Format

Send to

Choose Destination
Cancer Cell Int. 2019 Feb 28;19:46. doi: 10.1186/s12935-019-0766-5. eCollection 2019.

Establishment of primary human breast cancer cell lines using "pulsed hypoxia" method and development of metastatic tumor model in immunodeficient mice.

Author information

1
1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Lavrentiev Avenue, 8, Novosibirsk, 630090 Russia.
2
2Novosibirsk State University, Pirogova Str. 1, Novosibirsk, 630090 Russia.
3
3Institute of Molecular Biology and Biophysics, Siberian Branch, Russian Academy of Medical Sciences, Ac. Timakov Str. 2, Novosibirsk, 630117 Russia.
4
National Novosibirsk Regional Oncology Dispensary, Plakhotnogo Str. 2, Novosibirsk, 630000 Russia.
5
Novosibirsk Municipal Budgetary Healthcare Institution "Municipal Clinical Hospital #1", Zalessky Str. 6, Novosibirsk, 630047 Russia.

Abstract

Background:

Among 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.

Methods:

Breast 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.

Results:

The 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.

Conclusions:

The 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.

KEYWORDS:

Breast cancer; E-cadherine; EMT; MET; Mediastinum lymph node metastasis; Mice tumor models; Primary cultures; Pulsed hypoxia

Supplemental Content

Full text links

Icon for BioMed Central Icon for PubMed Central
Loading ...
Support Center