Evaluation And Mechanism Study On The Difference Of Imaging Effect Between NIRF Dye ICG And MHI-148 For Liver Cancer

[Research background] The treatment of hepatocellular carcinoma(HCC)is a comprehensive treatment based on surgery.Hepatectomy requires the removal of primary tumor and maximum preservation of normal liver tissue.However,in clinical surgery,it is difficult to accurately identify tumor tissue and its boundary by naked eye and palpation,which often leads to insufficient or excessive resection.Near infrared fluorescence(NIRF)imaging is a real-time,low-cost,non-invasive,high-sensitivity imaging technology,which has been widely studied in the application of guided surgery to remove tumors.Indocyanine green(ICG)is the first NIRF dye used in clinic,but its limitations have been exposed through long-term clinical practice and can not meet clinical needs.Therefore,it is necessary to develop new near-infrared fluorescent dyes to solve the existing shortcomings of ICG and improve the visualization effect of liver cancer.Heptamethrin cyanine dye MHI-148 is a new dye that has been reported recently and can be specifically concentrated in tumor tissue.Its excellent tumor-targeted imaging effect is expected to become a new imaging agent for guiding the resection of liver cancer.The mechanism of NIRF dye aggregation in liver cancer tissue is currently believed to be closely related to the proteins involved in drug transport on the liver cancer cell membrane.Transport proteins can be divided into two types: the proteins of uptake and efflux.Transmembrane uptake of substances are mainly mediated by organic anion transport peptides(OATPs),while liver cancer cells mainly excrete substances from cells through adenosine triphosphate-binding cassette proteins(ABCs).It is reported that OATP2B1 and ABCG2 plays an important role in the metabolism of many drugs and dyes.However,whether these two molecules regulate the metabolism of MHI-148 in liver cancer cells and whether they affect the imaging effect of MHI-148 in liver cancer is still unclear.[Objective] To compare the advantages and disadvantages of MHI-148 and the traditional near-infrared fluorescent dye ICG,clarify the specific parameters of MHI-148 fluorescent dye imaging,analyze the mechanism of MHI-148 only gathering in tumor tissue,break through the bottleneck of the existing NIRF dye application in real-time imaging technology,and provide new insights for the optimization of real-time imaging technology in hepatectomy.[Research methods] 1.Wild type C57 mice were used to inject the same dose of MHI-148 and ICG dye into the tail vein to monitor the body weight changes of the mice within 30 days.At the same time,blood samples were taken from the eyeball vein of the mice on the 7th and 30 th days to detect the biochemical indicators such as ALT,AST,CK,CREA.Besides dissect mice to obtain important tissues and organs for HE staining to observe morphological changes and evaluate the biocompatibility of the two dyes.2.The metabolic characteristics of MHI-148 and ICG used in human hepatoma cell lines SNU-739,SNU-368 and human normal hepatocyte THLE-2 were measured,and the parameters of the two dyes used in cell fluorescence imaging were compared.The mouse subcutaneous CDX model was established by subcutaneously inoculating the hepatoma cell line SNU-739 in nude mice.ICG and MHI-148 were injected into the mice through tail vein.The fluorescence intensity of the dye was measured at different time nodes to determine the metabolic process and the change trend of fluorescence intensity in vivo.The aggregation of the two fluorescent dyes in tumor tissue was observed,and the fluorescence intensity of liver cancer tissue and normal tissue was compared.So as to clarify the specific aggregation effect of MHI-148 dye in tumor tissue and the advantages and disadvantages of ICG dye.3.Acquire multiple sets of liver cancer transcriptome data from TCGA,GEO and other public databases,and evaluate the expression level of ABCG2 and OATP2B1 in tumor tissue and adjacent tissues in combination with the liver cancer tissue samples of our hospital.ABCG2 and OATP2B1 overexpression and interference stable cell lines were constructed in human hepatoma cell line SNU-739.The stable cell lines were imaged with MHI-148 to compare the changes of fluorescence intensity.A mouse subcutaneous CDX model was established by subcutaneously inoculating ABCG2 and OATP2B1 stable overexpression and interference with liver cancer cell lines in nude mice.After tumorigenesis,MHI-148 was injected through the caudal vein to conduct live imaging,and the effects of ABCG2 and OATP2B1 on fluorescence intensity were compared.[Research results] 1.After ICG and MHI-148 were injected into mice by tail vein,there was no statistical difference between the weight gain of mice and the control group within one month;There was no obvious abnormality in serum biochemical indexes and HE staining of tissue sections.2.The fluorescence intensity and imaging time of MHI-148 in hepatoma cell lines SNU-739 and SNU-368 were significantly higher than those of ICG.And the fluorescence intensity of MHI-148 reached the highest value at 60 minutes,then decreased slowly.The fluorescence intensity of ICG in THLE-2 of normal human hepatocytes is higher than that of MHI-148.Its fluorescence intensity reaches the highest value at 30 minutes,then it is basically completely discharged from the cells at 180 minutes.After ICG was injected into the tail vein of nude mice subcutaneously inoculated with hepatoma cell line SNU-739,the fluorescence signal first appeared in the liver region,and then transferred to the lower abdomen.There was no fluorescence signal in the subcutaneous transplanted tumor region;When MHI-148 was injected into the tail vein of nude mice for 1 hour,the subcutaneous transplanted tumor could display strong fluorescence,and the fluorescence signal could last for more than 24 hours.After the nude mice were killed,the heart,liver,lung,kidney,spleen,intestine and tumor tissue were taken out for live imaging.It was found that ICG was specifically ingested by the liver and then discharged into the intestine through bile and then discharged out of the body.The tumor tissue had no ICG fluorescence signal.But the MHI-148 specifically aggregates in tumor tissue.3.TCGA and GEO transcriptome data suggest that OATP2B1 is highly expressed in tumor tissue.And the transcription level of ABCG2 is low expressed in tumor tissue.Immunohistochemical results further confirmed that the expression of OATP2B1 was significantly increased and the expression of ABCG2 was remarkable decreased in liver cancer tissues.In vivo and in vitro experiments revealed that overexpression of OATB2B1 and interference with ABCG2 promoted the uptake of MHI-148 in liver cancer cells,whereas interference with OATP2B1 and overexpression of ABCG2 inhibited the accumulation of MHI-148 in liver cancer cells.[Conclusion] ICG and MHI-148 have good biocompatibility and can be used for imaging in vivo.After ICG is specifically ingested by normal hepatocytes,the dye is discharged into the intestine through bile and then passed out of the body.After entering the body,MHI-148 can specifically target into tumor tissue,with high fluorescence signal intensity and long imaging time.In hepatocellular carcinoma,OATP2B1 is highly expressed and mediates dye uptake,while ABCG2 is low expressed and mediates dye excretion.Therefore,the increase of dye intake and the decrease of dye excretion in liver cancer lead to the higher fluorescence signal of MHI-148 in tumor tissue.These results show that MHI-148 can replace ICG to achieve real-time tumor-specific imaging during liver cancer surgery,improve the effectiveness and safety of hepatectomy,and improve the prognosis of patients.