SUPPLEMENTARY INFORMATION

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1 doi: /nature15260 Supplementary Data 1: Gene expression in individual basal/stem, luminal, and luminal progenitor cells. Box plots show expression levels for each gene from the 49-gene differentiation signature in individual cells. P-values and fold change for each gene are shown in Supplementary Table 2. Black dots, single cells; red dots, single cells with no expression. B, basal; LP, luminal progenitor; L, luminal. Supplementary Data 2: Full heatmaps showing unsupervised hierarchical clustering 1

2 2 HCI-001 KIT NOTCH3 GATA3 CD10 NOTCH4 SNAI2 CDK1 BCL2 TGFB2 AR CAV1 BCL2L1 SKP2 LGR5 CCNE1 KRT5 TP63 COL1A2 JAG1 ERBB3 KRT19 ITGB1 CD24 CLDN4 CAV2 EMP1 CDH3 CDH1 PARP2 CCNB1 PROM1 ERBB2 MYCN ESR2 MUC1 PTEN ITGA6 MYLK PGR PLCB4 TP53 Basal/stemlike ( Low enriched) Luminal-like ( High enriched) Intermediate

3 3 SUPPLEMENTARY INFORMATION HCI-002 KIT CCNB1 MUC1 EMP1 CD24 CAV2 ITGB1 CDH1 GATA3 KRT19 PTEN PROM1 CDK1 TP63 ERBB2 PARP2 ERBB3 CLDN4 ERBB1 TP53 CCNE1 MYLK ITGA6 CDH3 COL1A2 NOTCH3 JAG1 PLCB4 ESR2 KRT5 SNAI2 CAV1 AR BCL2L1 SKP2 NOTCH4 BCL2 LGR5 TGFB2 PGR MYCN CD10 Basal/stem-like ( Low enriched) Luminal-like ( High enriched)

4 4 HCI-010 KIT CCNB1 MUC1 EMP1 CD24 CAV2 ITGB1 CDH1 GATA3 KRT19 PTEN PROM1 CDK1 TP63 ERBB2 PARP2 ERBB3 CLDN4 ERBB1 TP53 CCNE1 MYLK ITGA6 CDH3 COL1A2 NOTCH3 JAG1 PLCB4 ESR2 KRT5 SNAI2 CAV1 AR BCL2L1 SKP2 NOTCH4 BCL2 LGR5 TGFB2 PGR MYCN CD10 Basal/stem-like ( Low enriched) Luminal-like ( High enriched) Intermediate

5 HCI #453 (Resected animal) Red = 10.4% Red = 4.2% Luminal-like mets, post-resection (red) = 85.4% Basal/stem-like ( Low enriched) Intermediate Luminal-like ( High enriched) KIT CCNB1 MUC1 EMP1 CD24 CAV2 ITGB1 CDH1 GATA3 KRT19 PTEN PROM1 CDK1 TP63 ERBB2 PARP2 ERBB3 CLDN4 ERBB1 TP53 CCNE1 MYLK ITGA6 CDH3 COL1A2 NOTCH3 JAG1 PLCB4 ESR2 KRT5 SNAI2 CAV1 AR BCL2L1 SKP2 NOTCH4 BCL2 LGR5 TGFB2 PGR MYCN CD10 Clustering by PDX model PDX model HCI-001 HCI-002 HCI-010 CLDN7 CD24 CD44 PARP2 BRCA1 PTEN CD10 THY1 TGFB2 Supplementary Data 2: Full heatmaps showing unsupervised hierarchical clustering analyses shown in Extended Data Fig. 4 Supplementary Data 3: Gene expression in individual lung metastatic cells from each of the 5

6 SUPPLEMENTARY INFORMATION Supplementary Data 3: Gene expression in individual lung metastatic cells from each of the three PDX models (HCI-001, HCI-002, and HCI-010). Box plots show expression levels for each of the 53 genes differentially expressed between the models by Anova. Black dots, single cells; red dots, single cells with no expression. 6

7 DNA damage response and repair Chromatin modification and gene regulation Development and differentiation *NS *NS *NS Cell cycle Other Supplementary Data 4: Gene ontology (GO) enrichment analysis reveals several biological processes are differentially regulated in low-burden metastatic cells. GO enrichment analysis was performed to identify pathways that were more represented in the set of significantly differentially expressed genes than would be expected by chance alone. Pathways involving DNA damage response and repair, chromatic modification and gene regulation, development and differentiation, and cell cycle were identified. p-values are shown at the right of each bar. *NS, not significant. 7

8 SUPPLEMENTARY INFORMATION Supplementary Data 5: Gene expression in individual cells from tissues with low, intermediate, or high metastatic burden. Box plots show expression levels for each differentially expressed gene (p<0.05) in individual cells. P-values and fold change for each gene are shown in Extended Data Table 2. Black dots, single cells; red dots, single cells with no expression. 8

9 Supplementary Data 6: Kaplan-Meier analysis shows the prognostic relevance of 16 genes characteristic of low-burden metastatic cells. The prognostic value of each of the 55 genes characteristic of low-burden metastatic cells (Extended Data Table 2) was determined by Kaplan-Meier analysis using Km-plotter online software. 29 Km plots are shown as probability of distant metastasis-free survival (DMFS). 16 of 55 genes were found to be prognostic: High ACTA2, CHEK1, EPHA4, MYCN, NOTCH3, NOTCH4, SKP2, TGFB1, TGFB2, THY1 and TWIST1 expression was associated with increased risk of distant metastasis, and increased CDH1, ERBB3, MUC1 and PTEN expression was associated with reduced risk of metastasis. 9

10 SUPPLEMENTARY INFORMATION Supplementary Data 7: Gene expression in individual primary tumor cells and metastatic cells from the lung, lymph node, peripheral blood, bone marrow, and brain Supplementary Data 7: Gene expression in individual primary tumor and metastatic cells from the lung, lymph node, peripheral blood, bone marrow, and brain. Box plots show all genes (80) that were significantly differentially expressed (p<0.05, ANOVA) between at least one pair of tissues. The p-value and fold change for each gene and tissue pair and are shown in Supplementary Table 3. Each black dot represents expression in a single cell, and red dots represent non-expressing cells. LU, lung; LN, lymph node; BM, bone marrow; PB, peripheral blood (CTC); BR, brain. 11 Supplementary Data 8: Histogram plots show the distribution of cells yielded following tissue dissociation of lungs and lymph nodes from PDX animals. For consistency, any tissue digests which yielded an abnormal number of cells (>1 standard deviation from the mean) was excluded from the study. This allowed for comparison of the number of metastatic cells in different animals with reasonable consistency. 1 0 W W W. N A T U R E. C O M / N A T U R E

11 Number of animals Lung x = 6.6x10 6-1σ +1σ Number of animals σ Lymph node x = 1.7x σ Total cell number (x10 6 ) Total cell number (x10 6 ) Supplementary Data 8: Histogram plots show the distribution of cells yielded following tissue dissociation of lungs and lymph nodes from PDX animals. For consistency, any tissue digests which yielded an abnormal number of cells (>1 standard deviation from the mean) was excluded from the study. This allowed for comparison of the number of metastatic cells in different animals with reasonable consistency. 11