Elucidating and Pharmacologically Targeting Master Regulators of Stem-like Breast Cancer Cell State (CROP-Seq)

In many tumors, small subpopulations of stem-like cells can exist in drug-resistant states that are transient and epigenetically governed. Single-cell RNA sequencing (scRNA-seq) provides transcriptional profiles for individual cells which, although sparse and noisy, may provide insight into how they are regulated and how they may be targeted. We took a systems-biology approach to analyzing cancer stem-like cells (CSLCs) in breast tumors and predicting Master Regulator (MR) proteins responsible for governing the transcriptional state of these cells, thus revealing drug sensitivities that may be leveraged via combination therapy. The MRs were validated by pooled, CRISPR-KO mediated CROP-seq profiling of single cells representing either the CLSC or the differentiated breast cancer (BRCA) cells. For control samples, the four breast cancer cell lines (MCF7, HCC2157, HCC38, and HCC1143) were incubated, dissociated, and subjected to scRNA-seq (10X Genomics). For the CROPSeq screening, total 50 genes, identified as top 25 activated proteins in breast CSLCs and top 25 activated proteins in well-differentiated BRCA cells, were targeted with 3 sgRNAs/gene for HCC1143 and HCC38 cells. In addition, 15 intergenic sgRNAs experiments were included a negative controls. Cells were grown for 14 days of post sgRNA transduction before the scRNAseq (10X Genomics).