Single-cell analysis reveals the Comma-1D cell line as a unique model for mammary gland development and breast cancer

Abstract

The mammary epithelial tree contains two distinct populations, luminal and basal. The investigation of how this heterogeneity is developed and how it influences tumorigenesis has been hampered by the need to perform these studies using animal models. Comma-1D is an immortalized mouse mammary epithelial cell line that has unique morphogenetic properties. By performing single-cell RNA-seq studies we found that Comma-1D cultures consist of two main populations with luminal and basal features and a smaller population with mixed lineage and bipotent characteristics. We demonstrated that multiple transcription factors associated with the differentiation of the mammary epithelium in vivo also modulate this process in Comma-1D cultures. Additionally, we found that only cells with luminal features were able to acquire transformed characteristics after an oncogenic HER2 mutant was introduced in their genomes. Overall, our studies characterize at a single-cell level the heterogeneity of the Comma-1D cell line and illustrate how Comma-1D cells can be used as an experimental model to study both the differentiation and the transformation processes in vitro.

Insulin Receptor Loss Impairs Mammary Tumorigenesis in Mice

“…To gain further insight into the mechanism(s) behind reduced tumour initiation upon INSR deletion, we generated two independent INSR -deficient luminal COMMA1D mouse mammary cell lines (COMMA1D INSR (-/-)) and two accompanying wild-type controls (COMMA1D INSR (+/+)) using CRISPR/Cas9 ( Supplementary Figure 7A ) 44 . COMMA1D cells are immortalized but not transformed mammary epithelial cells isolated from Balb/C mice in mid-pregnancy and have been used to study oncogenic mammary transformation 45,46 . INSR deletion in COMMA1D cells did not affect net activation of the PI3K-Akt ( Supplementary Figures 7B, C ) and Ras-MAPK ( Supplementary Figures 7B, D ) signaling pathway components in response to insulin stimulation, likely due to the presence of IGF1R, as evidenced by a reduced but not depleted phospho-INSR/IGF1R signal in INSR -deficient cells ( Supplementary Figure 7B, E ).…”

“…CRISPR/Cas9 in COMMA1D murine mammary epithelial cellsCOMMA1D cells, a non-transformed murine mammary epithelial cell line, were cultured in COMMA1D media (DMEM/F12 media (Wisent Inc #319-005-CL) supplemented with 5% FBS (Wisent Inc #098150,), 20ng/ml EGF (Peprotech #AF-100-15), 0.5mg/ml Hydrocortisone (Sigma #H0888), 100ng/ml Cholera Toxin (Sigma C8052), 10ug/ml Insulin (Biogems #10-365), and 1% Penicillin/Streptomycin (Wisent Inc #450-201-EL)). Cells were stained with FITC anti-mouse Ly-6A/E (Sca-1; Biolegend #122504) and APC/Cy7 anti-mouse CD326 (Ep-CAM; Biolegend #118217) and sorted by FACS (Aria Fusion, BD Biosciences) to isolate the luminal (Ep-CAM high:Sca-1 low) and basal (Ep-CAM low:Sca-1 high) populations46 . Sorted luminal COMMA1D cells were then genetically modified to delete INSR using CRISPR/Cas9 as described in44 .Briefly, px458 (Addgene #62988), a plasmid containing a Cas9n D10A nickase mutant with a puromycin selection marker, was cloned to incorporate knock-out sgRNAs targeting INSR (Top: 5'-CAC-CGG-GTA-TAA-GTC-TCT-CAT-TTG-G-3', Bottom: 5'-AAA-CCC-AAA-TGA-GAG-ACT-TAT-ACC-C-3').…”