Preliminary Study Of The Inhibition Of ABCG2 Can Enhance The Aggregation Of MHI-148 Cyanine Dye In Hepatocellular Carcinoma

?Background?Hepatectomy is characterized by difficulty,high risk and poor safety.While we can clearly observe the tumor edge and greatly improve the quality of surgery throng intraoperative real-time imaging technology.Near-infrared fluorescent dyes(NIRF)have the ability of tumor aggregation,so we can observe the tumor range and boundary in real time through the fluorescence imaging system.The most representative NIRF dye is Indocyanine green(ICG),which has been used in gastric fundus angiography,limb lymph node imaging,lymph node markers of gastric cancer and breast cancer,etc.,since the 1990 s.In 2009,Ishizawa et al.reported for the first time that ICG molecular fluorescence imaging could be used to guide hepatectomy.Since then,the exploration of the diagnosis and intraoperative navigation of NIR fluorescence dye in hepatocellular carcinoma(HCC)has been gradually deepened.MHI-148 is a new type of NIRF dye,which has been discovered in recent years.However,the research on the targeted imaging ability of this dye in HCC is still not comprehensive.Therefore,in this study,we will further study the imaging ability,aggregation principle and biological safety of MHI-148 in HCC cell.Most studies considered that the absorbing of the tumor tissue accumulation of MHI-148 is by the membranes polypeptide which called OATP(Organic anion transporting polypeptide).In this study,we havealso found that the amount of MHI-148 gathered by HCC cell(SNU-739)is significantly reduced after the treatment of OATP inhibitor or the decrease of OATPs related protein expression.However,the efflux mechanism of MHI-148 in tumor cells is still not clear and need to be studied systematically.The ABC(ATP-bindingcassette transporter)protein familyis a large class of transmembrane proteins that transport their binding substrates out of the cell.ABCG2 have been shown to be involved in the excretion of many intracellular and exogenous substances and play an important role in the multi-drug resistance of tumor cells.In addition,studies have shown that when treated with the inhibitors of the protein,the aggregation of the dye,suchas Hoechst 33342,in the cells can significantly increase.Based on these studies,we will explore the role of these three efflux proteins in the efflux of MHI-148 in HCC cell and HCC tissues to fill the gaps in the studies on the efflux mechanism of NIR fluorescent dyes,and to provide a theoretical basis for the further construction of NIR fluorescent dyes that are difficult to be excreted by tumor tissues and with better tumor enrichment ability.?Objectives?1.The aggregation performance,subcellular localization and biological safety of near-infrared dye MHI-148 in HCC cells were investigated in vitro,providing a theoretical basis for the clinical trials of MHI-148 in guiding intraoperative navigation of HCC.2.Explore the role of ABCG2 exonucleotide proteins in the aggregation of MHI-148 in HCC cells and tissues,to fill the gaps in the studies on the effusion mechanism of NIR fluorescent dyes,and to provide a theoretical basis for the further construction of NIR fluorescent dyes that are difficult to effuse and have better tumor enrichment ability.?Methods?1.We incubated SNU-739 cells with concentrations of MHI-148 dye and selected for the best incubate concentration.2.We incubated L-02 cells and human HCC cellsin vitro,and the cells incubated with mhi-148 dyes were photographed under the confocal laser microscopy which equipped with ICG filters that the excitation/emission of which is 750?800 nm/820?860 nm.We analysedthe fluorescence by Image J software and then compared the differences of aggregation of MHI-148 dyes in normal hepatocytes and human HCC cells.3.We incubated SNU-739 cell with MHI-148 and mitochondrial or lysosomal tracers to explorethe localization of MHI-148 in HCC cells.4.We treated SNU-739 with OATP inhibitor and then observe the role of OATP transporter in the accumulation of HCC cell.5.Exploredthe biosafety of MHI-148 by cck-8 cell activity assay.6.We treated HCC cell with different concentrations of KO143 and then explored the effect of different concentration of KO143 on HCC cells.7.Constructed stable HCC cell lines which was interferencedor overexpressedof ABCG2.Andexploredthe role of ABCG2 in the efflux ofMHI-148 of HCC cells.8.Construct a xenograft mouse model and verify whether the decrease of ABCG2 expression can enhance the aggregation of MHI-148 in HCC tissues.?Results?1.Through vitro experiments,we found that the optimal incubation concentration of MHI-148 in HCC cells was 15?M,and the aggregation capacity of MHI-148 in HCC cells was significantly superior to that of normal liver cells L-02.2.We found that MHI-148 was mainly concentrated in mitochondria and lysosomes of HCC cells through co-location experiments.3.After inhibiting OATP protein using OATP inhibitor sulfobromophthalein sodium hydrate(BSP),we found that the aggregation capacity of MHI-148 in HCC cells was significantly reduced.4.We found that MHI-148 had certain toxicity through cell viability experiments of 293 T and L-02 cells.5.The treatment of 0.8?m KO143 leads to the increase of MHI-148 aggregation in HCC cells.6.We further verified that inhibition of ABCG2 can decrease the efflux of MHI-148 in HCC cells and HCC tissues,so as to increase the aggregation of MHI-148 in HCC cells and HCC tissues through vivo and vitro experiments.?Conclusion?This study confirmed that MHI-148 which with certain toxic and side effects can accumulate in HCC specifically,enter HCC cells through OATP protein,and stay in mitochondria and lysosomes.We preliminarily provedthattheinhibition of ABCG2 can increase the aggregation of MHI-148 in HCC cells and HCC tissues.