Linical animal studies showed promising immunogenicity, but none of the Rosiglitazone immuno conjugates succeeded in clinical trials, despite safe administration and proper immune responses [28]. Recent and ongoing vaccine trials are encouraging for future trials and the design of proper immunogens and immuno-conjugates remain the main challenge. Interestingly, ABO blood group IgM agglutinins/antibodies were observed to interact with PDAC O-GalNAc modified glycoproteins possibly affecting cancer onset [29]. Nevertheless, pancreatic carcinoma is one of the worlds’ most aggressive malignancies [30] and consequences of COSMC mediated Tn antigen expression in pancreatic carcinoma are not fully understood. Investigation of Tn modified glycoproteins and its impact on oncogenic properties is crucial to understand tumor biology and potential therapeutic options.ResultsDifferential expression of Tn antigen in human pancreatic carcinoma cell linesSeveral PDAC derived cell lines were available for Tn antigen screening using Western and Far-Western blot analysis. Besides commercially available PDAC cell lines such as Panc-1, BxPC3, MiaPaca2 and L3.6pl, patient derived cell lines PaCa 5061 [31], PaCa 5072 and PaCa 5167, and a Gemcitabine resistant sub-clone of the parental L3.6pl cell line, L3.6pl-res cells were used [32]. Analysis of Tn antigen expression in cell lysates was performed using distinct antibodies or glyco epitope recognizing lectins. Jurkat cells express mutated COSMC [13] and were used as Tn antigen positive control. Interestingly, PDAC cell lines displayed differential expression of the Tn antigen. Using the Tn antigen specific antibody MA1-90544, L3.6pl-Res and PaCa 5072 cells showed a strong positivity, whereas L3.6pl, PaCa 5061 and BxPC3 cells were weakly positive. Minimal signals were detectable in Panc-1, PaCa 5167 and MiaPaca2 cells (Fig. 1b). Similar results were obtained using VVL, HPA lectins and sialyl Tn antibody reflecting consistency (Additional file 1: Figure S1-S3). In order to induce the expression of Tn antigen in the minimal Tn antigen positive Panc-1 cell line and the moderate Tn antigen positive L3.6pl cell line, we performed a lentiviral-mediated knockdown of COSMC chaperone followed by single colony selection with puromycin. Knockdown of COSMC was highly efficient in Panc-1 cells, compared to cells transduced with a controlHofmann et al. Molecular Cancer (2015) 14:Page 3 ofFig. 1 (See legend on next page.)Hofmann et al. Molecular Cancer (2015) 14:Page 4 of(See figure on previous page.) Fig. 1 Expression of aberrant O-glycans in pancreatic cancer. a Biosynthesis of Tn antigen, PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/6833145 sTn antigen and Core1 and 3 structures. Tn antigen is composed of an O-glycosidic linked N-acetylgalactosamine (GalNAc) to the H group of serine/threonine (S/T). Tn antigen is either processed by core 1 T-synthase (C1GalT1) and its chaperone (COSMC), which transfers a galactose (Gal) to GalNAc-serine/threonine to form the T antigen also referred as core 1 structure or processed by transfer of a N-acetylglucosamine (GlcNAc) to form the core 3 structure. Tn antigen can also be modified by addition of a sialic acid (NeuAc). b Differential expression of Tn antigen in pancreatic carcinoma cell lines. Eight different PDAC cell lines were available for analysis. Western and Far-Western blot analysis of total cell lysates was performed using the Tn antigen specific antibody MA1-80055. Detection of HSC70 served as loading control. Jurkat cells were used as posi.