A better understanding of the features that define the interplay between cancer cells and immune cells is key to identify new cancer therapies 1 . Yet, focus is often given to those interactions that occur within the primary tumor and its microenvironment, while the role of immune cells during cancer dissemination in patients remains largely uncharacterized 2,3 . Circulating tumor cells (CTCs) are precursors of metastasis in several cancer types [4][5][6] , and are occasionally found within the bloodstream in association with non-malignant cells such as white blood cells (WBCs) 7,8 . The identity and function of these CTC-associated WBCs, as well as the molecular features that define the interaction between WBCs and CTCs are unknown. Here, we achieve the isolation and interrogation of individual CTC-associated WBCs, alongside with corresponding cancer cells within each CTC-WBC cluster, from multiple breast cancer patients and mouse models. Single-cell RNA sequencing reveals a specific pattern of WBCs attached to CTCs, with neutrophils representing the majority of the cases. When comparing the transcriptome profiles of CTCs that were associated to neutrophils with that of CTCs alone, we detect a number of differentially expressed genes that outline cell cycle progression, leading to a higher ability to efficiently seed metastasis. Additionally, we identify cell-cell junction and cytokine-receptor pairs that define CTC-neutrophil clusters, representing key vulnerabilities of the metastatic process. Thus, the association between neutrophils and CTCs fuels cell cycle progression within the bloodstream and expands the metastatic potential of CTCs, providing a rationale for targeting this interaction in breast cancer. 3/28 Main TextCirculating tumor cells (CTCs) are precursors of metastasis in various solid cancers including breast cancer 6 , and are occasionally found in association to white blood cells (WBCs) 7 . The role of CTC-WBC clusters in metastasis development as well as the principles that govern the interplay between CTCs and WBCs during blood-borne metastasis are largely uncharacterized.We first sought to determine the number and composition of CTC-WBC clusters in breast cancer patients and mouse models. We obtained blood samples from 70 patients with invasive breast cancer that discontinued their treatment due to progressive disease, as well as from five different breast cancer mouse models, and we enriched for CTCs using the Parsortix microfluidic device 9 (Extended Data Fig. 1a-e). Live CTCs were stained for cancer-associated cell surface markers EpCAM, HER2, and EGFR or imaged directly for the expression of GFP, as well as labeled for CD45 to identify WBCs (Fig. 1a and Extended Data Fig. 1f). Among 70 patients, 34 (48.6%) had detectable CTCs, with a mean number of 22 CTCs per 7.5ml of blood (Supplementary Tables 1 and 2). While the majority of CTCs were single (88.0%), we also detected CTC clusters (8.6%) and CTC-WBC clusters (3.4%) (Fig. 1b and Extended Data Fig. 1g,h). Similarly, we observed that CTC-...

SummaryThe 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, we profile the DNA methylation landscape 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 critical 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.

Osteosarcomas are aggressive bone tumours with a high degree of genetic heterogeneity, which has historically complicated driver gene discovery. Here we sequence exomes of 31 tumours and decipher their evolutionary landscape by inferring clonality of the individual mutation events. Exome findings are interpreted in the context of mutation and SNP array data from a replication set of 92 tumours. We identify 14 genes as the main drivers, of which some were formerly unknown in the context of osteosarcoma. None of the drivers is clearly responsible for the majority of tumours and even TP53 mutations are frequently mapped into subclones. However, >80% of osteosarcomas exhibit a specific combination of single-base substitutions, LOH, or large-scale genome instability signatures characteristic of BRCA1/2-deficient tumours. Our findings imply that multiple oncogenic pathways drive chromosomal instability during osteosarcoma evolution and result in the acquisition of BRCA-like traits, which could be therapeutically exploited.

As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy.

Cancer chromosomal instability (CIN) results from dynamic changes to chromosome number and structure. The resulting diversity in somatic copy number alterations (SCNA) may provide the variation necessary for cancer evolution. Multi-sample phasing and SCNA analysis of 1421 samples from 394 tumours across 24 cancer types revealed ongoing CIN resulting in pervasive SCNA heterogeneity. Parallel evolutionary events, causing disruption to the same genes, such as BCL9, ARNT/HIF1B, TERT and MYC, within separate subclones were present in 35% of tumours. Most recurrent losses occurred prior to whole genome doubling (WGD), a clonal event in 48% of tumours. However, loss of heterozygosity at the human leukocyte antigen locus and loss of 8p to a single haploid copy recurred at significant subclonal frequencies, even in WGD tumours, likely reflecting ongoing karyotype remodeling. Focal amplifications affecting 1q21 (BCL9, ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal and exhibited an illusion of clonality within single samples. Analysis of an independent series of 1024 metastatic samples revealed enrichment for 14 focal SCNAs in metastatic samples, including late gains of 8q24.1 (MYC) in clear cell renal carcinoma and 11q13.3 (CCND1) in HER2-positive breast cancer. CIN may enable ongoing selection of SCNAs, manifested as ordered events, often occurring in parallel, throughout tumour evolution.

Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological, and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine patient management (1). A discovery exome sequencing screen (n=17), followed by a prevalence screen (n=60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2, BPTF), cell division (STAG2, SMC1A, SMC1B), and DNA repair (ATM, ERCC2, FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated/lost in UBC, mainly in tumors of low stage/grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally-stable tumors and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a novel UBC tumor suppressor acting through mechanisms that are different from its role to prevent aneuploidy.

Hereditary mixed polyposis syndrome (HMPS) is characterised by the development of mixed morphology colorectal tumours and is caused by a 40 kb duplication that results in aberrant epithelial expression of the mesenchymal Bone Morphogenetic Protein antagonist, GREM1. Here we use HMPS tissue and a mouse model of the disease to show that epithelial GREM1 disrupts homeostatic intestinal morphogen gradients, altering cell-fate, that is normally determined by position along the vertical epithelial axis. This promotes the persistence and/or reacquisition of stem-cell properties in Lgr5 negative (non-expressing) progenitor cells that have exited the stem-cell niche. These cells form ectopic crypts, proliferate, accumulate somatic mutations and can initiate intestinal neoplasia, indicating that the crypt base stem-cell is not the sole cell-of-origin of colorectal cancer. Furthermore, we show that epithelial expression of GREM1 also occurs in traditional serrated adenomas, sporadic pre-malignant lesions with a hitherto unknown pathogenesis and these lesions can be considered the sporadic equivalents of HMPS polyps.

These data highlight an essential mechanism in asthma pathogenesis by demonstrating that ILC2s are responsible for bronchial epithelial TJ barrier leakiness through IL-13.