Journal: Theranostics

Article Title: Glycoproteogenomics characterizes the CD44 splicing code associated with bladder cancer invasion

doi: 10.7150/thno.67409

Figure Lengend Snippet: CD44-Tn and STn glycoproteoforms present high cancer specificity. A) Tn and STn antigens are not expressed in the healthy urothelium, are elevated in cancer, and increased in invasive tumors. The STn antigen, defined by affinity for the anti-tag-72 antibody [B72.3+CC49], is not expressed in the healthy urothelium and is elevated in cancer, being significantly overexpressed in muscle invasive bladder cancer (MIBC). The Tn antigen, based on VVA lectin immunoaffinity, is also not expressed in the healthy urothelium and is elevated in a subset of patients, specially at more advanced stages. B) CD44s colocalizes with Tn and STn antigens in MIBC. CD44 was diffusively expressed across the tumor section without a defined pattern. The STn antigen was observed both in superficial and invasive layers and the Tn antigen was found in scattered niches without a defined expression pattern. Immunohistochemistry also showed the co-localization of CD44 with Tn and STn positive areas in CD44s high tumors, exhibiting low amounts of other isoforms (according to RT-PCR, data not shown). The healthy urothelium expressed high amounts of CD44 and the Tn antigen is present in the cytoplasm of upper stratum umbrella cells while the STn antigen was not detected. C) In situ proximity ligation assays (PLA) supports CD44s-STn glycoproteoforms in MIBC and its presence within tumor invasive fronts. In situ proximity ligation assay showed close spatial proximity between CD44 and STn in the same cells, strongly supporting CD44-STn glycoproteoforms in tumors. This phenotype was mostly observed in invasive fronts of CD44s high tumors and was not detected in the healthy urothelium, suggesting cancer-specificity. D) Double staining immunofluorescence supports CD44s-Tn glycoproteoforms in bladder cancer. CD44s high tumors presented niches of cells co-expressing CD44 and Tn antigen, strongly suggesting CD44-Tn glycoproteoforms. This was not observed in the healthy urothelium. E) Glycosylation with Tn and STn antigens provides cancer specificity to CD44. CD44 was abundantly expressed in all studied healthy tissues. STn expression was either absent or low in secretions and cells facing the lumen of the respiratory, gastrointestinal, and colorectal tracts. Immunohistochemistry suggested STn and CD44 co-localization in the stomach, appendix, small intestine, colon, gallbladder, and white blood cells in MALT. PLA did not confirm these hypotheses, suggesting cancer specificity of CD44-STn glycoproteoforms. In healthy tissues, the Tn was restricted to the cytoplasm of goblet cells in the intestinal tract, Leydig cells in testicular tissue, pancreatic acini, hepatocytes, mucinous cells of the gastric epithelium, alveolar macrophages, and gallbladder epithelium. CD44 and Tn antigen co-expression was suggested in pancreatic tissue, testicle, and gallbladder, which was not confirmed by double staining immunofluorescence.

Article Snippet: FFPE bladder tumors and healthy tissue sections were screened by immunohistochemistry for CD44 and Tn and STn antigens, as previously described by Peixoto, A et al . . Tn antigen expression was evaluated using the biotinylated VVA lectin (Vector Laboratories, 40 mg/mL, 1 hour at 37 ºC; ) and the detection of STn and CD44 antigens were performed using the anti-tag-72 (B72.3 + CC49; Abcam, 0.5 mg/mL, overnight at 4 ºC; ) and anti-CD44 (1:5000, ab157107, Abcam; ) antibodies, respectively.

Techniques: Expressing, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction, In Situ, Ligation, Proximity Ligation Assay, Double Immunofluorescence Staining