SUMMARY Clusters of circulating tumor cells (CTC-clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC-clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC-clusters have 23-50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC-clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC-cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC-clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC-clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and while rare, they greatly contribute to the metastatic spread of cancer.

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-...

Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients with solid tumors. It has been proposed that the isolation, ex vivo culture, and characterization of CTCs may provide an opportunity to noninvasively monitor the changing patterns of drug susceptibility in individual patients as their tumors acquire new mutations. In a proof-of-concept study, we established CTC cultures from six patients with estrogen receptor–positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets. With optimization of CTC culture conditions, this strategy may help identify the best therapies for individual cancer patients over the course of their disease.