CD4+, CD8+, FoxP3+ and HSP60+ Expressions in Cellular Infiltrate of Canine Mammary Carcinoma in Mixed Tumor

Authors

  • Belarmino Eugênio Lopes-Neto Programa de Pós-graduação em Ciências Veterinárias, Laboratório de Imunologia e Bioquímica Animal, Faculdade de Veterinária (FAVET), Universidade Estadual do Ceará (UECE), Fortaleza, CE, Brazil.
  • Stephanie Caroline Bezerra Souza Programa de Pós-graduação em Ciências Veterinárias, Laboratório de Imunologia e Bioquímica Animal, Faculdade de Veterinária (FAVET), Universidade Estadual do Ceará (UECE), Fortaleza, CE, Brazil.
  • Lúcio Marinho Bouty Hospital Veterinário, Faculdade de Veterinária (FAVET), Universidade Estadual do Ceará (UECE), Fortaleza, CE, Brazil.
  • Glauco Jonas Lemos Santos Programa de Pós-graduação em Ciências Veterinárias, Laboratório de Imunologia e Bioquímica Animal, Faculdade de Veterinária (FAVET), Universidade Estadual do Ceará (UECE), Fortaleza, CE, Brazil.
  • Emanuele Silva Oliveira Laboratório de Genética Molecular, Universidade Federal do Ceará (UFC), Fortaleza.
  • José Cláudio Carneiro de Freitas Programa de Pós-graduação em Ciências Veterinárias, Laboratório de Imunologia e Bioquímica Animal, Faculdade de Veterinária (FAVET), Universidade Estadual do Ceará (UECE), Fortaleza, CE, Brazil.
  • Diana Célia Sousa Nunes-Pinheiro Programa de Pós-graduação em Ciências Veterinárias, Laboratório de Imunologia e Bioquímica Animal, Faculdade de Veterinária (FAVET), Universidade Estadual do Ceará (UECE), Fortaleza, CE, Brazil.

DOI:

https://doi.org/10.22456/1679-9216.80758

Keywords:

canine mammary glands, carcinoma in mixed tumor, T-lymphocytes, heat shock protein, immunolabeling.

Abstract

Background: Cancer is a complex process that receive many influences of the tumor microenvironment. The participation of immune system cells and proteins in tumor microenvironment is not yet completely understood. Thus, the aim of this study was to evaluate the infiltrate cellular, subpopulations of T-lymphocytes and HSP60 of canine mammary carcinoma in mixed tumor (CMCMT).

Materials, Methods & Results: Female dogs (n = 20) were selected after Canine mammary tumor (CMT) diagnosis and data were achieved throughout clinical-pathological information. Clinical staging was evaluated and tumor biopsies were processed by histology and cellular infiltrate was performed according criteria and grade. Survival curve were generated by Kaplan-Meier and the lymphocytic infiltrate were compared by Log-Rank followed Chi-Square χ². For immunolabeling it was used anti-CD4, anti-CD8, anti-FoxP3 and HSP60 monoclonal antibodies and were attributed scores from 0 to 3. Clinical-pathological relationship was analyzed using Spearman correlation. This study was approved by the Committee for Ethics in Research using Animals (CEUA-UECE), protocol 12247080-2. Our data showed a mean age of 9.3 years-old, the size of tumors presented more than 5 cm (50%), which were located in inguinal mammary glands (70%), and CMTs shows I (70%) and II (30%) grade. The cellular infiltrate was distributed both in peri and intratumoral regions, dispersed multifocally with moderate intensity and lymphocytes were the major populations found into tumors (n = 826 ± 220). In relationship to cellular infiltrate with CMT grade it was observed that lymphocytes (ρ = 0.28) and plasma cells (ρ = 0.22) showed a slight positive correlation, and an opposed negative correlation of neutrophils (ρ = -0.1) and macrophages (ρ = -0.38). CMT presents moderate lymphocytic infiltrate (< 800 lymphocytes), shows higher (P = 0.01) survival rates as compared to intense lymphocytic infiltrate (≥ 800 lymphocytes). FoxP3+ showed lower intensity while CD4+ and CD8+ expression were concentrated surrounding of lymphocytic infiltrate tumor region. HSP60+ was observed in the inflammatory and tumor cells.

Discussion: Our data are according to a greater risk to the development of breast tumor in old bitches, not castrated and before or after puberty, as well as the use of contraceptives based on progesterone and estrogen. In relation to size of tumor, these findings reinforce that there is a relationship of tumor size with a higher malignancy grade and with a worse prognosis. The predominant tumor location was in the inguinal breasts that is attributed to the high activity of the mammary glands to hormonal stimuli. CMT with low clinical staging are associated with greater overall survival of affected bitches. In relation to tumor microenvironment, it has been reported that heterogeneous populations of the immune system cells often infiltrate the mammary tumors, whose lymphocytes are the main cells. It is suggested that tumor lymphocytosis may be necessary for malignant behavior of the tumor microenvironment. On the other hand, macrophages and neutrophils play an important role that may favor or inhibit the tumor cells development in the tumor microenvironment. In our work, CD4, CD8 and FoxP3 labeling were distributed in peri and intratumoral regions, and consequently, these markers can be used as prognostic for CMT, as well as being a potential target for anticancer therapies. This is the first work that presents results about the participation of HSP60 in CMT, however this data needs further investigation. HSP60 participates as a potent activator of the immune system through its peptides and other HSP types were studied in mammary carcinomas in bitches and presenting results that indicate the association of these proteins with the carcinogenesis process.

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References

Carvalho M., Pires I., Prada J., Gregório H., Lobo L. & Queiroga F. 2016. Intratumoral FoxP3 expression is associated with angiogenesis and prognosis in malignant canine mammary tumors. Veterinary Immunology and Immunopathology. 178: 1-9.

Carvalho M., Pires I., Prada J. & Queiroga F. 2014. A Role for T-Lymphocytes in Human Breast Cancer and in Canine Mammary Tumors. BioMed Research International. 2014: 1-11.

Cassali G.D., Bertagnolli A.C., Ferreira E, Damasceno K.A., Gamba C.O. & Campos C.B. 2012. Canine mammary mixed tumours: a review. Veterinary Medicine International. 2012 (Article ID 274608): 7 p.

Cassali G.D., Lavelle G.E., Ferreira E. & Torres R. 2014. Consensus for the Diagnosis, Prognosis and Treatment of Canine Mammary Tumors - 2013. Brazilian Journal of Veterinary Pathology. 7: 38-69.

Castro S.V., Lobo C.H., Figueiredo J.R. & Rodrigues A.P. 2013. Proteínas de choque térmico HSP-70: Estrutura e atuação em resposta ao estresse celular. Acta Veterinaria Brasilica. 7: 261-271.

Ciocca D.R. & Calderwood S.K. 2005. Heat shock proteins in cancer: diagnostic, prognostic, predictive and treatment implications. Cell Stress and Chaperones. 10(2): 86-103.

Dias M.L., Andrade J.M., Castro M.B. & Galera P.D. 2016. Survival analysis of female dogs with mammary tumors after mastectomy: epidemiological, clinical and morphological aspects. Pesquisa Veterinária Brasileira. 36: 181-186.

Elinav E., Nowarski R., Thaiss C.A., Hu B., Jin C. & Flavell R.A. 2013. Inflammation-induced cancer: crosstalk between tumours, immune cells and microorganisms. Nature Reviews. 13: 759-771.

Elston C.W. & Ellis I.O. 1991. Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer experience from a large study with long-term follow-up. Histopathology. 19: 403-410.

Estrela-Lima A., Araujo M.S., Costa-Neto J.M. & Cassali G. 2010. Immunophenotypic features of tumor infiltrating lymphocytes from mammary carcinomas in female dogs associated with prognostic factors and survival rates. BMC Cancer. 10: 1-14.

Flohe S.B., Bruggmann J., Lendemans S., Nikulina M., Meierhoff G., Flohe S. & Kolb H. 2003. Human heat shock protein 60 induces maturation of dendritic cell versus a Th1-promoting phenotype. The Journal of Immunology. 170: 2340-2348.

Fonseca C.S. & Daleck C.R. 2000. Neoplasias mamarias em cadelas: influência hormonal e efeitos da ovario-histerectomia como terapia adjuvante. Ciência Rural. 30(4): 731-735.

Hanahan D. & Weinberg R.A. 2011. Hallmarks of cancer: the next generation. Cell. 144(5): 646-674.

Horiuchi Y., Tominaga M., Ichikawa M., Yamashita M., Jikumaru Y., Nariai Y., Nakajima Y., Kuwabara M. & Yukawa M. 2009. Increase of regulatory T cells in the peripheral blood of dogs with metastatic tumors. Microbioology. Immunolology. 53: 468-474.

Im K.S., Kim N.H., Lim H.Y., Kim H.W., Shin J.I. & Su J.H. 2014. Analysis of a New Histological and MolecularBased Classification of Canine Mammary Neoplasia. Veterinary Pathology. 52: 549-559.

Kim J.H., Chon S.K., Im K.S., Kim N.H. & Sur J.H. 2013. Correlation of tumor-infiltrating lymphocytes to histopathological features and molecular phenotypes in canine mammary carcinoma: A morphologic and immunohistochemical morphometric study. The Canadian Journal of Veterinary Research. 77: 42-149.

Matos A.J.F., Baptista C.S., Gärtner M.F. & Rutteman G.R. 2012. Prognostic studies of canine and feline mammary tumours: The need for standardized procedures. The Veterinary Journal. 193: 24-31.

Moulton J.E. 1990. Tumors of the mammary gland. In: Tumors in Domestic Animals. 3rd edn. Oakland: University of California Press, pp.518-549.

Nunes F.C. 2015. Diagnóstico, prognóstico e tratamento dos carcinomas de glândulas mamárias de cadelas atendidas no Hospital Veterinário da UFMG – Estudo retrospectivo (Dissertação). 67f. Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

Owen L.N. 1980. TNM Classification of tumors in domestic animals. World Health Organization. Geneva: World Health Organization, 53p.

Ribeiro L.G.R. 2012. Carcinoma inflamatório de mama em cadela: caracterização da resposta inflamatória, achados clínicos e anatomohistopatológicos (Dissertação). 142f. Universidade Federal da Bahia, Salvador, Brazil.

Romanucci M., Marinelli A., Sarli G. & Salda L.D. 2006. Heat shock protein expression in canine malignant mammary tumours. BMC Cancer. 6: 1-12.

Rudensky A.Y. 2011. Regulatory T Cells and FoxP3. Immunological Reviews. 242: 260-268.

Salas Y., Márquez A., Diaz D. & Romero L. 2015. Epidemiological Study of Mammary Tumors in Female Dogs Diagnosed during the Period 2002- 2012: A Growing Animal Health Problem. PLoS ONE. 10: 1-15.

Published

2017-01-01

How to Cite

Lopes-Neto, B. E., Souza, S. C. B., Bouty, L. M., Santos, G. J. L., Oliveira, E. S., Freitas, J. C. C. de, & Nunes-Pinheiro, D. C. S. (2017). CD4+, CD8+, FoxP3+ and HSP60+ Expressions in Cellular Infiltrate of Canine Mammary Carcinoma in Mixed Tumor. Acta Scientiae Veterinariae, 45(1), 8. https://doi.org/10.22456/1679-9216.80758

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