Modulation Of P2X₇Receptor In The Homing Of EPCs To Brain Glioma And The Growth Of Glioma

Background and ObjectivesEndothelial progenitor cells （EPCs） are a cell population that have high proliferationpotential and can differentiate to mature endothelial cells. EPCs in circulation play animportant role in repairing vessels and maintaining the stabilization of vasculature.Meanwhile, EPCs can mobilize from bone marrow and home to ischemia and angiogenesisarea in response to various physiological or pathological stimuli. The bone marrow derivedEPCs contribute to tumor-related angiogenesis in the manner of paracrine or postnatalneovasculature, which is referred to as vasculogenesis. Glioma is the most common primarytumor in central nervous system and one of the hypervascular tumors in human. During theprogression of glioma, a large number of angiogenesis are formed. Many researches havebeen shown that EPCs can home to the hot spot of neovascularization in tumor tissue andcontribute to the formation of tumor-related microvessels. Our previous study also found thatmost of the newborn microvessels are located in the peritumor area, where is the priorlocation of the transplanted EPCs. Based on this property, it is expected to detect tumorinvasion or satellite lesions via transplated labled EPCs by related radiological techenology,such as maganetic resonance imaging （MRI） scanning technology, thereby improving thelevel of clinical diagnosis and treatment. Our previous study confirmed that MRI can revealthe distribution of the magnetically labeled EPCs in glioma tissue in a short period. However,with the extension of time, MRI could not detect the transplanted cells since the concentrationof USPIO was lower in the glioma tissue which was resulted from the death of EPCs.Moreover, although most of the transplanted EPCs home to the glioma tissue, a few of thecells were found in the spleen of the rat. Therefore, if we want to use the EPCs as a probe todetect glioma or a vector to bring antitumor agents to the glioma, how to enhance theproliferation and targeting ability of transplanted EPCs is the most important problem toresolve. The ruined neurons or hyperplastic glial resulted from the development of brain glioma release a great quantity of ATP to extracellular compartment. ATP not only act as anintracellular energy source, but also as the ligand of P2receptors. P2X₇receptor （P2X₇R） iswidely distributed, as most bone marrow cells including mononuclear cells, are reported toexpress the P2X₇receptor. The activation of P2X₇receptor could modulating cell proliferation,differentiation, apoptosis and migration. So, it is possible that activation of P2X₇receptorcould promote the proliferation and targeting ability of EPCs. The tumor cells used in thepresent study is C6glioma cells, which is the most widely used cells in the brain gliomaresearch. Since P2X₇receptor also is reported to express in C6glioma cells, whetheractivating the P2X₇receptor of EPCs by systemic administration could affect the gliomagrowth remains unknown.To test above hypothesis, the present study was divided into two parts. Part one: Theexpression of P2X₇receptor in EPCs was determined by western blot and immunofluorescencestaining. Evaluated the effect of P2X₇R activation on cell proliferation, apoptosis andmigration through stimulating EPCs with BzATP, a P2X₇R agonist. The targeting ability oftransplanted EPCs after long-term suppression of P2X₇R by persistent BBG stimulation wasevaluated. Part two: The expression and distribution of P2X₇R in C6glioma cells wasconfirmed by western blot assay and immunofluorescence staining. The change of intracelluar[Ca2+]i in C6glioma cells after activation of P2X₇R with BzATP was detected to elucidate thefunctional expression of P2X₇R. C6glioma cells proliferation after stimulation with BBG wasmeasured by Ki-67staining, MTT assay and RNA interference to uncover the effect of P2X₇Ron C6cells growth. Glioma growth after long-term suppression of P2X₇R with BBGadministration. The effect of P2X₇R activation on proliferation and targeting ability of EPCsand the progression of glioma were determined to assess the feasibility and security ofenhancing the targeting ability and proliferation of EPCs by modulating P2X₇R.Materials and methodsThe whole research was composed of in vitro and in vivo study. The in vitro experimentswere conducted on cultured rat spleen-derived EPCs and C6glioma cells. For in vivoexperiments, rat brain glioma model was used.The effect of P2X₇R on proliferation, apoptosis, migration and targeting of ratspleen-derived EPCs 1．Healthy SD rat speen derived-mononuclear cells were isolated by density gradientcentrifugation, after3days cultured,non-attached cells were washed away by DPBS and thenattached cells were cultured for7days, the medium was changed every3days. EPCs werecharacterized as adherent cells were double positive for DiI-acLDL uptake and lectin bindingby immunofluorescence staining. Meanwhile, EPCs were also identified by specialmorphological characteristics, such as colony, line-like or tube-like structure.2．P2X₇receptor expression in EPCs was detected by western blot. Meanwhile, thedistribution of P2X₇receptor in EPCs was evaluated by immunofluoresence staining.3．Calcium imaging in EPCs after BzATP stimulation with different concentration in thepresence or absence of BBG were detected by fluorescence.4．The EPCs were divided into three groups including control group, BzATP interventiongroup and BzATP+BBG intervention group. All groups cultured in37℃,5%CO2incubatorfor24h, then examined the optical density value with microplate reader to reveal effect ofP2X₇R on the proliferation of EPCs.5．In order to confirm the influence of P2X₇R on apoptosis of EPCs, the7days culturedEPCs were stimulated with BzATP in the presence or absence of BBG. Then, the apoptosislevel of treated cells were measured by immunofluorescence staining for Annexin V or DAPI.6．The migration of EPCs with BzATP stimulation in the presence or absence of BBGwas assessed by scratch-wound migration assay.7．Brain glioma model was constructed using stereotaxic apparatus in healthy SD rat.The distributin of USPIO labeled-EPCs in the glioma tissue was evaluated by conventionalMR imaging and Perls staining.8．The expression of CXC chemokine ligand1（CXCL1） and monocyte chemoattractantprotein-1（MCP-1） in glioma tissue was detected using western blot assay.The effect of P2X₇receptor on the growth of brain glioma1．The expression of P2X₇R in C6glioma cells was detected using western blot assay,while the distribution of P2X₇R was determined by immunofluorescence staining.2．Activated P2X₇R in C6glioma cells with BzATP of different concentration anddectected the changes in intracellular [Ca2+]i after BzATP stimulation in the presence orabsence of BBG. The ratios of fluorescence reflected the effect of P2X₇antagonist on thechanges in intracellular [Ca2+]i. 3．C6glioma cells were cultured with BBG of different concentration in the presence orabsence of BzATP, suramin （a P2Y2receptor antagonist） and gefitinib （an EGFR inhibitor）,respectively. Then, the cells proliferation was measured by MTT assay and Ki-67staining toevaluate the effect of P2X₇R on C6glioma cells growth.4．C6glioma cells were cultured with BBG in the presence or absence of BzATP for24h.After that, the expression of P2X₇R, EGFR, p-EGFR and P2Y2receptor were detected bywestern blot, respectively.5．To knockdown the expression of P2X₇R in C6glioma cells, shRNA was used. AfterC6cells were transfected with shRNA, the changes in cells proliferation were determinedusing MTT assay.6．Western blot detected the expression of EGFR and p-EGFR protein in shRNA orempty vector transfected C6glioma cells.7．The shRNA or empty vector transfected C6glioma cells were cultured with BzATP for24h to confirm the specificity of BzATP on P2X₇R expression.8．Brain glioma model was constructed using stereotaxic apparatus in healthy SD rat andrandomly divided into control, BBG treatment, empty vector and P2X₇R shRNA treatmentgroup. The tumor growth of each group was determined using MRI, while the angiogenesis intumor of each group were evaluated using CT perfusion.9．The relative tumor size of each group was determined by Nissl staining, cellsproliferation was measured by Ki-67staining, the expression of VEGF or EGFR was detectedthrough immunohistochemical staining for VEGF and EGFR, respectively. Microvesseldensity of glioma tissue from different group was determined by immunohistochemicalstaining for CD34.10．The expression of EGFR, p-EGFR, P2X₇R, VEGF, HIF-1α, P2Y2and caspase-3protein in glioma tissue were determined using western blot assay.ResultsThe effect of P2X₇R on proliferation, apoptosis, migration and targeting of ratspleen-derived EPCs1．The lately-isolated MNCs were small and round, after3days cultured, attached cellsbecome bigger and more lucency. With the extension of time, the attached cells increased, appeared more spindle cells and exhibited the typical cobblestone morphology. After7dayscultured, fluorescence microscope confirmed that90%of the adherent cells were doublepositive for DiI-acLDL uptake and lectin binding.2．Both western blot and immunofluorescence confirmed the expression of P2X₇receptorin rat spleen-derived endothelial progenitor cells.3．BzATP stimulation induced a significant increase of intracellular [Ca2+]i in EPCs inthe manner of dose-dependent. While, BBG co-culture inhibited BzATP-induced [Ca2+]iincrease.4．P2X₇receptor activation induced by BzATP stimulation enhanced the proliferation ofEPCs which was inhibited by BBG.5．Although Annexin and PI staining demonstrated that the BzATP could induced morecells showing apoptosis feature, the difference between the cells with or without treatmentwas not significant.6．The migration of EPCs was promoted by BzATP stimulation, which could be reversedby BBG intervention.7．The MRI detected glioma in the cerebral parenchyma, which showed high intensity inT2-weighted imaging （T2WI） as well as enhancement at10days post-injection of C6gliomacells. T2WI revealed the low signal intensity in the periphery of tumor at1dayspost-transplantation of USPIO labeled-EPCs. Perls staining showed that there were numerousblue-stained cells accumulating in the tumor neovasculature region. Compared with theglioma tissue from the control group, there were less blue-stained cells in glioma tissue withBBG treatment.8．P2X₇R antagonist, BBG, inhibited the expression of CXCL1, but not MCP-1inglioma tissue.The effect of P2X₇receptor on the growth of brain glioma1．Protein expression of P2X₇receptor was detected both in glioma tissue and culturedC6glioma cells.2． BzATP dose-dependently induced a notable increase in intracellular [Ca2+]iconcentration in C6glioma cells, but this effect of BzATP could be suppressed by BBGtreatment.3．BBG with low concentration showed no effect on C6glioma cells growth, while BBG with higher concentration dose-dependently promoted the proliferation of C6glioma cells.This effect could be inhibited by EGFR inhibitor or P2Y2receptor antagonist, but not byBzATP co-culture.4．BBG stimulation significantly downregulated the exprsession of P2X₇R in C6gliomacells, which was abolished by BzATP. Contrary to the change of P2X₇R, the expression ofEGFR or p-EGFR protein and P2Y2receptor in C6glioma cells were markedly elevated byBBG stimulation.5．P2X₇R knockdown in C6glioma cells using shRNA significantly promoted theproliferation of the treated cells.6．P2X₇R knockdown in C6glioma cells dramatically upregulated the expression ofEGFR or p-EGFR and P2Y2receptor in the cells.7． Although BzATP partly restored the decreased P2X₇R expression in shRNAtransfected C6glioma cells, the values showed no significant difference.8．MRI could detect the glioma growth at about10days after C6glioma cells transplant.The tumor size was significantly increased during the observation. BBG treatment or P2X₇Rknockdown promoted the tumor growth by about40%and50%, respectively. In addition,compared to the control, the glioma tissue with or P2X₇R knockdown BBG treatmentexhibited higher rCBV and PS value.9．Long-term suppression of P2X₇R via BBG treatment or P2X₇R shRNA transfectioncould markedly promoted the proliferation of glioma cells and increased the tumor size andthe expression of EGFR and p-EGFR protein.10．Long-term inhibition of P2X₇R induced by BBG or P2X₇R shRNA markedlydownregulated the P2X₇R expression and increased the expression of EGFR、p-EGFR、VEGF、HIF-1α and P2Y2receptor. Although the protein expression of cleaved caspase-3wasdecreased either in the P2X₇R shRNA or BBG treatment group, the difference between thegroups was not significant.Conclusions1．Activation of P2X₇R in EPCs could promote the proliferation and targeting ability ofthe cells.2．The USPIO-labeled EPCs could home to angiogenesis region of the glioma. P2X₇R suppression could inhibit the accumulation of exogenous EPCs in glioma tissue, whichsuggests that is possible to change the targeting ability of EPCs via modulating P2X₇R.3．Suppression of P2X₇R can increase the expression of P2Y2receptor in C6glioma cellswhich could transactivate EGFR protein to promote the glioma growth.4．Suppression of P2X₇R in C6glioma cells can upregulate the expression of EGFR,HIF-α and VEGF protein which exerts a promoting effect on angiogenesis to further supportglioma growth.5．According to above findings, the present study suggests that activation of P2X₇R canmake the EPCs do better as a targeting and tracing probe for MR imaging through enhancingcell proliferation and targeting ability of EPCs. In addition, the P2X₇R in C6glioma cells mayexert a antitumor effect, so activation of P2X₇R in EPCs may not promote the glioma growth,which suggests a great biological security for P2X₇R modulation in EPCs.