| Background and purposeUrothelial carcinoma is the fourth for males and female sixth one of the most common malignant solid tumor,of which upper tract urothelial carcinoma(UTUC)5%to 10%of urothelial carcinoma.In western countries,the incidence of UTUC is estimated to be 2‰,and the peak age of the disease is 70-90 years old.The incidence of UTUC in males is three times higher than that in females.UTUC includes carcinogenesis of the urothelial transition from the calyces to the ureter.Like bladder cancer,UTUC can be classified as low malignancy potential papilloma,low grade,high grade papilloma,carcinoma in situ,and invasive urothelial carcinoma.Although both UTUC and bladder cancer are urothelial cancers with many similarities.And the expected consequences and tumor progression are different from bladder cancer according to existing literature reports and clinical experience.Cancer stem cells(CSCs)are a group of tumor cells with self-renewal,multidirectional differentiation potential,and the ability to initiate and reconstruct the phenotype of tumor tissue.In recent years,aldehyde dehydrogenase(ALDH)also gradually began to an important symbol of CSCs are recognized as a variety of tumors.In a recent study on bladder urothelial carcinoma,it was found that patients with high expression of ALDH1A1,ALDH1A2 and ALDH1A3 have a short survival period and poor expected consequences,and the proportion of high grade urothelial carcinoma is also relatively high.Therefore,it is speculated that the high expression of ALDH1 may also play an important role in the progression of upper tract urothelial carcinoma.Recently,the role of tumor immune microenvironment(TIME)in tumor progression has been paid more attention.Tumor cells stimulate the body’s immune system,causing immune cells to secrete pro-inflammatory cytokines.These proinflammatory cytokines can recruit more immune cells such as monocytes and T cells,the new immersion of immune cells in the Tumor microenvironment for immune mediated by differentiation inhibitory Tumor associated macrophage(TAMs)and Regulatory T cells(Regulatory cells,Tregs),which inhibit the immune system’s attack on Tumor,promote the progress of Tumor.Vitamin A is absorbed by cells and transformed into RA under the catalysis of enzymes such as ALDH.RA plays an important role in regulating immune balance.In the immune system,RA is well known for mediating intestinal T cells and B cells to express intestinal"homing molecules"and inducing Tregs and promoting cellular immune tolerance.Therefore,we speculated that ALDH expression may promote the differentiation of immune cells into immunosuppressive cells,leading to tumor immune escape.Currently,clinically,UTUC still lacks effective early diagnostic indicators and relatively poor prognosis,and there is still no broad consensus on standardized treatment.Therefore,in-depth discussion of the specific biological characteristics of UTUC,especially the indicators related to tumor progression,will provide an effective basis for the early diagnosis and treatment targets of UTUC.Method1.Tumor tissue samples(including cases with different histological grades and TNM stages)of UTUC patients in our hospital from 2011 to 2015 were collected,and medical records(including gender,age,TNM stage,degree of tumor differentiation,presence or absence of distant metastasis,etc.)were also collected.The case data of the patients were collected and followed up,and the prognosis of the patients after surgery was recorded.2.The collected fresh tissue samples were made into frozen sections for immunohistochemical staining to observe the content of CD163,a marker of ALDH1A1 and TAMs in tissues,and analyze its relationship with tumor grading.3.Tissue wax blocks collected from the department of pathology of our hospital were sliced and immunohistochemical staining was carried out to observe the content of ALDH1A1 and TAM marker CD163 in the tissue,and analyze its relationship with the prognosis of patients and tumor grading.4.Different levels of urinary epithelial cancer cell lines:T24 cell line,5637 cell line,and normal urinary cell line:Sv-huc cell lines were selected for flow detection to observe the difference in ALDH1A1 content.5.Urinary epithelial carcinoma cell lines were selected and sorted into ALDH1A1+and ALDH1A1-groups by flow technique,and the invasion and migration abilities of the clustered cells were detected by Tran swell.6.Urinary epithelial cancer cell lines were selected and transfected with siRNA to knock down the expression of ALDH1A1,thus forming the ALDH1A1 knockdown group and the Blank group was set at the same time.RNA and protein from Blank group and ALDH1A1 knockdown group were extracted respectively,and the transfection efficiency and ALDH1A1 expression content were detected by qrt-pcr western blot.Tran swell was used to detect the invasion and migration ability of clustered cells.7.T24 cell line of urothelial carcinoma was selected and treated with ALDH inhibitor,and blank group was set at the same time.After co-culture of the two groups with peripheral blood mononuclear cell(PBMC),the ability of cell induction and differentiation of TAM in the two groups was detected.Result1.A total of 30 UTUC specimens were collected from our hospital from 2011to 2015,including 15 high-level specimens and 15 low-level specimens,and all of them were followed up.2.Immunofluorescence results showed that ALDH1A1 was much higher in high-level UTUC tissues than in low-level tissues,and the proportion of immunosuppressive cells(CD163)in tumor stroma was also significantly higher than that in low-level tissues.3.The preliminary results of this study showed that ALDH1A1 expression rate and positive cell number in high-level UTUC tissues were significantly higher than those in low-level groups,and the differential expression of ALDH1A1 in 30 patients was significantly correlated with tumor level and prognosis of patients(p<0.005).4.After flow detection,it was found that the content of ALDH1A1 in T24 cells was the highest.After sorting,it was found that the invasion and migration ability of ALDH1A1~+group was significantly higher than that of ALDH1A1~-group.(P<0.05)the invasion and migration ability of cells treated with ALDH1A1 inhibitor was significantly lower than that of blank group.(P<0.05)the invasion and migration ability of cells in the experimental group after knocking down ALDH1A1 was significantly lower than that in the blank group.(P<0.05)5.After co-culture,TAMs cells(CD163)in ALDH1A1 inhibitor treatment group were significantly lower than those in Blank group(P<0.05).Conclusion1.The expression level of ALDH1A1 was significantly correlated with the clinical data such as tumor grade and survival period.2.ALDH1A1 highly expressed cell lines have higher invasion,migration and mediated differentiation of immunosuppressive cells. |
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