The Study Of A New NIR Fluorescent Probe Based On NTR Response In Recognition And Detecting Invasiveness Of HNSCC

Background:Head and neck squamous cell carcinoma(HNSCC)has high mortality,which is a common kind of cancer.Hypoxia is also a common phenomenon in head and neck squamous cell carcinoma.Hypoxia in HNSCC is a characteristic of tumor micro-environment caused by insufficient oxygen supply,and this phenomenon is closely related to tumorigenesis,malignant progression,tumor metastasis and resistance to therapies,and making HNSCC more aggressive.Therefore,detecting and estimating the degree of tumor hypoxia have great importance in diagnosing the invasiveness of HNSCC and predicting the clinical anticancer efficacies.Purpose:Nitroreductase(NTR)is highly expressed in HNSCC.Therefore,NTR can be used as an effective target to detect the hypoxic level of HNSCC.In this paper,a new near near-infrared(NIR)fluorescent probe HNT-NTR based on NTR response is proposed,and the ability of detecting NTR concentration in HNSCC is explored.Methods:1.A novel NIR fluorescent probe HNT-NTR was constructed and synthesized.It recognized NTR by converting nitro into amino group,and tested the material properties such as specificity,reaction time and detection extreme value of HNT-NTR.2.CCK-8 assay and histopathological method were used to detect the toxic effects of the probe on cells and organisms.3.Responses of HNT-NTR in vitro:(1)HNT-NTR was used to detect directly and distinguish different HNSCC cells under normoxia and hypoxia,and to judge whether it can be used for the detection of cell hypoxia;(2)HNSCC cells and ordinary cells(not tumor cells)were cultured into 3D spheroids to simulate the internal environment of HNSCC cells and the hypoxic state produced by the aggregation of HNSCC cells.The hypoxic situation of different cell microspheres was detected by HNT-NTR,and its ability to distinguish HNSCC cells from ordinary cells was judged again;(3)The microspheres of HNSCC cells were treated with chemotherapeutic drugs and their emission fluorescence intensity was measured to judge the detection ability of HNT-NTR for cell activity.4.Responses of HNT-NTR in vivo:HSC2 and Cal-27 were used to construct the tumor bearing mouse model,and HNT-NTR was used to detect the constructed HNSCC,reflecting the application effect of HNT-NTR in vivo.Results:1.HNT-NTR has the following outstanding characteristics:(1)HNT-NTR could detect NTR with obvious color change from yellow to orange by the naked eye;(2)HNT-NTR has prominent selectivity;(3)HNT-NTR with NIR emission may has negligible damage to living cells;(4)The molecular weight of HNT-NTR is small,which can enter the cell by diffusion and cause less damage to the cells.2.The results of CCK-8 assay and histopathological method confirmed that HNT-NTR had no biological toxicity and could be used in organisms in vivo.3.Responses of HNT-NTR in vitro:(1)Comparing the detection results of HNSCC cells under normoxic state and hypoxic state,it could be seen that HNT-NTR can directly distinguish the two cells;(2)In the detection of 3D HNSCC cell microspheres and ordinary cell microspheres,it could be seen that there was almost no fluorescence emission in ordinary cell microspheres,while HNSCC cell microspheres emitted strong fluorescence;(3)The fluorescence of microsphere of HNSCC cells treated with chemotherapeutic drugs was significantly weaker than that of before.The fluorescence of HSC2 was stronger than Cal-27.4.In vivo,HNT-NTR can detect HNSCC solid tumors in tumor bearing mouse,and the fluorescence of HSC2 tumor bearing mouse was stronger,and the maximum fluorescence occurred about 2-4 h after injecting the probe.Conclusions:In this paper,a novel fluorescent probe named HNT-NTR is developed,for detecting and visualizing NTR in complicated biological system.HNT-NTR recognized NTR by transforming nitro group to amino moiety.Meanwhile,HNT-NTR displayed fast response to NTR with high selectivity and sensitivity,distinct colorimetric changes(from yellow to orange)and NIR emission.HNT-NTR has plenty of strong points,such as remarkable fluorescence enhancement,good cell membrane permeability,low cytotoxicity,etc.Additionally,HNT-NTR can make a distinction between hypoxic HNSCC cells and other normoxia cells depended on the level of NTR particularly in vitro.In addition,HNT-NTR can directly and visually express HNSCC cells in vivo to distinguish them from other cells,as well as show the NTR concentration level in the tumor.Due to these advantages,HNT-NTR could detect the early formation and suspicious metastasis of tumor,and even can diagnose the invasiveness of tumor cells.