Abstract
Conditionally reprogrammed cells (CRCs) are epithelial cells that are directly isolated from patients’ specimens and propagated in vitro with feeder cells and a Rho kinase inhibitor. A number of these cells have been generated from biopsies of breast cancer patients, including ductal carcinoma in situ and invasive carcinomas. The characterization of their genomic signatures is essential to determine their ability to reflect the natural biology of their tumors of origin. In this study, we performed the genomic characterization of six newly established invasive breast cancer CRC cultures in comparison to the original patients’ primary breast tumors (PBT) from which they derived. The CRCs and corresponding PBTs were simultaneously profiled by genome-wide array-CGH, targeted next generation sequencing and global miRNA expression to determine their molecular similarities in the patterns of copy number alterations (CNAs), gene mutations and miRNA expression levels, respectively. The CRCs’ epithelial cells content and ploidy levels were also evaluated by flow cytometry. A similar level of CNAs was observed in the pairs of CRCs/PBTs analyzed by array-CGH, with >95% of overlap for the most frequently affected cytobands. Consistently, targeted next generation sequencing analysis showed the retention of specific somatic variants in the CRCs as present in their original PBTs. Global miRNA profiling closely clustered the CRCs with their PBTs (Pearson Correlation, ANOVA paired test, P<0.05), indicating also similarity at the miRNA expression level; the retention of tumor-specific alterations in a subset of miRNAs in the CRCs was further confirmed by qRT-PCR. These data demonstrated that the human breast cancer CRCs of this study maintained at early passages the overall copy number, gene mutations and miRNA expression patterns of their original tumors. The further characterization of these cells by other molecular and cellular phenotypes at late cell passages, are required to further expand their use as a unique and representative ex-vivo tumor model for basic science and translational breast cancer studies.
Highlights
Reprogrammed cells (CRCs) are epithelial cells that grow indefinitely without the need for transduction of exogenous viral or cellular genes [1]
Genome-wide copy number analysis was performed by array-CGH in all the six established breast cancer Conditionally reprogrammed cells (CRCs) and their corresponding primary breast tumors (PBT) analyzed
The average number of copy number alterations (CNAs) observed in these cells was 25.50±14.79, which was not significantly different from the average number of the CNAs observed in the PBT group (29.33±18.01) (Fig 2, Table 1)
Summary
Reprogrammed cells (CRCs) are epithelial cells that grow indefinitely without the need for transduction of exogenous viral or cellular genes [1]. A potential and direct clinical translation of the CRC model is the ability to assess for sensitivity a variety of chemotherapy drugs, allowing for the in vitro selection of the most likely effective drugs for a particular patient [3,10]. This unique possibility, offers a system where response to known drug therapies and/or novel therapeutic compounds can be directly tested on cells expanded from individual cancer patients. These steps would guarantee that future molecular and/ or functional downstream analysis using these CRCs can be consistently and reproducibly performed
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