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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - GlycoModels (3D glyco-engineered models to address the role of glycosylation in gastric cancer clinical management)

Teaser

Summary

Gastric cancer is an important health problem, being the fifth most common cancer and the third leading cause of cancer death. Its late diagnosis accompanied with a bad prognosis keeps this malignancy among the most deadly. After generations of cancer therapy based on chemical drugs causing severe side effects and limited efficiency, cancer treatment undergoes a paradigm shift towards targeted therapy, employing both monoclonal antibodies and small molecule inhibitors of receptor tyrosine kinase activity, as the most promising tool for the years to come. However, there are numerous patients that do not respond to these novel treatments due to mutations in downstream signalling pathways or to undefined molecular mechanisms. Protein glycosylation has been shown to modulate cellular receptor functions and alter antibody binding affinities and therefore foreshadows to play a major role in cancer therapy resistance.
The GlycoModels project aims to identify glycan alterations on cell receptors that lead to targeted therapy resistance of gastric tumours. Using cutting-edge gene editing techniques, gastric cancer cell lines will be glyco-engineered to express hallmark glycan epitopes and applied in 3D-culture systems as advanced models mimicking the tumour microenvironment. In-depth analysis of the 3D-GlycoModels, covering glycan characterisation of the cell receptors, disclosing activated signalling pathways, and transcriptomics will unveil our understanding on driving mechanisms in therapy resistance and the glycosylation patterns involved. The structural validation of these glycoforms in human samples will deliver new biomarkers to be used in personalised therapy selection and thus, improve the outcome of advanced gastric cancer patients.

Work performed

The GlycoModels project awarded to Dr. BalmaÃ±a aimed at unravelling the glycan modifications of gastric tumour cells using a 3D context to identify the glycan alterations on key cell receptors that lead to targeted therapy resistance in cancer patients.

Specifically, the researcher was involved in the development of gastric cancer cell lines overexpressing/silencing of different genes (e.g. ST6Gal1). The researcher also contributed in the implementation of the CRISPR/Cas9 technology in the host group. Dr. BalmaÃ±a successfully implemented novel 3D cell culture systems expected to have a great impact on the field. Also, and automated imaging analysis system was developed to standardized the analysis of the 3D spheroids. Dr. BalmaÃ±a characterised using lectins and glycan-targeting antibodies the glycophenotype of the 3D GlycoModels by flow-cytometry and immunofluorescence, including the expression of mucins. The features displayed by the 3D GlycoModels allowed to address the response to targeted therapy. The increased in sialylation was associated with resistance to tyrosine kinase inhibitors, such as crizotinib, in the 3D spheroids of gastric cancer cells. The activation of a large panel of receptor tyrosine kinases as well as the downstream signalling were analysed using arrays. The validation of potential glycan biomarkers in clinical samples was performed to corroborate that O-glycans truncation promoted the colocalization of the CD44v6 cancer-related glycoform with the receptor tyrosine kinase RON.

The work performed by Dr. BalmaÃ±a during the MSC fellowship lead to the publication of 8 scientific manuscripts and other 2 that are currently under revision. Besides, the work developed in the laboratory by the researched and the different collaborations of the project supervisor that involved Dr. BalmaÃ±a will also result in future publications.

BalmaÃ±a M, et al.
Molecules. 2018 Oct 30;23(11). doi: 10.3390/molecules23112815.

Freitas D*, BalmaÃ±a M et al. *equally contribution
Journal of Extracellular Vesicles, 2019, 8:1, 1621131, doi: 10.1080/20013078.2019.1621131

Albuquerque APB, BalmaÃ±a M, et al.
Biol Chem. 2018 Jun 27;399(7):661-672. doi: 10.1515/hsz-2018-0121.

Albuquerque APB, BalmaÃ±a M, et al.
Journal of Molecular and Clinical Medicine 2018, 1 (3): 127-134. doi: 10.31083/j.jmcm.2018.03.001

Duarte HO, BalmaÃ±a M, Mereiter S, OsÃ³rio H, Gomes J, Reis CA.
Int J Mol Sci. 2017 Oct 28;18(11). doi: 10.3390/ijms18112262.

Rodrigues JG, BalmaÃ±a M, et al.
Cell Immunol. 2018 Nov;333:46-57. doi: 10.1016/j.cellimm.2018.03.007.

Mereiter S, Martins ÃM, Gomes C, BalmaÃ±a M, et al.
FEBS Lett. 2019 May 11. doi: 10.1002/1873-3468.13432.

Mereiter S, BalmaÃ±a M, et al.
Cancer Cell. (Accepted).

The scientific manuscripts published within the GlycoModels project have been also summarised and published in the Portuguese Association for Cancer Investigation both in Portuguese and English to explain the relevance of the work to non-specialists.
The researcher of the GlycoModels project has participated in several events to disseminate the results to a non-specialist audience (European Researchers) and she has also engaged in activities to bring science closer to high-school students (Ciencia Viva no Laboratorio).

Final results

The GlycoModels project goes beyond the state-of-the art by producing original insights in the field of therapy resistance in cancer, adding a new mechanistical level of regulation based on the tumour glycosylation profile. On one hand, this work will unveil specific glycoforms involved in treatment response and on the other hand, provide the required models to improve our understanding of the role of glycosylation in carcinogenesis and cancer progression. The proposed methodology is innovative as it is the first time that glyco-engineered cell models will be used in 3D-cell culture to address the functions of cellular glycosylation in health and disease. This multidisciplinary approach will provide a unique collection of glyco-engineered gastric 3D-cell models becoming an unprecedented tool for the study of the role of glycosylation that will create a niche of research for the applicant, placing Dr. BalmaÃ±a in the forefront of research in the field of glycosylation in cancer. Furthermore, the strategy developed during this Marie Curie action will provide a novel approach that could afterwards be applied to different types of cancers, and even other diseases.
This proposal is intended to study the relation between the altered glycosylation of cell receptors and the sensitivity/resistance to targeted therapy in gastric cancer patients. Thus, it is expected that the results will provide clinicians with new tools to determine the best treatment for each patient. In this sense, the group is in close collaboration with the IPO-Porto Hospital and the clinicians will participate in the discussion of the results as well as contribute to design strategies that foresee clinical application of the identified glycomarkers.