Screening And Application Of Glycosylated Ligands For Colorectal Cancer Based On Lectin Microarray

Glycans/glycoconjugates such as glycoproteins,proteoglycans and glycolipids participate in a series of important physiological and pathological processes including cell differentiation and proliferation,cell-cell communication,immune response,and tumor growth and metastasis.Lectin,a kind of non-enzyme and non-antibody carbohydrate binding proteins,exhibits high specificity for saccharide moieties.And lectins can be used as specific biomarkers for early diagnosis and classification of tumors due to the differences in the expression of glycans on cell surface between tumor cells and normal cells.Therefore,sensitive and high-throughout analysis methods are great significant for investigating the interaction of glycans on cell surface with lectins.Due to the advantages of lower sample consumption,automation and high-throughput,microarrays have been widely used in the field of gene and protein.In particular,lectin microarray has become an important tool to study the expression of glycans on cell surface,cell classification,and the detection and capture of mutant cells.Here,a polyacrylamide(PAAM)hydrogel-based lectin microarray was fabricated for profiling glycan expression on cell surface of different cell lines including three common colorectal cancer(CRC)cell lines and one normal colon cell line,and the lectin exhibiting reasonable specificity with early colorectal cancer cells(SW480)was screened.Furthermore,the specific lectin was used as a glycosylated ligand for the diagnosis and treatment of early CRC.The main points of this thesis are outlines as follows:1.A PAAM hydrogel-based lectin microarray was prepared for profiling glycan expression on CRC cell surface,and uelx europaeus agglutinin-I(UEA-I)was identified to have specific binding affinity with SW480 cells through analyzing the interactions of glycans on cell surface of three common CRC cell lines(SW480,HCT116 and SW620)and one normal colon cell line(NCM460)with 27 different lectins.After conjugation of UEA-I with silica-coated NaGdF 4:20%Yb,2%Er@NaGdF 4 upconversion nanoparticles(termed as UCNP@SiO2-UEA-I),the follow-up in vitro and in vivo experiments provide further evidence on that UEA-I can serve as a glycosylated ligand to diagnose SW480 tumor by multimodal imaging including upconversion luminescence(UCL)imaging,T1-weighted magnetic resonance(MR)imaging,and X-ray computed tomography(CT)imaging.2.The PAAM-based lectin microarray modified with UEA-I was applied to evaluate five small molecule inhibitors against ?-1,2-fucosyltransferase(?-l,2-FucT)indirectly.Furthermore,fluorescein labeled uelx europaeus agglutinin-I(termed as FL-UEA-I)and lanthanide-doped upconversion nanoparticles modified with uelx europaeus agglutinin-I(termed as UCNP@SiO2-UEA-I)were served as fluorescence reporters to monitor the inhibitory effect of quercetin by the cell fluorescence imaging and the tumor UCL imaging,respectively.The effect of quercetin to cell proliferation and tumor growth were also obtained.These results verified the analysis result of lectin microarray,and quercetin can be used as a potential drug for the treatment of early CRC.3.A novel carboxy-terminated silica coated NaErF4:10%Yb@NaYF4:40%Yb@NaNdF4:10%Yb@NaGdF4:20%Yb upconversion nanoparticles(termed as UCNP@SiO2-COOH)with 808 nm near-infrared(NIR)exciation and bright 655 nm upconversion luminescence(UCL)emission was synthesized for realizing deep tissue imaging.After conjugated with various molecules including NH2-PEG3400-COOH,peptide D-SP5 and UEA-I,biodistribution,clearance pathway and tumor-targeting capacities of the UCNP@SiO2-COOH and corresponding bioconjugates(termed as UCNP@SiO2-PEG,UCNP@SiO2-D-SP5 and UCNP@SiO2-UEA-I,respectively)were investigated by tracking the UCL intensities of livers,kidneys and tumors.Both of in vitro and in vivo experimental results revealed that there was no significant difference for their in vivo biodistributions and clearance pathways,but the UCNP@SiO2-UEA-I exhibited much higher SW480 tumor targeting capacity than those of other nanoprobes.In particular,the as-prepared UCNP@SiO2-UEA-I could serve as an efficient optical nanoprobe to diagnose ultrasmall(c.a.3 mm3 in volume)subcutaneous SW480 tumor in Balb/c mouse.4.A cell-sorting strategy based on UEA-I modified Fe3O4 magnetic beads(termed as MB-UEA-I)was developed for efficiently and specifically capture ?-1,2-fucose overexpression circulating tumor cells(CTCs)from the cell spiking complete culture medium and whole blood.More than 90%of captured cells remained good viability and proliferation ability without detached from MB-UEA-I,which satisfied requirements of the downstream mutation detection and cell proliferation.Furthermore,combination with three-color immunocytochemistry(ICC)identification,MB-UEA-I was successfully applied to isolate and detect of CTCs with ?-1,2-fucose overexpression in peripheral bloods of CRC patients,which demonstrated the practicability of MB-UEA-I.The experimental results also indicated that primary CRC and metastatic CRC had different CTC characterization.