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Showing content from https://pubmed.ncbi.nlm.nih.gov/28474673/ below:

Single-cell RNA-seq enables comprehensive tumour and immune cell profiling in primary breast cancer

doi: 10.1038/ncomms15081. Single-cell RNA-seq enables comprehensive tumour and immune cell profiling in primary breast cancer Woosung Chung^{1 2}, Hye Hyeon Eum^{1 3}, Hae-Ock Lee^{1 4}, Kyung-Min Lee^{5 6}, Han-Byoel Lee^{5 7}, Kyu-Tae Kim¹, Han Suk Ryu⁸, Sangmin Kim⁹, Jeong Eon Lee⁹, Yeon Hee Park¹⁰, Zhengyan Kan¹¹, Wonshik Han^{5 7}, Woong-Yang Park^{1 2 4}

Affiliations

¹ Samsung Genome Institute, Samsung Medical Center, Seoul 06351, Korea.
² Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences &Technology, Sungkyunkwan University, Seoul 06351, Korea.
³ Department of Biomedical Sciences, Seoul National University Graduate School, Seoul 03080, Korea.
⁴ Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, Korea.
⁵ Department of Surgery and Cancer Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea.
⁶ Biomedical Research Institute, Seoul National University Hospital, Seoul 03080, Korea.
⁷ Department of Surgery, Seoul National University College of Medicine, Seoul 03080, Korea.
⁸ Department of Pathology, Seoul National University College of Medicine, Seoul 03080, South Korea.
⁹ Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Korea.
¹⁰ Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Seoul 06351, Korea.
¹¹ Oncology Research, Pfizer Inc., San Diego, California 92121, USA.

PMID: 28474673
PMCID: PMC5424158
DOI: 10.1038/ncomms15081

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Single-cell RNA-seq enables comprehensive tumour and immune cell profiling in primary breast cancer

Woosung Chung et al. Nat Commun. 2017.

doi: 10.1038/ncomms15081. Authors Woosung Chung^{1 2}, Hye Hyeon Eum^{1 3}, Hae-Ock Lee^{1 4}, Kyung-Min Lee^{5 6}, Han-Byoel Lee^{5 7}, Kyu-Tae Kim¹, Han Suk Ryu⁸, Sangmin Kim⁹, Jeong Eon Lee⁹, Yeon Hee Park¹⁰, Zhengyan Kan¹¹, Wonshik Han^{5 7}, Woong-Yang Park^{1 2 4} Affiliations

¹ Samsung Genome Institute, Samsung Medical Center, Seoul 06351, Korea.
² Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences &Technology, Sungkyunkwan University, Seoul 06351, Korea.
³ Department of Biomedical Sciences, Seoul National University Graduate School, Seoul 03080, Korea.
⁴ Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 16419, Korea.
⁵ Department of Surgery and Cancer Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea.
⁶ Biomedical Research Institute, Seoul National University Hospital, Seoul 03080, Korea.
⁷ Department of Surgery, Seoul National University College of Medicine, Seoul 03080, Korea.
⁸ Department of Pathology, Seoul National University College of Medicine, Seoul 03080, South Korea.
⁹ Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Korea.
¹⁰ Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Seoul 06351, Korea.
¹¹ Oncology Research, Pfizer Inc., San Diego, California 92121, USA.

PMID: 28474673
PMCID: PMC5424158
DOI: 10.1038/ncomms15081

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Abstract

Single-cell transcriptome profiling of tumour tissue isolates allows the characterization of heterogeneous tumour cells along with neighbouring stromal and immune cells. Here we adopt this powerful approach to breast cancer and analyse 515 cells from 11 patients. Inferred copy number variations from the single-cell RNA-seq data separate carcinoma cells from non-cancer cells. At a single-cell resolution, carcinoma cells display common signatures within the tumour as well as intratumoral heterogeneity regarding breast cancer subtype and crucial cancer-related pathways. Most of the non-cancer cells are immune cells, with three distinct clusters of T lymphocytes, B lymphocytes and macrophages. T lymphocytes and macrophages both display immunosuppressive characteristics: T cells with a regulatory or an exhausted phenotype and macrophages with an M2 phenotype. These results illustrate that the breast cancer transcriptome has a wide range of intratumoral heterogeneity, which is shaped by the tumour cells and immune cells in the surrounding microenvironment.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures
Figure 1. Intratumoral heterogeneity in primary breast…

Figure 1. Intratumoral heterogeneity in primary breast tumours.

( a ) Unsupervised PCA on the…

Figure 1. Intratumoral heterogeneity in primary breast tumours.

(a) Unsupervised PCA on the transcriptome, indicating a mixed distribution of intra- and interpatient cells. Individual cells are coloured yellow for luminal A, green for luminal B, blue for HER2, and red for TNBC tumours. This colour scheme is maintained throughout the manuscript. (b) Individual cells exhibiting gene expression heterogeneity for ER (ESR1), PR (PGR) and HER2 (ERBB2). The overall single-cell expression profiles agree with the bulk tumour expression profiles and the pathology results. (c) Haematoxylin and eosin staining on formalin-fixed paraffin-embedded slides. Microscopic findings indicated carcinoma and non-carcinoma cells, including tumour-infiltrating lymphocytes (TIL, 1–60%). Most of the TNBC tumours except BC10 were heavily infiltrated with lymphocytes, whereas luminal A tumours showed enrichment with carcinoma cells. Scale bar, 100 μm. (d) A part of the tumour tissue in c is magnified to show non-neoplastic cellular components as a representative. Scale bar, 25 μm.

Figure 2. Separation of carcinoma and non-carcinoma…

Figure 2. Separation of carcinoma and non-carcinoma cells.

( a ) Scheme of cell classification.…

Figure 2. Separation of carcinoma and non-carcinoma cells.

(a) Scheme of cell classification. (b) Hierarchical clustering of the chromosomal gene expression pattern separating the patient-specific carcinoma cell groups from the non-carcinoma cell cluster. Each row represents single cells and matched bulk tumours (triangle): the tumour groups are colour-coded as in Fig. 1a. For each chromosome, the chromosomal gene expression pattern was estimated from the moving average of 150 genes. These patterns implicate chromosomal amplification and deletion. (c) Unsupervised PCA showing the separation of carcinoma and non-carcinoma cell groups. (d) Carcinoma cells identified in a, scored low for stromal and immune signatures, whereas non-carcinoma cells scored high for immune signatures. Tumour score was inferred from the stromal and immune signature using ESTIMATE algorithm. Normal tissues represent 183 mammary tissue data from GTEx portal ( http://www.gtexportal.org/ ). Each box shows the median and interquartile range (IQR 25th–75th percentiles), whiskers indicate the highest and lowest value within 1.5 times the IQR and outliers are marked as dots. P value, Student's t-test (***P<0.001). (e) Representative gene expression in single cells for the immune (PTPRC, LAPTM5 and IL2RG), stromal (HTRA1, FBN1 and FAP) and epithelial (KRT19, CDH1 and EPCAM) cell types.

Figure 3. Removal of non-carcinoma cells reveals…

Figure 3. Removal of non-carcinoma cells reveals intrinsic tumour cell heterogeneity.

( a ) Centred…

Figure 3. Removal of non-carcinoma cells reveals intrinsic tumour cell heterogeneity.

(a) Centred correlation matrix for all single cells demonstrates low cell-to-cell correlations (Pearson's r) in tumours with lymph node metastases (left). After separation of carcinoma and non-carcinoma cells, cell-to-cell correlations within the same tumour group are increased (right). Each row and column represents single cells. In the colour panel on the far left side, grey represents tumour and light blue represents non-tumour cells. (b) Intratumoral correlations before (white boxes) and after (striped boxes) the removal of non-carcinoma cells (left). Each box shows the median and interquartile range (IQR 25th–75th percentiles), whiskers indicate the highest and lowest value within 1.5 times the IQR and outliers are marked as dots. Samples were ranked by mean value of cell-to-cell Pearson's correlation coefficient (right). (c) Unsupervised PCA on the transcriptome separating patient-specific tumour groups for only tumour cells.

Figure 4. Intrinsic tumour cell heterogeneity.

(…

Figure 4. Intrinsic tumour cell heterogeneity.

( a ) Subtype prediction of tumour cells as…

Figure 4. Intrinsic tumour cell heterogeneity.

(a) Subtype prediction of tumour cells as ER+, HER2+ or TNBC types using ER and HER2 module scores (R software package genefu, top left panel). Heatmap of subtype-specifying module scores (top right) and pie charts (bottom) demonstrating Intratumoral subtype heterogeneity in HER2 tumours. (b) Pearson's correlation coefficient (r) between aggressive cancer gene expression signatures (EMT, stemness and angiogenesis). Horizontal and vertical dashed lines indicate signature score cut-offs at top 5%. The linear regression result is drawn as a solid line. The coloured dots mark cells with high expression signatures for both axis.

Figure 5. Subtype-specific gene expression profiling at…

Figure 5. Subtype-specific gene expression profiling at single-cell resolution.

( a ) LRT based on…

Figure 5. Subtype-specific gene expression profiling at single-cell resolution.

(a) LRT based on zero-inflated data and heatmap analysis (upper panels) identifying differentially expressed genes among different subtype tumours at a single-cell level (left) or in bulk (right). GSVA analysis with subtype-related pathways also indicates differential activation of pathways at a single-cell level (lower panel). (b) HER2/HER3 downstream signalling pathways (PI3K/AKT, NF-kB and RAS/MEK/MAPK) are highly activated in BC04 HER2+ tumour cells. Expression of PI3K and NF-kB pathway genes was upregulated in the lymph node metastasis for BC03 ER+HER2+ tumour cells. Pathway activation was determined by the GSVA enrichment score. (c) TNBCtype analysis was applied only to TNBC tumours (BC07–BC11), which characterized individual cells as one or more of six different subtypes. Mixed subtype composition within a tumour indicates intratumoral heterogeneity comparable to intratumoral heterogeneity.

Figure 6. Identification of immune cell populations…

Figure 6. Identification of immune cell populations in the tumour microenvironment.

( a ) Immune…

Figure 6. Identification of immune cell populations in the tumour microenvironment.

(a) Immune cell clusters were characterized by gene ontology terms. The cluster-specific genes extracted by LRT test (left) were associated with B cells, T cells or macrophages (MØ), respectively (right). (b) Immunofluorescence staining (IF) for CD3 or CD20 showing the infiltration of T or B lymphocytes in tumour tissues. Scale bar, 20 μm. (c) Immunofluorescence staining results show significant correlations with gene expression in bulk tumour samples (Pearson's r, 0.66 for CD3 and 0.67 for CD20, P<0.05). The linear regression result is drawn as a dashed line. Number of captured single cells is marked with a colour key.

Figure 7. T-cell signatures in the tumour…

Figure 7. T-cell signatures in the tumour microenvironment.

Hierarchical clustering was performed using GSVA enrichment…

Figure 7. T-cell signatures in the tumour microenvironment.

Hierarchical clustering was performed using GSVA enrichment scores for gene sets for naive T cells, T-cell costimulation, regulatory cytokines and receptors, T-cell exhaustion and cytotoxicity (upper panel). Specific genes from used gene sets are presented in the lower panel.