Preliminary Study On Carbon Ions Irradiation To Inhibit The Proliferative Activity And Angiogenesis Of Rat C6 Glioma

Purposes:The proliferative activity and microcirculatory changes of rat C6glioma before and after carbon ion irradiation（CIR）were dynamically monitored by a combination of imaging and molecular pathology,and the inhibitory effects of carbon ions on glioma cell proliferation and angiogenesis were initially explored.Methods and materials:Eighty-five male SD rats were grown with C6 glioma cells in the right basal ganglia region,and 78 adult tumors were detected by energy-spectral CT-enhanced scanning 14 days after inoculation.Six tumor-bearing rats were randomly selected as pre-irradiation molecular and pathological controls,and the remaining 72 rats were randomly divided into 0 Gy group（n=24）,1 Gy group（n=24）and 2 Gy group（n=24）for CIR and underwent energy-spectral CT-enhanced scans at day 7,14 and 21 post-irradiation.After each scan,3 rats were randomly selected to collect brain tissue for histopathological staining;after the completion of energy-spectral CT-enhanced scans at day 7 and 14 after irradiation,3 rats were randomly selected to collect tumor tissues,and the expression of vascular-associated proteins HIF-1α,VEGFA,VEGFR-2 andα-SMA were measured by Western Blot（WB）.The energy spectrum data were analysed using AW4.7 GSI viewer software,and the 40-140 ke V single-energy CT values,the best single-energy CT value(CT _{70ke V}),and the iodine concentration value（IC）in the solid tumour region（R1）,the peripheral region（R2）and the contralateral normal brain tissue region（R3）were measured and recorded,and the slope of the energy spectrum curve(K_{（40-70 ke V）})was calculated.The percentages of immunohistochemically stained positive cells of Ki-67,HIF-1α,VEGF andα-SMA in brain tissues and the expression of vascular-associated proteins in tumor tissues were counted using Image J software.Pearson correlation test was used to analyze the correlation between CT quantification parameters of energy spectrum and Ki-67,HIF-1α,VEGF andα-SMA positive cell counts.Results:1.Survival analysis:The median survival times of the rats in the 0 Gy,1Gy and 2 Gy groups were 22 days（95%CI,18.61-25.40）,30 days（95%CI,24.16-35.84）and 33 days（95%CI,18.40-47.61）,respectively,the differences between the 1 Gy and 2 Gy groups and the 0 Gy group were statistically significant（p<0.05）2.Tumor volume detection:Before CIR,the tumor volumes in the 0 Gy,1 Gy and 2 Gy groups were 114.38±21.46 mm³,110.50±17.11 mm³and 106.61±15.61 mm³,respectively,and the differences were not statistically significant（p>0.05）.7 days after CIR,the tumor volumes gradually increased,and were 219.44±47.63 mm³,168.80±40.58 mm³and 159.72±34.90 mm³,respectively,with a significant increase in the 0 Gy group and a statistically significant difference compared to the 1 Gy and 2Gy groups（p<0.01）.At 14 days after CIR,the tumor volume in the 0 Gy group increased further（322.03±72.34 mm³）,while the tumor volume in the 1 Gy and 2 Gy groups did not progress significantly or decreased,169.84±72.15 mm³and121.17±43.53 mm³,respectively,with statistically significant differences between the0 Gy group and the 1 Gy,2 Gy group（p<0.001）.Only 6 rats in the 1 Gy group and 5rats in the 2 Gy group survived 21 days after CIR.The tumour volumes in the two groups were further reduced to 95.63±37.19 mm³and 108.69±71.23 mm³,respectively,with no statistically significant difference between them（p>0.05）.3.Energy-spectrum CT and pathological findings:Solid area（R1）:7 days after CIR,CT _{70 Ke V},IC and K_{（40-70 ke V）}decreased in the 2 Gy group and did not progress significantly in the 1 Gy group,and the difference was statistically significant compared with the 0 Gy group（p<0.05）;14 days after CIR,CT _{70 Ke V},IC and K_{（40-70ke V）}were significantly reduced and the difference was statistically significant compared to the 0 Gy group（p<0.001）.There was a strong positive correlation（r>0.68,p<0.05）between the values of all three groups of tumor solid zone energy spectrum parameters detected and the percentage of Ki-67,HIF-1α,VEGF andα-SMA positive cells.Ki-67 correlated most strongly with IC,with r>0.90 in the 0Gy group,r>0.85 in the 1 Gy group and r>0.86 in the 2 Gy group,all with p<0.001.Peritumoral area（R2）:The values of each energy spectrum parameter in the 1 Gy and 2 Gy groups decreased gradually at 7 and 14 days after CIR.The differences inCT _{70 ke V}and K_{（40-70 ke V）}were statistically significant in the 2 Gy group compared to the 0 Gy group 7 days after CIR（p<0.05）,and the differences in all energy spectrum parameters were statistically significant in both the 1 Gy and 2 Gy groups compared to the 0 Gy group 14 days after CIR（p<0.01）.Strong positive correlation between each energy spectrum parameter and Ki-67 proliferation index,VEGF andα-SMA positive cell percentage in the 0 Gy group（r>0.68,p<0.05）,with the strongest correlation between VEGF and IC values（r>0.88,p<0.001）;strong correlation between CT _{70 ke V},IC and Ki-67 proliferation index,HIF-1α,VEGF andα-SMA positive cell percentage in the 1 Gy group（r>0.70,p<0.01）;there was strong positive correlation between 2 Gy group IC and K_{（40-70 ke V）}and Ki-67 proliferation index,HIF-1α,VEGF andα-SMA positive cell percentage,respectively（r>0.64,p<0.05）.4.Western-blotting results:HIF-1α,VEGFA,VEGFR-2 andα-SMA protein expression were down-regulated in the 2 Gy group at 7 days after irradiation compared with the 0 Gy group in the same time point（p<0.05）;vascular-related protein expression was significantly down-regulated in the 1 Gy and 2 Gy groups 14days after irradiation（p<0.001）.Conclusion:1.CIR was able to inhibit the local growth of C6 glioma in rats and prolong the survival of tumour-bearing rats.2.The CT _{70 ke V},IC and K_{（40-70 ke V）}and Ki-67 value-added index,HIF-1α,VEGF,α-SMA positive cell expression and vascular-related protein expression were all reduced after CIR in the 1 Gy and 2 Gy groups of rats.CIR may control local tumor progression by inhibiting tumor cell proliferation and tumor angiogenesis.3.The quantitative parameters of the energy spectrum CT(CT _{70 ke V},IC and K_{（40-70 ke V）})correlate well with Ki-67,HIF-1α,VEGF,α-SMA and can indirectly reflect the inhibitory effect of CIR on the proliferative activity and angiogenesis of rat C6 glioma cells,enabling non-invasive dynamic detection.