N-glycosylation Profiling of Colorectal Cancer Cell Lines Reveals Association of Fucosylation with Differentiation and Caudal Type Homebox 1 (CDX1)/Villin mRNA Expression

Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between α2,3- and α2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.Colorectal cancer (CRC)¹ is a very prevalent and heterogeneous pathology with highly variable disease progression and clinical outcome among patients. It is the third most common cancer in men and the second most common in women (1) with a highly stage-specific patient survival (2). Treatments are often curative for patients with local disease stages (stage I-II), whereas a 5-year survival of only 13% is observed in patients with distant metastasis (stage IV) (2). As CRC is often asymptomatic in the first years, unfortunately, only 40% of the patients are diagnosed at stage I-II, thus pointing to the urgent need of sensitive diagnostic tools for early detection and consequently effective, curative treatment (3). In this context, understanding the complex mechanisms of CRC is an overriding condition for the development of new, more efficient means of detection, treatment, and prognosis of the disease.Altered glycosylation is a hallmark of cancer (4) and is known to occur with cancer progression (4, 5) as glycans are involved in many cancer-associated events such as adhesion, invasion, and cell signaling (6). As a result of altered glycan structures, cellular processes can be affected due to a change of interactions with glycan-binding proteins (7–9). Several CRC tissue-associated changes in N-glycans, O-glycans, and glycosphingolipid glycans have been reported and recently reviewed (7). For instance, N-glycans extracted from colorectal tumor tissues are characterized by an increase of sulfated glycans, (truncated) high-mannose-type glycans, and glycans containing sialylated Lewis type epitopes, while showing a decrease of bisection as compared with glycans from nontumor colorectal tissue of the same individuals (10). In accordance, elevated expression of sialyl Lewis A (NeuAcα2,3Galβ1,3[Fucα1,4]GlcNAc-R; NeuAc = N-acetylneuraminic acid, Gal = galactose, Fuc = fucose, GlcNAc = N-acetylglucosamine, R = rest) and pauci-mannosidic N-glycans (truncated high-mannose-type, Man1–4GlcNAc1–4GlcNAc; Man = mannose) was recently found to be correlated with poor prognosis in (advanced) colon carcinomas and N-glycomic profiling was successfully applied to distinguish colorectal adenomas from carcinomas (11).Due to limitations in accessibility of tumor materials and possibilities of in vivo studies on a large scale, cancer cell lines represent a relevant alternative and are widely used as model systems for studying the molecular mechanisms associated with cancer outcome and progression. Since the early 1960s, colorectal cancer cell lines have been established with HT29, LoVo, LS-180, LS-174T, and Co115 representing the first continuous cell lines derived from colon tumors and xenografts (12–14). Major benefits of cancer cell lines are their continuous availability, their fast growth, and relatively easy handling, making them suitable also for high-throughput screenings (15) and a large range of experimental possibilities (16). Of note, advantages and limitations of cell lines have been recently reviewed (15).In order to select suitable in vitro models, the characterization of molecular features and their comparison to tumor tissues are needed. A detailed Cancer Cell Line Encyclopedia was recently established containing a genomic dataset for 947 human cancer cell lines, from which 58 are colorectal cancer lineages (17). The Cancer Cell Line Encyclopedia includes data collections on genomic characterization, point mutation frequencies, DNA copy number, and mRNA expression levels. Comparison of these features between cell lines and primary tumors showed a high correlation in most cancer types, especially for colorectal cancer, suggesting that cell lines do represent tumor tissues quite reasonably at least on the genetic level. However, the number of publications characterizing cancer cell lines at a molecular level is far behind the number of articles using cancer cell lines as model systems (18), and only few studies have been conducted on whether in vitro cultured cell lines can serve as suitable models for human tumors (19–22). Furthermore, cell lines are well characterized genetically, but they are largely understudied with regard to their glycosylation profiles.Here, we developed and optimized a new analytical method for the more sensitive and higher throughput N-glycome profiling of cells. This method is based on the release of N-glycans in a 96-well plate format from a PVDF-membrane (23) starting from a low number of cells (250,000 cells), the chemical derivatization of released N-glycans enabling the stabilization and discrimination of α2,3- and α2,6-linked N-acetylneuraminic acids (24), followed by registration of the N-glycans by MALDI-TOF(/TOF)-MS. The method was applied to characterize the N-glycome of 25 different colorectal cell lines in a fast and robust manner, including biological and technical replicates for all the cell lines. We obtained the comprehensive N-glycan profiles of 21 cell lines derived from primary tumors, two from lymph node metastases, one from a lung metastasis, and one from ascites fluid to assess their potential as glycobiological tumor model systems. Cancer cell line glycosylation features were then correlated with cancer cell markers and phenotypes as well as glycosyltransferase expressions. This study provides new insights into colon-cancer-associated glycan changes and sets a basis for studies into the functions of N-glycans in CRC with cell lines as model systems.