/nrc138815229479 DwinellMBLügeringNEckmannLKagnoffMFRegulated production of interferon-inducible T-cell chemoattractants by human intestinal epithelial cellsGastroenterology1204959200110.1053/gast.2001.2091411208713 KawadaKHosogiHSonoshitaMSakashitaHManabeTShimaharaYSakaiYTakabayashiAOshimaMTaketoMMChemokine receptor CXCR3 promotes colon cancer metastasis to lymph nodesOncogene2646794688200710.1038/sj.onc.121026717297455 ZhangRZhangHZhuWPardeeABCoffeyRJJrLiangPMob-1, a Ras target gene, is overexpressed in colorectal cancerOncogene1416071610199710.1038/sj.onc.12009579129152 JiangZXuYCaiSCXCL10 expression and prognostic significance in stage II and III colorectal cancerMol Biol Rep3730293036201010.1007/s11033-009-9873-z19821051 LiZSLiQThe latest 2010 WHO classification of tumors of digestive systemZhonghua Bing Li Xue Za Zhi403513542011(In Chinese)21756837 MatsudaTSekiTOgawaraMMiuraRYokouchiMMurakamiMLevels of beta-thromboglobulin and platelet factor 4 in various diseases (author's transl)J Jpn Hematol Soc438718781980(In Japanese) SchimanskiCCSchwaldSSimiantonakiNJayasingheCGönnerUWilsbergVJungingerTBergerMRGallePRMoehlerMEffect of chemokine receptors CXCR4 and CCR7 on the metastatic behavior of human colorectal cancerClin Cancer Res1117431750200510.1158/1078-0432.CCR-04-119515755995 OttaianoAFrancoRAielloTalamanca ALiguoriGTatangeloFDelrioPNastiGBarlettaEFacchiniGDanieleBOverexpression of both CXC chemokine receptor 4 and vascular endothelial growth factor proteins predicts early distant relapse in stage II–III colorectal cancer patientsClin Cancer Res1227952803200610.1158/1078-0432.CCR-05-214216675573 CorrealePRotundoMSBottaCDel VecchioMTGinanneschiCLicchettaAConcaRApollinariSDe LucaFTassonePTumor infiltration by T lymphocytes expressing chemokine receptor 7 (CCR7) is predictive of favorable outcome in patients with advanced colorectal carcinomaClin Cancer Res18850857201210.1158/1078-0432.CCR-10-318622142823 LiJSunRTaoKWangGThe CCL21/CCR7 pathway plays a key role in human colon cancer metastasis through regulation of matrix metalloproteinase-9Dig Liver Dis434047201110.1016/j.dld.2010.05.01320609636 CabiogluNYaziciMSArunBBroglioKRHortobagyiGNPriceJESahinACCR7 and CXCR4 as novel biomarkers predicting axillary lymph node metastasis in T1 breast cancerClin Cancer Res1156865693200510.1158/1078-0432.CCR-05-001416115904 NakataBFukunagaSNodaEAmanoRYamadaNHirakawaKChemokine receptor CCR7 expression correlates with lymph node metastasis in pancreatic cancerOncology746975200810.1159/00013912618544997 SatoEFujimotoJToyokiHSakaguchiHAlamSMJahanITamayaTExpression of IP-10 related to angiogenesis in uterine cervical cancersBr J Cancer9617351739200710.1038/sj.bjc.660379017505511 AntonicelliFLorinJKurdykowskiSCXCL10 reduces melanoma proliferation and invasiveness in vitro and in vivoBr J Dermatol164720728201110.1111/j.1365-2133.2010.10176.x21155750 RobledoMMBartolomeRALongoNRodríguez-FradeJMMelladoMLongoIvan MuijenGNSánchez-MateosPTeixidóJExpression of functional chemokine receptors CXCR3 and CXCR4 on human melanoma cellsJ Biol Chem2764509845105200110.1074/jbc.M10691220011571298 Zipin-RoitmanAMeshelTSagi-AssifOShalmonBAviviCPfefferRMWitzIPBen-BaruchACXCL10 promotes invasion-related properties in human colorectal carcinoma cellsCancer Res6733963405200710.1158/0008-5472.CAN-06-308717409450 KawadaKHasegawaSMurakamiTItataniYHosogiHSonoshitaMKitamuraTFujishitaTIwamotoMMatsumotoTMolecular mechanisms of liver metastasisInt J Clin Oncol16464472201110.1007/s10147-011-0307-221847533 MetznerBHofmannCHeinemannCZimpferUSchraufstätterISchöpfENorgauerJOverexpression of CXC-chemokines and CXC-chemokine receptor type II constitute an autocrine growth mechanism in the epidermoid carcinoma cells KB and A431Oncol Rep614051410199910523720 HuBElinavEHuberSStrowigTHaoLHafemannAJinCWunderlichCWunderlichTEisenbarthSCMicrobiota-induced activation of epithelial IL-6 signaling links inflammasome-driven inflammation with transmissible cancerProc Natl Acad Sci USA11098629867201310.1073/pnas.130757511023696660 GuthrieGJRoxburghCSRichardsCHHorganPGMcMillanDCCirculating IL-6 concentrations link tumour necrosis and systemic and local inflammatory responses in patients undergoing resection for colorectal cancerBr J Cancer109131137201310.1038/bjc.2013.29123756867 VaiopoulosAGAthanasoulaKCHPapavassiliouAGNF-κB in colorectal cancerJ Mol Med (Berl)10291037201310.1007/s00109-013-1045-x23636511 de NietAde BruijneJPlat-SinnigeMJTakkenbergRBvan LierRAReesinkHWvan LeeuwenEMUpregulation of CXCR3 expression on CD8+ T cells due to the pervasive influence of chronic hepatitis B and C virus infectionHum Immunol74899906201310.1016/j.humimm.2013.04.01723643635 BjörkanderSHeidari-HamedaniGBremmeKGunnarssonIHolmlundUPeripheral monocyte expression of the chemokine receptors CCR2, CCR5 and CXCR3 is altered at parturition in healthy women and in women with systemic lupus erythematosusScand J Immunol77200212201310.1111/sji.1202123298254

CXCL10 and CXCR3 membranous and cytoplasmic staining in colorectal tumors. (A) Low CXCL10 expression was demonstrated in 19/71 samples in the recurrence group (RG) and 36/72 samples in the non-recurrence group (NRG). (B) Strong CXCL10 expression was observed in 27/71 samples in the RG and 10/72 in the NRG. (C) Low CXCR3 expression was demonstrated in 21/71 samples in the RG and 48/72 in the NRG. (D) Strong CXCR3 expression was demonstrated in 12/71 samples in the RG and 2/72 in the NRG. (E) Low CXCL10 expression was demonstrated in 6/10 normal tissue samples. (F) Negative CXCR3 expression was demonstrated in 10/10 normal tissue samples.

Survival curve of CXCR3 expression. (A) Patients with strong expression (n=14) exhibited significantly shorter disease-free survival (DFS) compared with those with low expression (n=69, P<0.0001, log-rank test) and negative expression (n=60, P<0.0001, log-rank test); low vs. negative expression, P=0.012. (B) Patients with strong expression also exhibited significantly shorter overall survival (OS) compared with those with low expression (P<0.0001) and negative expression (P<0.0001); however, there was no significant difference between low and negative expression (P=0.184).

Kaplan-Meier analysis of cancer-specific overall survival (OS) of relapsed patients with stage II colorectal cancer subdivided by the presence of inflammatory adhesions and CXCR3 expression (P<0.0001). Expression of CXCR3 (A) in cases without adhesions and (B) in cases with adhesions.

Comparison of clinicopathological characteristics in stage II colorectal cancer patients undergoing potentially curative resection.

A, Clinicopathological characteristics
Survival status Recurrence status
Variables Total (n=401) Alive (n=325) Deceased (n=76) P-valuea No recurrence Recurrence P-valuea
Gender 0.320 0.681
  Male 229 182 47 167 62
  Female 172 143 29 128 44
Age, years 0.341 0.721
  ≤60 199 166 33 149 50
  >60 202 159 43 146 56
Location 0.026b 0.101
  Colon 220 187 33 169 51
  Rectum 181 138 43 125 54
General classification 0.551 0.515
  Ulcerated 288 229 59 206 82
  Elevated 111 94 17 87 24
  Other 2 2 0 2 0
Tumor size, cm 0.031b 0.051
  ≤10 381 312 69 283 98
  >10 20 13 7 8 12
Stage 0.179 0.441
  IIA 71 59 12 55 16
  IIB 323 262 61 236 87
  IIC 7 4 3 4 3
Recurrence risk factors 0.009b <0.001b
  None 75 63 12 56 19
  1 276 229 47 208 68
  ≥2 50 33 17 31 19
Preoperative CEA 0.838 0.205
  Abnormal 28 23 5 18 10
  Normal 373 302 71 277 96
Anemia 0.731 0.952
  No 300 242 58 221 79
  Yes 101 83 18 74 27
Family history 0.528 0.295
  No 327 263 64 235 90
  Yes 74 62 12 58 16
Inflammatory adhesions 0.024b 0.214
  No 310 258 52 232 78
  Yes 91 67 24 63 28
B, Uni- and multivariate Cox proportional hazards model for overall survival in stage II colorectal cancer patients
Variables B SE Wald P-valuea Exp (B) 95% CI
Location −0.622 0.250 6.184 0.013b 0.537 0.329–0.877
Tumor size 0.455 0.213 4.562 0.033b 1.577 1.038–2.395
Recurrence risk factors 0.432 0.206 4.395 0.036b 1.540 1.029–2.307
Inflammatory adhesion 0.600 0.261 5.298 0.013b 1.823 1.093–3.039

P-values were obtained with the log-rank and χ2 test in Table IA and Cox regression in Table IB.

P<0.05, statistically significant. B, regression coefficient; SE, standard error; Exp, odds ratio; CI, confidence interval; CEA, carcinoembryonic antigen.

Correlation between the clinicopathological characteristics of relapsed patients and overall survival.

Characteristics P-valuea
Imflammatory adhesions 0.025b
General classification 0.314
Recurrence risk factors 0.194
Tumor size 0.516
Gender 0.487
Age 0.931
N stage 0.266
Preoperative CEA level 0.218
Preoperative Hb level 0.295
Tumor location 0.309
Family history 0.859

P-values were obtained with Cox regression.

P<0.05, statistically significant. CEA, carcinoembryonic antigen; Hb, hemoglobin.

Immunostaining for CXCL10 and CXCR3 expression in normal and CRC tissues.

CXCL10 CXCR3
Variable Negative Low Strong Negative Low Strong
CRC (n=143) 51 55 37 60 69 14
Normal tissue (n=10) 4 6 0 10 0 0
P-valuea 0.145 0.002b

P-values were obtained with the χ2 test.

P<0.05, statistically significant. CRC, colorectal cancer.

Immunostaining for CXCL10 and CXCR3 in colorectal cancer (negative expression samples are absent).

Variable Recurrence group (n=71) Non-recurrence group (n=72) P-valuea
CXCL10 0.001b
  Low expression 19 36
  Strong expression 27 10
CXCR3 <0.001b
  Low expression 21 48
  Strong expression 12 2

P-values were obtained with the χ2 test.

P<0.05, statistically significant.

Association between CXCL10/CXCR3 and prognosis

DFSa OSa
Variables Mean ± SE P-valueb Mean ± SE P-valueb
CXCR3 expression <0.001c <0.001b
  Negative 42.46±3.30 67.12±3.75
  Low 44.96±2.02 79.52±3.05
  High 11.29±2.59 25.21±4.52
CXCL10 expression <0.001c 0.181
  Negative 46.47±3.41 68.46±4.29
  Low 51.93±3.48 59.83±2.54
  High 28.66±3.25 61.79±5.62

DFS and OS calculated as time (months).

P-values were obtained with the log-rank test.

P<0.05, statistically significant. DFS, disease-free survival; OS, overall survival; SE, standard error.