Background:High risk of recurrence/progression bladder tumours is treated with Bacillus Calmette-Guérin (BCG) immunotherapy after complete resection of the tumour. Approximately 75% of these tumours express the uncommon carbohydrate antigen sialyl-Tn (Tn), a surrogate biomarker of tumour aggressiveness. Such changes in the glycosylation of cell-surface proteins influence tumour microenvironment and immune responses that may modulate treatment outcome and the course of disease. The aim of this work is to determine the efficiency of BCG immunotherapy against tumours expressing sTn and sTn-related antigen sialyl-6-T (s6T).Methods:In a retrospective design, 94 tumours from patients treated with BCG were screened for sTn and s6T expression. In vitro studies were conducted to determine the interaction of BCG with high-grade bladder cancer cell line overexpressing sTn.Results:From the 94 cases evaluated, 36 had recurrence after BCG treatment (38.3%). Treatment outcome was influenced by age over 65 years (HR=2.668; (1.344–5.254); P=0.005), maintenance schedule (HR=0.480; (0.246–0.936); P=0.031) and multifocallity (HR=2.065; (1.033–4.126); P=0.040). sTn or s6T expression was associated with BCG response (P=0.024; P<0.0001) and with increased recurrence-free survival (P=0.001). Multivariate analyses showed that sTn and/or s6T were independent predictive markers of recurrence after BCG immunotherapy (HR=0.296; (0.148–0.594); P=0.001). In vitro studies demonstrated higher adhesion and internalisation of the bacillus to cells expressing sTn, promoting cell death.Conclusion:s6T is described for the first time in bladder tumours. Our data strongly suggest that BCG immunotherapy is efficient against sTn- and s6T-positive tumours. Furthermore, sTn and s6T expression are independent predictive markers of BCG treatment response and may be useful in the identification of patients who could benefit more from this immunotherapy.

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Avaliaram-se a hematologia e a bioquímica sérica em equinos de concurso completo de equitação (CCE) em treinamento durante testes de esforço incremental em esteira ergométrica de alta velocidade. Foram utilizados 16 equinos em delineamento experimental inteiramente ao acaso com quatro tratamentos e quatro repetições em esquema de parcelas subdivididas, utilizando-se como fontes de variação nos tratamentos a idade e o histórico de treinamento em CCE. As parcelas foram constituídas pelos testes incrementais realizados nas fases inicial e final do treinamento. As subparcelas foram representadas pelos tempos de avaliação e coletas. Os equinos do grupo experimental novos iniciantes apresentaram valor médio do hematócrito de 43,24%, sendo inferior ao hematócrito do grupo adultos iniciantes, 45,63%, novos experientes, 46,39%, e competidores, 47,74%. Houve diferença (P<0,05) entre os testes físicos realizados nas fases inicial e final do treinamento, com redução na concentração plasmática de glicose, de 112 para 98,88mg/dL, nas concentrações séricas de creatinina, de 1,41 para 1,29mg/dL, e de proteínas totais, de 6,52 para 6,38g/dL, na contagem de monócitos, de 0,54 para 0,48 10³/mm³, e com aumento na concentração plasmática de lactato, de 3,31 para 3,79mmol/L, na concentração sérica de ácido úrico, de 1,44 para 1,77mg/dL, no hematócrito, de 44,19 para 46,90%, na concentração de hemoglobina, de 14,33 para 15,10g/dL, e na contagem de leucócitos totais, de 9,26 para 9,61 10³/mm³. O treinamento dos equinos de CCE aumentou o condicionamento físico dos equinos, com maior capacidade de metabolização do lactato após o exercício e aumento nos valores basais do hematócrito e da concentração de hemoglobina.

Rationale: Bladder cancer (BC) management demands the introduction of novel molecular targets for precision medicine. Cell surface glycoprotein CD44 has been widely studied as a potential biomarker of BC aggressiveness and cancer stem cells. However, significant alternative splicing and multiple glycosylation generate a myriad of glycoproteoforms with potentially distinct functional roles. The lack of tools for precise molecular characterization has led to conflicting results, delaying clinical applications. Addressing these limitations, we have interrogated the transcriptome and glycoproteome of a large BC patient cohort for splicing signatures. Methods: CD44 gene and its splicing variants were assessed by Real Time-Polymerase Chain Reaction (RT-PCR) and RNAseq in tumor tissues. The co-localization of CD44 and short O -glycans was evaluated by proximity ligation assay (PLA), immunohistochemistry and double-immunofluorescence. An innovative glycoproteogenomics approach, integrating transcriptomics-customized datasets and glycomics for protein annotation from nanoLC-ESI-MS/MS experiments, was developed and implemented to identify CD44 variants and associated glycosignatures. The impact of CD44 silencing on proliferation and invasion of BC cell lines and glycoengineered cells was determined by BrdU ELISA and Matrigel invasion assays, respectively. Antibody phosphoarrays were used to investigate the role of CD44 and its glycoforms in the activation of relevant oncogenic signaling pathways. Results: Transcriptomics analysis revealed remarkable CD44 isoforms heterogeneity in bladder cancer tissues, as well as associations between short CD44 standard splicing isoform (CD44s), invasion and poor prognosis. We further demonstrated that targeting short O -glycoforms such as the Tn and sialyl-Tn antigens was key to overcome the lack of cancer specificity presented by CD44. Glycoproteogenomics allowed, for the first time, the comprehensive characterization of CD44 splicing code at the protein level. The concept was applied to invasive human BC cell lines, glycoengineered cells, and tumor tissues, enabling unequivocal CD44s identification as well as associated glycoforms. Finally, we confirmed the link between CD44 and invasion in CD44s-enriched cells in vitro by small interfering RNA (siRNA) knockdown, supporting findings from BC tissues. The key role played by short-chain O -glycans in CD44-mediated invasion was also demonstrated through glycoengineered cell models. Conclusions: Overall, CD44s emerged as biomarker of poor prognosis and CD44-Tn/ Sialyl-Tn (STn) as promising molecular signatures for targeted interventions. This study materializes the concept of glycoproteogenomics and provides a key vision to address the cancer splicing code at the protein level, which may now be expanded to better understand CD44 functional role in ...