Supplementary MaterialsTable S1. CTCs (Turquoise, Brown) and Xenograft-Derived CTC Clusters (Green, Yellow), Related to Physique?4 mmc5.xlsx (61K) GUID:?5F1C065B-0597-4DB3-8DCA-D53E55110C48 Table S6. FDA-Approved Compounds Used in the Screen, Related to Physique?5 mmc6.xlsx (92K) GUID:?A87AA08F-30D6-4371-A778-54F1CFD71CA5 Table S7. Stemness-Related Genes Up- or Downregulated in BR16 and BRx50 as a Result of a 17-Day Treatment with 20? nM of Digitoxin or Ouabain, Related to Physique?6 mmc7.xlsx (78K) GUID:?14F590F1-E02E-4E17-8ABB-75E257D8627F Summary The ability of circulating tumor cells (CTCs) to form clusters has been linked to increased metastatic potential. Yet biological features and vulnerabilities of CTC clusters remain largely unknown. Here, A 803467 we profile the DNA methylation scenery of single CTCs and CTC clusters from breast cancer patients and mouse models on a genome-wide scale. We find that binding sites for stemness- and proliferation-associated transcription factors are specifically hypomethylated in CTC clusters, including binding sites for OCT4, NANOG, SOX2, and SIN3A, paralleling embryonic stem cell biology. Among 2,486 FDA-approved compounds, we identify Na+/K+ ATPase inhibitors that enable the dissociation of CTC clusters into single cells, leading to DNA methylation remodeling at crucial sites and metastasis suppression. Thus, our results link CTC clustering to specific changes in DNA methylation that promote stemness and metastasis and point to cluster-targeting compounds to suppress the spread of cancer. line) and single CTCs (line). TSS: Transcription Start Site; TES: Transcription End Site. (JCL) Hypergeometric gene set enrichment analysis of promoters (J), gene bodies (K) and super-enhancers (L) displaying 20% methylation difference (p?value? 0.01) in xenograft-derived CTC clusters compared to single CTCs. Gene sets with adjusted p value 0.05 are shown for promoters (J) and gene bodies (K). For super-enhancers (L), the top-20 significant gene sets with adjusted p value 0.05 are shown. Gene sets related to PRC2 activity are highlighted in red. (M) Histogram showing mapped reads in patient CTCs corresponding to a methylated cytosine (C) (red) or a thymine (T) (blue; corresponding to a bisulfite-converted, unmethylated cytosine) in representative regions that include binding sites for OCT4, SOX2, NANOG and SIN3A (shaded-orange box). n?= number of CpGs covered. Open in a separate window Physique?1 Whole-Genome Bisulfite Sequencing Analysis of CTCs from Breast Cancer Patients and Xenografts (A) Heatmap showing methylation variable regions with 80% methylation difference between patient-derived CTC clusters and single CTCs (false discovery rate [FDR]? 0.05). (B) Heatmap showing methylation variable regions with 70% methylation difference between xenograft-derived CTC clusters and single CTCs (FDR? 0.05). (C and D) Normalized enrichment score (NES) representing enrichment (NES 3.4) of transcription factor binding sites (TFBSs) in CTC cluster hypomethylated regions (blue) and single CTC hypomethylated regions (red) of patients (C) or xenografts (D), identified using i-and expression modules, significantly enriched in xenograft-derived Rabbit Polyclonal to SHC2 single CTCs. (DCF) Dot plots showing the percent of Ki67-positive single CTCs and Ki67-positive CTCs within CTC clusters, detected in BR16 xenograft-derived CTCs (D), LM2 xenograft-derived CTCs (E), and patient 3-derived CTCs (F). ?p? 0.05 by Students t test. Error bars represent SEM. ID?= Internal ID. (G) Representative pictures of BR16 xenograft-derived single CTCs and CTC clusters, stained A 803467 with Pan Cytokeratin (PanCK) (and modules. (B and C) Whisker plots showing the average methylation difference in CTC clusters relative to single CTCs, detected around the 5kb region upstream of the transcription start site of each target gene, in patient- (A) and xenograft-derived (B) CTCs. Hypomethylated genes in CTC clusters are represented with color, hypomethylated genes in single CTCs are represented with color. Transcription factors relative to target genes are shown within boxes. (D) Venn diagram showing the overlap between genes enriched in single CTCs of patient-derived (and and and and octagons). Altogether, our gene expression data both at the transcriptome-wide level and also focused on stem cell-related genes strongly supports the model proposed with the DNA methylation analysis, suggesting that when compared to single CTCs, CTC clusters are endowed with a stemness- and proliferation-related network centered A 803467 on the activity of key transcription factors including OCT4, SOX2, NANOG, and SIN3A. Activation of these programs may play a pivotal role in determining the metastasis-seeding ability of CTC clusters. Identification of FDA-Approved Cluster-Targeting Brokers Next, we sought to identify actionable vulnerabilities of CTC clusters, and to test whether the epigenetic and transcriptional features of clustered CTCs are reversible upon cluster dissociation into single cells. To this end, we evaluated 2,486 FDA-approved compounds (Table S6) for their ability to dissociate clusters of human breast CTC-derived cells obtained from two patients with breast malignancy (BR16 and BRx50), without affecting cellular viability. Cluster dissociation was assessed using an image-based high-content screening system and comparing CTCs treated with individual.