| Lung cancer is one of the malignant tumors with the highest incidence and mortality in the world.The pathological types of lung cancer are divided into Small-cell lung carcinoma and Non-small cell lung cancer（NSCLC）.Lung cancer has the characteristics of unclear early clinical symptoms and difficult diagnosis existing in the middle and late stage.Chemotherapy-based comprehensive treatment is the primary method to treat NSCLC.Perfluorocarbon（PFC）is a kind of fluorine-containing organic compounds in which the hydrogen atom in the hydrocarbon chain is replaced by fluorine atom.Perfluorooctyl bromide（PFOB）is a kind of PFC.The PFC nano-emulsion has a high capacity of dissolved oxygen,which can be used as an exogenous oxygen carrier to alleviate the tumors hypoxia.PFC can also be used as a probe of magnetic resonance imaging（MRI）for its characteristic of only existing in bone tissue.Previous studies of this project showed that when PFC nanoemulsion was used as nano probe for 19F-MRI imaging examination of lung cancer,it had higher accuracy and specificity for imaging diagnosis of lung cancer through intratracheal（IT）administration than traditional intravenous（IV）administration.It was also observed that PFC following intratracheal administration can inhabit the tumor volume of lung cancer significantly when combined with radiotherapy.This points out that PFC had an effect of sensitizing radiotherapy.Objective:Based on rabbit VX2 orthotopic lung carcinoma models following radiotherapy,IT PFOB,IT PFOB plus radiotherapy,we aim to study the mechanism of PFC sensitization radiotherapy through analyzing tumor tissue,and the effect of different treatment methods on urine and multi-organ tissue metabolomes by untargeted metabolomics.Method:The model of the VX2 orthotopic lung carcinoma was made in the experimental rabbits.The healthy rabbits without cancer and the rabbits with orthotopic lung carcinoma were divided into the healthy group（n=6）,model group（n=3）,radiotherapy group（n=4,5Gy）,IT PFOB probe group（n=4,0.5 mg/kg）,IT PFOB probe plus radiotherapy group（n=4,5Gy,0.5 mg/kg）.The probe was given on the 4th day after the enrollment,and radiotherapy was given on the 5th day.The volume of tumor was measured on CT on the first,7th and 14 th days after enrollment.Urine samples were collected on the 4th,5th,6th,7th and 8th days after enrollment.Samples of tumor tissue,heart,spleen,liver,lung,kidney,and muscle were collected after execution on the 15 th day.The collected urine and tissue samples were analyzed by using UHPLC-Q-TOF-MS.The peak extraction,noise reduction,normalization and MS/MS spectrometry identification were carried out by MS DIAL 4.48 and MS FINDER 3.50 software.The principal component analysis（PCA）,Partial least squares discrimination analysis（PLS-DA）,Heatmap was conducted by Metabonalyst 5.0 to analyze the differences amongst groups.T-test was conducted between radiotherapy group,IT PFOB probe group,IT PFOB probe plus radiotherapy group and model group.The differences compounds were screened by P<0.05.Based on HMDB database,the biological significance of the different compounds was analyzed,and the metabolic pathway was analyzed by Reactome pathway database.The results showed that the level of endogenous metabolites in multi tissues were affected by different treatment methods by using multi omics factor analysis（MOFA）,and a small amount of inference factors were used to capture the complete source of changes of multi-tissue,and the time series of urine fluid was analyzed by using Mfuzz.Result:CT results showed that the tumor volume growth rate of the combined group was significantly reduced than that of the model group after the 14 th day of the group.It indicated that the combination of IT PFOB probe and radiotherapy had the effect of significantly reducing the tumor volume and inhibiting the tumor growth.Metabonomic data of tumor tissue showed that compared with the model group,changes of metabolites levels in three treatment groups indicated that the enhanced oxidative stress stated.The relative expression level of the lipid peroxidation product Stearoylcarnitine-a fatty acid ester lipid molecule,had increased significantly in the radiotherapy group,IT PFOB probe and IT PFOB+RT group,suggesting that both radiotherapy and PFOB probe could increase the oxidative stress state in tumor.On this base,the relative expression level of Glutathione（GSH）had decreased significantly,suggesting an imbalance of redox state.Meanwhile,the relative expression levels of Guanine and Uridine had decreased significantly,suggesting the down-regulated of nucleotide metabolism.These results indicated that tumor cells are in a state of oxidative stress with a high level of ROS（Reactive Oxygen Species）,thus inhabiting tumor cell growth.The results of time cluster analysis of urine samples showed that the metabolites of three treatment groups were compared with those of the model group（P < 0.05）respectively,and seven different time patterns of urine expression were obtained by Mfuzz cluster analysis;The statistical results of different compounds classification and metabolic pathways under different time patterns showed that radiotherapy had less effect on the number of different compounds in urine（11 differential metabolites）,mainly increased the relative expression levels of oxidation products（3,5-dimethoxyphenol）and organic acids（2-methylbutyrylglycine and 6-hydroxymethylatonin）,which suggested the enhanced oxidative stress state of urine.PFOB probe had a significant effect on the amounts of different compounds in urine（105 differential metabolites）,mainly reducing the oxidation metabolites（4-pyridoxic acid,quinone）,organic acid metabolism（2-aminobenzoic acid）and nucleotide degradation（7-methylxanthine）,suggesting a reduced oxidative stress state of urine.In the combination group,the effect of probe before radiotherapy on urinary metabolism was similar to that in the radiotherapy group,and the number of different compounds had less effect（34 differential metabolites）,mainly increased the metabolism of oxidation products（3,5-dimethoxyphenol）and organic acid（6-hydroxy-6-enoylglycine,Nacetyl-l-phenylalanine）.Metabonomic data of normal organs tissues showed that lung,kidney and liver tissues were more affected by different treatment methods,while heart,spleen and muscle were less affected.When the lung tissue was directly exposed to radiotherapy and probe,the number of differential metabolites in the combined treatment group was more than that in the single treatment group.The increase of PC（15:0/15:0）and PC（14:0/18:2（9z,12z））levels indicated the enhancement of lipid peroxidation,while the increase of inosine and cytidine levels indicated the enhancement of nucleotide metabolism.The increase of antioxidant GSH suggested that the reduction reaction of ROS scavenging in lung tissue was enhanced,which indicated that probe plus radiotherapy enhanced the oxidative stress state of lung tissue.The contents of DNA methylation products 5-methylcytidine and 2-hydroxyadenine in kidney were significantly decreased after radiotherapy,which indicated the therapeutic effect of radiotherapy on tumor growth.The liver,as a detoxification organ,reflects the body’s response to tumor metabolism.Under the combined operation of probe and radiotherapy,the lipid peroxidation products Lysope（0:0/20:2（11z,14z））and Lyso PC（18:2（9z,12z）/0:0）decreased significantly,suggesting that the lipid peroxidation level decreased,which reflected the remission of liver oxidative stress state when tumor growth was inhibited.Conclusion:Based on un-targeted metabolomics,this study aims to analyze the changed metabolic levels of urine samples in time series and multiple organ tissues at the end point in rabbit VX2 orthotopic lung carcinoma models.The metabolome data in tumor tissue showed that both radiotherapy and PFOB probe could enhance the oxidative stress in tumors,in addition when combing radiotherapy and IT PFOB probe,oxygen carried by PFOB probe could synergistically increase the ROS produced by radiotherapy,and finally excessive ROS inhibited tumor growth.For the time series of urine samples,radiotherapy mainly increased the level of oxidants,enhancing the oxidative stress state of body.IT POFB probe mainly decreased the level of oxidative metabolites to weaken the oxidative stress state of the body.The combination of radiotherapy and PFOB probe could offset the inhibiting effect of PFOB probe to increase the oxidation metabolites levels,enhancing oxidative stress state of the body.In addition,the treatment effect on normal metabolism of organs has tissue specificity.For the lung tissue directly exposed to radiation and PFOB probe,ionizing radiation and oxygen supply synergistically increased the oxidative stress state of lung tissue;For the kidney and liver tissues,metabonomic data indicated that these tissues are greatly affected by tumor metabolism. |
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