Single cell transcriptomics reveals molecular subtype and functional heterogeneity in models of breast cancer

Abstract

Breast cancer has long been classified into a number of molecular subtypes that predict prognosis and therefore influence clinical treatment decisions. Cellular heterogeneity is also evident in breast cancers and plays a key role in the development, evolution and metastatic progression of many cancers. How clinical heterogeneity relates to cellular heterogeneity is poorly understood, so we approached this question using single cell gene expression analysis of well established in vitro and in vivo models of disease.\n\nTo explore the cellular heterogeneity in breast cancer we first examined a panel of genes that define the PAM50 classifier of molecular subtype. Five breast cancer cell line models (MCF7, BT474, SKBR3, MDA-MB-231, and MDA-MB-468) were selected as representatives of the intrinsic molecular subtypes (luminal A and B, basal-like, and Her2-enriched). Single cell multiplex RT-PCR was used to isolate and quantify the gene expression of single cells from each of these models, and the PAM50 classifier applied. Using this approach, we identified heterogeneity of intrinsic subtypes at single-cell level, indicating that cells with different subtypes exist within a cell line. Using the Chromium 10X system, this study was extended into thousands of cells from the MCF7 cell-line and an ER+ patient derived xenograft (PDX) model and again identified significant intra-tumour heterogeneity of molecular subtype.\n\nEstrogen Receptor (ER) is an important driver and therapeutic target in many breast cancers. It is heterogeneously expressed in a proportion of clinical cases but the significance of this to ER activity is unknown. Significant heterogeneity in the transcriptional activation of ER regulated genes was observed within tumours. This differential activation of the ER cistrome aligned with expression of two known transcriptional co-regulatory factors of ER (FOXA1 and PGR).\n\nTo examine the degree of heterogeneity for other important phenotypic traits, we used an unsupervised clustering approach to identify cellular sub-populations with diverse cancer associated transcriptional properties, such as: proliferation; hypoxia; and treatment resistance. In particular, we show that we can identify two distinct sub-populations of cells that may have denovo resistance to endocrine therapies in a treatment naive PDX model of ER+ breast cancer. One of these consists of cells with a non-proliferative transcriptional phenotype that is enriched for transcriptional properties of ERBB2 tumours. The other is heavily enriched for components of the primary cilia. Gene regulatory networks were used to identify transcription factor regulons that are active in each cell, leading us to identify potential transcriptional drivers (such as E2F7, MYB and RFX3) of the cilia associated endocrine resistant cells. This rare subpopulation of cells also has a highly heterogenous mix of intrinsic subtypes highlighting a potential role of intra-tumour subtype heterogeneity in endocrine resistance and metastatic potential.\n\nOverall, These results suggest a high degree of cellular heterogeneity within breast cancer models, even cell lines, that can be functionally dissected into sub-populations of cells with transcriptional phenotypes of potential clinical relevance.