Application Of Near Infrared Fluorescence Probe In Molecular Imaging Of Colon Cancer

Purpose Colon cancer is one of the common malignant tumor.Early colonoscopic diagnosis and early treatment has been associated with significantly improved survival.Currently,white light illumination is used to detect premalignant and malignant colonic lesions based on structural changes.The morphological changes of early malignant colonic lesions are less discriminatory and the biopsy lack of accuracy,leading to a high missed rate.The existing model of early diagnosis of cancer is inadequate,fluorescent endoscopic molecular imaging for us to provide a new direction.The aim of this study was to construct the near-infrared fluorescent probe ICG-Cetuximab using the near-infrared fluorescent dye indocyanine green labeled cetuximab,which is the target of epidermal growth factor receptor,and to further verify the application value of the probe in colon cancer fluorescence molecular imaging,and to provide a theoretical basis for the clinical diagnosis of early colon cancer with near infrared fluorescence imaging.Methods NH2-Reactive ICG has a succinimidyl ester group,and can make a covalent bond with an amino group of the cetuximab.Western blot was used to detect the expression of EGFR in each colorectal cancer cell line.Human colorectal cancer cells with high and low expression of EGFR(Lovo and SW620,respectively)were screened.Lovo and SW620 infected with luciferase gene were injected into the subcutaneous and colon wall of nude mice to construct the subcutaneous and orthotopic transplanted tumor model.ICG-Cetuximab were used to detect and quantify the fluorescent signal intensity of tumor tissue by in vivo dynamic fluorescent molecular imaging after injecting of the probe(200ug/200ul)to subcutaneous xenograft models ofcolon cancer.The orthotopic transplanted tumor specimens were incubated in ICG-Cetuximab solution at room temperature,full immersion,eluting unbound probe after ex vivo FLI.Then the pathological and immunohistochemical examination of the tumor was performed to confirm the expression of EGFR and compare with the results of FLI.Results 1.The ICG-Cetuximab probe was successfully constructed by covalently binding the succinimidyl ester group and the amino group.When the cetuximab was 200 ug,the percentage of cetuximab labeled ICG was 56.37±3.34% by UV-visible spectrophotometer.Finally,ICG Labeling-NH2 samples labeled with 200 ug protein were identified.2.Sequential fluorescence monitoring of subcutaneous transplanted tumor models(Lovo-GFP-Luc)after i.v.injection of ICG-Cetuximab(200ug/200ul)demonstrated the pharmacokinetics of ICG-Cetuximab.The labeled antibody was distributed throughout the body gradually accumulated in the subcutaneous transplanted tumor,24 h after injection at the tumor site reached a peak,the average fluorescence intensity is about 2.16±0.21 times before injection.48 h after the probe specific targeting effect is better,continuous imaging 168 h can still be observed fluorescence signal in the tumor site,compared with the normal tissue significantly.Residual unbound antibody was excreted mainly from the liver and intestine by 2 days after injection.In the two control groups,The probes was distributed throughout the body,then concentrated in the liver,48 h after injection almost excreted by liver and intestine completely.After the injection of the probe to 48 h continuous imaging,were not detected in the tumor specific fluorescence signal.The results of FLI and bioluminescence imaging after injection of 24 h showed that the probe was indeed concentrated in the tumor site.The orthotopic transplanted tumor of colon cancer was incubated with ICG-Cetuximab solution,ex vivo FLI results showed orthotopic colon cancer site has a strong fluorescence signal,and the tumor adjacent to the normal mucosa was obvious.After incubation for 5min and 15 min,the mean fluorescence signal intensity at the tumor wasabout 3.56±0.34 and 3.44±0.30 times higher than that of the normal mucosa respectively.The results of IHC showed that EGFR was significantly expressed in Lovo-GFP-Luc subcutaneous and orthotopic transplanted tumors,and no significant EGFR expression was observed in SW60-GFP-Luc subcutaneous xenografts.IHC results are in good agreement with the targeting binding of fluorescent probes to EGFR.Conclusion The ICG-Cetuximab,a fluorescent molecular probe targeting EGFR,was successfully constructed by covalently binding the ICG and Cetuximab approved by the FDA.The targeting and fluorescence efficiency of ICG-Cetuximab on colon cancer were verified by in vivo fluorescent molecular imaging after injecting probes to subcutaneous xenograft models.And further validated the application value of ICG-Cetuximab in colorectal cancer fluorescent molecular imaging.The ex vivo fluorescence imaging based on ICG-Cetuximab was used to visualize the tumor lesions and guide the biopsy.