Gene expression analysis of dissociated and FDG FACS-sorted mammary glands of 16 week old nulliparous AXL +/LacZ and AXL LacZ/LacZ mice (B6.129P2-AXLtm1Dgen strain, Jackson Labs)

The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony-formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl+ cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers, and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when coopted, maintains acquired stemness in breast cancer cells. Gene expression analysis of FDG FACS-sorted 16 week old nulliparous AXL +/LacZ and AXL LacZ/LacZ (B6.129P2-AXLtm1Dgen strain, Jackson Labs) was conducted using the Illumina MouseWG-6 v2.0 Expression BeadChip (BD-201-0202, BD-201-0602). AXL-expressing cells were enriched from dissociated mammary glands by FACS using the fluorogenic β -galactosidase substrate fluorescein Di-β-D-galactopyranoside (FDG; FluoReporter® lacZ Flow Cytometry Kit, Molecular Probes F-1930). All samples are pooled mammary glands from several individuals of the same genotype. The following sorting strategy were used: freshly dissociated, labelled cells were discriminated from the debris and gated in the forward scatter (FSC-A) and side scatter (SSC-A) plots. Subsequently, the forward scatter Width (FSC-W) versus FSC-A plots were used for doublet cell exclusion. Propidium iodide (PI) staining was used for dead cell discrimination and the APC channel was used as a dump channel for the APC conjugated lineage markers used. Thus, only single, viable, PI-negative, and APC-negative (CD45-, CD31-, CD11b-) cells were included in the final sort of FDG-high and FDG-low cells used for subsequent experiments. An FDG high gate (highest 5%) was used to enrich for AXL-expressing cells, while AXL-negative cells were sorted using an FDG low gate (lowest 65%). A post-sort analysis was performed to verify purity of the sorted cells. Log2 quantile normalized of the gene expression data were used for unsupervised hierarchical clustering, gene signature analysis and differentially expressed genes. Hierarchical clustering was performed using Pearson’s correlation as distance measurement. Gene signatures among cell population were determined by comparing the gene expression levels and available molecular signatures of the mammary cell subpopulations (Lim et al., 2009) using GSEA software package (Subramanian et al., 2005). Differentially expressed genes between AXL +/LacZ FDG high and AXL LacZ/LacZ FDG high cell groups were identified using Rank Product method. The genes with a p-value <0.01 and fold change >= 1.5 were considered as differentially expressed genes.