| BackgroundAt present,most lung cancer patients have been diagnosed at a late stage and have distant metastasis due to the less early symptoms,which seriously affects the choice of clinical treatment in clinic.Radiotherapy and chemotherapy,commonly used in clinic,which makes the treatment efficiency and patient survival rate significantly lower.The clinical treatment of lung cancer patients with different pathological types and stages is different,so the efficacy and survival rate are significantly different.Early diagnosis is crucial for the treatment and prognosis of lung cancer patients.At present,the commonly used diagnostic methods for lung cancer mainly include imaging examination,serum tumor marker detection,sputum cytology examination,lung puncture and fiberoptic bronchoscopy.These diagnostic methods have limitations in specificity and practicability.Therefore,it is urgent to find high-specific lung cancer-targeted molecular probes for early diagnosis of lung cancer diseases,and provide a basis for efficacy monitoring and individualized targeted therapy.In recent years,molecular imaging developed rapidly and made great achievements,especially nuclear medicine molecular imaging,which with high sensitivity,high specificity and easy clinical transformation.Radionuclide tumor molecular imaging,including receptor imaging,metabolic imaging and gene imaging,plays an important role in the early diagnosis of malignant tumors and the differential diagnosis of benign and malignant tumors,and has good clinical application value.Tumor radioimmunoimaging technology,as a high specificity imaging tracer technology,radionuclide combined with antibody,is suitable for monitoring and quantifying the expression level and distribution of target molecules in tumor tissues,which is helpful for early accurate diagnosis,prognosis and treatment evaluation of tumors.It has been used in imaging of many common tumors in clinic.The lung cancer targeting molecule has become a key factor for radioimmunoimaging establishment and it is basis for tumor imaging and therapeutic and clinical efficacy evaluation.At present,exploring new radionuclide labeled molecular probes has become the core of tumor imaging research.Such molecular probes should meet the requirements of high specificity,high sensitivity and high selectivity,suitable for clinical applicationAngiogenesis providing oxygen and nutrients to all tissues and cells of the body,involving in a variety of physiological and pathological processes,plays a key role in the development of solid tumors.Tumor neovascularization has atypical morphological features,which make the tumor tissue over-infiltrated and poorly perfused,and the hypoxic microenvironment can mediate the spread,invasion and metastasis of tumor cells.Tumor tissue,lacking oxygen and energy supply of blood vessels,will not be able to break through the critical tumor size for distant organ metastasis.Tumor neovascular imaging and treatment have become the hotspot of basic research in clinical tumors.Tumor angiogenesis involves mutations in many genes and expression of different cytokines and receptors.Recent studies have found that CD93 is a transmembrane glycoprotein,expressed in endothelial cells,stem cells and bone marrow cells.The most important expression is on the surface of endothelial cells,and endothelial cells are essential for neovascularization of tumors.Early studies have shown that CD93 was an immune molecule involved in the adhesion and migration of immune cells.The factors affecting angiogenesis include activators and inhibitors.Recent literature reports that CD93 is a new type of angiogenesis activator.It mainly affects the growth of tumors by promoting endothelial cell adhesion and accelerating tumor angiogenesis.High expression of CD93 can promote the growth of tumors and reduce the survival of hosts.In addition,the extracellular domain of CD93 on the membrane surface is prone to cleavage or shedding under inflammatory stimuli,forming soluble CD93 and promoting angiogenesis.Therefore,it is postulated that CD93 may be a key molecule in tumor neovascularization and can be used as a molecular marker for the diagnosis and prognosis of lung cancer,and it is expected to be used for early targeted diagnosis of tumors.Studies have shown that the expression of CD93 in cancer tissues is closely related to clinical pathological types and prognosis of patients.Early identification of CD93 expression in lung cancer tissues is very important for patients to choose targeted therapy.However,there is no relevant research report on the early detection and evaluation of tumors using CD93 as a molecular target.The high expression of CD93 molecule on endothelial cells of tumor vessels and low expression in non-proliferating endothelial cells highlight the potential clinical value of CD93 molecule in cancer diagnosis.To explore whether radionuclide labeled CD93 molecular probe can be used for targeted imaging of tumors is of great significance for evaluation of CD93 as a non-invasive diagnosis of tumor molecular targets.In this paper,we selected lung cancer with high clinical incidence and difficult early diagnosis as the research object.We used radionuclide labeled anti-CD93 antibody to preliminarily study the targeted monitoring of lung cancer with new CD93 molecular probe in two parts.Firstly,the expression of CD93 in non-small cell lung cancer cell line was studied in vitro,and a novel radionuclide molecular probe 125I-anti-CD93 mAb was prepared and identified,and we studied its binding specificity to lung cancer cells.Secondly,lung cancer-bearing mouse model was established to study the specificity of 125I-anti-CD93 mAb in targeting imaging of lung cancer tumors in vivo,and compared with the isotype imaging agent 125I-IgG and the non-specific imaging agent 18F-FDG.This paper aims to explore new target molecule for early diagnosis of lung cancer and provide a new way for early non-invasive diagnosis of lung cancer.Part One Preparation and identification of CD93 radionuclide molecular probeMethods1.Preparation of 125I-anti-CD93 mAb and 125I-IgGThe nuclide probes 125I-anti-CD93 mAb and 125I-IgG were labeled with Iodogen method,and PD-10 gel column was used for elution and purification.Radioactivity was measured to calculate the labeled rate and specific activity of the two tracers.Radiochemical purity was determined by paper chromatography.125I-anti-CD93 mAb and 125I-IgG protein peaks were mixed with saline and serum,respectively.The stability was measured by paper chromatography at room temperature at 24h,48h and 72h.2.Detection of the expression of CD93 in cells by RT-PCR and Western BlotThree kinds of lung cancer cell lines were routinely cultured:adenocarcinoma A549 cells,large cell carcinoma H460 cells,and squamous carcinoma H520 cells.RT-PCR was used to detect the expression of CD93 mRNA in three cell lines,Western Blot was used to detect the expression of CD93 protein in three cell lines,and the difference of CD93 expression among three lung cancer cell lines was analyzed.3.Binding assay of probes in vitro125I-anti-CD93 mAb group:three kinds of lung cancer cells(2×105/well)were inoculated into 24-well plate.Labeled antibody was added according to the concentration gradient(3-100nM).After a certain period of time,washed and the radioactivity counts of the cells were measured,and the differences of affinity(KD value)of each lung cancer cell line to 125I-anti-CD93 mAb were compared and analyzed.125I-IgG group:A549 lung cancer cells(2×105/well)with good growth condition were inoculated into 24-well plates.125I-IgG were added according to the concentration gradient(3-100nM).After a certain period of time,washed and the radioactivity counts of the cells were measured to analyze the affinity(KD value)of A549 lung cancer cell line to125I-IgG,and compared with 125I-anti-CD93 mAb A549 group.4.Binding blocking assay of probe in vitroA549 lung cancer cells(2×105/well)with good growth condition were inoculated into 24-well plates.Quantitative 125I-anti-CD93 mAb and anti-CD93 mAb were added(0,1.42,14.2,28.4nmol/L),after a certain time,washed and measured the radioactivity,and analyzed the blocking of the binding of between 125I-anti-CD93 mAb and A549 by non-labeled anti-CD93 mAb.Results1.The radionuclide probe 125I-anti-CD93 mAb was successfully prepared.The labeled rate was 91.37%,the specific radioactivity was 1096.44MBq/mg,and the radiochemical purity was 96.49%.The labeled rate of 125I-IgG was 90,24%,the specific radioactivity was 1082.88MBq/mg,and the radiochemical purity was 94.82%.Paper chromatography showed that both 125I-anti-CD93 mAb and 125I-IgG had good stability in saline solution and serum,and keep above 90%until 72h.2.Both RT-PCR and Western Blot showed that CD93 was high expressed in A549 cells and H460 cells,and low expressed in H520 cells.There was a significant difference between them.3.The cell-binding assay showed that the KD of 125I-anti-CD93 mAb in A549 cells,H460 cells and H520 cells was 27.09nmol/L,28.84nmol/L and 39.90nmol/L,respectively.The affinity of A549 cells and H460 cells was significantly higher than that of H520 cells.The Bmax values of A549 cells and H460 cells were 1679cpm/104 cells and 1780cpm/104 cells,which were significantly higher than that of H520 cells(756cpm/104 cells).Comparative analysis with the 125I-IgG group indicated that the 125I-anti-CD93 mAb specifically binds to A549 lung cancer cells.4.The cell binding blocking experiments showed that the non-labeled anti-CD93 mAb specifically blocked the binding of A549 cells to 125I-anti-CD93 mAb,and the amount of addition was proportional to the blocking rate.Part Two Study of CD93 radionuclide probe on tumor targeting in lung cancer-bearing miceMethods1.Establishment of lung cancer-bearing mice modelThree kinds of lung cancer cell lines:A549,H460 and H520 were cultured routinely.Female BALB/c nude mice,aged 4-6 weeks,were injected subcutaneously with 5×107ml cell suspension on the shoulder of the right forelimb,0.2ml/each,to establish lung cancer-bearing mice models.The whole process was aseptically operated and the tumor volume change was recorded.The experiment was performed when the maximum diameter of the tumor reached 0.8-1.0cm.2.Biodistribution of 125I-anti-CD93 mAb in three kinds of lung cancer-bearing miceThree kinds of lung cancer-bearing mice,thyroid blocked with 3%NaI solution 48h before,were injected with 125I-anti-CD93 mAb,0.37MBq/each by tail vein.And five mice were randomly selected from each group at 24h,48h and 72h after injection.Sacrificed,collected main organs,tissues,tumors and opposite muscle tissues to weigh and measure radioactivity(cpm).To calculate the percentage of radioactivity per gram of tissue or organ to standard source(%ID/g)and target to non-target radioactivity ratio(T/NT ratio).3.Dynamic phosphor-autoradiography of 125I-anti-CD93 mAb in three kinds of lung cancer modelThree kinds of lung cancer model mice were injected with 125I-anti-CD93 mAb,0.37MBq/mice,through tail vein separately after thyroid blocked with 3%NaI solution 48h earlier.Dynamic in vivo whole-body phosphor autoradiography was aquired at 24h,48h and 72h after injection to compare the accumulation of 125I-anti-CD93 mAb in the tumors of three kinds of lung cancer models.4.Biodistribution of 125I-anti-CD93 mAb and 125I-IgG in A549 lung cancer-bearing miceA549 lung cancer-bearing mice were randomly divided into two groups and were injected with 125I-anti-CD93 mAb or 125I-IgG through tail vein after thyroid blocked separately.Animals were sacrificed at 24h,48h and 72h after injection to collect blood,tumor,opposite normal muscle and other main organs,to study the biodistribution and calculate T/NT ratio in two groups of mice.5.Dynamic whole-body phosphorescent screen imaging with three imaging tracers in A549 tumor-bearing miceA549 lung cancer-bearing mice was randomly divided into three groups with thyroid blocked 48 hours earlier,model mice were injected with 125I-anti-CD93 mAb/125I-IgG,separately,0.37MBq/each to dynamic image at 24h,48h and 72h after injection.Another group of model mice was injected with '8F-FDG,37MBq/each through the tail vein,to study dynamic imaging at 30min,3h and 6h after injection.The radioactivity of 125I-anti-CD93 mAb,125I-IgG,and 18F-FDG at the tumor site was compared and analyzed using OptiQuantTM image analysis software.6.H&E staining and CD93 immunohistochemical staining of lung tumor tissueTumor tissues from the three groups of animals which have been used for dynamic whole-body phosphor-autoradiography were harvested,fixed into 4%paraformaldehyde and prepared paraffin sections.Hematoxylin-eosin staining was used routinely.Anti-CD93 antibody was used for immunohistochemical staining.The positive expression was observed and photographed under the microscope.Results1.The biodistribution of three kinds of lung cancer-bearing mice showed that the 125I-anti-CD93 mAb tracer were mainly metabolized by liver and kidney.The radioactivity count of tumors was the highest at 48h,and the radioactivity counts and the T/NT ratio of A549 and H460 groups at 48h were higher than the H520 group,and the difference between the two groups was statistically significant.2.The whole body dynamic phosphor screen imaging in lung cancer-bearing mice showed that the mice were weakly visible at 24h after injection of 125I-anti-CD93 mAb in the three groups,and the tumor imaging was the clearest at 48h.The radioactivity accumulation on tumor tissues in A549 group and H460 group,were significantly higher than that in H520 group.The semi-quantitative analysis showed that the ratio of radioactivity in A549 group and H460 group was higher than that in H520 group at 48h,the difference was statistically significant.3.The biodistribution in A549 lung cancer bearing mice showed that both tracers were metabolized by liver and kidney.Tumor radioactivity accumulation was obviously in 125I-anti-CD93 mAb group compared with 125I-IgG group.The radioactivity and T/NT ratio of 125I-anti-CD93 mAb group were significantly higher than those in 125I-IgG group.4.After injecting the probe into A549 lung cancer bearing mice,the tumour imaging was the most clear at 48 hours,and the radioactivity of 125I-anti-CD93 mAb group was significantly higher than that of 125I-IgG group.This indicated that the radioactivity of 125I-anti-CD93 mAb in the tumour location was the specific binding between 125I-anti-CD93 probe to CD93,rather than the non-specific binding of the homologous IgG Fc fragment.The 18F-FDG control group showed the best imaging at 3 hours after injection.The radioactivity ratio of tumors was 1.60±0.10,which was significantly lower than that of the CD93 probe injection group(3.34±0.18).This indicated that 125I-anti-CD93 mAb was clearer imaging than the non-specific imaging agent 18F-FDG in imaging A549 tumors.5.Pathological analysis of tumor tissues showed that malignant proliferation of tumor with neovascularization,and the positive rate of CD93 immunohistochemical staining were high among three kinds of tumors.The CD93 positive rate of CD93 in A549 group and H460 group were higher than that in H520 group,which was consist with the expression of CD93 analyzed in vitro with lung cancer cells.Conclusion1.The expression of CD93 was significantly different in three kinds of lung cancer cells.In non-small cell lung cancer,the expression of CD93 was higher in lung adenocarcinoma and large cell carcinoma cells,but lower in squamous cell carcinoma cells.2.The radionuclide tracer 125I-anti-CD93 mAb has good biological activity;125I-anti-CD93 mAb can specifically bind to lung cancer cells,and the affinity of lung cancer cells to 125I-anti-CD93 mAb is positively correlated with the expression of CD93.3.The bioldistribution of 125I-anti-CD93 mAb in A549 lung cancer model mice was consist with the results of whole body phosphorus screen dynamic imaging,which indicated that 125I-anti-CD93 mAb could be specifically accumulated on the tumor location in CD93 positive lung cancer bearing mice,and accumulation degree was positively correlated with the expression of CD93.The results provided a new possible molecular target for early targeted monitoring of CD93 positive tumors.innovation1.It was found that CD93 molecule was high expressed in lung adenocarcinoma and large cell carcinoma cells of non-small cell lung cancer,while CD93 molecule was low expressed in squamous cell carcinoma cells.2.The radionuclide labeled probe 125I-anti-CD93 mAb has good targeting ability in lung cancer both in vivo and in vitro.3.The radionuclide labeled probe 125I-anti-CD93 mAb has apparently lung cancer targeting ability compared with the isotype IgG and non-specific imaging agents.CD93 may be used as a new molecular target for lung cancer and has potential clinical application value. |
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