Circulating tumour cells in women with advanced oestrogen-receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer acquire a HER2-positive subpopulation after multiple courses of therapy1,2. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here we analyse circulating tumour cells from 19 women with ER+/HER2− primary tumours, 84% of whom had acquired circulating tumour cells expressing HER2. Cultured circulating tumour cells maintain discrete HER2+ and HER2− subpopulations: HER2+ circulating tumour cells are more proliferative but not addicted to HER2, consistent with activation of multiple signalling pathways; HER2− circulating tumour cells show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2+ and HER2− circulating tumour cells interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. Although HER2+ and HER2− circulating tumour cells have comparable tumour initiating potential, differential proliferation favours the HER2+ state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2− phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic circulating tumour cell-derived tumour models. Together, these results point to distinct yet interconverting phenotypes within patient-derived circulating tumour cells, contributing to progression of breast cancer and acquisition of drug resistance.
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Gene Expression Omnibus
Single-cell RNA-seq data have been deposited in the Gene Expression Omnibus under accession number GSE75367. Mass spectrometry raw data have been deposited in the MassIVE proteomics data repository under accession number MSV000079419 (https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp).
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We thank the patients who participated in this study. This work was supported by National Institutes of Health (NIH) 2RO1CA129933, the Howard Hughes Medical Institute, the Breast Cancer Research Foundation, the National Foundation for Cancer Research (DAH) and Wellcome Trust 102696 (C.B.), NIH Quantum 2U01EB012493 (M.T., D.A.H.), T32 CA009361, Susan G. Komen Foundation PDF16376429 (N.V.J.), K12 5K12CA087723 (A.B.) and T32GM007753 (R.Y.E.). We thank D. Dombrowski (NIH 1S100D1016372-01) for expert flow cytometry.
The authors declare no competing financial interests.
Nature thanks J. P. Medema and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 Patients with advanced ER+/HER2− breast cancer harbour discrete HER2+ and HER2− subpopulations.
a, CTCs freshly isolated from 19 patients with ER+/HER2− breast cancer were stained with HER2 (green) and EpCAM (yellow) and imaged using imaging flow cytometry. Bar graph shows the number of HER2+ (black) and HER2− (white) CTCs (median 22% HER2+ CTCs, range 4–58%). Supplementary Table 1 provides HER2+/HER2− ratios and each patient’s clinical history. b, scRNA-seq for ERBB2 expression at multiple time-points showing acquisition of HER2+ CTCs (Brx-82, Brx-42) over the course of progressive disease. Single asterisk (*) denotes patient expiration. Rx, sacituzumab (IMMU-132); Rx1, vinorelbine + trastuzumab; Rx2, eribulin. c, Distinct HER2+ and HER2− CTCs from 13 patients with triple-negative breast cancer (TNBC) determined by scRNA-seq (HER2− ≤ 0 RPM; HER2+ > 153, range 33–463). d, HER2 fluorescence in situ hybridization (FISH) analysis of metastatic tumours from patients, Brx-42, Brx-82 and Brx-142, shows no amplification of ERBB2 compared with HER2-amplified control (Supplementary Table 1 for tumour source data). HER2 (red); chromosome enumeration probe 17 (CEP17) (cyan); scale bar, 10 μm. Representative images from five independent fields are shown. e, Bright field and immunofluorescence (DAPI, blue; HER2, green) images of CTC lines, Brx-42, Brx-82 and Brx-142, demonstrate heterogeneity in HER2 expression. Scale bar, 100 μm (bright field); 20 μm (immunofluorescence). Representative images from three independent fields are shown. f, FACS analysis shows two distinct HER2+ and HER2− subpopulations in the CTC line Brx-42 (at initiation) compared with HER2− control. Representative data of two independent experiments are shown. g, HER2 FISH analysis of the HER2+ and HER2− subpopulations from CTC lines Brx-42, Brx-82 and Brx-142 shows that ERBB2 is not amplified. HER2-amplified SKBR3 cells shown as control. HER2 (red); CEP17 (green); scale bar, 10 μm. Representative images from five independent fields are shown.
a, Increased expression of the proliferation marker Ki67 (red) in the HER2+ subpopulation of CTC line Brx-142 (t-test, P < 0.0001), compared with the HER2− subpopulation, with no change in cleaved-caspase 3 (red). HER2+ cells (green); scale bar, 20 μm. Representative images from five independent fields are shown. b, FACS analysis for the apoptotic marker Annexin V-FITC shows no difference in apoptosis between the HER2+ and HER2− subpopulations of FACS-purified CTC line Brx-142. Representative data from two independent experiments are shown. c, Tumours initiated by HER2+ or HER2− CTCs (Brx-82: 200,000 cells) orthotopically injected into the mammary fat pad show differential growth rates; n = 8. d, Metastatic frequency of HER2+ and HER2− cultured CTCs (Brx-82: P = 0.05; Brx-142: P = 0.009) following orthotopic injection; n = 8. e, Limiting dilution experiments demonstrate comparable tumour initiating ability from 200 HER2+ and HER2− cultured CTCs (Brx-82, Brx-142); n = 8.
a, FACS-purified HER2+ and HER2− subpopulations from CTC line Brx-82 were monitored over 28 days to determine shifts in the composition of sorted populations. Representative data of two independent experiments are shown. b, Growth curves for HER2+ (red) and HER2− (blue) FACS-purified single cell clones from CTC line Brx-142; two-way ANOVA, P < 0.0001; n = 20. c, IHC HER2 staining of tumour xenografts derived from unlabelled HER2− and HER2+ CTCs showing acquisition/loss of HER2 (brown), respectively. Arrows indicate regions of HER2 acquisition/loss. Representative image from at least five independent fields; n = 8. ER+/HER2− and HER2-amplified breast cancers are shown below as controls. d, Low-magnification (landscape) view of HER2 IHC staining of tumour xenografts derived from mixed HER2+ and HER2− CTC cultures containing either GFP-tagged HER2+/HER2− cells (high magnification images are shown in Fig. 2f). Top: representative GFP-tagged HER2− cells give rise to GFP+/HER2+ cells (GFP: cytoplasmic red stain, HER2: cell surface brown stain). Bottom: GFP-tagged HER2+ cells produce GFP+/HER2− cells. Scale bar, 100 μm.
a, b, MS-based whole cell proteome profiles (6,349 proteins) comparing HER2+ and HER2− populations from CTC lines (Brx-42, Brx-82, Brx-142). Matched HER2+ versus HER2− proteomic differences show significant linear correlation (Pearson correlation coefficient = 0.71 between Brx-82 and Brx-42; Pearson correlation coefficient = 0.64 between Brx-142 and Brx-42); NI, normalized intensity; n = 2 per cell line are shown. c, Phospho-RTK array of HER2+ and HER2− populations of CTC cell lines Brx-142 and Brx-82 show increased phosphorylation of RTKs in the HER2+ population. Numbers denote the following: 1, HER2; 2, HER3; 3, HER4; 4, INSR; 5, EPHA1; 6, EPHA2; 7, EPHA10. Representative data from two independent experiments are shown. d, Volcano plot depicts genes enriched in HER2+ (red) and HER2− (blue) individual CTCs isolated from patients Brx-42 and Brx-82 and analysed by scRNA-seq; n = 22. e, Venn diagram showing PID pathway overlap of genes and proteins derived from scRNA-seq (Brx-42, Brx-82) and quantitative proteomics of HER2+ CTCs, respectively.
Extended Data Figure 5 Fifty-five panel drug screen shows differential drug sensitivities exhibited by HER2+ versus HER2− subpopulations.
a, Heat map showing percentage cell viability (represented as decimal) after 6 days of drug treatment of the HER2+ and HER2− subpopulations derived from CTC lines Brx-142 and Brx-82. Red and blue represent high and low drug sensitivities, respectively; n = 6. b, Lapatinib sensitivity of HER2+ (red) and HER2− (blue) subpopulations of CTC line Brx-82. MDA-231 (TNBC) and SKBR3 (HER2-amplified) are shown as controls. c, Chemosensitivity of HER2+ (red) and HER2− (blue) subpopulations of CTC line Brx-142. MDA-231 (blue) and SKBR3 (red) are shown as controls. d, Sensitivity of HER2+ (red) and HER2− (blue) subpopulations of CTC line Brx-142 to Notch inhibition with Notchi1 (BMS-708163) and Notchi2 (RO4929097). MDA-231 and SKBR3 cells are shown as controls. a–d, Representative of at least two independent experiments for each condition; n = 6.
a, Western blot analysis of HER2+ and HER2− subpopulations from CTC lines Brx-142 and Brx-82 show increased NOTCH1 in HER2− cells. β-Actin is shown as control. Immunofluorescence analysis and scRNA-seq of NOTCH1 (red) and HER2 (green) shows inversely correlated expression in CTC lines (Brx-142, Brx-82). b, Ectopic expression of constitutively active Notch intracellular domain (ICD) or NRF2 results in increased expression of the Notch1 ligand JAG1 but does not alter HER2 expression. Representative data of two independent experiments are shown; s.e.m. (error bars). c, siRNA-mediated inhibition of HER2 in Brx-42 HER2+ CTCs, and lapatinib-mediated inhibition of HER2 in SKBR3 cells results in dose-dependent increases in the expression of genes involved in Notch signalling (NOTCH1, JAG1, DLL1, HES1, HEY1, HEY2). Representative data of two independent experiments are shown; s.e.m. (error bars). d, Inhibition of HER2 using lapatinib or siRNA knockdown in Brx-82 HER2+ CTCs increases the expression of NRF2-driven cytoprotective genes downstream of the Notch pathway. Representative data of two independent experiments are shown; s.e.m. (error bars). e, Quantitation of the interconversion of HER2+ cells from single-cell clones into 5- to 9-cell and >10-cell clusters following treatment with 10mM H2O2; t-test, P < 0.05; n = 10. f, Paclitaxel treatment of mice with tumours derived from Brx-142 FACS-purified HER2+ CTCs, demonstrating a reduction in CTCs, and HER2− CTCs with no change in counts; t-test P < 0.05; NS, not significant. g, Paclitaxel treatment of mice with mammary xenografts derived from parental CTC line Brx-142 showing initial tumour response, followed by recurrent tumour growth. IHC analysis and quantitation of the recurrent tumour shows greatly reduced HER2+ (brown stain) cell composition in the Paclitaxel drug treated (T, 3 weeks post-treatment) tumour compared with the untreated tumour U, and the recovered tumour (R, 5 weeks post-treatment). Bar indicates duration of drug treatment (Rx). Scale bar, 100 μm; two-way ANOVA, P < 0.0001; n = 6. Representative images from five independent fields per tumour are shown and quantified; t-test, P < 0.001. h, Dual GFP (red, cytoplasmic stain) and HER2 (brown, cell surface stain) IHC of tumour xenografts derived from mixed GFP-tagged HER2+ and untagged HER2− CTC cultures demonstrating enhanced conversion from GFP+/HER2+ to GFP+/HER2− after 4 weeks of paclitaxel treatment; t-test, P < 0.0001; n = 6. Scale bar, 100 μm. Arrows indicate interconverting cells. Representative images from five independent fields per tumour are shown. i, Mouse tumour xenografts derived from the CTC line Brx-142 treated with a combination of the Notchi3 (LY-414575) and paclitaxel shows diminished tumour relapse; n = 6. Bar indicates treatment duration.
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Jordan, N., Bardia, A., Wittner, B. et al. HER2 expression identifies dynamic functional states within circulating breast cancer cells. Nature 537, 102–106 (2016). https://doi.org/10.1038/nature19328
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