Multidimensional phenotyping of breast cancer cell lines to guide preclinical research

Cell lines are extremely useful tools in breast cancer research. Their key benefits include a high degree of control over experimental variables and reproducibility. However, the advantages must be balanced against the limitations of modelling such a complex disease in vitro. Informed selection of cell line(s) for a given experiment now requires essential knowledge about molecular and phenotypic context in the culture dish.We performed multidimensional profiling of 36 widely used breast cancer cell lines that were cultured under standardised conditions. Flow cytometry and digital immunohistochemistry were used to compare the expression of 14 classical breast cancer biomarkers related to intrinsic molecular profiles and differentiation states: EpCAM, CD24, CD49f, CD44, ER, AR, HER2, EGFR, E-cadherin, p53, vimentin, and cytokeratins 5, 8/18 and 19.This cell-by-cell analysis revealed striking heterogeneity within cultures of individual lines that would be otherwise obscured by analysing cell homogenates, particularly amongst the triple-negative lines. High levels of p53 protein, but not RNA, were associated with somatic mutations (p\ua0=\ua00.008). We also identified new subgroups using the nanoString PanCancer Pathways panel (730 transcripts representing 13 canonical cancer pathways). Unsupervised clustering identified five groups: luminal/HER2, immortalised ('normal'), claudin-low and two basal clusters, distinguished mostly by baseline expression of TGF-beta and PI3-kinase pathway genes.These features are compared with other published genotype and phenotype information in a user-friendly reference table to help guide selection of the most appropriate models for in vitro and in vivo studies, and as a framework for classifying new patient-derived cancer cell lines and xenografts

Docetaxel/doxorubicin chemotherapy of MCF10CA1a is unaffected by <i>EGFR</i> mutation or in combination with afatinib.

<p>Cells were cultured in the presence of EGF and treated with docetaxel/doxorubicin in a 5:1 ratio (DD) alone or in combination with the half IC₅₀ dose of afatinib (DDA) for seven days. Cell survival was assessed using the MTS assay and the IC₅₀ dose was reported using the docetaxel concentrations. CA: MCF10CA1a-EV, WT: MCF10CA1a-<i>EGFR</i>-WT, GS: MCF10CA1a-<i>EGFR</i>-GS, DEL: MCF10CA1a-<i>EGFR</i>-DEL.</p

Overexpression of wild type or mutant <i>EGFR</i> increases the growth of MCF10CA1a mammary fat pad xeongrafts.

<p>Female BALB/c nude mice were injected in the mammary fat pad with 5x10⁶ cells from the indicated cell line and tumour formation was monitored with bioluminescent imaging. CA: MCF10CA1a-EV, WT: MCF10CA1a -<i>EGFR</i>-WT, GS: MCF10CA1a -<i>EGFR</i>-GS, DEL: MCF10CA1a -<i>EGFR</i>-DEL <b>A.</b> Representative bioluminescent images of individual mice taken on day 14 and day 43. <b>B.</b> Plot of the increase in luciferase signal in each group of mice (*p<0.05, **p<0.01, ****p<0.0001, n = 5, vs CA control, One-Way ANOVA). <b>C.</b> Representative bioluminescent images of individual mice taken on day 49 post injection in an independent cohort of mice. <b>D.</b> Plot of the magnitude of luciferase signal in each group of mice at day 49 post injection (**p<0.01, n = 5).</p

Expression of the <i>EGFR</i>-GS and—DEL mutant proteins enhances MCF10CA1a EGF-independent growth but not sensitivity to gefitinib.

<p><b>A.</b> Cells were cultured in the presence (+ EGF) or absence (- EGF) of EGF and their proliferation assessed using real-time imaging on the Incucyte Zoom and measuring the increase in confluency over 4–5 days. The doubling times of the cell lines were calculated from the rates of proliferation from 36–60 hours (24 hour period). While there was no difference in proliferation between the cell lines in the presence of EGF, in the absence of EGF all three <i>EGFR</i> constructs reduced MCF10CA1a doubling time (*p<0.05, **** p<0.0001^,, unpaired T-test, n = 6). <b>B.</b> Representative growth curves for cells grown in EGF-free media with measurements of confluency taken every 6 hours for up to 102 hours. <b>C-D.</b> Calculated IC₅₀ values of gefitinib for inhibition of proliferation (<b>C</b>) or cell toxicity (<b>D</b>) from cells treated for 48 hours in various doses of gefitinib and monitored in real-time using the Incucyte Zoom (** p<0.01, unpaired T-test, n = 3). <b>E</b>-<b>G.</b> Calculated mean IC₅₀ values from cells treated for seven days in various doses of gefitinib (<b>E</b>, n = 2), afatinib (<b>F</b>, n = 4) or cetuximab (<b>G</b>, n = 1) and cell death determined using the MTS assay. CA: MCF10CA1a-EV, WT: MCF10CA1a-<i>EGFR</i>-WT, GS: MCF10CA1a-<i>EGFR</i>-GS, DEL: MCF10CA1a-<i>EGFR</i>-DEL.</p

<i>EGFR</i>-DEL expression enhances anchorage-independent growth of MCF10A cells in the absence of EGF.

<p>Cells were cultured in media containing growth factor-reduced Matrigel with 2% horse serum in the absence (- EGF) or presence of 5 ng/ml EGF (+ EGF). At 13 days spheroid formation assessed was assessed with phase contrast microscopy. Scale bars = 400 μm.</p

Circos plot summarizing the gene amplification and loss differences between MCF10A and MCF10CA1a.

<p>The outer band details chromosome number and position, middle band represents MCF10A and the inner band represents MCF10CA1a. In each case, values plotted are from copy number analysis of Illumina Human Omni 2.5M SNP array data. Red regions highlight gene amplification while green regions represent gene loss.</p

Frequency of each class of mutations in MCF10A and MCF10CA1a cells.

<p>Frequency of each class of mutations in MCF10A and MCF10CA1a cells.</p

Montolío Durán, Estrella (2010), Estrategias de comunicación para mujeres directivas, Barcelona, Departament de Treball de la Generalitat de Cataluya y Fondo social Europeo, pp. 138.

Background Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown. Materials and Methods Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed. Results Mutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer. Conclusions Mutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance