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Breast cancer tissue slices as a model for evaluation of response to rapamycin

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Abstract

Rapamycin is a selective inhibitor of the mammalian target of rapamycin (mTOR), a regulator kinase that integrates growth factors signaling via the phosphoinositide-3-kinase pathway and that has emerged as a novel therapeutic modality in breast cancer (BC). We propose a pre-clinical “ex-vivo” personalized organotypic culture of BC that preserves the microenvironment to evaluate rapamycin-mediated gene expression changes. Freshly excised ductal invasive BC slices, 400 μm thick (n=30), were cultured in the presence or absence (control) of rapamycin (20 nM) for 24 h. Some slices were formalin-fixed for immunohistochemical determinations and some were processed for microarray analysis. Control slices in culture retained their tissue morphology and tissue viability (detected by BrdU uptake). The percentage of proliferating cells (assessed by Ki67) did not change up to 24 h of treatment. Immunohistochemical evaluation of p-AKT, p-mTOR, p-4EBP1 and p-S6K1 indicated that AKT/mTOR pathway activation was maintained during cultivation. For microarray analysis, slices were divided into two groups, according to the presence/absence of epidermal growth factor receptor-type 2 and analyzed separately. Limited overlap was seen among differentially expressed genes after treatment (P<0.01) in both groups suggesting different responses to rapamycin between these BC subtypes. Ontology analysis indicated that genes involved in biosynthetic processes were commonly reduced by rapamycin. Our network analysis suggested that concerted expression of these genes might distinguish controls from treated slices. Thus, breast carcinoma slices constitute a suitable physiological tool to evaluate the short-term effects of rapamycin on the gene profile of individual BC samples.

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Acknowledgments

We are indebted to Dr. C. Krumdieck (University of Alabama, Birmingham, Ala., USA) for the kind donation of the Krumdieck tissue slicer to our institution. We also thank Dr. Igor Moyses Longo Snitcovsky for critical suggestions, Dr. Fiorita G. L. Mundim for helping with the immunohistochemical determinations, Mrs. Maria Jose Gonçalves Benevides for secretarial help and Mrs. Cristina Piñeiro Grandal for figure edition.

S.H.G.G. provided the tumor samples, prepared the tissue slices, collected all clinical data and was involved in drafting the manuscript. M.L.H.K. performed the culture, microarray and RT-PCR experiments. R.A.R. performed the microarray determinations. H.B. and R.A.R. performed the microarray data analysis. S.N., F.A.S. and A.F.L.W. participated in the immunohistochemistry studies. L.L. carried out the analysis of the co-expression network. J.C.S.G. provided clinical support for patient recruitment. M.A.A.K.F., M.L.H.K. and F.S.P. carried out the statistical analysis. M.M.B. was responsible for the study conception. M.M.B. and M.A.A.K.F. were involved in study design and manuscript preparation.

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Authors and Affiliations

  1. Instituto Brasileiro de Controle do Câncer, Av. Alcântara Machado 2576, CEP 03102-002, São Paulo, SP, Brasil

    Stana Helena Giorgi Grosso & João Carlos Guedes Sampaio Góes

  2. Departamento de Radiologia e Oncologia, Disciplina de Oncologia (LIM-24), Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Arnaldo 455, Sala 4123, CEP 01246-903, São Paulo, SP, Brasil

    Maria Lucia Hirata Katayama, Rosimeire Aparecida Roela, Maria Aparecida Azevedo Koike Folgueira, Fátima Solange Pasini & M. Mitzi Brentani

  3. Central, Divisão de Patologia, Laboratório de Imuno-histoquímica, Instituto Adolfo Lutz, Av. Dr. Arnaldo 355, 7º Andar Cerqueira César, CEP 01246-902, São Paulo, SP, Brazil

    Suely Nonogaki

  4. Hospital A C Camargo, Rua Professor Antônio Prudente 211, 01509-010, São Paulo, Brasil

    Fernando Augusto Soares

  5. Departamento de Psiquiatria (LIM-23), Faculdade de Medicina da Universidade de São Paulo, Rua Ovideo Pires de Campos S/Nº, São Paulo, Brasil

    Helena Brentani

  6. Laboratório de Bioinformática e Bioestatística, Centro Internacional de Pesquisa e Ensino (CIPE), Hospital AC Camargo, Rua Taguá 440, CEP 01508-010, São Paulo, Brazil

    Leandro Lima

  7. Departamento de Patologia, Universidade Federal de São Paulo, Rua Botucatu 740, CEP 04023-900, São Paulo, Brasil

    Angela Flávia Logullo Waitzberg

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  1. Stana Helena Giorgi Grosso
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  2. Maria Lucia Hirata Katayama
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  3. Rosimeire Aparecida Roela
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  5. Fernando Augusto Soares
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  6. Helena Brentani
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  7. Leandro Lima
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  8. Maria Aparecida Azevedo Koike Folgueira
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  10. Fátima Solange Pasini
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  11. João Carlos Guedes Sampaio Góes
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  12. M. Mitzi Brentani
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Corresponding author

Correspondence to M. Mitzi Brentani.

Additional information

S.H.G.G., M.L.H.K. and R.A.R. contributed equally to this work.

This work was supported by FAPESP 2009/10088-7 and CNPQ.

The authors declare that they have no competing interests.

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Grosso, S.H.G., Katayama, M.L.H., Roela, R.A. et al. Breast cancer tissue slices as a model for evaluation of response to rapamycin. Cell Tissue Res 352, 671–684 (2013). https://doi.org/10.1007/s00441-013-1608-8

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