| Background & Objective:Tumor recurrence and metastasis is a multi-step, multi-factor and sequential complex process. Firstly, epithelial-mesenchymal transition (EMT) of the tumor cells makes them easier to fall off from the primary tumor, after entering the blood circulation, the tumor cells will be called circulating tumor cells (CTCs). However,most of the CTCs in the blood system will be in apoptosis,and only a few can resistanoikis to survival. The living CTCs will experience the reverse process of the EMT,namely the mesenchymal-epithelial transition (MET), and eventually colonize the parenchymal organ to form the recurrence or metastasis.Recent studies have shown that chemokine ligand 8 (CXCL8), also known as IL-8, is related to the occurrence and development of tumors, including colorectal cancer (CRC) as well as the recurrence and metastasis of CRC. CXCL8 was the first chemotactic factor that had been found, and it was also the first one to be proven to have angiogenic effects. The corresponding biological effects of CXCL8 are mainly made by binding with its receptors, CXCR1 and CXCR2. They are seven transmembrane G protein coupled receptors, and about 80% of them are homologous sequences. They can not only promote the growth and proliferation of colorectal cancer cells, but also induce EMT, anoikis resistance, angiogenesis of them. Most of the clinical research and basic research data has showed that CXCL8 and its receptors played a role in recurrence and distant metastasis process of colorectal cancer, what’s more, our previous studies had also confirmed that CXCL8 was the critical factor of EMT and anoikis resistance of colorectal cancer cells. However, how CXCL8 and its receptors induce the "signal homing" mechanism of CTCs is not very clear.The purpose of this study was to establish the MACS sorting and four-color immunofluorescence staining method for the enrichment and identification of CTCs as well as the CXCR1/2 phenotype on CTCs in peripheral blood of patients with CRC.Analyzing the correlation between the detected CTCs or the positive rate of CXCR1/2 on CTCs and the clinical parameters or the prognosis of patients with CRC, we can evaluate the feasibility’ of the detected CTCs or the positive rate of CXCR1/2 on CTCs in the clinical prognosis of CRC patients and create a divided layer for screening patients, which may provide new ideas for the establishment of a new strategy to reduce the recurrence and metastasis of CRC.Methods:1. The expression of CXCR1/2 in various CRC cell lines was detected by western blotting, flow cytometry, immunofluorescence of cell smear. The higher and lower CXCR1/2 expressional cell lines were screened for the future research.2. We incorporated a certain number of CRC cells to the peripheral blood of healthy volunteers and established the specific method of sorting, identification of CTCs and the CXCR1/2 phenotypes of CTCs, according to the recovery rate of the incorporated CRC cells.(1) Using Ficoll-Paque PLUS density gradient separation method and Magnetic Activated Cell Separation (MACS) removal sorting method to separate the mononuclear cells from the healthy volunteers’ peripheral blood, which has been incorporated with different numbers of colorectal cancer cells.(2) The collected cells will be stained by four-color immunofluorescence experiments after cleaning. The recovery rate of incorporated CRC cells was calculated under the fluorescence microscope.(3) The specific scheme was established when the recovery rate was above 70%,and then used to detect the blood samples of clinical CRC patients.3. Detection and identification of CTCs in the 22 normal people, 18 patients with intestinal polyps and 129 patients with colorectal cancer. The positivity of CTCs in patients with colorectal cancer was defined according to the CTC count from different patients.4. The clinical significance of the detection of CTCs and the expression of CXCR1/2 on CTCs in 129 CRC patients. According to the CTCs and the expression of CXCR1/2 on CTCs, the patients were divided into various groups to study the significance of clinical pathological factors in the early prognosis of patients with different groups.Results:1. SW480 cell line and Lovo cell line were screened to be incorporated into the mononuclear cells to establish CTCs model..2. The recovery of various incorporation levels were all more than 70%, by using the specific method of sorting,identification of CTCs and the CXCR1/2 phenotypes of CTCs, which meant that the method was feasible. And the phenotypes of recycled SW480 cells or Lovo cells were both CK+/CD45-/DAPI+/CXCR1/2+ or CK+/CD45-/DAPI+/CXCR1/2-. However,most of the recycled SW480 cells were CK+/CD45-/DAPI+/CXCR1/2-,. and most of the. recycled Lovo cells were CK+/CD45-/DAPI+/CXCR1/2+.3. CTCs were detected in 7 of the 22 normal persons, and 15 of them were not detected. The quantity was ranged from 0 to 2, with an average of 0.5±0.7. CTCs were detected in 12 of the 18 intestinal polyp patients, and 6 of them were not detected. The quantity was ranged from 0 to 23, with an average of 4.9±7.5. CTCs were detected in 118 of the 129 originating CRC patients, .and 11 of them were not detected. The quantity was ranged from 0 to 84, with an average of 22.7±20.4. The percent of patients of each group (normal persons, patients with colorectal polyps and colorectal cancer) that had a CTC count greater than each of the cutoff values (CTCs > 0,1, or 2 per 5 mL) were calculated. For each cutoff value, the positive incidence rates increased with disease severity. Using > 2 CTCs as a cutoff,0% of the healthy patients had CTC counts higher than the cutoff. The percentage of patients. with CTC counts > 2 were 33% and 79.8% for patients with polyps and CRC,respectively. Because of these results, we used a CTC cutoff of > 2 per 5 mL of blood as the CTC positive, which included 103 patients. The CTC positive patient was considered the high expression of CXCR1/2 on CTCs when more than 50% of CTCs expressed CXCR1/2; otherwise,considered as low expression of CXCR1/2 on CTCs.All CRC patients were followed up for 1～2 years, 30 of them occurred early recurrence / metastasis, and the other 99 patients not occurred. The early recurrence /metastasis rate was about 23%.5. In the 129 originating CRC patients, the χ2 text showed that the positive CTC was associated with the early recurrence / metastasis after operation within two years(χ2 = 4.419, P = 0.039). However, univariate and multivariate COX regression analyses showed that only the preoperative CEA level (HR = 2.189, P = 0.044),clinical stage (HR = 3.165, P = 0.005), lymph node metastasis (HR = 4.557, P ＝0.002) were the risk factors of early recurrence / metastasis patients. Whether CTCs was positive or not was not an independent risk factor for early prognosis of patients with colorectal cancer.6. In the 103 CTCs positive patients, the expression of CXCR1/2 on CTCs was associated with the tumor size (χ2 = 7.663, P = 0.009), tumor grade (χ2 = 7.174, P =0.009), and lymph node metastasis (χ2 = 5.598, P = 0.028). Univariate and multivariate COX regression analyses showed that the expression of CXCR1/2 on CTCs (HR = 3.667, P = 0.022), clinical stage (HR = 2.688, P = 0.023) and lymph node metastasis (HR = 3.428, P = 0.028) were three risk factors of early recurrence /metastasis in the 103 CTCs positive patients.7. The 129 originating CRC patients were divided into circulating CRC CXCR1/2 high expression group (the higher CTCCXCR1/2 patients) and circulating CRC CXCR1/2 low expression group (the lower CTCCXCR1/2 or CTCs negative patients) according to the expression of circulating CRC CXCR1/2. Univariate and multivariate COX regression analyses showed that the expression of circulating CRC CXCR1/2 (HR = 3.522, P = 0.011), clinical stage (HR = 2.256, P = 0.046), lymph node metastasis (HR = 3.533, P = 0.013) were three risk factors of early recurrence /metastasis in the 129 patients. The high expression of CXCR1/2 on CTCs was an independent risk factor for early prognosis of patients with colorectal cancer.8. Kaplan-Meier survival analyses showed that whether CTCs was positive or not was not associated with disease-free survival (DFS) of patients with CRC (Log Rank = 2.977, P = 0.088). The difference between CTCsCXCR1/2 expression and DFS was significant, high CTCsCXCR1/2 group had a shorter DFS than the low CTCsCXCR1/2 group (Log Rank = 20.420, P < 0.01). In the 129 originating CRC patients, circulating CRC CXCR1/2 high expression group had a shorter disease-free survival (DFS) than the circulating CRC CXCR1/2 low expression group, with significant difference (Log Rank = 26.622, P < 0.01).[Conclusions](1) This study established the MACS sorting and DAPI/CK/CD45/CXCR1/2 four-color immunofluorescence staining method, which can be used for theidentification of CXCR1/2 phenotype on CTC. (2) According to the expression ofCXCR1/2 on CTCs, patients with CRC can be stratified to assess the risk of early recurrence / metastasis. Targeting CXCL8-CXCR1/2 signal axis was expected to become a new treatment strategy for colorectal cancer. |
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