Screening And Application Of A Novel Probe To Specifically Target Heparin-like Epitopes On Tumor Cells

Cancer remains the second cause of mortality globally.Early diagnosis and appropriate treatment are important to improve the survival rate and prognosis of patients.Currently,target therapy that target molecules overexpressed or specifically expressed by tumor cells is considered to be the most promising therapeutic strategy,however,the heterogeneity and mutation of tumor cells often lead to the failure of target therapy,and the expression of target molecules have great individual differences,and a single target is prone to off-target in the treatment process.Therefore,there is an urgent need to enrich new targets for multitarget therapy of tumors.Glycosaminoglycans(GAGs)are widely distributed on the surface and in extracellular matrix of mammalian cells.The synthesis of GAGs is not driven by template and has a highly spatiotemporal specificity,so abnormal expression of GAGs is associated to tumorigenesis and are potential targets.In this thesis,the gene sequence of rVAR2 was used as a template for random mutation,a mutant library was constructed by phage display technology,and tumor GAGs(such as HepG2-GAGs)were used as target molecules to establish a new method for specific screening and identification of probes for the specific structures of tumor GAGs.Based on the investigation of the specificity of the probe and the structural characteristics of its epitopes,the potential application value of the probe in tumorous therapy and diagnosis was further explored.The main achievements are summarized as follows:1.Screening and specificity analysis of tumorous GAGs-binding probes:in order to screen probes that specifically recognize tumorous GAGs,a new strategy was established.rVAR2 gene sequence was randomly mutated by error-prone PCR polymerase to construct mutant gene library,and the mutants were expressed by phage display technology.Next,magnetic beads modified with HepG2-GAGs were used for high-throughput targeted screening of probes.After multiple rounds of panning,the obtained mutant genes were expressed in E.Coli,and the binding capacity of mutant clones to HepG2-GAGs was investigated by binding assay.The highest binding mutant was selected to perform further binding assay with different commercial GAGs at the molecular level,and its gene was sequenced to compare the difference with template protein,then,the effect of altered sequences on its binding capacity was investigated.Finally,we obtained a mutant protein that specifically bound to Hep and named it VAR2HP.SPR analysis showed that VAR2HP had a KD of 38 nM with Hep and 30 nM with HepG2GAGs.The gene sequence of VAR2HP contained 1953 bp,encoded a 651 amino acid protein with a theoretical molecular weight of 73.07 KDa.Compared with rVAR2,VAR2HP has an additional amino acid fragment L292C293Y294T295D296K297L298E299L300N301 and an additional N312,which participated in the specific binding of VAR2HP to Hep.The successful acquisition of VAR2HP indicates that the strategy of targeted screening of probes with specific binding capacity to other GAGs is feasible and has important practical value through random mutation of identified GAGs recognition probe gene sequence.2.Structural characteristic analysis of epitopes recognized by VAR2HP:since it is difficult to obtain sufficient amounts of cellular HepG2-GAGs for fine structure analysis,in this study,commercial Hep that can also interact with VAR2HP with high affinity were used to analyze the structural characteristics of epitopes recognized by VAR2HP.First,Hep was partially degraded and separated with gel filtration chromatography to prepare a series of Hep oligosaccharides(UDP2 to UDP16)with different degrees of polymerization.Competitive inhibition assay showed that UDP10 was the minimal oligosaccharide which strongly inhibited the binding of VAR2HP to Hep.Next,UDP 10 were further separated by anion exchange HPLC,and 11 subfractions(P10-1 to P10-11)with successively increasing charge density were obtained.Competitive inhibition assay showed that with the increase of charge density,the inhibition of these subfractions on the binding of VAR2HP to Hep gradually increased until P10-7 the inhibition increased significantly slow or even fluctuate.These results indicate that P10-7 is the minimal Hep structure with the lowest charge density that can interact with VAR2HP with high affinity.The disaccharide composition analysis of these 11 fractions showed that the content of trisulfated HexA2S(1-4)GlcNS6S increased with increase of oligosaccharide charge density.Finally,the P10-7,which was the fraction with the least charge and high affinity bound to VAR2HP,and was sequenced by enzymatic approach,and its basic sequence was HexA(1-4)GlcNS-HexA2S(1-4)GlcNS6S-HexA(1-4)GlcNS6S-HexA2S(1-4)GlcNS6SHexA2S(1-4)GlcNS6S.These results indicated that the minimal epitope of VAR2HP was decasaccharide containing at least three discontinuous trisulfated HexA2S(1-4)GlcNS6S,which was different from those epitopes recognized by the identified probes for Hep/heparan sulfate(HS)chains.VAR2HP is a novel probe for Hep/HS recognition.3.Investigation on expression of VAR2HP-recognized epitopes on cell surface and in vivo:to compare the expression of VAR2HP epitopes on normal and tumorous cells,VAR2HP was first biotinylated(VAR2HP-Bio)and fluorescence-labeled(VAR2HP-FITC),and then the binding capacities of VAR2HP on the surface of various immobilized cells and living cells were analyzed by cell ELISA and flow cytometry.The results showed that VAR2HP weakly bound to normal cells,but strongly bound to various tumor cells.Moreover,the binding of VAR2HP on tumor cell surface could be strongly inhibited by the pretreatment of cells with heparinases(Hepases)or the addition of exogenous Hep,indicating that the Hep-like structure expressed on the surface of tumor cells was involved in binding to VAR2HP.Further,we visualized and confirmed the specific binding of VAR2HP to Hep-like structures on the surface of tumor cells by laser confocal microscopy assay.These results indicate that the Hep-like epitopes recognized by VAR2HP are highly expressed in variety of tumor cells,and VAR2HP is a potential broadspectrum probe to target different tumor cells.We analyzed the disaccharide composition of HS chains in GAGs expressed by different cells,and results showed that HexA2S(1-4)GlcNS6S was expressed in all kinds of cells used in the experiment,but higher level of HexA2S(14)GlcNS6S was expressed in various tumor cells strongly bound to VAR2HP.These results indicate that the expression level of HexA2S(1-4)GlcNS6S in cells is one of the important factors that determine the binding capacity to VAR2HP.In order to further study the expression of VAR2HP epitopes in vivo,we first injected VAR2HP-FITC into mice through the tail vein,and investigated the expression and distribution of VAR2HP epitopes in various organs by observing the fluorescence intensity of each organ.The results showed that VARA2HP still had the ability to bind organs after it existed in complex internal environment for more than 6 hours.VAR2HP-FITC relatively strongly bound to liver,stomach and intestine,but was weakly bound to heart,kidney and spleen.After injecting VAR2HP-FITC,the fluorescence staining of various organs from the group pretreated with Hepases was significantly weakened,while that from the group pretreated with CSase ABC was not inhibited significantly,indicating that VAR2HP epitopes had different expression and distribution in various organs.VAR2HP is a stable protein probe and can maintain its bioactivity to specifically recognize Hep-like structure in complex in vivo environment.Subsequently,by extracting GAGs from various organs and analyzing their disaccharide composition of Hep/HS,we found that Hep/HS expressed in liver,kidney and stomach contained relatively more trisulfated Hex2S(1-4)GlcNS6S,which was consistent with the detection that VAR2HP preferred to binding liver,kidney,stomach and intestine.These results indicate that these organs contain more VAR2HP epitopes.Next,to investigate the targeting of VAR2HP to tumors in vivo,VAR2HP-FITC was injected into lung tumor-bearing mice constructed with murine breast cancer cell line 4T1 cells via tail vein,in vivo imaging results showed that VAR2HP had high fluorescence intensity in hepatic and intestinal region,and tumorigenic lung region.After the lungs were taken for fluorescence detection,the results showed the fluorescence intensity of tumorous nodules in lung was significantly higher than that of adjacent tissues,suggesting that VAR2HP is promising to be used as a selectively targeting tumor probe and antitumor drug carrier in complex physiological environment in vivo.4.Preliminary study on the application potential of VAR2HP in targeted therapy:to investigate the effect of VAR2HP as drug carrier on enhancing the anti-tumor activity of drugs by targeting the overexpressed Hep-like epitopes in tumorous cells,we first analyzed the endocytosis of VAR2HP after binding to cells,laser confocal analysis showed that VAR2HP could rapidly bind to cellular surface and be internalize into the nucleus within 45 min,suggesting that VAR2HP has the potential to be used as a carrier to deliver drugs into target cells.Subsequently,VAR2HP was coupled with the broad-spectrum anticancer drug doxorubicin hydrochloride(DOX)(VAR2HP-DOX),and the targeting of VAR2HP-DOX to tumor cells was analyzed by flow cytometry.The results showed VAR2HP-DOX rapidly bound to cells within 10 min.Although the content of DOX in VAR2HP-DOX was only one-tenth of that of DOX group,its fluorescence intensity was significantly higher than that of DOX group,indicating that VAR2HP could promote drug enrichment in tumor cells.Next,we analyzed the effect of VAR2HP,VAR2HP-DOX and DOX on the viability of different cells,a MTT assay showed that VAR2HP weakly inhibited cell proliferation and higher inhibited tumor cells when acted on a variety of cell lines alone.DOX had a strong lethal effect on all tested cells including normal and tumorous cells,but the lethal effect on 4T1 cells was more significant.The lethal effect of VAR2HP-DOX on normal cells was significantly lower than that on tumor cells.After treatment for 24 h,the IC50 value of VAR2HP-DOX on 4T1 cells was 5.01±0.27 μg/ml,and the dosage of DOX was reduced by 30 times compared with DOX group,while the IC50 value could not be calculated for normal HEK 293T cells at the experimental concentration.These results showed that VAR2HP as drug carrier had excellent targeting to tumorous cells,which was conducive for reducing the damage of the drug to normal cells.To further study the antitumor activity of VAR2HP-DOX in vivo,VAR2HP,VAR2HP-DOX and DOX were subcutaneously administered to 4T1 cells-tumor-bearing mice,results showed VAR2HP-DOX could significantly inhibit tumor growth,and VAR2HP-DOX group still achieved a similar therapeutic effect to DOX group even when the dose of DOX was reduced by 16 times compared with that of DOX alone.Additionally,during the treatment process,the experimental group injected with VAR2HP-DOX had significantly lower weight loss than DOX group,indicating that VAR2HP could significantly reduce the damage of DOX to normal tissues by specifically targeting tumor tissues,and was an ideal drug carrier for tumor targeted therapy.5.Preliminary investigation of VAR2HP for tumor diagnosis:Biotinylated VAR2HP(VAR2HP-Bio)was used as a probe to conduct immunohistochemical staining analysis on paraffin sections of different clinical tumor tissues.The results showed that the VAR2HP-Bio staining of liver cancer and breast cancer tissues were significantly stronger than that of paracancer tissues,and the staining could be competitively inhibited by added exogenous Hep,suggesting that Hep-like epitopes recognized by VAR2HP were overexpressed in clinical liver cancer and breast cancer tissues,and these epitopes could be used as potential target molecules for tumor diagnosis and targeted therapy.Furthermore,we investigated the expression of VAR2HP epitopes in the serum of clinical patients with HCC and the potential of VAR2HP epitopes as a target molecule for diagnosis of HCC.Firstly,we used VAR2HP-modified Sepharose 4 Fast Flow to isolate Hep/HS from clinical serum samples of normal person(NL),hepatitis patients(CH)and HCC patients(HCC)respectively and analyzed disaccharide composition.The results showed that the total Hep/HS content in serum of HCC patients reached 331.61 μg/ml,which was significantly higher than those in NL(192.20 μg/ml)and CH(197.29μg/ml),and Hep-like structure recognized by VAR2HP only existed in HCC serum with a content of 36.28μg/ml,indicating that Hep/HS was not only overexpressed in HCC serum but also contained specific VAR2HP epitopes,which could be used as serum marker for the diagnosis of HCC.Based on these findings,we developed a diagnostic method for HCC according to the principle of fluorescence energy transfer(FRET).Thus,we quantified VAR2HP-recognized epitopes in serum by detecting the recovery of GO-caused fluorescence quenching after adding clinical serum for HCC diagnosis.The results showed that the residual fluorescence intensity after adding HCC serum was higher than those after adding NL serum and CH serum,and could be significantly reduced after the serum was pretreated with Hepases,indicating that HCC serum contained more VAR2HP epitopes,which inhibited the fluorescence quenching of VAR2HP-FITC by GO.To facilitate to calculate the detection rate of this method,the residual fluorescence intensity of VAR2HP-FITC quenched by GO in PBS buffer was used as the control group.The data were shown as increased fold relative to the control group.When using the increased fold of CH serum residual fluorescence intensity plus double standard deviation as the lower limit,the detection rate of HCC reached 59.97%.The detection rate of Glypican-3 and AFP as serum markers for HCC are also about 60%,indicating that Hep/HS with special structure in serum can be used as a new marker for HCC diagnosis and is feasible.This study provides a new detection method for HCC diagnosis.To sum up,this study establishes a new strategy and technology platform that can be used for the purposeful screening of specific probes for tumor-related GAGs.By using this platform,the gene sequences of identified GAGs-binding proteins can be used as templates,and through random mutation combined with phage display and direct panning of target GAGs,the specificity of existing GAG probes can be optimized realized and new specific probes for GAGs with specific structure can be purposefully screened,which can not only meet the urgent need for specific probes in the structural and functional studies and detection of complex GAGs,but also provide urgently needed specific probes for the diagnosis and targeted therapy of GAGs-related diseases.In this thesis,the obtained probe VAR2HP specifically bound to Heplike structure can be used as a new biocompatibility probe not only for the specific detection of Hep in vitro and in vivo,but also for targeting Hep-like epitopes overexpressed on the surface of various tumor cells、VAR2HP and its epitopes,as tumor GAGs-specific probes of and tumor tissue and serum markers,respectively,have important potential application value in tumor diagnosis and targeted therapy.