Fixed precipitates were washed with the IMDM media, post-fixed with 1% solution of osmium tetroxide and dehydrated

Fixed precipitates were washed with the IMDM media, post-fixed with 1% solution of osmium tetroxide and dehydrated. 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 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 nonmalignant 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-positiveup 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, Daclatasvir these cells have lost HER3 and decreased HER2 membrane receptors. Three of the Serpinf2 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 Daclatasvir 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 culturesinduced and originaldont show tumor initiating capacity. Electronic supplementary material The online version of this article (10.1186/s12935-019-0766-5) contains supplementary material, which is available to authorized users. fibroblastoid-like morphology, epithelial-like morphology Primary cell culture preparation Breast tumor tissue was isolated and processed in a sterile manner. Tissues were washed extensively with 1?PBS with 200 U/mL penicillin, 200?g/mL streptomycin, and 500?mg/mL amphotericin without centrifugation. For collagenase treatment tissue specimens were mechanically dissociated using a scalpel with removal of vascular material and transferred to a solution of 20?mg/mL collagenase I (Gibco BRL Co., Invitrogen) in DMEM media and incubated at 37?C for 15?h on a shaking incubator (Grant Bio, Keison Products, UK). Specimens dissociated into single cells were washed with 10?excess of phosphate-buffered saline (PBS) and cell pellet was collected by centrifugation at 300for 5?min. Cells were plated in IMDM with 10% FBS and, after cell adhesion, 10?M Rho-associated protein kinase Daclatasvir (ROCK) inhibitor was added to the culture medium for 1?h. Next, the media was replaced with fresh complete IMDM media. At the next passages, cells were cultured in complete IMDM media supplemented with epithelial cell growth supplement (#6622, Cell Biologics, Chicago, IL, USA), Mito?+?Serum Extender (BD BiosciencesDiscovery Labware, San Jose, CA, USA), 2?mM?l-glutamine, 100 U/mL penicillin, 100?g/mL streptomycin, Daclatasvir and 250?mg/mL amphotericin B and were cultivated in 6-well plates at 37?C in a humidified atmosphere containing 5% CO2. When 70C80% confluence was reached, cells were harvested using 0.05% trypsin/ethylenediaminetetraacetic acid (Sigma-Aldrich) and sub-cultured for further experiments. In the case of collagenase-free method, mechanically dissociated tissue specimens were put into IMDM media with 10% FBS, supplemented with Mito?+?Serum Extender (BD BiosciencesCDiscovery Labware, San Jose,.