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The reciprocal interactions between astrocytes and prostate cancer cells represent an early event associated with brain metastasis

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Abstract

Tumor establishment, growth, and survival are supported by interactions with microenvironment components. Here, we investigated whether the interactions between prostate cancer cells and cortical astrocytes are associated to a potential role for astrocytes in tumor establishment. We demonstrate that astrocytes interact in vitro with prostatic cancers cells derived from different metastatic sites. Astrocytes and their secreted extracellular matrix, stimulate DU145 cell (a brain-derived prostate tumor cell line) proliferation while inhibiting cell death and modulating the expression of several genes related to prostate cancer progression, suggesting the activation of EMT process in these cells. In contrast, DU145 cells and their conditioned medium inhibited cell proliferation and induced cell death of astrocytes. On the other hand, the astrocytes were unable to significantly induce an increment of LNCaP cell (a lymph node-derived prostate tumor cell line) proliferative activity. In addition, LNCaP cells were also unable to induce cell death of astrocytes. Thus, we believe that DU145 cells, but not LNCaP cells, present an even more aggressive behavior when interacting with astrocytes. These results provide an important contribution to the elucidation of the cellular mechanisms involved in the brain microenvironment colonization.

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Abbreviations

EMT:

Epithelial mesenchymal transition

TGF-β:

Transforming growth factor beta

CNS:

Central nervous system

ECM:

Extracellular matrix

FBS:

Fetal bovine serum

24-CM:

Conditioned medium collected after 24 h of culture

48-CM:

Conditioned medium collected after 48 h of culture

PBS:

Phosphate buffered saline

GFAP:

Glial fibrillary acidic protein

CK:

Cytokeratin

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

V T :

Tangential velocity

V S :

Spreading velocity

MMP:

Matrix metalloproteinase

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Acknowledgments

The authors thank Dr. Vivaldo Moura Neto for discussion and comments on the study; Dr. Jane Faria and Morgana Ferreira Sobrinho for the encouragement; and Maria Aparecida de Oliveira Domingos and Grasiella Ventura for technical assistance. This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Tecnológico (CNPq), and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

Conflict of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

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Authors and Affiliations

  1. Programa de Pesquisa em Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

    Eliane Gouvêa de Oliveira Barros, Antonio Palumbo Jr., Pedro Lucas Prado Mello, Rômulo Medina de Mattos, Julianna Henriques da Silva, Bruno Pontes, Rackele Ferreira do Amaral, Flavia Regina Souza Lima, Celia Yelimar Palmero, Leandro Miranda-Alves & Luiz Eurico Nasciutti

  2. LPO/COPEA, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

    Bruno Pontes & Nathan Bessa Viana

  3. Instituto de Física, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

    Nathan Bessa Viana

  4. Programa de Carcinogênese Molecular, Centro de Pesquisas (CPQ), Instituto Nacional de Câncer (INCA), Rio de Janeiro, Brazil

    Nathalia Meireles da Costa

  5. Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

    Christina Maeda Takiya

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  1. Eliane Gouvêa de Oliveira Barros
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Correspondence to Luiz Eurico Nasciutti.

Electronic supplementary material

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10585_2014_9640_MOESM1_ESM.tif

Electronic Supplementary Material Fig. 1 Characterization by immunofluorescence and cell proliferation and cell death analysis of astrocytes and LNCaP cells in co-culture. (A)The cells were incubated for 24 and 48 h in medium supplemented with 0.5 FBS, and immunostained with anti-GFAP and human anti-cytokeratin. The astrocytes (red) LNCaP cells (white). In co-culture, LNCaP cells formed small clusters arranged over the astrocytes monolayer. (B) Quantification of cell number present in co-culture by flow cytometry. (C) TUNEL assay performed in co-culture of astrocytes and LNCaP cells. DAPI (blue), nuclei staining. Scale bar, 200 µm (TIFF 5792 kb)

10585_2014_9640_MOESM2_ESM.tif

Electronic Supplementary Material Fig. 2 Cell viability analysis of astrocytes and DU145 cells isolated and in co-culture. Cell viability was measured by the MTT colorimetric assay after 24 (A) and 48 (B) h of culture in supplemented medium with 10 %, 0.5 %, and without FBS. Note that after 48 h in 0.5 % FBS, the viability of astrocytes begins to be rescue. The results are expressed as the means ± CI of three independent experiments. CI = confidence interval. (*)p < 0.05, (**) p < 0.01, (***) p < 0.001 (TIFF 2144 kb)

10585_2014_9640_MOESM3_ESM.tif

Electronic Supplementary Material Fig. 3 Schematic model of co-culture and cell sorting. (A) Schematic model of co-culture of astrocytes labeled with Green CMFDA and unlabeled DU145 cells. (B) Separation of cells by flow cytometry. Note that there are differences in fluorescence emission peaks of labeled astrocytes and unlabeled DU145 cells (TIFF 2228 kb)

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Supplementary material 5 (DOCX 34 kb)

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de Oliveira Barros, E.G., Palumbo, A., Mello, P.L.P. et al. The reciprocal interactions between astrocytes and prostate cancer cells represent an early event associated with brain metastasis. Clin Exp Metastasis 31, 461–474 (2014). https://doi.org/10.1007/s10585-014-9640-y

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  • DOI: https://doi.org/10.1007/s10585-014-9640-y

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