ALDH1A1~+CD44~+ Gastric Cancer Cells Population Is Associated With Tumor Stem Cells Property

Backgrounds:Despite decades of efforts and a diversity of treatments,gastric cancer is still a disease with poor prognosis.The problem mainly lies in the unclear etiological factors and pathogenetic mechanisms in gastric cancer.Research found some malignant tumors cells'biological characteristics are very similar to stem cells.These phenomena might be explained by the so-called tumor-initiating cells or cancer stem cell(CSC)theory.Accumulating evidence has proposed that CSC plays a responsible role for cancer progression and relapse.In recent researches,more and more solid organ tumors' CSC were discovered and approved.However,either whether gastric cancer possesses cancer stem cells or biomarker of gastric cancer stem cell is still unclear.Aldehyde dehydrogenase 1 A1(ALDH1A1)and CD44 positive cells have recently been suggested as a marker for cancer stem cells or cancer stem-like cells in many human malignancies,but the exact function of ALDH1A1 and CD44 in gastric tumor tissue and cell lines remains largely unknown.The purpose of this study is to investigate the function,biological characteristics and stem cell characteristics of ALDH1A1 and CD44 positive cells in vitro and in vivo.Methods:1.The expression of ALDH1A1 and CD44 in 66 human gastric cancer tissue and 29 normal gastric tissues were evaluated by immunohistochemical method;2.The expressions of ALDH1A1 and CD44 of gastric cancer cell lines MKN28,SGC7901 and MKN45 were investigated by immunofluorescence method;3.Magnetic Activated Cell Sorting(MACS)and fluorescence activated cell sorting(FACS)were used to sorting different phenotype of gastric cancer cell lines.The cells were divide into five groups:Group A,ALDH1A1+CD44+;Group B,ALDH1A1+CD44-;Group C,unsorted cells,Group D,ALDH1A1-CD44+;Group E,ALDH1AI1-CD44-;4.Detected different group cells' by chemoresistance,Flat colony forming assay,Soft agar clonogenic assay and transwell invasion assay;5.Western Blot assay was used to detected CD133,OCT4,ABCG2 and MMP2 protein expression in different groups;6.Tumorigenicity of different cells was detected in BALB/C nude mice.Results:1.The ALDH1A1 and CD44 expressed in most gastric carcinoma specimens,The outcomes of ALDH1A1+ expression in well-differentiated,moderately-differentiated and poor-differentiated gastric tumor tissues of patients are 18/21,27/30,14/15,respectively.The mean rate of ALDH1A1+ expression in the gastric tumor tissues was 89.4%(59 of 66).The expression rate in none-cancer tissues is 24.1%(7/29);CD44+ expression in well-differentiated,moderately-differentiated and poor-differentiated gastric tumor tissues of patients are19/21,28/30,14/15,respectively.The mean rate of CD44 expression in the gastric tumor tissues was 90.9%(61 of 66),The expression rate in none-cancer tissues is 41.4%(12/29).2.ALDH1A1 and CD44 express differently in different gastric carcinoma cell lines,the positive rate of ALDH1A1 expressions is 2.3%,3.4%,6.3%,respectively.the positive rate of CD44 is 3.1%,5.2%,7.1%,respectively.3.Isolated cells were checked by immunofluorescence cell staining and flow cytometer,except SGC7901 cell line,MKN28 and MKN45 cell lines positive rate was both above 95%.4.Different groups show different resistant to 5-Fu,when concentrations is at 100ug/ml group A with MKN28 or MKN45 inhibition ratio is 21.4%and 16.7%,other groups are all above 59.4%.5.The Flat colony forming efficiency in the ALDH1A1+CD44+ cells from MKN28 and MKN45 is 36%and 42%.Soft agar colony forming efficiency is 4.3%and 4.9%.6.Transwell invasion assay,the number of ALDH1A1+CD44+ cells is 265.8 and 293.8 in lower surface of the membrane,the number of cell between 68.8-164.3 in other groups.7.The expression of CD133,OCT4,ABCG2 and MMP2 in the ALDH1A1+CD44+ cells from MKN28,MKN45 are 1.9,0.9,1.4,1.5 fold and 2.2,2.6,1.8,2.1 fold compared with unsorted isogenic cells.8.The tumorigenicity of ALDH1A1+CD44+ cells from MKN28 is 3/5,4/5,5/5 when cell number were 1.5×103,1.5×104 and 1.5×105.The tumorigenicity of the ALDH1A1+CD44+ cells from MKN45 is 3/5,4/5,5/5 when cell number were 1.5×103,1.5×104 and 1.5×105 in athymic mouse.All the ALDH1A1+CD44+ cells show higher oncogenicity compared with the isogenic cells.Conclusions:1.The ALDH1A1 and CD44 express differently in different stages of human gastric cancer cells.2.The ALDH1A1+CD44+ cells from gastric carcinoma cell lines have higher drug resistance,more colony forming efficiency and invasion ability.3.The expression of CD133,OCT4,ABCG2,MMP2 in the ALDH1A1+CD44+ cells are higher than the isogenic cells.4.The xenograft tumor formation in vivo of the ALDH1A1+CD44+ cells is higher than the isogenic cells.5.The ALDH1A1+CD44+ cells have the characters of stem cells.ALDH1A1 and CD44 may be the candidate marker for stem cells of gastric carcinoma.