Single-cell RNA-sequencing of migratory breast cancer cells: discovering genes associated with cancer metastasis

Abstract

Considerable evidence suggests breast cancer metastasis arises from cells undergoing epithelial-to-mesenchymal-transition (EMT) and cancer stem-like cells (CSCs). Using a microfluidic device that enriches migratory breast cancer cells with enhanced capacity for tumor formation and metastasis, we identified genes differentially expressed in migratory cells by high-throughput single-cell RNA-sequencing. Migratory cells exhibited overall signatures of EMT and CSCs with variable expression of marker genes, and they retained expression profiles of EMT over time. With single-cell resolution, we discovered intermediate EMT states and distinct epithelial and mesenchymal sub-populations of migratory cells, indicating breast cancer cells can migrate rapidly while retaining an epithelial state. Migratory cells showed differential profiles for regulators of oxidative stress, mitochondrial morphology, and the proteasome, revealing potential vulnerabilities and unexpected consequences of drugs. We also identified novel genes correlated with cell migration and outcomes in breast cancer as potential prognostic biomarkers and therapeutic targets to block migratory cells in metastasis.

Figure 1.. Selection of migratory breast cancer cells for single-cell RNA-Seq.

(a) Workflow of the presented work. Microfluidic migration was performed to isolate migratory cells. The isolated cells were validated functionally and then processed for single-cell RNA-Seq. (b) The different clonal morphologies of migratory and wild-type cells, including MDA-MB-231, SUM159, and SUM149. The clones (day 3) derived from wild-type cells are connected, while the ones from migratory cells are disseminated. (scale bar: 50 μm) (c-e) Microfluidically sorted migratory cell populations have distinct migratory behaviors as compared to wild-type ones. Graphs show positions of individual cells (after migration of 24 hours) and box plot and whiskers summaries for migration of (c) MDA-MB-231, (d) SUM159 and (e) SUM149 cells toward 10% fetal bovine serum (FBS) media. (n = 600 channels). *** refers to P < 0.001.

Figure 2.. tSNE clustering demonstrates the distinct gene expression profiles of migratory and wild-type breast cancer cells.

tSNE plot of single-cell transcriptome analysis for migratory (red color) and wild-type (blue color) cells of four breast cancer cell lines: (a) SUM149, (b) SUM159, (c) MDA-MB-231, and (d) GUM36. X-axis represents tSNE1, and Y-axis represents tSNE2. Each dot represents a cell. Two populations of the same cell line are clearly separated, indicating distinct gene expression profiles.

Figure 3.. Migratory breast cancer cells express more mesenchymal/EMT related genes and less epithelial related genes than wild-type ones.

(a) Dot plot shows the expression of 13 genes among 8 cell populations (migratory and wild-type of 4 cell lines). Larger dot means higher percentage of single cells expressing that gene, and smaller dot means lower percentage of single cells expressing that gene. Grey color represents the lowest expression, and red color represents the highest expression. The expression is logarithmically normalized. (b, c) Gene expression and clustering of migratory and wild-type SUM149 cells. X-axis represents tSNE1, and Y-axis represents tSNE2. Each dot represents one cell. Red color represents high (90th percentile) expression of a gene, and grey color represents low (10th percentile) expression of a gene. The expression is logarithmically normalized. (b) The expression of epithelial genes: keratin 18 (KRT18), keratin 8 (KRT8), keratin 7 (KRT7), epithelial cell adhesion molecule (EpCAM), and cadherin 1 (CDH1). (c) The expression of mesenchymal and EMT genes: cadherin 2 (CDH2), vimentin (VIM), fibronectin 1 (FN1), integrin beta 1/CD29 (ITGB1), hypoxia inducible factor 1 alpha (HIF1A), transcription factor SOX-4 (SOX4), snail transcriptional repressor 1 (SNAI1), snail transcriptional repressor 2 (SNAI2), Zinc finger E-box-binding homeobox 1 (ZEB1), and Zinc finger E-box-binding homeobox 2 (ZEB2). Migratory SUM149 cells express more mesenchymal and EMT genes as compared to wild-type ones yet exhibit significant cellular heterogeneity.

Figure 4.. Migratory breast cancer cells express more CSC related genes than wild-type ones.

(a) Dot plot shows the expression of 13 genes among 8 cell populations (migratory and wild-type of 4 cell lines). Larger dot means higher percentage of single cells expressing that gene, and smaller dot means lower percentage of single cells expressing that gene. Grey color represents the lowest expression, and red color represents the highest expression. The expression is logarithmically normalized. (b-d) Gene expression and clustering of migratory and wild-type SUM149 cells. X-axis represents tSNE1, and Y-axis represents tSNE2. Each dot represents one cell. Red color represents high (90th percentile) expression of a gene, and grey color represents low (10th percentile) expression of a gene. The expression is logarithmically normalized. (b) The expression of MET-CSC genes: pan-aldehyde dehydrogenase isoforms (Pan-ALDH), aldehyde dehydrogenase 1 family member A3 (ALDH1A3), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). (c) The expression of MET-CSC genes: CD44 and CD24. (d) The expression of stem-cell related genes: CD29/ITGB1, polycomb complex protein BMI-1 (BMI1), thymus cell antigen 1 (THY1), ATP-binding cassette super-family G member 2 (ABCG2/CD338), Wnt family member 5A (WNT5A), pan-NOTCH isoforms (Pan-NOTCH), NOTCH homolog 2 (NOTCH2), transcription factor SOX-9 (SOX9), GATA binding protein 3 (GATA3), and epidermal growth factor receptor (EGFR). Overall, migratory SUM149 cells express more stemness related genes than wild-type ones.

Figure 5.. tSNE clustering demonstrates the cellular heterogeneity of migratory SUM149 cells.

(a) tSNE plot of single-cell transcriptome analysis suggests two populations among migratory SUM149 cells: mesenchymal-like (red color) and epithelial-like (blue color) cells. X-axis represents tSNE1, and Y-axis represents tSNE2. Each dot represents a cell. Two populations are clearly separated, indicating distinct gene expression profiles. (b) EMT-like CSCs (CD44 high), MET-like CSCs (ALDH1A3 high), and dual positive CSCs (both CD44 and ALDH1A3 high) on the tSNE plot. epithelial-like CSCs are generally distributed in the epithelial cluster, and mesenchymal-like CSCs are distributed in the mesenchymal cluster. (c, d) Gene expression and clustering of migratory SUM149 cells. Each dot represents one cell. Red color represents high (90th percentile) expression of a gene, and grey color represents low (10th percentile) expression of a gene. The expression is logarithmically normalized. (c) The expression of epithelial/MET-CSC genes: Pan-ALDH, ALDH1A1, ALDH1A3, CDH1, EPCAM, KRT7, KRT8, KRT18. (d) The expression of mesenchymal/EMT-CSC genes: CD44, CD24, VIM, CDH2, FN1, ITGB1, HIF1A, Enhancer of Zeste Homolog 2 (EZH2), SNAI1, SNAI2, ZEB1, and ZEB2.

Figure 6.. Sequencing of migratory breast cancer cells identifies new markers for prognosis prediction, treatment guidance, and mechanistic studies.

(a) Dot plot shows the expression of 10 genes among 8 cell populations (migratory and wild-type of 4 cell lines). Larger dot means higher percentage of single cells expressing that gene, and smaller dot means lower percentage of single cells expressing that gene. Grey color represents the lowest expression, and red color represents the highest expression. The expression is logarithmically normalized. Migratory breast cancer cells express more listed genes as compared to wild-type ones. (b-e) The Kaplan-Meier plot shows that high levels of (b) Anillin Actin Binding Protein (ANLN), (c) Small Nuclear RNA Activating Complex Polypeptide 1 (SNAPC1), (d) Poly(A) Binding Protein Cytoplasmic 3 (PABPC3), and (e) Centromere Protein F (CENPF) expression correlate with reduced relapse-free survival (RFS) in breast cancer.