| PurposeThe existing digestive endoscopic system has significantly improved the diagnosis and treatment of digestive tract tumors.However,in the diagnosis of early diagnosis of digestive tract,there are still problems that are difficult to find,such as difficulty in finding small lesions,difficulty in border recognition,and limitations of biopsy.In response to these problems,we have carried out research on new equipment for endoscopic molecular imaging and new diagnostic techniques.High-resolution microendoscopy(HRME)developed in the early stage can significantly improve the accuracy of early diagnosis of digestive tract early cancer,but the HRME imaging spectrum is single,and the clinical application is limited.Therefore,our team developed a new multispectral fluorescence microendoscopy(MFE).The purpose of this study was to combine microscopic endoscopic imaging with fluorescent targeting molecular probes and apply MFE at the cellular level.Multi-spectral molecular imaging of colon cancer lesions at the living level of animals and exploring new methods for high-resolution instant immunohistochemical imaging.MethodImmunohistochemistry was used to detect the expression of CD44,EGFR,αvβ3 and CD133 in colon cancer cell line HCT116.The software calculates the IOD value to evaluate the expression levels of the four target proteins,and selects two relatively high-expressed target proteins as fluorescent probes to bind the target.The colon cancer cells HCT116 were divided into: FITC-anti-CD133 group,AF680-anti-αvβ3 group,and PBS group according to the type of probe applied.The self-developed MFE was used to molecularly image the colon cancer cell HCT116,and the ratio of the average fluorescence intensity of the tumor cells to the background signal(signal-to-noise ratio)in each group of MFE images was calculated.Using fluorescence microscopy as the gold standard,fluorescence imaging of colon cancer cells HCT116 was performed using fluorescence microscopy to evaluate the feasibility of MFE multi-spectral molecular imaging at the cellular level.A nude mouse model of subcutaneous tumor was constructed,and a small animal imager was used to perform fluorescence imaging of tumor-bearing mice.Animal models were divided into: FITC-anti-CD133 group,AF680-anti-αvβ3 group,and PBS group according to the type of probe.Images were acquired at different time points,the signal-to-noise ratio of the tumors in each group of images was calculated,and the optimal imaging time point of fluorescence imaging and the targeting of fluorescent probes in animal models were evaluated.To further evaluate the targeting of fluorescent probes,small animal imagers were used to image the isolated tissues(tumor,heart,liver,spleen,lung,kidney and small intestine)of tumor-bearing mice at the optimal time point,and the probe set was calculated.Signal to noise ratio of isolated tissue in PBS groupMFE imaging of subcutaneous tumors of tumor-bearing mice injected with fluorescent probes by tail vein.Animal models were divided into: FITC-anti-CD133 group,AF680-anti-αvβ3 group,and PBS group according to the type of probe.Evaluate the feasibility of MFE for multispectral molecular imaging at the in vivo level of animals.The drug delivery method was changed.MFE was used to image the subcutaneous tumor of tumor-bearing mice with local fluorescent probes.The signal-to-noise ratio of tumors in each group of MFE images was calculated to evaluate the effect of different routes of administration on MFE imaging.The tissue type of the tumor specimens of nude mice was detected by HE staining.Immunohistochemistry was used to detect the expression of target proteins in nude mouse tumor specimens.Statistical methods were used to compare differences between groups.If P < 0.05,the difference was statistically significant.ResultsImmunohistochemistry confirmed the expression of four target proteins in HCT116 cell line.Semi-quantitative analysis showed that the two target proteins of CD133 and αvβ3 were relatively high expression,and they were used as molecular imaging targets.MFE was used to image the colon cancer cell HCT116.In the MFE images of the FITC-anti-CD133 group and the AF680-anti-αvβ3 group,there were obvious fluorescence signals on the surface of the tumor cells,and the ratio of the average fluorescence intensity to the background signal(signal-to-noise ratio)were 10.137±1.534,13.709±1.672;there was no obvious fluorescence signal in the corresponding PBS group,and the signal-to-noise ratio of tumor cells were 1.585±0.185 and 2.068±0.227,respectively.Fluorescence microscopy images showed blue-stained tumor cell nuclei and green or red fluorescent signals on the cell surface,while the corresponding PBS group had no obvious fluorescence signal on the cell surface.In vivo fluorescence imaging of tumor-bearing mice in the two probe groups,the tumor signal-to-noise ratio were 1.346±0.005 and 1.830±0.029 after 24 hours of tail vein injection.In the matched PBS group,fluorescence signal wasn’t displayed in the MFE image.The ratios were 1.013 ± 0.003 and 1.016 ± 0.003,.In the fluorescence imaging of nude mice in vitro,the fluorescence signal-to-noise ratio of the gallbladder was the highest in the FITC-anti-CD133 group,the ratio of tumor was 2.031±0.038.The highest ratio of fluorescence signal to noise ratio in the AF680-anti-αvβ3 group was 2.506 ± 0.018.The corresponding fluorescence signal ratios of the tumors in the PBS group were 1.372±0.018 and 1.401±0.019.MFE was used to image the subcutaneous tumor of tumor-bearing mice injected with fluorescent probes in the tail vein.The image was characterized by uniform and high-brightness fluorescent signals throughout the field of view.The signal-to-noise ratios of the tumors in the FITC-anti-CD133 group and the AF680-anti-αvβ3 group were 6.765±0.141 and 4.738±0.150,respectively.There was no obvious fluorescence signal in the corresponding PBS group,and the tumor signal-to-noise ratio were 2.711±0.203,2.359 ± 0.196.After local spraying of the fluorescent probe,the MFE image of the tumor-bearing subcutaneous tumor of the tumor-bearing mouse is characterized by the fact that the fluorescent signal is star-shaped and evenly distributed on the surface of the tumor,resembling a cell.The signal-to-noise ratios of the tumors in the FITC-anti-CD133 group and the AF680-anti-αvβ3 group were 7.047±0.168 and 5.905±0.154.There was no obvious fluorescence signal in the corresponding PBS group,the ratio were 2.498±0.125,1.763±0.043.HE staining results showed that the tumor was colon cancer tissue.Histochemical staining of tumor immunohistochemistry showed expression of CD133 and αvβ3.The ratio between the above probe group and the corresponding PBS group was different,and both were statistically significant(P<0.05).Conclusion1.Multi-spectral molecular imaging studies of colon cancer tissue using MFE showed that CD133 and αvβ3 target proteins could be used as molecular imaging targets for colon cancer cell line HCT116.CD133 was first used for molecular imaging of microendoscopic devices.2.Multi-spectral molecular imaging of two targets of the same colon cancer cell line HCT116 was achieved at the cellular level using a self-developed microscopic endoscopic device MFE.MFE is allowed to conduct studies of multi-spectral molecular imaging at the living level.3.At the level of living animals,the self-developed microscopic endoscopic device MFE was used to achieve multi-spectral molecular imaging of two target colon cancer xenografts in nude mice,which proved that MFE has the feasibility of simultaneous multi-spectral imaging and real-time immunohistochemistry in vivo.4.The differences of MFE images in subcutaneous xenografts of nude mice were compared.The results showed that the MFE images after local spraying of probes were more effective.Local spraying of fluorescent probes may be the best mode of administration for future clinical application of MFE. |
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