Breast cancer tissue slices as a model for evaluation of response to rapamycin
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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.
KeywordsBreast cancer Rapamycin Ex-vivo model AKT/mTOR pathway Co-expression network Human
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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