Abstract
Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO "SimpleCell" (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer.
Highlights
From the ‡Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark; §IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Rua Dr Roberto Frias s/n, 4200 – 465 Porto, Portugal; ¶Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Rua Dr Antonio Bernardino de Almeida 4200 – 072 Porto, Portugal; ʈQOPNA, Department of Chemistry of the University of Aveiro, Campus Universitario de Santiago 3810 –193 Aveiro, Portugal; **Department of Pathology, Portuguese Institute of Oncology, Rua Dr Antonio Bernardino de Almeida 4200 – 072 Porto, Portugal; ‡‡Faculty of Medicine of the University of Porto, Al
We first characterized the Oglycoproteome and including the secretome of two gastric cancer cell lines, AGS and MKN45, using our SimpleCell (SC) discovery platform where we identified a total of 499 O-glycoproteins (1236 O-glycosites)
It is our hypothesis that cancer cells produce and shed/ secrete O-glycoproteins with truncated cancer-associated Oglycans that can be detected in circulation, and that such O-glycoproteins may serve as biomarkers with higher specificity
Summary
Generation and Characterization of AGS and MKN45 SimpleCells—We targeted the COSMC gene in two human gastric cell lines, AGS and MKN45, using zinc finger nuclease (ZFN) precise gene editing as previously described [26, 29, 30]. The digestion was quenched with 1% trifluoroacetic acid (TFA), and the resulting peptides were desalted with C18 stage tips and dried in a SpeedVac. The digest was neuraminidase treated (New England Biolabs, Ipswich, MA), diluted in 2 ml LAC A buffer and injected onto a pre-equilibrated 2.6 m long VVA agarose (Vector Laboratories, Burlingame, CA) column, similar to the system described previously [28, 34, 35]. Paraffin sections were dewaxed, rehydrated, antigen retrieval was performed as described previously [38], and endogenous peroxidase blocked before sections were incubated for 30 min at 37 °C with the Blocking Solution (Olink Bioscience, Uppsala, Sweden). Conjugated MAbs to CD44v6 (1:50) and STn (1:200) (labeled with PLA probes) were diluted in PBS with 5% BSA and with 1:20 of assay reagent (Olink Bioscience, Uppsala, Sweden) and slides incubated for 1 h at room temperature. Images were acquired using a Zeiss Axio cam MRm and the AxioVision Rel. 4.8 software
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