Tetraspanin co029 expression as a tumor biomarker for monoclonal antibodies preparation: antigenic assessment in colorectal cancer cells

ABSTRACT

Introduction: The identification of innovative cancer biomarkers is a very relevant ongoing quest. Moreover, their role in cancer diagnosis and clinical management has been radically changed in the last few years with the major emphasis on cancer molecular classification, therapeutic target identification, and therapeutic protocol responsivity. tetraspanins are a family of transmembrane proteins correlated with tumor stage, tumor type and patient outcome affecting cell growth, morphology, invasion, and metastasis.

Methods: We expressed the 31kDa transmembrane human tetraspanin co029 antigen in Escherichia coli expression host cells using Gateway^® platform. Western blotting and ELISA techniques, together with gene sequencing, confirmed the identity of TSP co029 recombinant protein. Forty clones producing antibodies against TSP co029 were obtained. These antibodies were incubated with human colorectal cancer cells in different conditions. ELISA and immunohistochemistry were also performed.

Results: The expressed tetraspanin had an appropriate conformation and antigenic integrity to produce antibodies with affinity to the native TSP co029 biomarker. The affinity of the purified recombinant protein and antibodies were confirmed by western blotting, florescent staining of human colorectal cancer cells in fluorescence and confocal microscopies and by ELISA and immunohistochemistry.

Conclusion: Our data showed that the recombinant protein and antibodies produced in this study allowed the confirmation of tetraspanin co029 protein presented on the surface of human colorectal cancer cells.

RESUMO

Introdução: A identificação de biomarcadores de câncer inovadores é uma busca contínua muito relevante. Além disso, seu papel no diagnóstico do câncer e no manejo clínico mudou radicalmente nos últimos anos, com grande ênfase na classificação molecular do câncer, na identificação do alvo t Western blotting erapêutico e na responsividade do protocolo terapêutico. As tetraspaninas são uma família de proteínas transmembrana correlacionadas com o estágio do tumor, tipo de tumor e evolução do paciente, que afetam o crescimento celular, morfologia, invasão e metástase.

Métodos: Expressamos o antígeno de tetraspanina co029 humana transmembrana de 31kDa em células hospedeiras de expressão de Escherichia coli usando a plataforma Gateway^®. As técnicas de Western blotting e ELISA, juntamente com o sequenciamento de genes, confirmaram a identidade da proteína recombinante TSP co029. Obtiveram-se quarenta clones produtores de anticorpos contra TSP co029. Esses anticorpos foram incubados com células de câncer colorretal humano em diferentes condições. ELISA e imunohistoquímica também foram realizados.

Resultados: A tetraspanina expressa tinha uma conformação adequada e integridade antigênica para produzir anticorpos com afinidade para o biomarcador TSP co029 nativo. A afinidade da proteína recombinante purificada e dos anticorpos foram confirmados por western blotting, coloração fluorescente de células de câncer colorretal humano em microscopias fluorescentes e confocais, e por ELISA e imunohistoquímica.

Conclusão: Nossos dados mostraram que a proteína recombinante e os anticorpos produzidos neste estudo permitiram a confirmação da proteína tetraspanina co029 apresentada na superfície de células de câncer colorretal humano.

Keywords:

Tetraspanins - Recombinant proteins - Antibodies - Monoclonal - Biomarkers, Tumor

Descritores:

Tetraspaninas - Proteínas recombinantes - Anticorpos monoclonais - Biomarcadores tumorais

Research ethics committee

This project was approved by the Ethical Research Committee of Fiocruz for animal use (CEUA-LM1/18) according to the International Guiding Principles for Biomedical Research Involving Animals developed by the Council for International Organizations of Medical Sciences and by the Ethical Committee on Research with Human Beings 34778, TTMB 0002/2019 additional term.

AUTHORSHIP

RFQG, LAC and JVA drafted the manuscript and carried out the assays, and analysis and interpretation of the data. RFQG, ANR and MMDAC conceived the study, designed the study protocol and critically revised the manuscript for intellectual content. LAC, CAC, JVA, MLCP, NGC, WJJ, VCFS, ASSM and MSMV carried out the experiments. All authors read and approved the final manuscript.

FUNDING

Financial support was provided by The Brazilian National Council for Scientific and Technological Development (CNPq), Merieux Foundation and the Oswaldo Cruz Foundation (Fiocruz). Research scholarships were provided by The Coordination for the Improvement of Higher Education Personnel (CAPES), Minas Gerais Research Foundation (Fapemig) and Fiocruz at the Fiocruz Postgraduate Program in Health Sciences and the Federal University of Minas Gerais (UFMG) Postgraduate Program in Adult Health.

CONFLICTS OF INTEREST

The authors have declared that no competing interests exist.

Publication History

Received: 03 June 2021

Accepted: 03 September 2021

Article published online:
09 March 2022

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