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.

Metastasis is the major driver of cancer deaths and begins when cancer cells invade surrounding tissues. Invasion and metastasis have been proposed to initiate following loss of the intercellular adhesion protein, E-cadherin (E-cad) 1,2 , based upon inverse correlations between in vitro migration and E-cad levels 3. This hypothesis is inconsistent, however, with the observation that most breast cancers are invasive ductal carcinomas (IDC) and express E-cad in primary tumors and metastases 4. To resolve this discrepancy, we tested the genetic requirement for E-cad in metastasis using murine and human models of both luminal and basal IDC. Here we show that E-cad promotes metastasis in IDC. While loss of E-cad increased invasion, it also reduced cancer cell proliferation and survival, circulating tumor cell number, seeding of cancer cells in distant organs, Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Summary Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation.

Human glioblastoma (GBM) is a highly aggressive, invasive and hypervascularised malignant brain cancer. Individual circulating tumour cells (CTCs) are sporadically found in GBM patients, yet it is unclear whether multicellular CTC clusters are generated in this disease and whether they can bypass the physical hurdle of the blood-brain barrier.  Here, we assessed CTC presence and composition at multiple time points in 13 patients with progressing GBM during an open-label phase 1/2a study with the microtubule inhibitor BAL101553. We observe CTC clusters ranging from 2 to 23 cells and present at multiple sampling time points in a GBM patient with pleomorphism and extensive necrosis, throughout disease progression. Exome sequencing of GBM CTC clusters highlights variants in 58 cancer-associated genes including ATM, PMS2, POLE, APC, XPO1, TFRC, JAK2, ERBB4 and ALK. Together, our findings represent the first evidence of the presence of CTC clusters in GBM.

This study was undertaken to test the hypothesis that Sjogren’s syndrome Antigen A (SSA)/Ro60-reactive T cells are activated by peptides originating from oral and gut bacteria. T cell hybridomas generated from HLA-DR3 transgenic mice recognized 3 regions on Ro60, with core epitopes mapped to amino acids 228-238, 246-256 and 371-381. BLAST analysis identified several mimicry peptides, originating from human oral, intestinal, skin and vaginal bacteria, as well as environmental bacteria. Amongst these, a peptide from the von Willebrand factor type A domain protein (vWFA) from the oral microbe Capnocytphaga ochracea was the most potent activator. Further, Ro60-reactive T cells were activated by recombinant vWFA protein and whole E. coli expressing this protein. These results demonstrate that peptides derived from normal human microbiota can activate Ro60-reactive T cells. Thus, immune responses to commensal microbiota and opportunistic pathogens should be explored as potential triggers for initiating autoimmunity in SLE and Sjögren’s syndrome.

Objectives Autoantibodies reactive with Ro52 are often found in sera of Sjögren’s syndrome (SS) patients. This study was undertaken to investigate the role of Ro52-induced immune responses in pathogenesis of SS. Methods New Zealand Mixed (NZM) 2758 mice were immunized with Ro52 in alum adjuvant. Control mice were immunized either with Maltose binding protein (MBP) or injected with alum alone. Mice were monitored for anti-Ro52 antibody, sialoadenitis and pilocarpine induced salivation. Antibody binding to salivary gland (SG) cells was analyzed in vivo and in vitro by immunofluorescence. Sera from immunized mice were passively transferred into untreated or alum injected NZM2758 mice. Results By day 30 post-immunization, Ro52 immunized mice generated immunoprecipitating anti-Ro52 antibodies and they had the maximum drop in saliva production. Both Ro52 immunized and control mice showed evidence of mild sialoadenitis. However, only Ro52 immunized mice had antibody deposition in their SG. Passive transfer of Ro52-immune sera induced SG dysfunction in recipient mice, only if the recipients were primed with alum. In vitro, antibodies from Ro52-immune sera were internalized by a SG cell line and this uptake was inhibited by Cytochalasin D treatment. Conclusion Our data shows for the first time that antibodies induced by Ro52 are capable of inducing SG dysfunction, and that this phenomenon is dependent on the activation of innate immunity. The mouse model described in this study implies that autoantibody deposition in the SG might be an important step in the induction of xerostomia and pathogenesis of SS.

Cancer cells can be released from a cancerous lesion and migrate into the circulatory system, from whereon they may form metastases at distant sites. Today, it is possible to infer cancer progression and treatment efficacy by determining the number of circulating tumor cells (CTCs) in the patient's blood at multiple time points; further valuable information about CTC phenotypes remains inaccessible. In this article, a microfluidic method for integrated capture, isolation, and analysis of membrane markers as well as quantification of proteins secreted by single CTCs and CTC clusters is introduced. CTCs are isolated from whole blood with extraordinary efficiencies above 95% using dedicated trapping structures that allow co‐capture of functionalized magnetic beads to assess protein secretion. The patform is tested with multiple breast cancer cell lines spiked into human blood and mouse‐model‐derived CTCs. In addition to immunostaining, the secretion level of granulocyte growth stimulating factor (G‐CSF), which is shown to be involved in neutrophil recruitment, is quantified The bead‐based assay provides a limit of detection of 1.5 ng mL−1 or less than 3700 molecules per cell. Employing barcoded magnetic beads, this platform can be adapted for multiplexed analysis and can enable comprehensive functional CTC profiling in the future.