Effect Of Acidic Tumor Microenvironment On Invasion And Migration Of Glioma Cells Through CD44 And Its Mechanism

Glioblastoma is one of the most invasive and migrative primary intracranial tumor which has the worst prognosis.Even though maximal surgical resection followed by radiotherapy and subsequent temozolomide treatment have benefited patients to some extent,the median survival of patients is now only 16~18 months.Cancer cells have the tendency to favor metabolism via glycolysis rather than the much more efficient oxidative phosphorylation pathway which is the preference of most other cells of the body,even in aerobic conditions.Accounting for the specific metabolic way of glucose of cancer cells,extracellular solid tumor pH has been determined to be significantly more acidic than in normal tissues and may decrease as tumor size increases.The intracellular PH of normal tissues is more acidic comparing to the extracellular PH.On the contrary,the extracellular PH is more acidic comparing to the intracellular PH of cancer cells.And,the closer to the core zone of tumor mass,the more acidic it would be.Electrode measurements of PH in human brain tumors have been as low as 5.9 with a mean around 6.8,whereas normal brain tissue has a pH of 7.1.Acidic tumor microenvironment can enhance the stem phenotype of glioma stem cells,promote the capacities of invasion and migration,promote the autophagy and suppress immunoreaction of glioma cells.CD44 is a transmembrane molecule with several isoforms.CD44 is overexpressed in many tumors including glioma cells and promotes tumor formation through interactions with the tumor microenvironment.Research has shown that the expression of CD44 increases with the degree of malignancy of glioma and that is involved with the capacities of invasion and migration of glioma cells.Besides,as the receptors of hyaluronic acid and osteopontin,CD44 plays an important role in glioma cell's invasion and migration.Recently,it was pointed that hypoxia condition could upregulate the expression of intracellular and extracellular domain of CD44 and eventually enhance the stem phenotype of glioma cells.Data has shown that CD44 correlates with a poorer prognosis of glioma patients.Little is known about the correlation between the acidic microenvironment and the invasion and migration of glioma cells and the role CD44 plays within it.In this study,we used the methods of titration with HCl and NaOH to establish a faintly acidic environment in vitro so as to examine whether the acidic conditions could affect the morphology of U87 and U251 glioma cell lines and then we confirmed whether acidic conditions could affect the expression of CD44 and the potential mechanism.We utilized sh-RNA to knock down the expression of CD44 to examine its effect on invasion and migration of U87 and U251 cells and to further examine that acidic tumor microenvironment affect the capabilities of glioma cells by upregulating the expression of CD44 and the internal mechanism.This study mainly investigated the role and underlying mechanism of the effect of acidic microenvironment on invasion and migration of glioma cells through CD44,including three parts:1.The expression level of CD44 correlates with the pathology grade and histological origin of glioma and it is associated with patient's prognosis.French?Lee and Sun databases From R2: Genomics Analysis and Visualization Platform shows that the mRNA expression level of CD44 not only correlates with pathology grade but also with its histological origin.TCGA,French mRNA and clinical data was retrieved to perform Kaplan-Meier survival cure,which shows that high expression of CD44 correlates with a poor prognosis.By analyzing TCGA-GBM database,we found that the expression level of CD44 is higher comparing with the other subtypes of GBM.2.The effect of tumor acidic microenvironment on capabilities of invasion and migration of U87 and U251 glioma cells and the effect on the expression of CD44 and HIF-2?.We utilized the methods of titration to build a acidic environment(PH=6.4)in vivo.By using transwell assay,we examined the differences of capabilities of invasion and migration between acidic group(PH=6.4)and normal group(PH=7.4).Western blot was adopted to examine the expression of CD44 and HIF-2?.3.CD44 regulates the capabilities of invasion and migration through Src.We utilized sh-RNA to knock down the expression of CD44 and examined the effect on invasion and migration.Western blot was used to examine the level of phosphorylation of acidic group and control,proteins correlate with invasion and migration like N-Ca,Vimentin and MMP-2 in sh-NC group and sh-CD44 group.Conclusion:1.The expression level of CD44 in glioma has positive correlation with histology level and those originated from astrocytoma tends to have a higher expression than olidendroma.Comparing with other subtypes,the expression level of CD44 tends to be higher in mesenchymal subtype of GBM.The expression level of CD44 corelate with a poor prognosis of patients.2.U87 cell cultured at PH 6.4 was in nest-like merisis while U251 cell cultured at different PH show no differences.Acidic tumor microenvironment upregulates the expression of CD44 by upregulating the expression of HIF-2?.3.Acidic tumor microenvironment enhances the capabilities of invasion and migration of glioma cells.Knock down the expression of CD44 reduces the capabilities of invasion and migration of glioma cells.The possible mechanism of upregulated capabilities of invasion and migration in tumor microenvironment could correlate with the upregulated level of phosphorylation of Src.