Stromal cell diversity associated with immune evasion in human triple‐negative breast cancer

Resource13 August 2020free access Source DataTransparent process Stromal cell diversity associated with immune evasion in human triple-negative breast cancer Sunny Z Wu The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Daniel L Roden The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Chenfei Wang Department of Data Sciences, Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Harvard T.H. Chan School of Public Health, Boston, MA, USA Search for more papers by this author Holly Holliday The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Kate Harvey The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia Search for more papers by this author Aurélie S Cazet The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Kendelle J Murphy The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Brooke Pereira The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Ghamdan Al-Eryani The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Nenad Bartonicek The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Rui Hou Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Nedlands, Perth, WA, Australia Search for more papers by this author James R Torpy The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Simon Junankar The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Chia-Ling Chan Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Chuan En Lam Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Mun N Hui The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia Chris O'Brien Lifehouse, Camperdown, NSW, Australia Search for more papers by this author Laurence Gluch The Strathfield Breast Centre, Strathfield, NSW, Australia Search for more papers by this author Jane Beith Chris O'Brien Lifehouse, Camperdown, NSW, Australia Search for more papers by this author Andrew Parker St Vincent's Hospital, Darlinghurst, NSW, Australia Search for more papers by this author Elizabeth Robbins Royal Prince Alfred Hospital, Camperdown, NSW, Australia Search for more papers by this author Davendra Segara St Vincent's Hospital, Darlinghurst, NSW, Australia Search for more papers by this author Cindy Mak Chris O'Brien Lifehouse, Camperdown, NSW, Australia Search for more papers by this author Caroline Cooper Pathology Queensland, Princess Alexandra Hospital, Brisbane, Qld, Australia Southside Clinical Unit, Faculty of Medicine, University of Queensland, Brisbane, Qld, Australia Search for more papers by this author Sanjay Warrier Department of Breast Surgery, Chris O'Brien Lifehouse, Camperdown, NSW, Australia Royal Prince Alfred Institute of Academic Surgery, Sydney University, Sydney, NSW, Australia Search for more papers by this author Alistair Forrest Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Nedlands, Perth, WA, Australia RIKEN Center for Integrative Medical Sciences, Yokohama, Japan Search for more papers by this author Joseph Powell Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia UNSW Cellular Genomics Futures Institute, University of New South Wales, Sydney, NSW, Australia Search for more papers by this author Sandra O'Toole The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Australian Clinical Laboratories, Northern Beaches Hospital, Frenchs Forest, NSW, Australia Search for more papers by this author Thomas R Cox orcid.org/0000-0001-9294-1745 The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Paul Timpson The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Elgene Lim The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia St Vincent's Hospital, Darlinghurst, NSW, Australia Search for more papers by this author X Shirley Liu Department of Data Sciences, Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Harvard T.H. Chan School of Public Health, Boston, MA, USA Search for more papers by this author Alexander Swarbrick Corresponding Author [email protected] orcid.org/0000-0002-3051-5676 The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Sunny Z Wu The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Daniel L Roden The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Chenfei Wang Department of Data Sciences, Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Harvard T.H. Chan School of Public Health, Boston, MA, USA Search for more papers by this author Holly Holliday The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Kate Harvey The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia Search for more papers by this author Aurélie S Cazet The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Kendelle J Murphy The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Brooke Pereira The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Ghamdan Al-Eryani The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Nenad Bartonicek The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Rui Hou Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Nedlands, Perth, WA, Australia Search for more papers by this author James R Torpy The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Simon Junankar The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Chia-Ling Chan Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Chuan En Lam Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Mun N Hui The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia Chris O'Brien Lifehouse, Camperdown, NSW, Australia Search for more papers by this author Laurence Gluch The Strathfield Breast Centre, Strathfield, NSW, Australia Search for more papers by this author Jane Beith Chris O'Brien Lifehouse, Camperdown, NSW, Australia Search for more papers by this author Andrew Parker St Vincent's Hospital, Darlinghurst, NSW, Australia Search for more papers by this author Elizabeth Robbins Royal Prince Alfred Hospital, Camperdown, NSW, Australia Search for more papers by this author Davendra Segara St Vincent's Hospital, Darlinghurst, NSW, Australia Search for more papers by this author Cindy Mak Chris O'Brien Lifehouse, Camperdown, NSW, Australia Search for more papers by this author Caroline Cooper Pathology Queensland, Princess Alexandra Hospital, Brisbane, Qld, Australia Southside Clinical Unit, Faculty of Medicine, University of Queensland, Brisbane, Qld, Australia Search for more papers by this author Sanjay Warrier Department of Breast Surgery, Chris O'Brien Lifehouse, Camperdown, NSW, Australia Royal Prince Alfred Institute of Academic Surgery, Sydney University, Sydney, NSW, Australia Search for more papers by this author Alistair Forrest Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Nedlands, Perth, WA, Australia RIKEN Center for Integrative Medical Sciences, Yokohama, Japan Search for more papers by this author Joseph Powell Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia UNSW Cellular Genomics Futures Institute, University of New South Wales, Sydney, NSW, Australia Search for more papers by this author Sandra O'Toole The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Australian Clinical Laboratories, Northern Beaches Hospital, Frenchs Forest, NSW, Australia Search for more papers by this author Thomas R Cox orcid.org/0000-0001-9294-1745 The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Paul Timpson The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Elgene Lim The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia St Vincent's Hospital, Darlinghurst, NSW, Australia Search for more papers by this author X Shirley Liu Department of Data Sciences, Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Harvard T.H. Chan School of Public Health, Boston, MA, USA Search for more papers by this author Alexander Swarbrick Corresponding Author [email protected] orcid.org/0000-0002-3051-5676 The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia Search for more papers by this author Author Information Sunny Z Wu1,2, Daniel L Roden1,2, Chenfei Wang3, Holly Holliday1,2, Kate Harvey1, Aurélie S Cazet1,2, Kendelle J Murphy1,2, Brooke Pereira1,2, Ghamdan Al-Eryani1,2, Nenad Bartonicek1,2, Rui Hou4, James R Torpy1,2, Simon Junankar1,2, Chia-Ling Chan5, Chuan En Lam5, Mun N Hui1,6, Laurence Gluch7, Jane Beith6, Andrew Parker8, Elizabeth Robbins9, Davendra Segara8, Cindy Mak6, Caroline Cooper10,11, Sanjay Warrier12,13, Alistair Forrest4,14, Joseph Powell5,15, Sandra O'Toole1,2,16, Thomas R Cox1,2, Paul Timpson1,2, Elgene Lim1,2,8, X Shirley Liu3 and Alexander Swarbrick *,1,2 1The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia 2St Vincent's Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, NSW, Australia 3Department of Data Sciences, Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Harvard T.H. Chan School of Public Health, Boston, MA, USA 4Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Nedlands, Perth, WA, Australia 5Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia 6Chris O'Brien Lifehouse, Camperdown, NSW, Australia 7The Strathfield Breast Centre, Strathfield, NSW, Australia 8St Vincent's Hospital, Darlinghurst, NSW, Australia 9Royal Prince Alfred Hospital, Camperdown, NSW, Australia 10Pathology Queensland, Princess Alexandra Hospital, Brisbane, Qld, Australia 11Southside Clinical Unit, Faculty of Medicine, University of Queensland, Brisbane, Qld, Australia 12Department of Breast Surgery, Chris O'Brien Lifehouse, Camperdown, NSW, Australia 13Royal Prince Alfred Institute of Academic Surgery, Sydney University, Sydney, NSW, Australia 14RIKEN Center for Integrative Medical Sciences, Yokohama, Japan 15UNSW Cellular Genomics Futures Institute, University of New South Wales, Sydney, NSW, Australia 16Australian Clinical Laboratories, Northern Beaches Hospital, Frenchs Forest, NSW, Australia *Corresponding author. Tel: +61 2 9295 8500; E-mail: [email protected] EMBO J (2020)39:e104063https://doi.org/10.15252/embj.2019104063 See also: F Brod & R Fässler (October 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single-cell RNA sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states: the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell signalling predictions, we provide evidence that stromal-immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs. Synopsis This single-cell gene expression resource deciphers the composition of triple-negative breast cancer (TNBC) stroma, revealing distinct subclasses of cancer-associated fibroblasts (CAFs) and perivascular-like (PVL) cells. These signatures are informative on tumour aetiology and potential strategies for development of targeted therapies. Single-cell analysis of primary TNBC highlights clusters of stromal and immune cell types. TNBC stroma is comprised of myofibroblast-like CAFs, inflammatory-like CAFs, differentiated PVL and immature PVL cells. Stromal subclasses differ in surface markers, spatial localisation in tissue, ECM functions, and predicted cellular crosstalk with immune cells. Inflammatory-like CAF and differentiated PVL cells are associated with cytotoxic T-cell dysfunction and exclusion in independent TNBC-patient cohorts. Introduction Heterotypic interactions between stromal, immune and malignant epithelial cells play important roles in solid tumour progression and therapeutic response. Cancer-associated fibroblasts (CAFs) play an integral part in the tumour microenvironment (TME) and can influence many aspects of carcinogenesis including extracellular matrix (ECM) remodelling, angiogenesis, cancer cell proliferation, invasion, inflammation, metabolic reprogramming and metastasis (Kalluri, 2016). Recent studies have described roles for CAFs in mediating immune suppression and chemoresistance, establishing CAFs as novel and attractive targets for anti-cancer therapies in advanced breast cancer (Brechbuhl et al, 2017; Cazet et al, 2018; Costa et al, 2018; Givel et al, 2018; Su et al, 2018). Despite their well-described roles in cancer biology, CAFs remain enigmatic: limited studies suggest phenotypic heterogeneity, plasticity and functional diversity, with both tumour-promoting and tumour-suppressive properties (Kalluri, 2016). The multi-faceted nature of CAFs suggests that they are comprised of diverse subpopulations, and an improved understanding of stromal heterogeneity may explain how CAFs contribute to the dynamic complexity and functional malleability of the tumour ecosystem. CAFs of the tumour parenchyma are routinely studied using a handful of markers including α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), CD90 (THY-1), platelet-derived growth factor receptor α and β (PDGFRα and PDGFRβ), podoplanin (PDPN) and fibroblast-specific protein 1 (FSP-1, also named S100A4) (Kawase et al, 2008; Kisselbach et al, 2009; Neri et al, 2015; Kalluri, 2016). However, these markers are not necessarily co-expressed, nor specific to the fibroblast lineage (Costa et al, 2018). For instance, α-SMA not only identifies CAFs with a myofibroblast morphology but also serves as a general marker for myoepithelial cells and perivascular cells. α-SMA+ cells in the breast tumour stroma can also arise from different mesenchymal lineages including resident fibroblasts, smooth muscle cells and pericytes (Ronnov-Jessen et al, 1995). In addition, FSP-1 is also expressed in macrophages, other immune cells and even cancer cells (Osterreicher et al, 2011). Thus, a categorical definition of cancer-associated stromal cells and specific cell surface markers remains challenging and is urgently needed (Kalluri, 2016). Three broad CAF subtypes have been recently profiled in mouse models of pancreatic ductal adenocarcinoma (PDAC) (Ohlund et al, 2017; Biffi et al, 2018; Elyada et al, 2019). These are characterised by a myofibroblast-like (myCAFs) phenotype, inflammatory properties (iCAFs) and antigen-presenting capabilities (apCAFs) (Ohlund et al, 2017; Biffi et al, 2018; Elyada et al, 2019). Although little is known about the mechanistic role and clinical relevance of iCAFs and apCAFs, an accumulation of the myCAF marker α-SMA has been shown to correlate with poor outcome in breast and pancreatic cancer (Yamashita et al, 2012; Sinn et al, 2014). We have shown that targeting Hedgehog-activated CAFs, which have a myofibroblast-like phenotype in ECM regulation, results in markedly improved survival, chemosensitivity and reduced metastatic burden in pre-clinical models of TNBC (Cazet et al, 2018). In addition, myofibroblast-like CAFs have been shown to contribute to an immunosuppressive microenvironment by attracting T-regulatory cells in breast and ovarian cancer (Costa et al, 2018; Givel et al, 2018). While these studies point towards the therapeutic targeting of myofibroblast-like CAFs, genetic ablation of α-SMA+ cells in a mouse model of PDAC resulted in more aggressive tumours and reduced mouse overall survival, indicating complex stromal functionalities across distinct tissue sites (Ozdemir et al, 2015). Recent advances in single-cell RNA sequencing (scRNA-Seq) have overcome some of the technical hurdles in the investigation of cellular heterogeneity amongst complex tissues such as carcinomas. Recent patient studies have dissected the TME in head and neck squamous cell carcinomas and lung tumours, revealing new insights into stromal and immune subsets associated with disease progression (Puram et al, 2017; Lambrechts et al, 2018). Single-cell studies of human breast cancers have been limited to immune cells, while studies in mouse models have revealed four subclasses of CAFs (Bartoschek et al, 2018). Although CAFs from human breast carcinomas have been profiled by flow cytometry and bulk sequencing, comprehensive single-cell profiling has yet to be performed in TNBC patients (Costa et al, 2018). TNBC is an aggressive breast cancer subtype, which is lacking in effective targeted therapeutic options. It is clinically defined by negative status for targetable hormone receptors (oestrogen receptor and progesterone receptor) or HER2 amplification. Studies in mice and humans have demonstrated that TNBC progression can be influenced by stromal cells; however, a comprehensive understanding of the stromal hierarchy is yet to be established (Brechbuhl et al, 2017; Cazet et al, 2018; Costa et al, 2018; Givel et al, 2018; Su et al, 2018). To investigate this in more detail, we performed unbiased high-throughput scRNA-Seq to profile the TME directly in patient tumour tissues. In addition to CAFs, we identified stromal cells with a perivascular-like (PVL) profile, which were not necessarily associated with blood vessels. Our study focuses exclusively on CAFs and PVL cells, which we collectively refer to as “stroma”. Using orthogonal methods, we found that functions previously ascribed to CAFs as unitary cell types are actually performed by specialised subsets of stromal cells with distinct morphological, spatial and functional properties (Bartoschek et al, 2018). In addition, by sampling cells from the entire TME, we were able to predict paracrine signalling between stromal and immune cell subsets. From this, we analysed large patient gene expression datasets to show significant association between inflammatory-like CAFs and differentiated-PVL cells with immune evasion. Our human TNBC single-cell datasets provide a new taxonomy of human cancer-associated stromal cells, which we envisage can be used to further develop TME-directed therapies. Results Composition of triple-negative breast cancers at cellular resolution We performed scRNA-Seq on primary breast tumours collected from five patients (Fig EV1A and B) using a marker-free approach. Fresh tissues were dissociated into single-cell suspensions prior to single-cell capture on the Chromium controller (10× Genomics) and sequencing on the NextSeq 500 (Illumina) (Figs 1A and EV1C). In total, we sequenced 24,271 cells, with an average of 4,854 cells per patient (Fig EV1D). A total of 28,118 genes were detected with an average of 1,658 genes expressed, and 6,215 unique molecular identifiers (UMIs) detected per cell (Fig EV1E–H). Data from individual tumours were integrated and clustered using canonical correlation analysis (CCA) in Seurat (Satija et al, 2015). Click here to expand this figure. Figure EV1. Clinical pathological features and overview of single-cell RNA sequencing metrics A. Clinical and pathological features of patient age, breast cancer subtype, tumour grade, Ki67 status, treatment history and TIL count of the 5 primary breast carcinoma samples analysed in the study. B. Representative haematoxylin–eosin (H&E)-stained sections for each patient analysed by single-cell RNA sequencing in this study. C. Quality control metrics as generated by the Cellranger software (10X Genomics). D. Number of cells that passed quality control and filtering using EmptyDroplets per patient. E. Number of cells that passed quality control and filtering using EmptyDroplets per cell type and patient. F–H. Boxplots showing the number of detected genes (F), UMIs (G) and proportion of mitochondrial counts (H) per cell type across all five tumour samples, respectively. Data points represent individual cells. The central band and boxes correspond with the median and lower/upper quartiles, respectively. Whiskers extend to the lowest or largest value no further than 1.5 × the interquartile range, with points outside this range considered outliers. Download figure Download PowerPoint Figure 1. Cellular composition of five triple-negative breast carcinomas Schematic highlighting the application of our single-cell RNA sequencing experimental and analytical workflow for primary patient tissue. UMAP visualisation of 4,986 epithelial cells aligned using canonical correlation analysis in Seurat. Cells are coloured by their cell type annotation (left) and patient of origin (right). Log-normalised expression of markers for epithelial (EPCAM), mature luminal epithelial (ESR1), myoepithelial (KRT5, KRT14 and ACTA2) and proliferating cancer cells (MKI67). UMAP visualisation of 19,285 stromal and immune cells aligned and visualised as represented in (B). Log-normalised expression of markers for fibroblasts (PDGFRB, THY1, COL1A1, ITGB1 and S100A4), endothelial cells (PECAM1), T cells (CD3D), CD8 T cells (CD8A), T-regulatory cells (FOXP3), B cells (MS4A1), myeloid cells (CD68) and plasma cells (JCHAIN). Proportion of cell types across each patient. Download figure Download PowerPoint Epithelial cells (Fig 1B and C) and stromal-immune cells (Fig 1D and E) were first annotated through the expression of canonical cell type gene markers. This revealed four major cell states within the epithelial compartment (Fig 1B and C), including a major cluster of 4,095 cancer cells (16.9% of all cells; EPCAM+, ESR1−) and a second cluster of 614 cancer cells with high proliferation (2.5%; MKI67+). The remaining two smaller epithelial clusters had gene expression features consistent with normal luminal (277 cells, 0.9%; EPCAM+, ESR1+) and myoepithelial cells (212 cells, 0.9%; EPCAMlo, KRT5+, KRT14+ and ACTA2+). Neoplastic or normal status of these cell clusters was confirmed by inferring genome copy number alterations over large genomic regions using InferCNV (Appendix Fig S1) (Patel et al, 2014). In addition to marker genes, stromal and immune clusters were further classified through scoring against published cell type signatures from the XCell database with an area under the curve approach (AUCell) (Fig EV2A; Aibar et al, 2017; Aran et al, 2017). In the immune compartment (Fig 1D and E), we identified 7,990 T lymphocytes (32.9%; CD3D), 1,245 B cells (5.1%; MS4A1), 1,955 plasma cells (8.1%; JCHAIN) and 4,606 myeloid cells (19.0%