| Background:Gastric cancer is one of the most common cancers in China and a leading cause ofmortality in many areas. Nowadays surgical ablation is the major way for gastric cancertherapy. In China, about90%cases of gastric cancer, when diagnosed, were in aggressivestage. Even after radical resection, about50%of patients would die because of relapse ormetastasis. Peritoneal metastasis is the major metastasis and death cause in gastric cancercases. The study of cancer stem cells (CSCs) has deeply influenced the understanding ofcarcinogenesis and supplied a new idea to control cancer. CSCs are a population of cellsthat retains the ability to self-renew while possessing the ability to differentiate intoprogeny and play an important role in cancer generation, development, relapse andmetastasis. At present the isolation and purification of CSCs are conducted in the followingways:1. Sorting with flow cytometry or magnetic beads depending on specific CD markers;2. Isolating by using the CSCs’ capability to form clone spheres continually;3.Isolating byusing the biological characteristics of CSCs, such as the ability to enject Hoechst33342(asknown as SP cells). Though confirmed in some studies, gastric cancer stem cells, comparedto other cancers, still remain unknown in many aspects because of the lack of widelyaccepted methods of isolating and concentrating. Since little is known about the surfacemarker of gastric cancer stem cells, concentrating CSCs on the basis of their biologicalcharacteristics and then testing the phenotypes and the functions of the concentrated cellgroups are worthy of exploring. Therefore, this study tried to concentrate stem cells on thebasis of the CSCs’ resistance to chemotherapy, and then further explore the differences ofthe effects of candidate markers on the CSCs’ purification.Methods:(1) Detect the expression pattern of CD44and CD133in gastric cancer tissues andlymph nodes as well as stemness molecular Sox-2and EMT-related marker E-cadherin.(2) Sort CD44hi and CD133+SGC7901cells and test CSCs-related capability such as clone formation, sphere formation and tumorigenic in NOD/SCID mice.(3) Treat SGC7901by different doze of5-FU and VCR respectively and then detectthe change of CD44and CD133by flow cytometry and immunofluorescence staining.Besides, stemness genes and EMT-related genes were tested by RT-qPCR. CSCs-relatedcapability was explored similar to part2.Results:(1) CD44was extensively expressed on cancer cells in primary gastric cancer tissuesand metastasis lymph nodes. Compared to CD44, CD133expression was limit. But thesetwo molecules shared some co-expression area. Besides, there was some area where CD44or CD133co-expressed with Sox-2. However, CD44and E-cadherin are distributed indifferent region.(2) The ratio of CD44positive cells in SGC7901cultivated alone was about90%,while the ratio of CD133positive is only about1%. The10%of the CD44, with the highestexpression,(marked as CD44hi) and CD133+appeared to be more power in clone formation,sphere formation and tumorigenic than unsorted SGC7901.(3) After the treatment of5-FU and VCR, cells became bigger, and flow cytometry,immunofluorescence staining and RT-qPCR showed that CD44expression increased in thecells treated with5-FU and VCR. Besides, the shape and mRNA expression profile showedthe change of EMT after VCR treatment, such as the decrease of E-cadherin and theincrease of vimentin. However, the change of Oct-4and Sox-2didn’t show significantdifferent. VCR treatment could enhance sphere formation, but the capability of cloneformation and tumorigenic didn’t increase after5-FU or VCR treatment.Conclusion:(1) CD44or CD133are co-expressed with Sox-2in gastric cancer cells, whichindicates the existence of GCSCs in cancer tissues. Moreover, CD44and E-cadheringeneral are distributed in different regions, which indicate the link of GCSCs and EMT.(2) The CD44hiand CD133+subpopulation in SGC7901shows the CSCs-relatedcharacteristic including clone formation, sphere formation and tumorigenic in NOD/SCIDmice.(3) The short treatment of5-FU and VCR influences CSCs-related phenotypes andearlier than CSCs-related characteristic, which may be concerned with the inhibition effect of chemotherapeutic on cancer cells including CSCs. However, the EMT change couldhappen in the early stage after VCR treatment. |
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