| BackgroundHepatocellular carcinoma(HCC)is the most common type of primary liver cancer.In90% of cases,HCC usually occurs in the setting of chronic cirrhosis or disease.In China,the 5-year overall survival(OS)rate for patients with HCC is less than 13%.HCC is insidious in its onset,with few obvious symptoms in the early stages of HCC,and most HCC is diagnosed at an advanced stage,and there is no curative treatment for advanced HCC,probably because the liver cancer cells upregulate their resistance to chemotherapeutic agents.Therefore,the diagnosis and staging of HCC is particularly important in order to maximise the effectiveness of treatment and improve the prognosis of HCC.Endoplasmic reticulum stress(ERS)is a mechanism of self-protection in hepatocellular carcinoma cells and possibly a cause of drug resistance in hepatocellular carcinoma cells.Glucose regulatory protein 78(GRP78)/ Bi P is a multifunctional protein and a marker protein of endoplasmic reticulum stress.The expression level of GRP78 in hepatocellular carcinoma cells is largely representative of the level of endoplasmic reticulum stress in hepatocellular carcinoma cells.SP94(SFSIIHTPILPL)is a peptide that binds specifically to hepatocellular carcinoma(HCC),and its coupled polymer shows a 104-fold higher affinity for HCC than for normal hepatocytes.This means that we can design targeted molecular probes for GRP78 and analyse the level of endoplasmic reticulum stress in hepatocellular carcinoma cells by imaging to evaluate the impact of endoplasmic reticulum stress on the efficacy of hepatocellular carcinoma treatment.ObjectiveGRP78 is overexpressed in a multitude of solid tumours and is a well-characterised target for imaging or treatment of tumours,as well as a marker of endoplasmic reticulum stress.To investigate the development of a novel GRP78-responsive radiotracer([68Ga]SP94),evaluate its therapeutic performance for diagnostic micro-PET imaging of GRP78 expression in HCC and analyse the impact of endoplasmic reticulum stress on drug resistance in tumour cells.Methods1.Cell and probe testing experiments1.1 Western blotting to detect the expression of GRP78 in HCC cells and human normal hepatocytes1.2 Annexin V-FITC/PI,Western blottin to detect endoplasmic reticulum stress resistance to lenvatinib-induced apoptotic properties in Hep G2 cells.1.3 Western blotting to analyse the relationship between endoplasmic reticulum stress and GPR78 expression levels and to evaluate the effect of lenvatinib on GPR78 expression levels1.4 Solid phase synthesis of the labelled precursor DOTA-SP94 and radiolabelling with68 Ga Cl3 to obtain the [68Ga]SP94 molecular probe.1.5 Identification and calculation of the radiochemical purity of the [68Ga]SP94molecular probe by radio-HPLC.1.6 To test the stability of the [68Ga]SP94 molecular probe in PBS solution,saline,5%HAS solution and mouse serum and perform Radio-HPLC analysis.1.7 Calculation of the lipid-water partition coefficient Log P of the [68Ga]SP94molecular probe in n-octanol/PBS buffer as determined by ?-counter.1.8 ?-counter measurement of the cellular uptake rate of [68Ga]SP94 after incubation with Hep G2 and LO2 cells,respectively.2.Animal experiments2.1 Pharmacokinetic assay of [68Ga]SP94The concentrations and clearance rates of [68Ga]SP94 in the blood of mice with Hep G2 tumours were measured at various time points.2.2 Micro-PET imaging of [68Ga]SP94Mice carrying Hep G2 tumours of approximately 250 mm3 in volume were randomly divided into two groups of four mice each.Mice in the experimental group were injected with [68Ga]SP94 only and mice in the control group were injected with[68Ga]SP94 and its blocker(DOTA-SP94).The ability of [68Ga]SP94 to target Hep G2 cells with high GRP78 expression was assessed by Micro-PET imaging and biodistribution assays and its distribution in tumour tissue,heart,liver,spleen,kidney,lung,intestine,pancreas and muscle at different time points and These results were expressed as percent of injected dose per gram(ID%/g).The ratio of [68Ga]SP94uptake in tumour vs muscle(T/M),in tumour vs blood(T/B)and in tumour vs liver(T/L)in the experimental and control groups at different time points were examined to further assess the targeting of [68Ga]SP94 to Hep G2 cells.2.3 Micro-PET imaging of [68Ga]SP94 to assess lenvatinib efficacy test Hep G2 tumour-bearing mice carrying a volume of approximately 250mm3 were randomly divided into treatment and non-treatment groups.Tunicamycin was used as an endoplasmic reticulum stress inducer.The treatment groups were lenvatinib group and(lenvatinib + tunicamycin)group,and the non-treatment groups were control group and tunicamycin group,with 5 mice in each group.Hep G2 tumour-bearing mice in the treated and non-treated groups underwent micro-PET imaging using [68Ga]SP94 on days 7 and days 14 of lenvatinib or tunicamycin intake,and tumour tissue uptake of[68Ga]SP94 was quantified and GRP78 expression levels were assessed in each group.Uptake results were expressed as(ID%/g).Tumour volumes from days 0 to days 14 in treated and non-treated Hep G2-bearing mice were measured and analysed in comparison with [68Ga]SP94 uptake to assess the efficacy of [68Ga]SP94 uptake on lenvatinib treatment and calculate the tumour inhibition rate.2.4 Western blotting of GRP78,apoptosis-associated protein(bax)and apoptosis-negative protein(bcl-2)expression levels in tumour tissues of treated and non-treated Hep G2-bearing mice after 14 days and comparison with the uptake of[68Ga]SP94,to explore the association between [68Ga]SP94 assessment of endoplasmic reticulum the effect of stress on lenvatinib drug sensitivity.2.5 Immunohistochemical techniques were used to detect the expression levels of GRP78,P53,VEGF and Glut-1 in the tumour tissues of treated and non-treated Hep G2tumour-bearing mice after 14 days and to analyse the association between these levels and the uptake of [68Ga]SP94.Results1.Cell and probe testing experiments1.1 Western blotting detected significantly higher expression of GRP78 in HCC cells than in human normal hepatocytes.1.2 Apoptosis rate by flow cytometry and expression of anti-apoptotic and apoptotic proteins by western blotting showed significant resistance of endoplasmic reticulum stress to lenvatinib-induced apoptosis in Hep G2 cells.1.3 Western blotting results showing that endoplasmic reticulum stress significantly increases GPR78 expression levels.Lenvatinib will increase the GPR78 expression level of Hep G2 cells to some extent,but will inhibit the effect of endoplasmic reticulum stress.1.4 A molecular probe for [68Ga]SP94 was obtained by solid-phase synthesis of the labelled precursor DOTA-SP94 and radiolabelled with 68 Ga Cl3.1.5 Radio-HPLC identified and calculated the radiolabelling purity of the [68Ga]SP94molecular probe to be >99%,with excellent radiolabelling purity and high yield,which is very suitable for micro-PET imaging.1.6 The [68Ga]SP94 molecular probe did not dissociate or decompose significantly in PBS solution,saline,5% HAS solution and mouse serum,indicating that [68Ga]SP94has good stability in vivo and in vitro.1.7 The lipid-water partition coefficient of the [68Ga]SP94 molecular probe in n-octanol/PBS buffer was 2.36 ± 0.002;indicating that [68Ga]SP94 has a high hydrophilicity.1.8 The uptake of [68Ga]SP94 by Hep G2 cells was significantly higher than that of[68Ga]SP94 by LO2 cells at all time points,and the rate of uptake of [68Ga]SP94 by Hep G2 cells was also significantly higher than that of [68Ga]SP94 by LO2 cells.The results of the cell binding affinity assay showed that Hep G2 cells had a high affinity for[68Ga]SP94.2.Animal experiments2.1 Pharmacokinetic studies of [68Ga]SP94 have shown that [68Ga]SP94 is rapidly distributed to tissues and organs and is rapidly cleared from the blood.2.2 Micro-PET imaging tests of [68Ga]SP94Higher tumour uptake of [68Ga]SP94 was observed in the experimental group of Hep G2 tumour-bearing mice(i.e.injected with [68Ga]SP94 only)for a period of 0.5hours post-injection and then gradually diminished.In contrast,in control mice(i.e.injected with both [68Ga]SP94 and DOTA-SP94),the radiotracer was concentrated in the kidney throughout the scan,and tumour tissue was barely observed,indicating the specific targeting ability of [68Ga]SP94 on GRP78 and its value in guiding clinical applications.The results of the biodistribution assay showed that the uptake of[68Ga]SP94 reached a maximum of 3.53 ± 0.447 %ID/g at 0.5 h post-injection and was then slowly cleared from the tumour.The highest uptake of [68Ga]SP94 was shown in the kidney,indicating that most of it was excreted mainly through the urinary system.Meanwhile,slight [68Ga]SP9 uptake was observed in the liver,suggesting that a small proportion of it was metabolised by the liver.The(T/B)and(T/M)of the experimental group were 1.75±0.235 and 4.40±1.690 at 0.5h,1.49±0.207 and 4.41±0.803 at 1h,and1.41±0.155 and 3.96±0.497 at 2h,respectively,indicating that the probe has a good signal-to-noise ratio in Hep G2 tumours.2.3 Micro-PET imaging of [68Ga]SP94 to assess the efficacy of lenvatinib Micro-PET imaging results showed significantly higher uptake of [68Ga]SP94 by tumours in the tunicamycin group than in the control group at days 0,7 and 14 in the non-treatment group.The uptake of [68Ga]SP94 was lower in the lenvatinib group than in the(tunicamycin+lenvatinib)group within the treatment group.These results showed a consistent trend with the expression levels of GRP78 in tumour tissues of all groups as well as in Hep G2 cells in vitro.The tumour volumes in the treated group were much smaller than those in the non-treated group,while the tumour volumes in the(tunicamycin+lenvatinib)group were all larger than those in the lenvatinib group in the treated group.The tumour suppression rates in the lenvatinib group were 62.95% on day7 and 70.22% on day 14 compared to the control and tunicamycin groups respectively(Table 3),which were greater than those in the lenvatinib+tunicamycin group.2.4 Western blotting results showed that the ratio of bcl-2 vs bax in the treated group was much smaller than that in the non-treated group.The ratio of bcl-2 vs bax in the treated group(tunicamycin + lenvatinib)was greater than that in the lenvatinib group.2.5 Immunohistochemistry showed that the expression of P53,VEGF and GLUT-1 in the treated group was much lower than that in the non-treated group.In the treated group,the expression of P53,VEGF and GLUT-1 was lower in the lenvatinib group than in the(tunicamycin + lenvatinib)group.The trend of GRP78 expression levels in each group was consistent with the trend of [68Ga]SP94 uptake in each group.ConclusionThe [68Ga]SP94 molecular probe developed in this study showed strong affinity for Hep G2 cells with high GRP78 protein expression and showed highly specific accumulation in GRP78-expressing Hep G2 tumours.The uptake of [68Ga]SP94 in tumor tissues in all groups was consistent with the expression levels of GRP78 detected in in vitro experiments in all groups,and the results of the comparative analysis of its uptake in all groups with the experimental results of therapeutic efficacy in all groups suggest that [68Ga]SP94 can be used to some extent to evaluate changes in endoplasmic reticulum stress levels in Hep G2-bearing mice and the effect of endoplasmic reticulum stress on the sensitivity of hepatocellular carcinoma cells to chemotherapeutic drugs. |
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