| 【Background】Gastric cancer is one of the most common malignant tumor with high incidence in the worldwide. Early detection and a positive intervention measures in time will be able to significantly improve patient survival, and as a result of improving the quality of life.Researchers have found that angiogenesis and vasculature play important roles in the growth and metastasis of solid tumors. With the improvement of the detection techniques,the study for diagnosis of tumor blood vessels has been gradually further. Additionally, it has been reported that tumor vessels have many different characteristics from normal vessels,which was caled "heterogeneity", to detect these molecular by specific manners will provide a new way of thingking. Although researchers have done much work in this field, the current detection method is still not perfect, this is mainly due to the relevant inspection technology of the deficiencies and the lack of specificity of tumor vasculartargeting molecules. Usually, MVD is used to evaluate angiogenesis of tumor tissue, but because this method requires a biopsy and embedding slice processing, which can cause organization perforation, bleeding and time consuming, limits its further application. In recent years, Confocal Laser Endomicroscopy has realised visualization of tumor angiogenesis quantitative detection and real-time dynamic tracking by observing fluorescent contrast medium short peptide or antibody probe.GEBP11 is a new nine amino acid vascular homing peptide, which was screened and identified using phage display technology. In our previous work,131 I was used to prepare the radioactive nuclide probes, 131I-GEBP11, then, SPECT and cherenkov imaging confirmed that it has good ability in vivo tumor vascular targeting property and imaging.These data showed that GEBP11 could serve as an important candidate for tumor molecular imaging. So this study is proposed to explore the CLE in the potential of FITC- GEBP11 in molecular imaging by cells in vitro and in vivo tumor tissues and tissue specimens, aims to rovide a safe and efficient candidate molecules.【Objectives】1. To evaluate the imaging effort of FITC-GEBP11 to vessels of gastric cancer.2. To explore the feasibility of the new mode CLE in the diagnosis of gastric cancer with FITC-GEBP11.【Methods】1. Preparation of fluorescein probe of FITC-GEBP11, then FACS,immunofluorescence staining and CLE of cells were conducted to confirm the binding specificity of peptide GEBP11 with Co-HUVECs.2. Afterinjection of FITC-GEBP11 by tail vein, whole body and organs in vitro were used to explore the best concentration and imaging time of GEBP11 through detecting the highest fluorescence signal.3. CLE was performed on SGC7901 gastric carcinoma-bearing mice to identify the targeting ability of GEBP11 peptide to tumor tissues and the imaging feasibility by CLE.4. Ex vivo fluorescent imaging of tumor tissues cryosections was conducted to confirm the binding specificity of GEBP11 in gastric cancer tissues; HE staining was used to distinguish the tissue types.5. After incubation with FITC-GEBP11, CLE was used to explore its feasibility in the local application of human tissue.6. Ex vivo fluorescent imaging of human tissues cryosections was conducted to confirm the binding specificity of GEBP11 in gastric cancer tissues; HE staining was used to distinguish the tissue types.【Results】1. Specific binding of FITC-GEBP11 with cells in vitro.1) In vitro FACS analysis results confirmed that GEBP11 had a stronger binding to co-HUVECs than HUVECs, whereas only an absent-to-mild binding to SGC7901 cells.2) Immunofluorescent staining and CLE of unfixed cell suspensions of cells confirmed that, Co-HUVECs showed a strong cellular signal compared with the other cells.mainly combined in the cell membrane and cytoplasm, while HUVECs, SGC7901 cell or FITC-URP peptide revealed no specific fluorescence signal following the same experimental protocol.2. CLE imaging of FITC-GEBP11 in gastric carcinoma-bearing mice.1) Both subcutaneous and orthotopic rodent cancer models were successfully generated without any adverse events.2) Whole body and ex vivo organs fluorescent imaging revealed that high activity was mainly observed from the tumor sample of both models after 24 h post-injection of FITC-GEBP11 in both rodent models, and at the dose of 1 ug/g weight.3) The best imaging of tumors was shown 24 h Specificity of fluorescent signal was observed by CLE in both models after injection of FITC-GEBP11 by tail vein,while no fluorescence signal was detected in the group of FITC-URP.4) The results from ex vivo fluorescent imaging of cryosections were consistent with in vivo CLE findings in both models, and FITC-URP showed no specific fluorescent signal; The H&E staining of the tumor specimens showed that was gastric cancer tissues.3. CLE imaging of FITC-GEBP11 in human gastric cancer tissues in vitro.1) A total of 56 samples from 28 patients with gastric cancer were analyzed by CLE.2) Specificity of fluorescent signal was observed by CLE in human tissues after injection of FITC-GEBP11, while no fluorescence signal was detected in the group of FITC-URP.3) The results from ex vivo fluorescent imaging of cryosections were consistent with in vivo CLE findings, and FITC-URP showed no specific fluorescent signal; The H&E staining of the tumor specimens showed the histologic type.4) After incubation with FITC-GEBP11, specific fluorescent signals(from+to+++)were observed in 26/28 samples, which were confirmed as gastric cancer by histopathology. In contrast, negative(0;n=20) or weak signals(+;n=8) were found in non-cancerous tissues, while no significant signals were detected in the controls. The results of the nonparametric test showed a significant difference between neoplastic and non-neoplastic tissues(p<0.01).The consistency between the degree of differentiation of human samples and semi-quantitative assessment of in vivo GEBP11 expression using CLE was also evaluated and yielded a Spearman’s r of 0.69(p < 0.001). The results from the cryosections of human specimens correlated well with the CLE observations. Additionally, the H&E staining of these specimens correlated well with the in vivo observations(Kappa = 0.64; 95% CI 0.35–0.84).【Conclusion】1. In vitro cell CLE molecular imaging was performed with FITC-GEBP11. The results confirmed that, FITC-GEBP11 could combine to the membrane and cytoplasm of HUVECs, which privide the experimental basi for follow-up experiments.2. After FITC-GEBP11 was injected into the mice via tail vein, it could specificity poly in the area of tumor; CLE could better detect the fluorescent signal in the tumor tissues. This suggests that it is feasible using FITC-GEBP11 for targeted molecular imaging by CLE in gastric cancer.3. After FITC-GEBP11 incubating with fresh human gastric cancer tissues in vitro, it could specificly bind with tumor blood vessels; CLE colud differ the differentiation degree by detecting the fluorescent signal which binded to the vessels of tumor. This suggests that local application of short peptide fluorescent probes, that could be a new method of imaging diagnosis of gastric cancer by targeting the blood vessels. |
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