| Objective To evaluate the changes of PET/CT image characteristics of Lewis Pulmonary Carcinoma Mice before and after radiotherapy,and to analyze the therapeutic effects of stereotactic ablation radiotherapy(SABR)and conventional fraction radiotherapy(CFRT);the related mutated genes after SABR were screened and the related signal pathways were analyzed,so as to provide preliminary research basis and direction guidance for the further application of radiogenomics in stereotactic ablation radiotherapy of lung cancer.Methods Lewis lung cancer cell line(LLC)was purchased for resuscitation and subculture.6-week-old female C57BL/6 mice with an average body weight of 20±2g were selected and subcutaneously injected with 1×10~6Lewis lung cancer cells per mouse.Four groups were randomly assigned according to stereotactic ablative radiotherapy regimen:group Model(0Gy);group 2Gy:conventional fraction radiotherapy group(2Gy×18F);group 8Gy:stereotactic ablative radiotherapy group(8Gy×3F);group 16.4Gy:stereotactic ablative radiotherapy group(16.4Gy×1F),10 mice in each group;The tumor size was about 0.8cm*0.8cm for PET/CT,each mouse was intraperitoneally injected with about 100u Ci 18F-FDG imaging agent.30min later,the mice were anesthetized by inhalation ether,and the anesthetized mice were placed flat on micro PET/CT bed,the limbs were fixed with adhesive tape and placed in the PET/CT imaging system for 15min collection.3D mode was used to collect the images,and the coronal cross-section sagittal plane tomography images were reconstructed by filtering and back projection method for analysis.The images were fused with the same machine for further image analysis.Micro PET-CT positioning,PET imaging images and Pinnacle3 3D treatment planning system were used for image fusion to delineate the target area and plan design,and 50%SUVmax was used as reference to determine the target area during radiotherapy,0.2m L,4%chloral hydrate anesthesia was used,the same postural fixation method was used,and IGRT image guidance was used to verify the position repeatability.The three radiotherapy groups had the same bioequivalent dose of 36Gy for continuous irradiation with an interval of more than 12 hours.After stereotactic ablative radiotherapy,PET/CT imaging examination was performed again to evaluate the efficacy of stereotactic ablative radiotherapy.Tumor tissue samples of mice in each group were collected and stored in-80℃refrigerator.Tumor tissue samples were subjected to HE staining Masson staining and Tunel test,respectively,to analyze the changes of pathological tissues in different radiotherapy groups,and detect the expression of AKT,PI3K,Cytochrome c and cell apoptosis.The group 8Gy with significant therapeutic effect and less tissue necrosis and the group Model were selected:the stereotactic ablative radiotherapy group 8Gy was used for second-generation sequencing genomic detection.DNA and RNA were extracted from frozen tissue of fresh tumors,and Weighted gene co-expression network analysis,KEGG pathway enrichment analysis,clustering analysis and other methods are proposed to select candidate genes from GEO lung cancer genome-wide expression database,the key gene LGR5,which maintains the characteristics of tumor stem cells,was selected for q-PCR experiment to verify and analyze its expression in different radiotherapy groups,to provide preliminary gene screening for subsequent imaging genomics studies in stereotactic ablative radiotherapy of lung cancer.Results 1.Results of body weight and tumor of mice in different treatment groups:(1)there was no significant difference in body weight between SABR(group 8Gy and group 16.4Gy)and group Model within one month,but CFRT(group 2Gy)body weight decreased significantly,P<0.01;(2)After 7 days of radiotherapy,the tumor growth rate of lung cancer mice was significantly controlled,P<0.01;SABR(group 8Gy and group 16.4Gy)had better tumor control effect than CFRT(group 2Gy),P<0.01.2.The comparison of the two PET-CT images showed that:(1)compared with the first PET-CT,the tumors of mice in the second PET-CT showed varying degrees of enlargement and increased tumor metabolism level;(2)The second PET-CT showed that compared with the group Mode,tumor size and metabolic capacity of mice after radiotherapy were significantly reduced,and tumor growth rate was significantly controlled;The local tumor control effect of SABR(group 8Gy and group16.4Gy)were better than that of CFRT(group 2Gy),and the treatment effect of the group8Gy was slightly better than that of the group 16.4Gy,with no significant difference.3.Pathological staining results showed that:(1)HE staining:After radiotherapy,the lung tumor cells were in various shapes and irregular arrangement,and the necrotic tissue increased compared with the group Model;SABR(group 8Gy and group 16.4Gy)showed more obvious necrosis area of lung tumor tissue,especially in group 16.4Gy,most tumor cells showed necrosis.(2)Masson staining:Radiotherapy destroyed the cell structure of lung tumor tissue and reduced the tumor parenchymal cells.Compared with the group Model,the fibrotic tissue was increased and the fibrosis level was significantly higher.Moreover,the fibrosis of SABR(group 8Gy and group 16.4Gy)was more obvious than that of conventional segmentation radiotherapy(group 2Gy),and no significant difference was observed between the group 8Gy and group 16.4Gy.4.Apoptosis of tumor cells in different radiotherapy groups:(1)Immunofluorescence test results showed that PI3K and AKT were highly expressed in the tumor tissues of the group Model,and the expression levels of both PI3K and AKT were decreased in the lung tumor tissues after radiotherapy.The expression levels of SABR(group8Gy and group 16.4Gy)were decreased more significantly than those of CFRT(group 2Gy).There was no significant difference between group 8Gy and group 16.4Gy.(2)Cyt C showed low expression in the group Model,but high expression in the radiotherapy group.The expression level of SABR(group 8Gy and group 16.4Gy)was higher than that of CFRT(group 2Gy),and there was no significant difference between group 8Gy and group 16.4Gy.Tunel test showed that the apoptosis of lung tumor cells after radiotherapy was more significant than that of the model group,and the efficacy of SABR(group 8Gy and group16.4Gy)was better than that of CFRT(group 2Gy).There was no significant difference between the group 8Gy and group 16.4Gy,which was consistent with the above results.5.Results of genomic analysis:We selected the Top 20 mutated genes in lung tumor tissues after SABR for analysis,and most of the mutated genes were missense mutations,all mutation types were SNP,and there were many T and C mutations.KEGG results indicated that the Top20 genes were enriched in different pathways,and the pathways closely related to the mutant genes were extracellular matrix receptor interaction pathway and MAPK signaling pathway.Combined with transcriptome sequencing,the top 30 mutant genes with transcriptional differences were analyzed,and cluster analysis results showed that transcription levels of CHRNG gene,GM20234 gene and LGHG3 gene significantly increased,and the transcription levels of SLC1A2,LGR5,GM2004,ARHGAP20 and MOGAT1 genes were significantly decreased after SABR.6.Q-PCR showed that LGR5 m RNA was highly expressed in the model group and significantly decreased in the radiotherapy group,P<0.01,which was consistent with the results of cluster analysis.Conclusion SABR is superior to CFRT in controlling tumor growth rate,promoting apoptosis and inhibiting proliferation of lung tumor cells.The effect of group 8Gy was slightly better than group 16.4Gy.The mutations after SABR were closely related to extracellular matrix receptor interaction pathway and MAPK signaling pathway.After SABR,the transcription levels of CHRNG,GM20234 and LGHG3 genes were significantly increased,while the transcription levels of SLC1A2,LGR5,GM2004.In the future,we will focus on the above genes and conduct in-depth studies in combination with radiomics. |
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