| BACKGROUNDColorectal cancer ?CRC? is one of the common malignant tumor in our country.The incidence rate of CRC in China is increasing faster and the patients' age is younger than ever during the past decades. Metastasis is the main cause of the death of CRC patients.Epithelial mesenchymal transition ?EMT? is closely related to cancer metastasis.The tumor microenvironment may induce the occurrence of EMT in tumor cells. The inflammatory component is an essential part of the malignant microenvironment.Many studies have certificated that there were a lot of macrophages infiltrated in pulmonary fibrosis, and consequently induce EMT through invoking the activity of fibroblast and myofibroblast. Interestingly, we discovered a large amount of macrophages accumulated around the invasive CRC. In tumor microenvironment,whether macrophages induce EMT of CRC is unknown. In most malignant tumors,tumor associated macrophages ?TAM? are the most common inflammatory cells infiltrated in tumor mesenchyma. They could facilitate tumor growth, invasion,metastasis and angiogenesis. Owing to phase and tissue specifity, TAM functions vary by type, stage and location of tumor. The role of TAM in the microenvironment of CRC remains unknown.OBJECTIVEThis study investigated the link between EMT and macrophages infiltrated in tumor microenvironment. In this study, we will identify the probable factors of EMT induction, certificate the key regulatory molecules and markers for EMT in the tumor microenvironment of CRC. This study will not only give us a better understanding of the molecular mechanism of malignant tumor formation, development and metastasis theoretically, but also play an important role in the diagnosis and therapy of malignant tumor practically.METHODS1 The distribution, classification and prognosis of TAM in colorectal cancerImmunohistochemical analysis was used to detect the infiltration density ofCD68+,NOS2+and CD163+ in CRC cases and paried adjacent normal tissue. Then with the clinicopathologic features and follow-up data, the relationships between the infiltration density of CD68+ and CD163+ macrophages in CRC microenvironment and lymph node metastasis and the survival time of CRC patients were explored. The effect of macrophage infiltration density on the prognosis of CRC patients was analyzed.2 The screening of differentially expressed protein of TAM by 2D-DIGEDifferential in-gel electrophoresis ?DIGE? was used to screen the differentially expressed protein between THP-1 and M2 THP-1 cells after co-cultured with CRC cells. All special spots were selected for mass spectrum identification.3 The screening and validation differentially secreted cytokines of TAM by cytokine microarrayCytokine microarray was used to screen the differentially cytokines in culture supernatant between human colorectal mucosa macrophages and tumor associated macrophages. Western blotting and ELISA were used to test the differentially expressed protein and secretion level, respectively.4 Effects of TAM on EMT in CRC?1?The differentiation of human peripheral blood mononuclear cells to macrophagesPeripheral blood mononuclear cells ?PBMC? from CRC patients were isolated from full blood by Ficoll-Hypaque density gradient centrifugation. After washed, adherent cells were harvested. Then the adherent cells were stimulated with LPS ?20 ng/ml?and IFN-y ?20 ng/ml? for 3 days to generate M1-polarized cells and IL-4 ?20 ng/ml?and IL-13 ?20 ng/ml? for 3 days to generate M2-polarized cells.?2?The differentiation of THP-1 cells to macrophagesTHP-1 cells was treated phorbol ester ?PMA, 10 ng/ml? for 24 h, washed and stimulated with LPS ?20 ng/ml? and IFN-y ?20 ng/ml? for 3 days to generate M1-polarized cells and IL-4 ?20 ng/ml? and IL-13 ?20 ng/ml? for 3 days to generate M2-polarized cells.?3?The indirect co-cultures model of CRC cells and macrophagesMacrophage were seeded into the upper insert of a six-well transwell apparatus?0.4p?m pore size, Corning, Lowell, MA?. After 4h, SW480 or SW620 cells were seeded in the lower co-culture compartment and then the morphology of CRC cells were observed.?4?The effect of M2 macrophages on proliferation of CRC cellsCell Counting Kit-8 ?CCK-8? assay was used to determine SW480 or SW620 cells viability at 0,1,2, 3,4, 5 and 6 days after co-cultured with macrophages for 48 h. The in vitro growth curve was drawed.?5? The effect of M2 macrophages on invasion ability of CRC cellsFor the invasion assay,1 ×105 SW480 or SW620 cells were plated in the top chamber of the transwell with a matrigel-coated polycarbonate membrane. 5×104 macrophages with 10% FBS were added to the lower chamber as a chemoattractant.After incubation for 24 and 48 hours, cells on the lower surface of the membrane were fixed, stained and counted.?6? The effect of M2 macrophages on EMT of CRC cellsThe real-time fluorescence quantification, immunofluorescence and western blotting were used to detect the expression of EMT related genes and proteins in SW620 cells After co-cultured with macrophages.5 The relationship between CRC metastasis and galectin-1 from TAM in tumor microenvironment?1? 10 cases fresh CRC tissue was to conduct frozen section. The colocalization of CD68 and galectin-1 was detected by immunofluorescence.?2? Immunohistochemical analysis was used to detect the expression of galectin-1 in 215 cases and paried adjacent normal tissue. Then with the clinicopathologic features and follow-up data, we explored the relationship between the expression of galectin-1 and lymph node metastasis and the survival time of CRC patients. The expression of galectin-1 in tumor microenvironment on the prognosis of patients was determined further.?3? The siRNA ?small interferring RNA? against galectin-1 was designed and synthesized. Then the siRNA was transfected to the M2 THP-1 cells. Galectin-1 expression was detected by QRT-PCR and western blotting. The secretion level of galectin-1 was determined by ELISA.?4? The effect of TAM on proliferation, adhesion, invasion and migration of CRC cellsCCK-8 assay, adhesion assay and transwell invasion assay were used to determine the effect of galectin-1 on proliferation, adhesion, invasion and migration abilities of SW620 cells,separately.?5? The immunofluorescence and western blotting were used to detect the effect of galectin-1 on expression of EMT related proteins of SW620 cells.6. Statistical analysesStatistical analyses were performed using SPSS13.0 software package. Statistics data were expressed as the mean±SD. Cross-tabulations were analyzed with Fischer's exact test and linear relationships with the exact linear-by-linear association test.Kaplan-Meier survival analysis was used to estimate cancer-specific survival, and comparisons between groups were performed with the log-rank test. Multivariate survival analyses were performed by using Cox proportional hazard models. The correlation analyses were used nonparametric bivariate correlation. The identification of different subtypes of macrophages,transwell assay,the expression of EMT related genes, the expression and secretion of galectin-1 were determined using one-way analysis of variance, followed by the least significant different ?LSD?/Tamhane's T2 test. The CCK8 assay was analyzed using multiple factor-multilevel factorial design and analysis of variance ?ANOVA?. The fluorescence of EMT related proteins of CRC cells after cocultured with macrophages was used 2 samples T test.P<0.05 was considered statistically significant.RESULTS1 The distribution, classification and prognosis of TAM in CRCCD68+/CD163+ macrophages were seldom observed in mesenchyma of normal colorectal mucosa. However, a large amount of CD68+/CD163+ macrophages were detected in the invasive front of CRC. NOS2+ macrophages were more observed in normal colorectal mucosa than in mesenchyma of colorectal cancer tissue. The expression intensity of CD68+/CD163+TAM varied according to different region of tumor and the majority of CD68+/CD163+ TAM distributed in the mesenchyma of tumor. The correlation analysis indicated that the expression of CD68 and CD 163 was positive related ?r=0.352, P<0.001?.Using Log-Rank analysis method to compare the survival curve of different groups,we found that the expression of CD68 intratumor was significantly correlated with overall survival of CRC patients?Log rank=7.191,P=0.007?.Using Log-Rank analysis method to compare the survival curve of different groups,we found that the expression of CD 163 peritumor in CRC was significantly correlated with overall survival ?Log rank=4.211, P=0.040? of CRC patients. In Multivariate Cox proportional hazard models analysis, CD163+ TAM in peritumor served as a prognostic indicator for CRC patients ?P=0.010?. The relative risk of CD 163 was higher than 1 and suggested an increased infiltration of CD163+macrophages in peritumor front was highly significantly associated with an bad prognosis ?P<0.001?.2 The screening of differentially expressed protein of TAM by 2D-DIGEThe results of 2D-DIGE showed that there were many different protein spots between THP-1 cells and M2 macrophages co-cultured with CRC cells. 72 differently-expressed protein spots were screened by analyzing the amount of protein expression. Compared with THP-1 cells, we found 45 protein spots were up-regulated,27 were down-regulated. 21 protein spots including 2 secreted proteins were identified in M2 THP-1 cells.3 The screening and validation differentially secreted cytokines of TAM by cytokine microarrayTo identify the secreted factors of TAM in CRC microenvironment, we use RayBio L-Series 1000 Antibody Array that simultaneously detects changes in the levels of 1000 proteins, we profiled the culture medium proteins of TAM in 6 CRC patient samples. We identified 195 proteins that were significantly modulated in TAM,compared with NM. Of these, 188 showed persistent upregulation and 7 downregulation in TAM.Incombination with the result of 2D-DIGE, we selected galectin-1 as a candidate.Western blot was used to detect the expression of galectin-1 in different macrophages.The results showed that the expression of galectin-1 in M2 THP-1 or M2 PBMC cells was significantly higher than that in control cells. ELISA assay was adopted to measure the amount of galectin-1 secreted by different subtypes of macrophages. The result indicated that SW620, THP-1 and PBMC cells hardly secreted galectin-1 whereas M2 THP-1 and M2 PBMC cells secreted higher level of galectin-1 than control group ?P<0.001?. The secretion level of TMA was ?117.47±9.52? ng/ml,which was higher than ?24.19±3.97? ng/ml in NM.4 Effects of TAM on EMT in CRC?1? Peripheral blood-derived monocyte from healthy persons could be differentiated into different subtypes of macrophages by cytokines. After cytokines treatment, the majority of macrophages ?more than 95%? were certificated CD68+ macrophages through immunofluorescence detection.?2? CD68 expression in different subtypes of PBMC cells was detected by immunofluorescence. We found that low or no expression of CD68 was found in PBMC cells. However, CD68 was highly expressed in M1 and M2 macrophages.?3? CCK8 assay was adopted to check the proliferation of SW480 and SW620 cells after co-cultured with macrophages. The result suggested that from the second day, a significantly decreased proliferation was found in SW480/M1 cells compared with that in SW480/Med cells ?P<0.001?. There was no significant difference in proliferation of SW480/M2 and SW480/Med cells ?P>0.05?. From the second day, a significantly decreased proliferation was found in SW620/M1 cells compared with that in SW620/Med cells ?P=0.011?. There was no significant difference in proliferation of SW620/Un and SW620/Med cells ?P>0.05?. From the third day, a significantly increased proliferation was found in SW620/M2 cells compared with that in SW620/Med cells ?P<0.001?.?4? The results of invasion assay indicated that after co-cultured with PBMC macrophages,the number of invaded SW480 and SW620 cells increased dramatically?F=272.184,P<0.001; F=115.052,P<0.001?. The number of invaded SW480 and SW620 cells increased after co-cultured with tumor associated macrophages?F=340.014, P<0.001; F=82.564, P<0.001?. Consequently, the results revealed that macrophages could enhance the invasive ability of CRC cells.?5? After co-cultured with M2 macrophages,the mRNA expression of epithelial markers, including E-cadherin, a-catenin, and P-catenin, in SW480 cells were down-regulated ?P<0.001,P<0·001,P=0.003? whereas the expression of mesenchymal marker vimentin was up-regulated ?P<0.001? . Transcriptional factors snail and slug were up-regulated as well ?P<0.001,P<0.001?. After co-cultured with M2 macrophages, the mRNA expression of epithelial markers, including E-cadherin,a-catenin, ?-catenin and y-catenin in SW620 cells were down-regulated ?P<0.001,P=0.002, P<0.001, P=0.003? whereas the expression of mesenchymal marker vimentin was up-regulated ?P<0.001? . Transcriptional factors snail slug and twist were up-regulated as well ?P<0.001, P<0.001, P=0.001?. Immunofluorescence was used to detect whether EMT occured in CRC cells after co-cultured with M2 macrophages. The results indicated that epithelial markers E-cadherin, ZO-1 and?-catenin were down-regulated significantly ?t=5.95, P=0.004; =11.66, P<0.001;t=11.33, P<0.001? while mesenchymal markers N-cadherin and vimentin,transcriptional factors snail, slug and TCF8 were up-regulated ?t=7.30, P=0.002;t=7.21,P=0.002; t=7.23,P=0.002?.5 The relationship between CRC metastasis and galectin-1 from TAM in tumor microenvironment?1? Co-localization of CD68 and galectin-1 was obviously detected in CRC tissue by using double immunofluorescence staining. Galectin-1 was detected around TAM,which indicated TAM expressed and secreted galectin-1.?2? The expression of galectin-1 in CRC tissue ?83.7%? was significantly higher than that in normal colorectal mucosa ?18.6%? ?P<0.001?. We also discovered that in CRC tissue, galectin-1 was mainly located around cancer cells, which varied in different cancer tissue regions. The gradient distribution of galectin-1 was found in extracellular matrix of CRC. Galectin-1 expression in invasive front was significantly higher than that in the center of CRC tissue. The expression of galectin-1 showed no obvious correlation with gender, age, tumor size, degree of differentiation and invasion?P>0.05?,but showed a close link with lymph node metastasis?P=0.043? and clinical stage?P=0.035?.Expression of galectin-1 was associated with lymph node metastases, distant metastasis and Dukes' stages ??2=16.859,P<0.001; ?2=5.272, P=0.022; ?2=20.148,P<0.001?. No significant association was found between galectin-1 expression and the patients' age, gender, tumor size, differentiation and invasive depth ??2=0.556,P=0456; ?2?1.746, P=0.186; ?2=1.143, P=0.285; ?2=0.174, P=0.917; ?2=3.524,P=0.172?.Using Log-Rank analysis method to compare the survival curve of different groups,we found that the overall survival in galectin-1 low expression group was better than galectin-1 high expression group ?Log Rank=9.644, P=0.002?. In Multivariate Cox proportional hazard models analysis,galectin-1 served as a prognostic indicator for CRC patients ?P=0.011?. The relative risk of galectin-1 was higher than 1 and suggested an increased expression of galectin-1 at the tumor front was highly significantly associated with an bad prognosis ?P=0.002?.?3? Real-time fluorescence quantification PCR was employed to check the interference rate of galectin-1 in M2 THP-1 cells. The interference rates of Sil and Si2 were 66% and 73% and had a significant difference with mock cells ?P<0.001?.The protein expression of galectin-1 were down-regulated in M2 macrophages after galectin-1 interferenced by western blotting. The secreted level of galectin-1 in the supernatant of two interferenced groups were found dramatically lower than that in control groups by ELISA ?P<0.001?.?4? The effect of galectin-1 on proliferation of CRC cells was examined by CCK8 assay. The result showed that after treated with 1.0 ?g/ml galectin-3 for 48 h, the OD value of SW620 cells was ?0.36±0.04? and the control was ?0.26±0.04?. There was a significant difference between the two groups ?P=0.001?. But galectin-1 had no obvious promoting effect on proliferation of SW620 cells.?5? Cell counting technique was used to detech the effect homogeneity adhesion assay. The fibronectin adhesion rate of SW620 cells treated with 1.0?g/ml galectin-1 was higher than control group ?P<0.001?. The results indicated that galectin-1 could increase fibronectin adhesion of CRC cells.?6? The results of invasion assay indicated that after treated with 1.0 ?g/ml galectin-1, the number of invaded SW620 cells was ?20.00±2.83? and was higer than that of ?3.50±1.05? of control group ?P<0.001?.?7? The results of immunofluorescence assay and western blotting showed that the expression of the epithelial markers, including E-cadherin, ZO-1 and ?-catenin,decreased while mesenchymal markers N-cadherin and vimentin and transcriptional factors snail, slug and TCF8 increased in SW620 cells after galectin-1 treatment. The results indicated that galectin-1 could induce EMT of CRC cells.CONCLUSION1. CD68 and CD 163 could be served as independent indiciators for CRC.2. TAM induced EMT in CRC and promoted the proliferation and invasive abilities of CRC cells.3. TAM promoted the proliferation,heterogeneous adhesion,movement and invasion of CRC cells through the secretion of galectin-1. Galectin-1 expression was significantly associated with lymph node metastasis, distant metastasis and Dukes'stages.INNOVATION IN THE STUDIES1. We demonstrated that CRC TAMs were certified primarily a macrophage subpopulation with M2 phenotype. M2 TAM was associated with progression and poor survival in CRC.2. This is the first time to propose that galectin-1 was secreted by TAM in the microenvironment of CRC. We confirmed that galectin-1 played an important role in promoting invasion and metastasis of CRC cells.3. We demonstrated that CD326 is the receptor of galectin-1 and galectin-1 from TAM promoted induced EMT of CRC via CD326. |
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