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-...

Single-cell analyses have revealed extensive intra-and inter-patient cancer heterogeneity 1 , but complex single-cell phenotypes and their spatial context are not yet reflected in the histologic stratification that is the foundation of many clinical decisions. Here, we used imaging mass cytometry 2 to simultaneously quantify 35 biomarkers resulting in 720 highdimension immunohistochemistry pathology images of tumor tissue from 352 breast cancer patients for whom long-term survival data were available. Spatial, single-cell analysis identified tumor and stromal single-cell phenotypes, their organization and heterogeneity, and enabled categorization of breast cancer cellular architecture based on cellular composition and tissue organization. Our analysis revealed multi-cellular features of the tumor microenvironment and novel breast cancer subgroups associated with distinct clinical outcomes. Thus, spatially resolved, single-cell analysis can characterize intra-tumor phenotypic heterogeneity in a disease-relevant manner with the potential to inform patient-specific diagnosis. MainHistologic and phenotypic differences between tumors guide cancer diagnosis, prognosis, and selection of treatment. Currently, breast cancer patients are graded based on tumor structure and cellular morphology, and subcategorized when more than 1% of tumor cells contain hormone receptors or more than 10% express high levels of HER2 or have amplified HER2 [3][4][5] . This leaves a large portion of cells uncharacterized even though additional molecular subclasses and morphologic features have been identified as prognostic [6][7][8][9] . It is clear that clonal evolution and spatially distinct tumor microenvironments drive inter-and intra-patient cellular