Integrative multiomics enhancer activity profiling identifies therapeutic vulnerabilities in cholangiocarcinoma of different etiologies.

OBJECTIVES: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. DESIGN: Integrative multiomics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining and single-cell spatial transcriptomics were used to explore the immunogenicity of diverse CCA. RESULTS: We identified three distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumour growth in in vitro and in vivo models. The first group (ESTRO), with mostly fluke-positive CCAs, displayed activation in estrogen signalling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive to oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intratumour differences in AA mutation load were correlated to intratumour variation of different immune cell populations. CONCLUSION: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumourigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits.

An allosteric pan-TEAD inhibitor blocks oncogenic YAP/TAZ signaling and overcomes KRAS G12C inhibitor resistance.

The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance.

PBRM1-deficient PBAF complexes target aberrant genomic loci to activate the NF-κB pathway in clear cell renal cell carcinoma.

PBRM1 encodes an accessory subunit of the PBAF SWI/SNF chromatin remodeller, and the inactivation of PBRM1 is a frequent event in kidney cancer. However, the impact of PBRM1 loss on chromatin remodelling is not well examined. Here we show that, in VHL-deficient renal tumours, PBRM1 deficiency results in ectopic PBAF complexes that localize to de novo genomic loci, activating the pro-tumourigenic NF-κB pathway. PBRM1-deficient PBAF complexes retain the association between SMARCA4 and ARID2, but have loosely tethered BRD7. The PBAF complexes redistribute from promoter proximal regions to distal enhancers containing NF-κB motifs, heightening NF-κB activity in PBRM1-deficient models and clinical samples. The ATPase function of SMARCA4 maintains chromatin occupancy of pre-existing and newly acquired RELA specific to PBRM1 loss, activating downstream target gene expression. Proteasome inhibitor bortezomib abrogates RELA occupancy, suppresses NF-κB activation and delays growth of PBRM1-deficient tumours. In conclusion, PBRM1 safeguards the chromatin by repressing aberrant liberation of pro-tumourigenic NF-κB target genes by residual PBRM1-deficient PBAF complexes.

Aberrant JAK-STAT signaling-mediated chromatin remodeling impairs the sensitivity of NK/T-cell lymphoma to chidamide.

BACKGROUND: Natural killer/T-cell lymphoma (NKTL) is a rare type of aggressive and heterogeneous non-Hodgkin's lymphoma (NHL) with a poor prognosis and limited therapeutic options. Therefore, there is an urgent need to exploit potential novel therapeutic targets for the treatment of NKTL. Histone deacetylase (HDAC) inhibitor chidamide was recently approved for treating relapsed/refractory peripheral T-cell lymphoma (PTCL) patients. However, its therapeutic efficacy in NKTL remains unclear. METHODS: We performed a phase II clinical trial to evaluate the efficacy of chidamide in 28 relapsed/refractory NKTL patients. Integrative transcriptomic, chromatin profiling analysis and functional studies were performed to identify potential predictive biomarkers and unravel the mechanisms of resistance to chidamide. Immunohistochemistry (IHC) was used to validate the predictive biomarkers in tumors from the clinical trial. RESULTS: We demonstrated that chidamide is effective in treating relapsed/refractory NKTL patients, achieving an overall response and complete response rate of 39 and 18%, respectively. In vitro studies showed that hyperactivity of JAK-STAT signaling in NKTL cell lines was associated with the resistance to chidamide. Mechanistically, our results revealed that aberrant JAK-STAT signaling remodels the chromatin and confers resistance to chidamide. Subsequently, inhibition of JAK-STAT activity could overcome resistance to chidamide by reprogramming the chromatin from a resistant to sensitive state, leading to synergistic anti-tumor effect in vitro and in vivo. More importantly, our clinical data demonstrated that combinatorial therapy with chidamide and JAK inhibitor ruxolitinib is effective against chidamide-resistant NKTL. In addition, we identified TNFRSF8 (CD30), a downstream target of the JAK-STAT pathway, as a potential biomarker that could predict NKTL sensitivity to chidamide. CONCLUSIONS: Our study suggests that chidamide, in combination with JAK-STAT inhibitors, can be a novel targeted therapy in the standard of care for NKTL. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02878278. Registered 25 August 2016, https://clinicaltrials.gov/ct2/show/NCT02878278.

RAD21 amplification epigenetically suppresses interferon signaling to promote immune evasion in ovarian cancer.

Prevalent copy number alteration is the most prominent genetic characteristic associated with ovarian cancer (OV) development, but its role in immune evasion has not been fully elucidated. In this study, we identified RAD21, a key component of the cohesin complex, as a frequently amplified oncogene that could modulate immune response in OV. Through interrogating the RAD21-regulated transcriptional program, we found that RAD21 directly interacts with YAP/TEAD4 transcriptional corepressors and recruits the NuRD complex to suppress interferon (IFN) signaling. In multiple clinical cohorts, RAD21 overexpression is inversely correlated with IFN signature gene expression in OV. We further demonstrated in murine syngeneic tumor models that RAD21 ablation potentiated anti-PD-1 efficacy with increased intratumoral CD8+ T cell effector activity. Our study identifies a RAD21-YAP/TEAD4-NuRD corepressor complex in immune modulation, and thus provides a potential target and biomarker for precision immunotherapy in OV.

Giredestrant reverses progesterone hypersensitivity driven by estrogen receptor mutations in breast cancer.

ESR1 (estrogen receptor 1) hotspot mutations are major contributors to therapeutic resistance in estrogen receptor-positive (ER+) breast cancer. Such mutations confer estrogen independence to ERα, providing a selective advantage in the presence of estrogen-depleting aromatase inhibitors. In addition, ESR1 mutations reduce the potency of tamoxifen and fulvestrant, therapies that bind ERα directly. These limitations, together with additional liabilities, inspired the development of the next generation of ERα-targeted therapeutics, of which giredestrant is a high-potential candidate. Here, we generated Esr1 mutant-expressing mammary gland models and leveraged patient-derived xenografts (PDXs) to investigate the biological properties of the ESR1 mutations and their sensitivity to giredestrant in vivo. In the mouse mammary gland, Esr1 mutations promote hypersensitivity to progesterone, triggering pregnancy-like tissue remodeling and profoundly elevated proliferation. These effects were driven by an altered progesterone transcriptional response and underpinned by gained sites of ERα-PR (progesterone receptor) cobinding at the promoter regions of pro-proliferation genes. PDX experiments showed that the mutant ERα-PR proliferative program is also relevant in human cancer cells. Giredestrant suppressed the mutant ERα-PR proliferation in the mammary gland more so than the standard-of-care agents, tamoxifen and fulvestrant. Giredestrant was also efficacious against the progesterone-stimulated growth of ESR1 mutant PDX models. In addition, giredestrant demonstrated activity against a molecularly characterized ESR1 mutant tumor from a patient enrolled in a phase 1 clinical trial. Together, these data suggest that mutant ERα can collaborate with PR to drive protumorigenic proliferation but remain sensitive to inhibition by giredestrant.

Transcriptional Regulation of the Hippo Pathway: Current Understanding and Insights from Single-Cell Technologies.

The Hippo pathway regulates tissue homeostasis in normal development and drives oncogenic processes. In this review, we extensively discuss how YAP/TAZ/TEAD cooperate with other master transcription factors and epigenetic cofactors to orchestrate a broad spectrum of transcriptional responses. Even though these responses are often context- and lineage-specific, we do not have a good understanding of how such precise and specific transcriptional control is achieved-whether they are driven by differences in TEAD paralogs, or recruitment of cofactors to tissue-specific enhancers. We believe that emerging single-cell technologies would enable a granular understanding of how the Hippo pathway influences cell fate and drives oncogenic processes, ultimately allowing us to design better pharmacological agents against TEADs and identify robust pharmacodynamics markers of Hippo pathway inhibition.

Chromatin Rewiring by Mismatch Repair Protein MSH2 Alters Cell Adhesion Pathways and Sensitivity to BET Inhibition in Gastric Cancer.

Mutations in the DNA mismatch repair gene MSH2 are causative of microsatellite instability (MSI) in multiple cancers. Here, we discovered that besides its well-established role in DNA repair, MSH2 exerts a novel epigenomic function in gastric cancer. Unbiased CRISPR-based mass spectrometry combined with genome-wide CRISPR functional screening revealed that in early-stage gastric cancer MSH2 genomic binding is not randomly distributed but rather is associated specifically with tumor-associated super-enhancers controlling the expression of cell adhesion genes. At these loci, MSH2 genomic binding was required for chromatin rewiring, de novo enhancer-promoter interactions, maintenance of histone acetylation levels, and regulation of cell adhesion pathway expression. The chromatin function of MSH2 was independent of its DNA repair catalytic activity but required MSH6, another DNA repair gene, and recruitment to gene loci by the SWI/SNF chromatin remodeler SMARCA4/BRG1. Loss of MSH2 in advanced gastric cancers was accompanied by deficient cell adhesion pathway expression, epithelial-mesenchymal transition, and enhanced tumorigenesis in vitro and in vivo. However, MSH2-deficient gastric cancers also displayed addiction to BAZ1B, a bromodomain-containing family member, and consequent synthetic lethality to bromodomain and extraterminal motif (BET) inhibition. Our results reveal a role for MSH2 in gastric cancer epigenomic regulation and identify BET inhibition as a potential therapy in MSH2-deficient gastric malignancies. SIGNIFICANCE: DNA repair protein MSH2 binds and regulates cell adhesion genes by enabling enhancer-promoter interactions, and loss of MSH2 causes deficient cell adhesion and bromodomain and extraterminal motif inhibitor synthetic lethality in gastric cancer.

Targeting enhancer reprogramming to mitigate MEK inhibitor resistance in preclinical models of advanced ovarian cancer.

Ovarian cancer is characterized by aberrant activation of the mitogen-activated protein kinase (MAPK), highlighting the importance of targeting the MAPK pathway as an attractive therapeutic strategy. However, the clinical efficacy of MEK inhibitors is limited by intrinsic or acquired drug resistance. Here, we established patient-derived ovarian cancer models resistant to MEK inhibitors and demonstrated that resistance to the clinically approved MEK inhibitor trametinib was associated with enhancer reprogramming. We also showed that enhancer decommissioning induced the downregulation of negative regulators of the MAPK pathway, leading to constitutive ERK activation and acquired resistance to trametinib. Epigenetic compound screening uncovered that HDAC inhibitors could alter the enhancer reprogramming and upregulate the expression of MAPK negative regulators, resulting in sustained MAPK inhibition and reversal of trametinib resistance. Consequently, a combination of HDAC inhibitor and trametinib demonstrated a synergistic antitumor effect in vitro and in vivo, including patient-derived xenograft mouse models. These findings demonstrated that enhancer reprogramming of the MAPK regulatory pathway might serve as a potential mechanism underlying MAPK inhibitor resistance and concurrent targeting of epigenetic pathways and MAPK signaling might provide an effective treatment strategy for advanced ovarian cancer.

Genomic and epigenomic EBF1 alterations modulate TERT expression in gastric cancer.

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.

Integrated paired-end enhancer profiling and whole-genome sequencing reveals recurrent CCNE1 and IGF2 enhancer hijacking in primary gastric adenocarcinoma.

OBJECTIVE: Genomic structural variations (SVs) causing rewiring of cis-regulatory elements remain largely unexplored in gastric cancer (GC). To identify SVs affecting enhancer elements in GC (enhancer-based SVs), we integrated epigenomic enhancer profiles revealed by paired-end H3K27ac ChIP-sequencing from primary GCs with tumour whole-genome sequencing (WGS) data (PeNChIP-seq/WGS). DESIGN: We applied PeNChIP-seq to 11 primary GCs and matched normal tissues combined with WGS profiles of >200 GCs. Epigenome profiles were analysed alongside matched RNA-seq data to identify tumour-associated enhancer-based SVs with altered cancer transcription. Functional validation of candidate enhancer-based SVs was performed using CRISPR/Cas9 genome editing, chromosome conformation capture assays (4C-seq, Capture-C) and Hi-C analysis of primary GCs. RESULTS: PeNChIP-seq/WGS revealed ~150 enhancer-based SVs in GC. The majority (63%) of SVs linked to target gene deregulation were associated with increased tumour expression. Enhancer-based SVs targeting CCNE1, a key driver of therapy resistance, occurred in 8% of patients frequently juxtaposing diverse distal enhancers to CCNE1 proximal regions. CCNE1-rearranged GCs were associated with high CCNE1 expression, disrupted CCNE1 topologically associating domain (TAD) boundaries, and novel TAD interactions in CCNE1-rearranged primary tumours. We also observed IGF2 enhancer-based SVs, previously noted in colorectal cancer, highlighting a common non-coding genetic driver alteration in gastric and colorectal malignancies. CONCLUSION: Integrated paired-end NanoChIP-seq and WGS of gastric tumours reveals tumour-associated regulatory SV in regions associated with both simple and complex genomic rearrangements. Genomic rearrangements may thus exploit enhancer-hijacking as a common mechanism to drive oncogene expression in GC.

HNF4α pathway mapping identifies wild-type IDH1 as a targetable metabolic node in gastric cancer.

OBJECTIVE: Gastric cancer (GC) is a leading cause of cancer mortality. Previous studies have shown that hepatocyte nuclear factor-4α (HNF4α) is specifically overexpressed in GC and functionally required for GC development. In this study, we investigated, on a genome-wide scale, target genes of HNF4α and oncogenic pathways driven by HNF4α and HNF4α target genes. DESIGN: We performed HNF4α chromatin immunoprecipitation followed by sequencing across multiple GC cell lines, integrating HNF4α occupancy data with (epi)genomic and transcriptome data of primary GCs to define HNF4α target genes of in vitro and in vivo relevance. To investigate mechanistic roles of HNF4α and HNF4α targets, we performed cancer metabolic measurements, drug treatments and functional assays including murine xenograft experiments. RESULTS: Gene expression analysis across 19 tumour types revealed HNF4α to be specifically upregulated in GCs. Unbiased pathway analysis revealed organic acid metabolism as the top HNF4α-regulated pathway, orthogonally supported by metabolomic analysis. Isocitrate dehydrogenase 1 (IDH1) emerged as a convergent HNF4α direct target gene regulating GC metabolism. We show that wild-type IDH1 is essential for GC cell survival, and that certain GC cells can be targeted by IDH1 inhibitors. CONCLUSIONS: Our results highlight a role for HNF4α in sustaining GC oncogenic metabolism, through the regulation of IDH1. Drugs targeting wild-type IDH1 may thus have clinical utility in GCs exhibiting HNF4α overexpression, expanding the role of IDH1 in cancer beyond IDH1/2 mutated malignancies.

VHL substrate transcription factor ZHX2 as an oncogenic driver in clear cell renal cell carcinoma.

Inactivation of the von Hippel-Lindau (VHL) E3 ubiquitin ligase protein is a hallmark of clear cell renal cell carcinoma (ccRCC). Identifying how pathways affected by VHL loss contribute to ccRCC remains challenging. We used a genome-wide in vitro expression strategy to identify proteins that bind VHL when hydroxylated. Zinc fingers and homeoboxes 2 (ZHX2) was found as a VHL target, and its hydroxylation allowed VHL to regulate its protein stability. Tumor cells from ccRCC patients with VHL loss-of-function mutations usually had increased abundance and nuclear localization of ZHX2. Functionally, depletion of ZHX2 inhibited VHL-deficient ccRCC cell growth in vitro and in vivo. Mechanistically, integrated chromatin immunoprecipitation sequencing and microarray analysis showed that ZHX2 promoted nuclear factor κB activation. These studies reveal ZHX2 as a potential therapeutic target for ccRCC.

VHL Deficiency Drives Enhancer Activation of Oncogenes in Clear Cell Renal Cell Carcinoma.

Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Roles for noncoding cis-regulatory aberrations in ccRCC tumorigenesis, however, remain unclear. Analyzing 10 primary tumor/normal pairs and 9 cell lines across 79 chromatin profiles, we observed pervasive enhancer malfunction in ccRCC, with cognate enhancer-target genes associated with tissue-specific aspects of malignancy. Superenhancer profiling identified ZNF395 as a ccRCC-specific and VHL-regulated master regulator whose depletion causes near-complete tumor elimination in vitro and in vivoVHL loss predominantly drives enhancer/superenhancer deregulation more so than promoters, with acquisition of active enhancer marks (H3K27ac, H3K4me1) near ccRCC hallmark genes. Mechanistically, VHL loss stabilizes HIF2α-HIF1ß heterodimer binding at enhancers, subsequently recruiting histone acetyltransferase p300 without overtly affecting preexisting promoter-enhancer interactions. Subtype-specific driver mutations such as VHL may thus propagate unique pathogenic dependencies in ccRCC by modulating epigenomic landscapes and cancer gene expression.Significance: Comprehensive epigenomic profiling of ccRCC establishes a compendium of somatically altered cis-regulatory elements, uncovering new potential targets including ZNF395, a ccRCC master regulator. Loss of VHL, a ccRCC signature event, causes pervasive enhancer malfunction, with binding of enhancer-centric HIF2α and recruitment of histone acetyltransferase p300 at preexisting lineage-specific promoter-enhancer complexes. Cancer Discov; 7(11); 1284-305. ©2017 AACR.See related commentary by Ricketts and Linehan, p. 1221This article is highlighted in the In This Issue feature, p. 1201.

Epigenomic Promoter Alterations Amplify Gene Isoform and Immunogenic Diversity in Gastric Adenocarcinoma.

Promoter elements play important roles in isoform and cell type-specific expression. We surveyed the epigenomic promoter landscape of gastric adenocarcinoma, analyzing 110 chromatin profiles (H3K4me3, H3K4me1, H3K27ac) of primary gastric cancers, gastric cancer lines, and nonmalignant gastric tissues. We identified nearly 2,000 promoter alterations (somatic promoters), many deregulated in various epithelial malignancies and mapping frequently to alternative promoters within the same gene, generating potential pro-oncogenic isoforms (RASA3). Somatic promoter-associated N-terminal peptides displaying relative depletion in tumors exhibited high-affinity MHC binding predictions and elicited potent T-cell responses in vitro, suggesting a mechanism for reducing tumor antigenicity. In multiple patient cohorts, gastric cancers with high somatic promoter usage also displayed reduced T-cell cytolytic marker expression. Somatic promoters are enriched in PRC2 occupancy, display sensitivity to EZH2 therapeutic inhibition, and are associated with novel cancer-associated transcripts. By generating tumor-specific isoforms and decreasing tumor antigenicity, epigenomic promoter alterations may thus drive intrinsic tumorigenesis and also allow nascent cancers to evade host immunity.Significance: We apply epigenomic profiling to demarcate the promoter landscape of gastric cancer. Many tumor-specific promoters activate different promoters in the same gene, some generating pro-oncogenic isoforms. Tumor-specific promoters also reduce tumor antigenicity by causing relative depletion of immunogenic peptides, contributing to cancer immunoediting and allowing tumors to evade host immune attack. Cancer Discov; 7(6); 630-51. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 539.

Epigenomic profiling of primary gastric adenocarcinoma reveals super-enhancer heterogeneity.

Regulatory enhancer elements in solid tumours remain poorly characterized. Here we apply micro-scale chromatin profiling to survey the distal enhancer landscape of primary gastric adenocarcinoma (GC), a leading cause of global cancer mortality. Integrating 110 epigenomic profiles from primary GCs, normal gastric tissues and cell lines, we highlight 36,973 predicted enhancers and 3,759 predicted super-enhancers respectively. Cell-line-defined super-enhancers can be subclassified by their somatic alteration status into somatic gain, loss and unaltered categories, each displaying distinct epigenetic, transcriptional and pathway enrichments. Somatic gain super-enhancers are associated with complex chromatin interaction profiles, expression patterns correlated with patient outcome and dense co-occupancy of the transcription factors CDX2 and HNF4α. Somatic super-enhancers are also enriched in genetic risk SNPs associated with cancer predisposition. Our results reveal a genome-wide reprogramming of the GC enhancer and super-enhancer landscape during tumorigenesis, contributing to dysregulated local and regional cancer gene expression.

Epigenomic Consequences of Coding and Noncoding Driver Mutations.

Chromatin alterations are integral to the pathogenic process of cancer, as demonstrated by recent discoveries of frequent mutations in chromatin-modifier genes and aberrant DNA methylation states in different cancer types. Progress is being made on elucidating how chromatin alterations, and how proteins catalyzing these alterations, mechanistically contribute to tissue-specific tumorigenesis. In parallel, technologies enabling the genome-wide profiling of histone modifications have revealed the existence of noncoding driver genetic alterations in cancer. In this review, we survey the current knowledge of coding and noncoding cancer drivers, and discuss their impact on the chromatin landscape. Translational implications of these findings for novel cancer therapies are also presented.

Tumor cells are dislodged into the pulmonary vein during lobectomy.

OBJECTIVE: Intraoperative tumor shedding may facilitate tumor dissemination. In earlier studies, shed tumor cells were defined primarily by cytomorphological examination, and normal epithelial cells could not always be distinguished from tumor cells. We sought to accurately identify tumor cells using single-cell sequencing and determine whether these cells were mobilized into the circulation during pulmonary lobectomy. METHODS: Forty-two blood samples collected from the tumor-draining pulmonary vein at the end of lobectomy procedures were analyzed. Arrays of nanowells were used to enumerate and retrieve single EpCAM(+) cells. Targeted sequencing of 10 to 15 cells and nested polymerase chain reaction of single cells detected somatic mutations in shed epithelial cells consistent with patient-matched tumor but not normal tissue. RESULTS: The mean number of EpCAM(+) cells in video-assisted thoracoscopy (VATS) lobectomy (no wedge) specimens (n = 16) was 165 (median, 115; range, 0-509) but sampling cells from 3 patients indicated that only 0% to 38% of the EpCAM(+) cells were tumor cells. The mean number of EpCAM(+) cells in VATS lobectomy (wedge) specimens (n = 12) was 1128 (median, 197; range, 47-9406) and all of the EpCAM(+) cells were normal epithelial cells in 2 patients sampled. The mean number of EpCAM(+) cells in thoracotomy specimens (n = 14) was 238 (median, 22; range, 9-2920) and 0% to 50% of total EpCAM(+) cells were tumor cells based on 4 patients sampled. CONCLUSIONS: Surgery mobilizes tumor cells into the pulmonary vein, along with many normal epithelial cells. EpCAM alone cannot differentiate between normal and tumor cells. On the other hand, single-cell genetic approaches with patient-matched normal and tumor tissues can accurately quantify the number of shed tumor cells.

Functional analysis of single cells identifies a rare subset of circulating tumor cells with malignant traits.

Ample evidence supports genetic and functional heterogeneity in primary tumors, but it remains unclear whether circulating tumor cells (CTCs) also exhibit the same hierarchical organization. We examined the functional diversity of viable, single CTCs using an array of subnanoliter wells (nanowells). The compartmentalization of single cells by nanowells allowed clonal comparison and mapping of heterogeneity of single cells or preformed clusters of cells. By measuring the short-term viability, invasiveness and secretory profiles of individual CTCs, it was evident that only a rare subset of CTCs possessed malignant traits indicative of metastatic potential in late-stage, progressing metastatic castration-resistant prostate cancer (mCRPC) patients. These CTCs were resistant to anoikis after being in the circulation, were invasive in their epithelial state, or secreted proteases capable of cleaving peptide substrates. Every CTC observed, however, did not exhibit such metastatic potential, suggesting that enumeration of CTCs alone may be insufficient to understand metastasis or stratify patients.