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Cell Line Of Breast Cancer

Comparing Cell Lines With Organoids

Understanding how cancer cell lines evolve in the lab, and what to do about it.

Fig. 5

Comparing CCLE breast cancer cell lines with patient-derived organoids using gene expression data. a Pair-wise comparison of subtype-specific TC analysis results. In the lower-left plots, each dot is an established organoid, with the two axis representing transcriptome similarity of the organoid with MET500 breast cancer samples of the two intersecting subtypes. The upper-right shaded values are the corresponding pair-wise spearman rank correlation values of each pair. b Boxplot of transcriptome similarity of CCLE breast cancer cell lines and organoids. P-values are computed with the two-sided Wilcoxon rank-sum test. In each box, the central line represents the median value and the bounds represent the 25th and 75th percentiles . The whiskers encompass 1.5 times the interquartile range. c For each subtype, the most-correlated organoid has significantly higher expression correlation with MET500 breast cancer samples of that subtype than the most-correlated cell line. P-values are computed with the two-sided Wilcoxon rank-sum test. In each box, the central line represents the median value and the bounds represent the 25th and 75th percentiles . The whiskers encompass 1.5 times the interquartile range. Source data are provided as a Source Data file

Breast Cancer Cell Lines Are Crude Models Of Breast Cancer

Despite the considerable roles played by breast cancer cell lines in deciphering the mechanisms underlying tumor initiation and evolution, cell lines are still viewed as crude models of tumors and could not capture all tumor features and heterogeneity.

The clonal population comprising of any single cell line could not well capture the heterogeneity of breast tumors at the intra-tumoral level. Carcinogenesis is a sequential process through many clinical and pathological stages including a typical hyperproliferation, local invasion, invasive carcinomas and ultimately metastatic disease, and is believed to be accompanied with a sequential acquisition of various genetic and epigenetic alterations in a single cell followed by clonal selection and expansion. The establishment of cell lines, on the other hand, may eliminate some types of tumor cells initially present in the tissue samples under the environmental stress created by the cultivation condition. For instance, cells unable to grow properly on plastic or need specific factors secreted by tumor microenvironment may be eliminated during the cultivation process. Thereby, how well a permanent breast cancer cell line could model the tumor intra-heterogeneity becomes a questionable issue.

Proliferative Migratory And Invasive Potential

The proliferative potential of 4Z-B-1 cells at P55 was assessed via the CCK-8 assay, and SK-BR-3 was used as a control. As shown in Figure 3A, 4Z-B-1 cells grew faster than SK-BR-3 cells, with a PDT of 40 h. The DNA content of 4Z-B-1 and SK-BR-3 cells was determined by flow cytometry, and the distribution of the cell phases was analyzed . Compared with SK-BR-3 cells, 4Z-B-1 cells were distributed more in S phase and G2/M phase and less in G0/G1 phase. Western blotting assays also showed that the expression level of CyclinE1 was higher in 4Z-B-1 cells than SK-BR-3 cells .

Figure 3. Proliferative, migratory and invasive potential of the 4Z-B-1 cell line. Growth curves of 4Z-B-1 and SK-BR-3 cells determined by the CCK8 assay. Distribution of 4Z-B-1 and SK-BR-3 cells determined by flow cytometry and statistical analysis. Expression of CyclinE1 in 4Z-B-1 and SK-BR-3 cells assessed by Western blotting. Representative micrographs and statistical analysis of migrated and invasive 4Z-B-1 and SK-BR-3 cells. Representative micrographs and statistical analysis of the invasive area of 4Z-B-1, SK-BR-3, MCF-7, and MDA-MB-231 mammospheres in Matrigel. Expression of EMT markers in 4Z-B-1, SK-BR-3, MCF-7, and MDA-MB-231 cells determined by Western blotting. ****p< 0.0001. Magnification, ×100 ×40.

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What Is The Time Frame Of An Application And What Do The Various Stages Consist Of

As soon as we know of your interest in obtaining cells from the Cell Bank we send you and Initial Enquiry Form.

This is a simple form for you to give us a short description of your project, what type of samples you may need and what methods you will be using them for.

On this form we ask when you will be available for a call. We then set up the call with you to discuss your needs in more detail. After this we will send you a Cell Application Form to put down the actual cell requirements as discussed in the phone call.

We endeavour to have these stages completed in a week to 10 days. As soon as we receive the completed Cell Application Form from you we fill out the MTA and DSA forms and send them on to you. For most requests the cells are already banked down and available once the paperwork is completed.

A few requests require bespoke cell preps and the time frame for these will be discussed in the phone call. Our aim is to have the cells sent out to you within 12 weeks of your initial contact. If the paperwork is completed quickly we have achieved this in as little as 3 weeks.

Establishment And Characterization Of Seven Human Breast Cancer Cell Lines Including Two Triple

Three dimensional cell culture of breast cancer cells (MCF
  • Affiliations: Korean Cell Line Bank, Laboratory of Cell Biology, Cancer Research Institute, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea, Department of Surgery, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea, Division of Hematology and Medical Oncology, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea, Department of Pathology, Seoul National University College of Medicine, Seoul 110-744, Republic of Korea
  • Pages: 2073-2081
  • This article is mentioned in:

    Abstract

    Introduction

    Breast cancer is a heterogeneous disease, and it haslong been appreciated that tumors with different biologicalfeatures have different clinical outcomes and responses to therapy.At present, prognosis and treatment selection in breast cancer arebased on characterization of tumor growth factor receptor statusinvolving estrogen receptor , progesterone receptor andC-erbB2. These markers can be used to define four functional groupsof tumors: i) hormone receptor-positive ii) C-erbB2-negative iii)hormone receptor-negative, C-erbB2-negative and iv) C-erbB2 overexpressing tumors with or withouthormone-receptor expression .

    Materials and methods

    Cell line establishment andmaintenance
    Growth properties and morphology invitro
    Nucleic acid isolation and cDNAsynthesis
    DNA profiles
    Expression of ER-, PR, C-erbB2, COX-2and E-cadherin genes
    Western blot analysis
    Taxol cytotoxicity assay

    Results

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    Mtor Pathway Signaling And Sensitivity To Mtor Inhibitor Everolimus And Dual Pi3k And Mtor Inhibitor Bez235

    The phosphorylation status of AKT, p70S6K, rpS6, and ERK in the NZBR lines was examined . NZBR1 showed the lowest degree of phosphorylated AKT expression, yet both NZBR1 and NBZR3 had comparable levels of phosphorylated p70S6K . On the other hand, both NZBR2 and NZBR4 contained low levels of phosphorylated p70S6K, indicating that they had low mTOR pathway utilization. NZBR2 and NZBR4 showed a low degree of phosphorylated rpS6, whereas NZBR1 had low levels of phosphorylated ERK.

    Figure 4. Signaling pathway usage and growth inhibitory concentrations for NZBR cell lines exposed to different drugs. Signaling pathway usage as measured by phosphorylation of AKT, p70S6K, rpS6 and ERK. Immunoblots with antibodies specific for phosphorylated proteins and their respective total protein are indicated. Tubulin is the loading control. IC50 values for everolimus, and NVP-BEZ235, shown as the mean ± standard error of triplicate experiments. Cell lines are identified as estrogen receptor positive, HER2 positive and triple negative .

    Two out of the three triple-negative NZBR cell lines were resistant to everolimus, with IC50 values of over 500 nM in a 3-day 3H-thymidine incorporation assay. The ER+ HER2+ NZBR3 cells showed high sensitivity to everolimus , yet the triple-negative NZBR2 with low mTOR pathway utilization also showed increased everolimus sensitivity . Significant differences between NZBR2 and the other three cell lines in BEZ235 sensitivity were also observed.

    Initial Characterization Of Nz Breast Cancer Cell Lines

    The cell lines were characterized by cellular DNA content, hormone receptor expression and tamoxifen sensitivity. The lines were all aneuploid and three of the four lines were triple-negative with no expression of estrogen receptor, progesterone receptor and HER2 . The ER+ NZBR3 cell line was sensitive to tamoxifen with an IC50 value of 60 nM . The triple-negative cell lines were relatively resistant to tamoxifen with IC50 values of > 1000, 390, and > 1000 nM, respectively.

    Table 1. Source, clinical and pathological features of tumors used to derive the New Zealand Breast Cancer cell lines, and DNA ploidy.

    Figure 1. Receptor expression, and growth inhibitory concentrations for NZBR cell lines exposed to tamoxifen. Relative expression of estrogen receptor , progesterone receptor , HER2 and EGFR in NZBR1, NZBR2, NZBR3, and NZBR4 breast cancer cell lines. Actin is shown as loading control. IC50 values for tamoxifen, shown as the mean ± standard error of triplicate experiments. The highest tamoxifen concentration used is indicated where 50% growth inhibition was not reached.

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    Sensitivity To Her2 Targeting Agents

    Since 4Z-B-1 cells were identified as HER2-positive cells and originated from a Herceptin-resistant patient, we tested the sensitivity of the cells to HER2-targeting agents, including trastuzumab, pertuzumab, lapatinib, pyrotinib and a combination of trastuzumab and pertuzumab. The sensitivity of several other HER2-positive breast cancer cell lines, including SK-BR-3, MDA-MB-453, BT-474, ZJU-0725, ZJU-1127, and ZJU-0327, was also tested for comparison. 4Z-B-1 cells showed sensitivity to trastuzumab and the combinations of trastuzumab and pertuzumab and lapatinib and pyrotinib but showed resistance to pertuzumab alone . In contrast, the ZJU-0725, ZJU-1127, and ZJU-0327 cell lines, which were identified as basal-like HER2-positive breast cancer cell lines, showed resistance to all of these HER2-targeting agents .

    Figure 4. Sensitivity to HER2-targeting agents and expression of associated proteins. Comparison of sensitivity to trastuzumab, pertuzumab, the combination of trastuzumab and pertuzumab, lapatinib and pyrotinib between the 4Z-B-1 cell line and other HER2-positive breast cancer cell lines. Expression of HER2-targeting agent sensitivity-associated genes in the 4Z-B-1 cell line and other HER2-positive breast cancer cell lines.

    Clinically Relevant Biomarkers Exhibit Heterogenous And Dynamic Expression In Bc Cell Lines

    Cell Membrane Biomarkers in Triple-Negative Breast Cancer (TNBC) cells from transcriptomic analysis

    Interestingly, HER2+ circulating tumour cells isolated from an ER+/HER2 breast cancer patient were shown to spontaneously interconvert from HER2 and HER2+, with cells harbouring a phenotype producing daughters of the opposite one52. To check if cell-cycle phase could explain the observed heterogeneity in the MDA-MB-361 cell line, we computationally predicted the cell cycle phase of each cell in both the HER2 and HER2+ subpopulations from single cell transcriptomics data53. A higher proportion of HER2 cells was predicted to be in S/G2/M phases when compared to HER2+ cells . This result is consistent with previous observations that report cell cycle arrest in G2/M phase following HER2 inhibition54.

    These results show that heterogeneity in the expression of clinically relevant biomarkers is present even in cell lines and that it can also be dynamic and of a non-genetic nature.

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    Tgca Bulk Expression Dataset And Cell

    Raw bulk expression data and relative patient clinical information were collected from the Genomic Data Commons portal by using the TCGAbiolinks package. Then, raw counts were normalized using the EdgeR package into R statistical environment 3.6. Bisque tool was used to estimate the cell-line composition from the patients bulk gene expression profile. Specifically, we applied the ReferenceBasedDecomposition function with parameters: bulk.eset set to the bulk gene expression dataset in log2 scale sc.eset set to our single-cell BC atlas with normalized raw counts rescaled in log2 use.overlap set to FALSE and set to the marker genes across the 32 BC cell lines estimated by using the function findClusterMarkers of gficf package. As in the original manuscript describing the Bisque tool, only marker genes with an FDR< 0.5 and Log2 fold change greaten then 0.25 were used for deconvolution purpose.

    Mtt Assay And Colony Formation Assay

    Cells were cultured in 96-well plates at 5000 cells per well, 24h after transfection. The 3–2, 5-diphenyl-tetrazolium bromide assay was used to determine cell viability 24 h, 48 h, 72 h, and 96 h after the cells were seed. Absorbance at 570 nm was measured using an automatic microplate reader. . Next, the cells were cultured for 10 days, and colonies were counted. The experiment was performed in triplicate. Data are expressed as mean ± standard deviation .

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    Scanning Electron Microscopy And Transmission Electron Microscopy Examination

    For SEM, mammospheres formed in low attachment 6-well plates or cells growing on cover glass were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer supplemented with 2% sucrose at room temperature for 1 h. The cells were washed once with cacodylate buffer and fixed with 1% osmium tetroxide in cacodylate buffer for 90 min. After being dehydrated through graded ethanol solutions, the sample cells were dried, coated with gold with an ACE200 coating system and imaged under a Nova Nano 450 microscope .

    For TEM, mammospheres were fixed with 2.5% glutaraldehyde at room temperature for 1 h, postfixed in 1% osmium tetroxide, dehydrated in a graded series of alcohol solutions, and embedded. Ultra-thin sections were cut, stained with uranyl acetate and lead citrate, and visualized under a Tecnai G2 spirit microscope .

    Cell Lines And Reagents

    Table 1 from Breast Cancer Cell Line Classification and Its Relevance ...

    SK-BR-3, MDA-MB-453, HBL-100, MCF-7, T-47D, BT-474, MDA-MB-231, BT-549, MDA-MB-468, HCC1937, and HS-578-T cell lines were purchased from the American Type Culture Collection . HBL-100 and SK-BR-3 cells were maintained in McCoys 5a modified medium. MCF-7 cells were maintained in Eagles minimum essential medium supplemented with 0.01 mg/ml insulin. HCC-1937, T-47D, and BT-549 cells were maintained in RPMI-1640 medium supplemented with 0.023 U/ml and 0.2 U/ml insulin. MDA-MB-231, MDA-MB-453 and MDA-MB-468 cells were maintained in Leibovitzs L-15 medium. BT-474 cells were maintained in Hybri-Care Medium supplemented with 1.5 g/L sodium bicarbonate. HS-578-T cells were maintained in DMEM supplemented with 0.01 mg/ml insulin. HER2 positive cell lines ZJU-0725, ZJU-1127 and ZJU-0327 were established by us and identified as basal like HER2 positive breast cancer cell lines and were maintained in RPMI 1640 medium. The ZJU-0725, ZJU-1127 and ZJU-0327 cell lines showed high invasiveness and potent resistance to chemotherapy in our previous study . All cells were cultured in medium supplemented with 10% FBS in a humidified incubator at 37°C. MDA-MB-231, MDA-MB-453 and MDA-MB-468 cells were maintained in room air, while the other cell lines were maintained in 5% CO2.

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    Evaluation Of Culture Purity Cell Cycle Distribution And Expression Of Cd24/cd44 By Flow Cytometry

    To evaluate cell purity, cells were fixed for 15 min in 4% formaldehyde, permeabilized in precooled 90% methanol for 10 min, blocked in 10% goat serum, and then incubated with an anti-cytokeratin -fluorescein isothiocyanate antibody diluted with PBS supplemented with 0.5% bovine serum albumin for 1 h. The cells were incubated in Alexa Fluor®-conjugated secondary antibody diluted with incubation buffer for 30 min and then counted by flow cytometry on a FACScan instrument .

    To analyze cell cycle distribution, cells in the exponential growth phase were processed according to the manufacturers instructions and then sorted by flow cytometry.

    To determine the expression of CD24/CD44, cells were suspended in 100 l PBS supplemented with 10 l PE mouse anti-human CD24 and FITC mouse anti-human CD44 antibodies. After incubation for 30 min at 4°C in the dark, the cells were examined by flow cytometry on a FACScan instrument .

    Boyden Chamber Invasion Assays

    Assays were performed in modified Boyden chambers with 8 μm pore filter inserts for 24-well plates . Filters were coated with 12.5 μl of ice-cold 20% basement membrane extract . Epithelial cells were added to the upper chamber in 300 μl of serum-free medium. For the invasion assay, 7.5 à 104 cells were seeded on the 20% Matrigel-coated filters and incubated for 24 hr. The lower chamber was filled with 300 μl of full medium. After incubation, epithelial cells on the underside of the filter were fixed with 2.5% glutaraldehyde in PBS and stained with 0.5% toluidine blue in 2% Na2CO3. Cells that remained in the gel or attached to the upper side of the filter were removed with cotton tips. Cells on the underside of the filter were counted using light microscopy. Assays were performed in triplicate or quadruplicate. The results were expressed as an average ± one standard deviation.

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    Establishment And Characterization Of A Her2

    • 1Key Laboratory of Cancer Prevention and Intervention, The Second Affiliated Hospital, School of Medicine, Cancer Institute, China National Ministry of Education, Zhejiang University, Hangzhou, China
    • 2Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, China
    • 3Key Laboratory of Biotherapy of Zhejiang Province, Biomedical Research Center, Hangzhou, China
    • 4The Affiliated Cixi Hospital, Wenzhou Medical University, Ningbo, China

    Drug resistance is a daunting challenge in the treatment of breast cancer, making it an urgent problem to solve in studies. Cell lines are important tools in basic and preclinical studies however, few breast cell lines from drug-resistant patients are available. Herein, we established a novel HER2-positive breast cancer cell line from the pleural effusion of a drug-resistant metastatic breast cancer patient. This cell line has potent proliferative capability and tumorigenicity in nude mice but weak invasive and colony-forming capability. The molecular subtype of the cell line and its sensitivity to chemotherapeutics and HER2-targeting agents are different from those of its origin, suggesting that the phenotype changes between the primary and metastatic forms of breast cancer.

    Characterization Of Stromal And Epithelial Facs Subpopulations Of The Normal Breast

    Cancer: from a healthy cell to a cancer cell

    To understand the relationship between cell lines and the different subpopulations of the normal breast, we FAC-sorted and expression-profiled stromal, MaSC/BiP, pL, and mL subpopulations from at least three independent reduction mammoplasties as previously described by Lim et al. . As shown in Fig. 2b and Supplemental material, ~35 and ~25 % of lineage-negative cells fall in the stromal- and MaSC/BiP-enriched groups, respectively, whereas only ~10 % of the cells are identified in the pL and mL-enriched groups. To further complement these results, we also analyzed six independent reduction mammoplasties using CD24 and CD44 cell surface markers. As shown in Fig. 2c, 50, 22, and 4.6 % of the sorted cells were CD24/CD44+, CD24/CD44, and CD24+/CD44+ in one sample. However, the CD24+/CD44+ subpopulation was not always identified .

    Fig. 2

    Our previously described differentiation score predictor was based on genomic data of the MaSC/BiP-, pL- and mL-enriched subpopulations of Lims et al. . To validate this differentiation model, we estimated the differentiation status of our newly sorted cell subpopulations . As expected, the MaSC/BiPs showed the lowest scores , the mLs showed the highest scores , and the pLs showed a slightly higher differentiation status than Lims pL . Overall, high correlation coefficients between EpCAM and CD49f gene and protein expression was observed in the four normal-sorted fractions .

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