Study On The Expression And Function Of CD169~+ Tumor Associated Macrophages In Colorectal Cancer

Chapter? Isolation and subsequently analysis of human colonic lamina propria mononuclear cellsObjective: Intestinal mucosal lamina propria is the place where immune cells settle and exert immune response to exogenous or endogenous pathogen,as well as cancer cells.The effective method of isolation lamina propria mononuclear cells(LPMC)is necessary to analyze the function of intestinal immune cells.Hence,we isolated LPMCs at different enzyme concentrations or at the same concentrations at different time points by using Liberase TM.The best way to isolate LPMCs was established,which laid the foundation for the follow-up experiment.Method: 1?In order to explore optimum concentration and time of digestion for enzyme,we preinstalled four concentration gradient: 50?g/ml,100?g/ml,150?g/ml and 200?g/ml.Moreover,enzyme digestion initial time point was 15 min and at an interval for 5 min.The harvested LPMCs were counted under microscope,and then,determined the optimal digestion time for each concentration gradient.2?After that,we isolated LPMCs at the optimal time for four concentration gradient.Following trypan blue staining,we counted dead cells under microscope and calculated the percentage of dead cells to determine the optimal concentration of enzyme digestion.3?Finally,we labeled LPMC cells with antibody conjugated fluorescein and detected specific antigen expressed on the surface of immune cells with flow cytometry.At the same time,we extracted the peripheral blood mononuclear cells(PBMC)from the patients with the corresponding mucosal tissue as comparison.Result: 1?In order to harvest maximal LPMCs,ending time point for four concentration gradient was: 50?g/ml for 50 min,100?g/ml for 40 or 45 min,150?g/ml for 25 or 30 min and 200?g/ml for 20 min.The number of LPMCs in the Liberase TM 150?g / ml with digesting ending time point at 25 or 30 min was significantly higher than that in the other three groups(P <0.05).2?The percentage of dead cells in group was significantly lower than that in the other three groups when the concentration of Liberase TM at 150?g /ml(P <0.05).There was no significant difference between other three groups(P>0.05).Furthermore,morphological features of LPMCs in 150?g /ml group was better than other group.The number of dead cells was significantly lower than that of the other three groups(P <0.05).3?Phenotypic analysis of LPMCs revealed that the digestion procedure had no adverse effect on the surface marker of CD3,CD4,CD8,CD45,Lin CD127 and CD14.Conclusion: Morphological identification and detection of the specific surface ant igen of the immune cells showed that the optimal procedure for isolation LPMCs was Liberase TM at a concentration of 150?g/ml and digestion terminated time was 25 min.Chapter ? Study on the expression and function of CD169~+ tumor associated macrophages in colorectal cancer patientsObjective: Tumor associated macrophage(TAM)has been shown to play a crucial role in tumor immunity,however the mechanism of TAM on colorectal cancer(CRC)has not been entirely elucidated.The expression and distribution of CD169~+ TAM cells in peripheral blood and tumor tissues of patients with CRC were investigated by immunohistochemistry,molecular biology and cytology.The relationship between CD169~+TAM cells and the clinical indexes of CRC patients was analyzed.The effect of activated CD169~+TAM cells on CRC immune reaction regulation was explored.Method: 1?The expression of CD169 on peripheral monocyte and tumor tissue TAM of CRC patient was detected by flow cytometry.Moreover,subsets of CD14~+CD169~+ monocytes/macrophages were further analysis.The distribution and expression of CD169~+TAM cells was detected by immunofluorescence assay in CRC tumor tissues.2 ? LPS ~+ PMA stimulated CD14~+CD169~+ monocytes/ macrophages in vitro.CD14~+CD169~+ IL-10~+ M2 cells and CD14~+CD169~+IL-12~+ M1 cells were detected by flow cytometry in peripheral blood and tumor tissues of CRC patients.3?Total RNA were extracted from tumor tissues.Relative m RNA expression level of CD169,IL-4,IL-10,IL-12 and IL-13 were explored by q RT-PCR.The expression of CD169~+TAM cell-related serum cytokine in CRC and health controls was investigated.4?According to the TNM staging system,CRC patients were stratified into early and advanced stage.The distribution pattern of CD14~+CD169~+TAM in early and advanced stage of CRC patients was detected.At the same time,the correlation between frequency of CD14~+CD169~+TAM and serum level of CEA was analyzed.5?Advanced CRC patients underwent postoperative chemotherapy were followup to study the effect of chemotherapy on the frequency of CD14~+CD169~+TAM.Result: 1 ? The frequency of circulating and tumor tissues CD14~+CD169~+TAM were significant higher than heathy controls.Moreover,the subset of CD14~+CD169~+CD163~+ and CD14~+CD169~+CD206~+M2 cells were increased in CRC patients.The immunofluorescence assay showed that CD14~+CD169~+TAM infiltrate major in tumor interstitial.2?In vitro activation of CD14~+CD169~+TAM cells found that the frequency of CD14~+CD169~+IL-10~+ cells significantly increased in circulating and tumor tissue.Activated CD169~+TAM secret large amount of IL-10.The percentage of CD169~+TAM was correlated positively with serum levels of IL-10.3?The transcript levels of IL-4,IL-10 and IL-13 m RNA in M2 cells were also increased,and the relative expression levels of CD169 m RNA were correlated positively with IL-4,IL-10 And IL-13.4?According to TNM staging,the number of CD169~+TAM cells in circulating and tissue in advanced stage was significantly higher than that in early stage of CRC patients.The frequency of circulating CD169~+TAM were correlated positively with tissue CD169~+TAM with no regard to early or advanced stage which indicated that CD169~+TAM infiltrated into tumor tissue and played their role in immune response to tumor cells.In addition,the proportion of CD169~+ TAM was correlated positively with serum level of CEA.5?Advanced CRC patients conducted postoperative chemotherapy showed that the proportion of CD169~+TAM cells gradually returned to normal level as chemotherapy proceed.Conclusion: Our study showed that CD169~+TAM infiltrated into tumor microenviroment polarized toward to M2 type and inhibit immune response to tumor cells.Dynamic detection of CD169~+TAM patients in circulating and tumor tissue may have potential value for monitoring tumor recurrence and prognosis.