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Establishment and characterization of two primary breast cancer cell lines from young Indian breast cancer patients: mutation analysis.

ABSTRACT: Two novel triple negative breast cancer cell lines, NIPBC-1 and NIPBC-2 were successfully established from primary tumors of two young breast cancer patients aged 39 and 38 years respectively, diagnosed as infiltrating duct carcinoma of breast. Characterization of these cell lines showed luminal origin with expression of epithelial specific antigen and cytokeratin 18 and presence of microfilaments and secretary vesicles, microvilli, tight junctions and desmosomes on ultra-structural analysis. Both the cell lines showed anchorage independent growth and invasion of matrigel coated membranes. Karyotype analysis showed aneuploidy, deletions and multiple rearrangements in chromosomes 7, 9, X and 11 and isochromosomes 17q in both the cell lines. P53 mutational analysis revealed no mutation in the coding region in both the cell lines; however NIPBC-2 cell line showed presence of heterozygous C/G polymorphism, g.417 C?>?G (NM_000546.5) resulting in Arg/Pro allele at codon 72 of exon 4. Screening for mutations in BRCA1&2 genes revealed presence of three heterozygous polymorphisms in exon 11 of BRCA1 and 2 polymorphisms in exons 11, and14 of BRCA2 gene in both the cell lines. Both the cell lines showed presence of CD 44+/24-breast cancer stem cells and capability of producing mammosphere on culture. The two triple negative breast cancer cell lines established from early onset breast tumors can serve as novel invitro models to study mechanisms underlying breast tumorigenesis in younger age group patients and also identification of new therapeutic modalities targeting cancer stem cells.

SUBMITTER: Pandrangi SL

PROVIDER: S-EPMC4016554 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Establishment and characterization of two primary breast cancer cell lines from young Indian breast cancer patients: mutation analysis.

Pandrangi Santhi Latha SL Raju Bagadi Sarangadhara Appala SA Sinha Navin Kumar NK Kumar Manoj M Dada Rima R Lakhanpal Meena M Soni Abha A Malvia Shreshtha S Simon Sheeba S Chintamani Chintamani C Mohil Ravindar Singh RS Bhatnagar Dinesh D Saxena Sunita S

Cancer cell international 20140205 1

Two novel triple negative breast cancer cell lines, NIPBC-1 and NIPBC-2 were successfully established from primary tumors of two young breast cancer patients aged 39 and 38 years respectively, diagnosed as infiltrating duct carcinoma of breast. Characterization of these cell lines showed luminal origin with expression of epithelial specific antigen and cytokeratin 18 and presence of microfilaments and secretary vesicles, microvilli, tight junctions and desmosomes on ultra-structural analysis. Bo ...[more]

PMID: 24502646

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Project description:BackgroundAmong breast cancer (BC) patients the outcomes of anticancer therapy vary dramatically due to the highly heterogeneous molecular characteristics of BC. Therefore, an extended panel of BC cell lines are required for in vitro and in vivo studies to find out new characteristic of carcinogenesis and metastasis. The purpose of this study was to develop patient-derived BC cell cultures and metastatic tumor models representing a tool for personal therapy and translational research.MethodsBreast cancer cells were prepared by optimizing technique from tumor samples. We used real-time RT-PCR, flow cytometry, western blotting, cytotoxicity assay, karyotyping and fluorescent and electron microscopy analyses to characterize the established cell lines. BC xenografts in scid mice were used for in vivo tumorigenicity studies.ResultsThe technique of preparing primary cells was optimized and this resulted in a high output of viable and active proliferated cells of nine patient-derived breast cancer cell lines and one breast non-malignant cell line. High E-cadherine and EpCAM expression correlated positively with epithelial phenotype while high expression of N-cadherine and Vimentin were shown in cells with mesenchymal phenotype. All mesenchymal-like cell lines were high HER3-positive-up to 90%. More interesting than that, is that two cell lines under specific culturing conditions (pulsed hypoxia and conditioned media) progressively transformed from mesenchymal to epithelial phenotypes displaying the expression of respective molecular markers proving that the mesenchymal-to-epithelial transition occurred. Becoming epithelial, these cells have lost HER3 and decreased HER2 membrane receptors. Three of the established epithelial cancer cell lines were tumorigenic in SCID mice and the generated tumors exhibited lobules-like structures. Ultrastructure analysis revealed low-differentiate phenotype of tumorigenic cell lines. These cells were in near-triploid range with multiple chromosome rearrangements. Tumorigenic BrCCh4e cells, originated from the patient of four-course chemotherapy, initiated metastasis when they were grafted subcutaneous with colonization of mediastinum lymph nodes.ConclusionsThe developed BC cells metastasizing to mediastinum lymph nodes are a relevant model for downstream applications. Moreover, our findings demonstrate that pulsed hypoxia induces transformation of primary fibroblastoid breast cancer cells to epithelial-like cells and both of these cultures-induced and original-don't show tumor initiating capacity.

Dataset Information

Establishment and characterization of two primary breast cancer cell lines from young Indian breast cancer patients: mutation analysis.

Publications

Establishment and characterization of two primary breast cancer cell lines from young Indian breast cancer patients: mutation analysis.

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