Heterogeneity Of Epidermal Growth Factor Receptor Expresses In Glioblastoma's Areas And Its Association With Tumors' Invasion Ability

Objectives To explore EGFR's(epidermal growth factor receptor) association with C6 glioma cells' invasiveness capacity.and the difference invasiveness of GBM's different regions. Methods C6 cells were dealed with epidermal growth factor(EGF) or Gefitinib. Then cells were divided into three groups,which were control group, EGF group and Gefitinib group. Cell invasion assay and cell wound-healing assay were used to evaluated the influence of EGFR on C6 cells' s invasion and migration ability. One-way analysis of variance was used to statistical analyzed experimental data.C6 glioma cells were injected into the right caudate nucleus of Sprgue-Dawley rats by stereotactic technique. After inoculation rats' common condition and body weight were observed. At the same time Bederson grading system was used to evalute neural function of rats. Then we won't kill rats until 28 days after inoculation, we should get rats' brain tissues to observe tumor's growth status. HE staining was used to observing C6 glioma's morphology. Immunohistochemical and immunofluorescence technique were used to observe the expression situation of vimentin,tubulin and EGFR in tumors. Then Image-Proplus 6 software was used to measure the optical density of these proteins in the brain tissue sections' tumors' area, tumors' edge and the normal brain tissue, and one-way analysis of variance way was used to statistical analyzed the different of the optical density of each protein in the three regions. Results During the invasion assay, the invasion number of C6 cells in EGF group,control group and Gefitinib+EGF group were 74±7,30±8,52±8, respectively. One-way analysis of variance was used to statistical analyzed experimental data. There was significant different between the three groups(F=73.59, P<0.001), by pairwise comparison, there was also significant different in each two groups of three groups(P<0.0001). As cell wound-healing assay showed that C6 cells' migration distance of EGF group,control group and Gefitinib+EGF group were 50933.33±1662.24mm and 12966.67 ± 2365.51mm,1623.33 ± 515.01mm and 4300 ± 840.18mm,1310.00 ± 570.00mm and 2340.00 ± 841.49mm at 24 h and 36 h respectively. Using One-way analysis of variance to statistical analyzed showed that t There was significant different between the three groups(F=11.83, P<0.001;F=41.06, P<0.001,respectively at 24 h and 36h). There was also significiant different between EGF group and control group, as well as EGF group and Gefitinib+EGF group(P<0.001) at 24 h and 36 h,but there was not different between Gefitinib+EGF group and control group at 24 h and 36h(P>0.05).After inoculation surgery, the general codition and Bederson score of the SD rats in the control group had no obvious change than before. But for vaccinated group,in earlier period, the SD rats were performance same as before. During middle period, the weight-loss of rats was obvious and they ate and drank less than before.In later period the symptoms includes hemiplegia, epilepsy, dyscrasia and so on. When the study was done, the Bederson score of these rat was significantly reduced than before. HE staining showed that the C6 cells were arranged long fusiform, there were pale necrosis aeras around the tumor boundary.The vimentin's immunohistochemistry examination showed that it expression in tumor cell's cytoplasm as brown. The OD value of tumor parenchymal is 8711.33±3138.35, tumor boundary's OD value is 3952.63±1525.29 and normal brain tissue's OD value is 1257.94 ± 960.39. There was significant different between the three groups(F=48.93,P<0.001), by pairwise comparison, there was also significant different in each two groups of three groups(P<0.0001). So vimentin was highest expressed in tumor parenchymal.For tubulin the result showed that it expression in normal brain cell 's cytoplasm as brown.The OD value of tumor parenchymal is 223.44±57.69, tumor boundary's OD value is 3280.82±1447.74 and normal brain tissue's OD value is 10808.52±6389.99. There was significant different between the three groups(F=31.04,P<0.001),by pairwise comparison, there was also significant different in each two groups of three groups(P<0.0001). So tubulin was highest expressed in brain tissue.As for EGFR the result showed that it expressed brown colour as different levels in normal brain tissue, tumor tissue and tumor boundary. The OD value of tumor parenchymal is 14123.09±9397.98, tumor boundary's OD value is 22101.43±9705.92 and normal brain tissue's OD value is 7122.46±5294.36. There was significant different between the three groups(F=12.01,P<0.001), by pairwise comparison, there was also significant different in each two groups of three groups(P=0.027,P<0.05). Then EGFR was highest expressed in tumor boundary. Immunofluorescence test showed that vimentin expressed in tumor tissue, tubulin expressed in normal brain tissue, in addition at the edge of tumor both ptoteins expressed there. EGFR mainly expressed in tumor tissue, and most of its espression regional were same with vimentin, which located at tumor boundary. Conclusions EGFR promotes the invasion and migration ability of C6 cells. EGFR was highest expressed in tumour's boundary. So the situation that EGFR advanced GBM's invasion may has regional advantage. This research contributed to the understanding of GBM biological characteristics, and provided the experimental basis for the diagnosis and treatment.