Franco, H. | bioRxiv Science

bioRxiv2023

Tamoxifen Response at Single Cell Resolution in Estrogen Receptor-Positive Primary Human Breast Tumors

In ER+/HER2- breast cancer, multiple measures of intra-tumor heterogeneity are associated with worse response to endocrine therapy. To investigate heterogeneity in response to treatment, we developed an operating room-to-laboratory pipeline for the collection of live human tumors and normal breast specimens immediately after surgical resection for processing into single-cell workflows for experimentation and genomic analyses. We demonstrate differences in tamoxifen response by cell type and identify distinctly responsive and resistant subpopulations within the malignant cell compartment of human tumors. Tamoxifen resistance signatures from 3 distinct resistant subpopulations are prognostic in large cohorts of ER+ breast cancer patients and enriched in endocrine therapy resistant tumors. This novel ex vivo model system now provides a foundation to define responsive and resistant sub-populations within heterogeneous tumors, to develop precise single cell-based predictors of response to therapy, and to identify genes and pathways driving resistance to therapy.

cancer biology↗

bioRxiv2020

CRISPR-based knockin mutagenesis of the pioneer transcription factor FOXA1; optimization of strategies for multi-allelic proteins in cancer cells

Precise genome engineering of living cells has been revolutionized by the introduction of the highly specific and easily programmable properties of CRISPR-Cas9 technology. This has greatly accelerated research into human health and has facilitated the discovery of novel therapeutics. CRISPR-Cas9 is most widely employed for its ability to inactivate, or knockout, specific genes, but can be also used to introduce subtle site-specific substitutions of DNA sequences that can lead to changes in the amino acid composition of proteins. Despite the proven success of CRISPR-based knockin strategies of genes in typical diploid cells (i.e. cells containing two sets of chromosomes), precise editing of cancer cells, that typically have unstable genomes and multiple copies of chromosomes, is more challenging and not adequately addressed in the literature. Herein we detail our methodology for replacing endogenous proteins with intended knockin mutants in polyploid cancer cells and discuss our experimental design, screening strategy, and facile allele-frequency estimation methodology. As proof of principle, we performed genome editing of specific amino acids within the pioneer transcription factor FOXA1, a critical component of estrogen and androgen receptor signaling, in MCF-7 breast cancer cells. We confirm proper levels of mutant FOXA1 protein expression and intended amino acids substitutions via western blotting and mass spectrometry. In addition, we show that mutant allele-frequency estimation is easily achieved by TOPO cloning combined with allele-specific PCR, which we later confirmed by next-generation RNA-sequencing. Typically, there are 4 - 5 copies (alleles) of FOXA1 in breast cancer cells making the editing of this protein inherently challenging. As a result, most studies that focus on FOXA1 mutants rely on ectopic overexpression of FOXA1 from a plasmid. Therefore, we provide an optimized methodology for replacing endogenous wildtype FOXA1 with precise knockin mutants to enable the systematic analysis of its molecular mechanisms within the appropriate physiological context.

genetics↗