Computational protocol: Lunatic Fringe and p53 Cooperatively Suppress Mesenchymal Stem-Like Breast Cancer

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Protocol publication

[…] Mouse mammary tissues were dissociated using Collagenase/Hyaluronidase solution (StemCell Technologies) and single cell suspensions were generated according to manufacturer's instructions. Lineage-depleted mammary epithelial cells were prepared using an EasySep mouse mammary stem cell enrichment kit (StemCell Technologies). Flow cytometry was performed by standard procedures using anti-mouse CD24-PE and anti-CD49f-FITC (StemCell Technologies). Fluorescence was recorded using Gallios Flow Cytometer (Beckman Coulter) and analyzed with Kaluza flow cytometry analysis software. Mammary tissues from three animals per genotype were analyzed with similar results. Percentages of stem cell-enriched cell populations in each genotype were quantitated. Lineage-depleted mammary epithelial cells were cultured for mammosphere formation for 7 days as previously described . [...] Human breast cancer gene expression data set GSE18229 (n = 372) was downloaded from GEO (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE18229). Data were z-transformed and applied quantile normalization by preprocessCore package from Bioconductor in R software. Gene expressions were summarized by average value if multiple probes mapping to the same gene. Preprocessed METABRIC breast cancer expression data (n = 2509) was downloaded from cBioportal web portal (http://www.cbioportal.org/). Differential expressions of LFNG, NOTCH3, JAG1, HES1 and HEY1 among different breast cancer subtypes were analyzed by one way ANOVA test. p values were calculated by comparing expression means across all subtypes. For the survival analysis related to LFNG expression (probeset 228762_at), we used online tool (http://www.kmplot.com/) to perform univariate Cox regression analysis and Kaplan–Meier survival curves with lower quartile as cutoff. The hazard ratio with 95% confidence and p values were calculated from 1402 patients with overall survival, 3955 patients with recurrence free survival, and 1747 patients with post progression survival information for breast cancer. [...] For mouse experiments, statistical analyses were performed using SigmaPlot (Systat Software) by applying the Log-rank test to compare survival data and the unpaired two-tailed t-test for all other comparisons. […]

Pipeline specifications

Software tools Kaluza, preprocessCore, cBioPortal, SigmaPlot
Applications Miscellaneous, Flow cytometry
Organisms Mus musculus, Homo sapiens