## 4 Discussion  
Glycosylation is the most abundant post-translational modification, and the majority of eukaryotic cells express cell surface glycans [29]. Knowledge about glycome has increased in the past few years. Indeed, the role of glycans in cancer have been highlighted by the fact that alterations in glycosylation regulate the development and progression of cancer. Thus, glycans serve as important biomarkers. We have developed SA-imprinted fluorescent nanoprobes to enable SA-detection, for use as novel tools in cancer diagnostics [20]. The correlation of SA-MIP binding and EpCAM expression on the breast cancer cell lines presented in this study show an additive level of phenotyping of breast cancer cells, together with the CD44 biomarker, which showed a reverse correlation. High expression levels of EpCAM in tumors are often associated with proliferation and a more aggressive phenotype with respect to overall survival and appearance of metastasis [6]. Identification of EpCAM glycosylation sites or structures may be a crucial step to define the potential role of EpCAM in breast cancer cells [30]. Various cancer cells are lacking EpCAM expression, therefore CellSearch approaches are limited [31]. Indeed, carcinoma cells undergoing epithelial-to-mesenchymal transition can at least partially downregulate epithelial cell-specific molecules such as EpCAM [32]. Several glycosylated markers could be of interest for study, including CD24, a highly glycosylated mucin-like antigen that has recently emerged as a novel oncogene and metastasis promoter in breast carcinoma [33]. Our results show that breast cancer cells expressing \(\alpha\)-2,3 or \(\alpha\)-2,6 glycosidic linkage can bind the SA-MIPs to various degrees. The fluorescence confocal microscopy results show the binding pattern of the SA-MIPs to the different cell types. For flow cytometry analysis, we could characterize the binding pattern of MAL I, SNA, and SA-MIPs to all six cell lines. In order to validate that binding of the MIPs to cells is mediated by direct interaction with SA, the MIPs were pre-incubated with pentavalent SA-derivatives. These derivatives were designed to block attachment of cossackievirus A24V and adenovirus 37 viruses to host cells [28]. The viruses use multivalent attachment, and these defined SA-derivatives are efficacious in cell based infection and attachment assays in contrast to the SA monosaccharide that need to be used in mM concentrations to observe week inhibition. We hypothesized that these derivatives, with their flexible and long spacers, should be capable of binding to the MIPs and thereby reduce cell binding. Indeed, the results showed that the binding of the SA-MIPs to the cell line T47D could be inhibited by the addition of two different SA-derivatives. Because of their efficient inhibition ability, SA-derivatives can be used for specificity tests for SA-MIPs. The two SA-derivatives used in this study were designed from multivalent ligands intended for topical administration for the viruses CVA24v and HAdV-37 that utilize SA for cell attachment [28]. Others have shown that SA-imprinted nanoparticles could selectively bind with the SA overexpressed in DU145 cancer cells [25]. In their hands, DU145 cells, but not HeLa cells, bound the SA-imprinted nanoparticles, revealing a specificity in binding. Moreover, unmodified nanoparticles did not show any binding to the two cell lines. Interestingly, it has also been demonstrated that SA-MIPs could specifically target cancer cells over normal cells, and that the addition of SA could compete out the SA-MIP binding to the cancer cells [26; 34]. These results support our observations using the SA-derivatives, although we cannot completely reduce the binding to SA-expressing cells. The majority of breast cancers carry Tn (GalNAc-Ser/Thr) within the same cell and in close proximity to extended glycan T (GalB1,3 GalNAc), the addition of Gal to the GalNAc being catalyzed by the T synthase [35].