Research Article: Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

Date Published: July 4, 2008

Publisher: BioMed Central

Author(s): Renata A Sobral, Suzana T Honda, Maria Lucia H Katayama, Helena Brentani, M Mitzi Brentani, Diogo FC Patrão, Maria Aparecida AK Folgueira.

http://doi.org/10.1186/1751-0147-50-27

Abstract

In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro.

Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 μM for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples ≥ 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors.

Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction ≥ 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed.

Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.

Partial Text

Human and canine malignant mammary tumors share some epidemiological and clinicopathological features. Incidence in both species increases with age, lifetime exposure to endogenous or exogenous estrogens is a common risk factor, and the majority of malignant mammary gland tumors arises from epithelial tissue [1-3]. In addition, some prognostic factors are similar for both species, such as clinical stage, tumor size, histological type and grade, however adjacent lymph node involvement is still a matter of discussion [1,4-7]. Furthermore, estrogen receptor expression, proliferation index evaluated by PCNA, Ki67 expression, or S-phase rate, have also been correlated to prognosis in canine mammary tumors [5,6], and immunohistochemical detection of Bcl2, p53 and cytokeratins, in human and canine tumors and corresponding adjacent tissues, have been similar [8].

Tumor samples were obtained from 38 dogs undergoing mastectomy at the “Hospital da Faculdade de Medicina Veterinária da Universidade Metodista de São Paulo (UMESP)”, São Bernardo do Campo, SP, Brazil, from March 2005 to January 2006. This study was approved by the Institutional Ethics Committee and animal owners signed the informed consent. Median age of patients was 10.4 y and 55% and 18.4% of them were mixed and poodle breeds, respectively. Eight patients were previously spayed.

Based on the previous established response criterion, a reduction in the cell number ≥ 21.7% upon doxorubicin treatment, nine samples were considered responsive to doxorubicin and 29 non-responsive (Table 2). In addition, considering the 38 samples treated and untreated, a mean reduction of 13.6% in the cell number (P < 0.001, Mann-Whitney test) was observed upon treatment. Tumor slices cultured in vitro may be an interesting model to evaluate drug response as it preserves some of the in vivo characteristics, as the epithelial mesenchymal relationship. An important issue is to guarantee a proper diffusion of oxygen and nutrients to the entire slice, as passive diffusion occurs through only 200 μm. In our study, tumor thickness varied between 300–400 μm and each tumor slice was placed on wells filled with culture medium, allowing them to float; conditions which, were previously shown to be appropriate to organ culture [19-22]. Our data suggest that short term culture of mammary tumor slices seems to be an interesting model to evaluate doxorubicin activity. However, parallel comparisons between in vitro and in vivo drug responses to establish their exact correlation are needed. Moreover, our results on the expression of a few genes emphasize the need to obtain a more detailed gene expression profile, associated with chemotherapy response in canine tumors. RAS participated in the design of the study, sample collection, tissue slice culture, PCR assays, and helped to draft the manuscript. STH participated in sample collection, tissue slice culture, and cell counting. MLHK participated in the design of the study, tissue slice culture. PCR assays and revised the manuscript for important intellectual content. HB participated in the design of the study and performed statistical analysis and data interpretation. MMB participated in the design of the study and revised the manuscript for important intellectual content. DFCP performed statistical analysis. MAAKF participated in the design of the study, data interpretation and helped to draft the manuscript.   Source: http://doi.org/10.1186/1751-0147-50-27

 

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