Limiting Self-Renewal of the Basal Compartment Induces Differentiation and Alters Evolution of Mammary Tumors

The goal of the study was to assess the role of PKA activation in mammary development and the development and progression of mammary tumors. The scRNA-Seq experiment was specifically designed to plot the evolution of cell subpopulations within mammary glands from the hyperplasia stage as they progress to fully-blown tumors. Results: PKA activation resulted in tumors that were more benign, exhibited reduced metastatic propensity, loss of tumor-initiating potential and increased sensitivity to chemotherapy. Longitudinal single cell profiling at the hyperplasia and tumor stages uncovered an altered path of tumor evolution whereby PKA curtails the emergence of aggressive subpopulations. Overall design: MMTV-PyMT WT and PKA activated mice were sacrificed at two time points - 8-9 weeks (hyperplasia) or 22 weeks (tumor). Hyperplastic mammary glands and tumors within the same mouse were processed for longitudinal assesment of tumor development. Single-cell RNA-Seq was carried out at the hyperplasia and tumor stages of WT tumors and those with activated PKA signaling. Single cell RNA-seq libraries were be prepared using the Chromium Single Cell 3' v2 kit (10x Genomics) and sequenced on an Illumina NextSeq500 High Output 26bp x 98bp run to generate an average of 50,000 reads/cell. Following cell-type specific markers were used for the cells classification, Basal cells: Krt14, Krt5, Snai2; Luminal Mature cells: Pgr, Prlr, Foxa1, Gpx3, Esr1; Luminal Progenitor cells; CD14, Kit, Lalba; Lumino-basal: Krt14, Krt8, CD14; EMT-like: Trim29, Vim, Cldn6, Sox4. We identified and classified cell-types using Monocle2's classifyCells function. Transition of gene expression kinetics was performed by integrative Pseudotime analysis using Monocle2.