An Experimental Study Of GLUT1-targeted Small Molecule Probe In Imaging Of Oral Squamous Cell Carcinoma

Objective: Oral squamous cell carcinoma(OSCC)is one of the most common malignant tumors in the world.Squamous cell carcinoma located in oral cavity is difficult to detect early and excise precisely.Early detection and complete resection of OSCC are very important for survival and prognosis of patients.Molecular imaging(MI),a new subject dedicated to in vivo imaging,is increasingly being applied in clinical imaging research.The purpose of this study is to find and validate new targets for OSCC imaging,and to synthesize novel molecular probes for OSCC optical imaging,to provide a new strategy for OSCC screening and intraoperative imaging.Materials and Methods: Bioinformatics analysis showed that glucose transporter 1(GLUT1)was highly expressed in OSCC.To further validate the potential of GLUT1 as an imaging target for OSCC,immunohistochemical staining and H-score analysis were performed on the patient samples and orthotopic CAL27-f LUC mouse tumor,and flow cytometry(FCM)and western blot(WB)were performed on oral squamous cell lines(HSC3 and CAL27-f LUC)and human normal oral keratinocytes(HOK).The target probe WZB117-IR820 was synthesized by linking the near infrared region I dye new indocyanine green(IR820)to GLUT1 small molecule inhibitor WZB117.The target probe WZB117-FITC was synthesized by linking the 5-carboxyfluorescein(5-FAM)to WZB117.Mass spectrometry,in vitro fluorescence intensity and optical stability of the targeted probe were detected.The biosafety of the targeted probe was evaluated by in vitro cytotoxicity test and in vivo toxicity test in nude mice.In vitro,FCM and confocal microscopy were used to detect the uptake of targeted probes by OSCC cells.Confocal laser endomicroscopy(CLE)was used to detect the uptake of targeted probes by orthotopic CAL27-f LUC mouse tumor tissues.The biological distribution of fluorescent probe in orthotopic CAL27-f LUC mouse tumor model was detected within 24 h by IVIS(?) Spectrum CT.Near infrared surgical equipment was used to perform fluorescence navigation surgery on tongue cancer bearing mice,and bioluminescence imaging(BLI)and pathological examination was performed to verify whether the tumor tissue was completely removed.Finally,fresh OSCC sample of patients were stained with targeted fluorescence probe and fluorescent imaging was acquired.Results: Bioinformatics analysis determined that GLUT1 was highly expressed in OSCC tissues compared to normal tissues.Immunohistochemical staining was performed on the patient’s specimens,and H-score was calculated in OSCC tissues and adjacent normal tissues,which demonstrate that GLUT1 overexpressed in patient’s tumor tissues.Flow cytometry and western blot assay showed that GLUT1 was highly expressed in OSCC cells compared with normal HOK cells.In tongue tumor models,immunohistochemical staining and H-score results showed that GLUT1 was highly expressed in tumor tissues compared with normal tongue tissue.The mass spectrometry results showed that we successfully attached the fluorescent dye to WZB117.The stability of WZB117-IR820 was well.The dissolved WZB117-IR820 remained clear after 60 days of storage at room temperature,without precipitation,and the average fluorescence intensity remained unchanged.The results of cytotoxicity test and in vivo toxicity test in nude mice showed that WZB117-IR820 had good biosafety.In vitro probe uptake experiments showed that compared with human normal oral keratinocyte(HOK),OSCC cells took more targeted probes and showed stronger average fluorescence intensity.In preclinical animal model studies,targeted probes have shown good targeting performance for OSCC.We successfully used the WZB117-IR820 targeted probe to perform fluorescence navigation surgery on tongue cancer bearing mice.BLI imaging and histopathology confirmed that there was no tumor residual at the surgical margin after fluorescence navigation.After staining fresh human biospecimens with WZB117-IR820,precise delineation of OSCC tumor areas was achieved,and the fluorescence intensity of OSCC tumor tissue was significantly higher than that of adjacent normal tissue.Conclusion: We verified GLUT1 as a new optical imaging target of OSCC by bioinformatics analysis and experimental analysis,and on this basis,we successfully synthesized OSCC targeting probes WZB117-IR820 and WZB117-FITC targeting GLUT1.In vivo and in vitro experiments verified that the targeted probe has a good targeting effect on OSCC tissues,and successfully imaging OSCC tumor tissues in fresh specimens through local application.In summary,we synthesized a new optical imaging probe targeting GLUT1 for OSCC,and proved that the optical imaging of OSCC can be successfully performed using this optical probe.This study provides a preliminary experimental basis for the clinical transformation of WZB117-IR820,which is of great significance for the application of WZB117-IR820 in the early detection,fluorescence guided surgery and surgical margin evaluation of OSCC in the future.