Investigating The Overexpressed Chemokines And Their Effect On T Cells Mobility In Esophageal Squamous Cancer Patients

ObjectiveEsophageal squamous cell carcinoma (ESCC) is worldwide and particularlycommon in certain regions of Asia. Even though the technology has advancedrecent years, the therapies used to cure ESCC show no significant achievementwith the5year survival less than20%. Many studies have shown that the abilityof T cells infiltrating to tumor sites associates with patients’ prognosis. CD8+Tcells play an important role in clearing tumor cells and extraneous materials whileregulatory T cells depress the ability of effector T cells. It is clinically meaningfulto investigate the molecular mechanism of lymphocytes homing to tumor sites. Itwas reported that in the malignant melanoma patients, chemokine cooperated withtheir receptors play an important role in T cells distribution. However, themechanism of T cells homing to ESCC tumor sites has not been revealed. In thisstudy, we investigated the overexpressed chemokine ligands in the tumor sites andthe expression of chemokine receptors on T cells from esophagus cancer patients’samples. We also analyzed the associations among chemokine and T cell markers.IHC was used to verify the expression of chemokine and investigate the mainsource of chemokine ligands. Transwell assay was performed to examine theeffect of chemokine-receptor axis on T cells mobility in vitro. CIK therapy is anew one used clinically, however, some patients showed no response to it. Animalassay revealed that the number of CIK cells successfully homing to tumor sitesmay determine the efficacy of the therapy. Investigating the mechanism of T cellshoming to ESCC may lay an important foundation for improve the CIK therapy.MethodsThe expression of chemokine CCL2/CCL4/CCL5/CCL8/CCL11/CCL17/CCL20/CCL21/CCL22/CXCL10/CXCL12and T cell markers CD4/CD8/FoxP3/IL-10/Perforin/Granzyme B was examined in tumor samples and adjacent ones ofESCC patients. IHC was used to verify the expression of chemokine in protein level and analyze the main source of them. Peripheral blood mononuclear cell(PBMC) and tumor infiltrating lymphocytes (TIL) were obtain usingFicoll-Plaque and then restored in-80°C fridge for use. T cell populations and theexpression of chemokine receptors in PBMC and TIL were detected using Flowcytometry. Using MACS to purify T cells and then Transwell was performed toinvestigate the effect of chemokine-receptor axis on T cells mobility.Results1. According to RT-PCR data, chemokine CCL4/CCL20/CXCL10wereoverexpressed in tumor sites compared with adjacent samples of ESCC patientwhile other chemokine showed no significant difference. IHC results verifiedthe result and tumor cells were the main ones to secret them.2. We found that chemokine CCL4and CXCL10had positively correlations withCD8/Perforin/Granzyme B while CCL20was positively associated with CD4/FoxP3and IL-10. These results reminded that chemokine CCL4/CCL20/CXCL10may have important roles in recruiting T cells to tumor sites.3. Flow data revealed that, compared with PBMC, CD8+T cells andCD4+Foxp3+T cells were accumulated while total CD4+T cells decreased inTIL. What is more, the percent of chemokine receptor CCR5/CCR6andCXCR3on T cell population were higher in TIL.4. Transwell result showed that recombinant CCL4/CCL20and CXCL10couldenhance the mobility of T cells in vitro; and analysis of the lower chambershowed that CCL4/CXCL10mainly recruit CD8+T cells while CCL20predominantly recruit CD4+Foxp3+T cells.ConclusionChemokine CCL4/CCL20/CXCL10were overexpressed in tumor sites ofESCC patients and could affect the mobility of CD8+T and CD4+Foxp3+T cellsthat expressed their receptors.