Glycan Profiling On The Cancer Cell Surface By Lectin Microarray

ObjectiveGlycans and their conjugates are involved in various biological events, including cell recognition, cell adhere, communication, immune response. Altered glycosylation is closely associated with the occurrence, development, progression and metastasis of cancer. Thus, the investigation of these glycosylation changes may reveal insight into glycan effect in the cancer progression.Due to their ability to bind reversibly with specific carbohydrate structures, lectins have extensively been used as "Translators" to translate the information of the sugar code. Traditional lectin-based analytical methods are not able to attain comprehensive information in a high-throughput fashion in one experiment.A lecin microarray system was constructed which could detect glycans on cell surface throughly. The advantage is mainly:combining lectins with high-throughput, specific and sensitive array, cells are automatically captured by lectin microarray, which makes it possible to analyze glycans on cell surface in a direct and rapid manner. The technology implements successfully the high-throughput analysis of glycans, and further gain more comprehensive biological information.The purpose of our study is to demonstrate the accuracy, reproducibility and specificity of our lectin microarray. Then we applied this system to examine glycan differences upon cell surface of cancer lines, normal mice and hepatoma mice tissues, and gain their glycan expression profilings. This work could greatly contribute to choose specific cancer-related glycan markers.Methods1,The accuracy, reproducibility and specificity of lectin microarrayThe accuracy: The glycan binding profiling of PANC-1 was detected by lectin microarray. According to references, glycan expression of PANC-1 cell surface was analyzed and contrasted with array results.Two representative lectins (SBA, LTL) were choosen as probes. After FITC labeled and single cell suspension reacted, their fluorescent signals were analyzed by flow cytometry. The result was contrasted with array results.The reproducibility:Identical PANC-1 cell samples were hybridized to different lectin microarray slides on different dates. Fluorescence values for all spots were used to assess the spot variability and reproducibility.The specificity:The array-linked covalently lectins and glycan specificity were validated by lactose inhibition assay:The specificity of array which captures cells was validated by ELISA assay:2,Detecting glycan profiling of cancer cell surface by lectin microarrayCancer cell lines were detected by lectin microarray in order to analyze glycan expression. The cell lines included:hepatic cells (L-02,BEL7402,BEL7404),gastric cells (GES-1,SGC7901,MGC,MKN,BGC),nonsmall-cell lung cancer cells (H460,A549),colon carcinoma cells(HT29).3,Detecting glycan changes on the cell surface between hepatoma and normal tissues by lectin microarray.To obtain though-out information of glycan changes in hepatocarcinoma progress, we used lectin microarray to observe H22 cell line and mouse liver cell surface glycan difference between normal and hepatocarcinoma mice. After marine hepatocarcinoma model established, the changes were analyzed by array scanning and appearance observation. At last, we obtained the statistical data of glycan changes during hepatoma progression.Results 1,The accuracy, reproducibility and specificity of lectin microarrayThe accuracy:The array results correlated well with prior reports on PANC-1 glycan expression. The flow cytometry data correlated well with microarray binding. Those indicated that our microarray has a good characteristic of accuracy.The reproducibility:It was found that standard deviations for all binding spots was within 10% of the mean value, all replicates considered. Those indicated that our microarray has a good characteristic of reproducibility.The specificity:The array-linked covalently lectins and glycan was specific.That the array which captures cells was specific. Those indicated that our microarray has a good characteristic of specificity.2,Detecting glycan profiling of cancer cell surface by lectin microarrayThe accessible cell-surface glycans varied significantly from one tissue to another. These cancer cell lines shared the same sugar characterization, including Sialic acid, GlcNAc/Glucose, GalNAc/Galactose, Mannose. Additionally, Those cells surface possibly expressed little al-3 Fucose, and it may have no or littleal-3 Fucose on gastric cancer cells.3,Detecting glycan changes on the cell surface between hepatoma and normal tissues by lectin microarray.The result showed that Sia, GluNAc, GalNAc, Man and Gal increased on hepatocarcinoma cell surface. No apparent evidence showed that the Fucose takes part in the cancer developing progression in our expriments.ConclusionsOur microarray has a good characteristic of accuracy, reproducibility and specificity. Lectin microarray provides dynamic, direct and high-thoughput detection for cell-surface glycans, which is an effective tool for analyzing cell-surface glycome profilings in cell development and metastasis.The cell-surface sugars of cancer cells from different tissues increased, including Sialic acid, Lewisx,GlcNAc/Glucose, GalNAc/Galactose and Mannose. These glycans may be potential tumour-related markers.The membrane sugars of hepatoma tissue enhanced significantly, including Sialic acid, Lewisx GlcNAc/Glucose, GalNAc/Galactose and Mannose. These glycans and related glycoprotein may have certain impacts during hepatoma occurrence and progression.