Resistance to mesenchymal reprogramming sustains clonal propagation in metastatic breast cancer.

The acquisition of mesenchymal traits is considered a hallmark of breast cancer progression. However, the functional relevance of epithelial-to-mesenchymal transition (EMT) remains controversial and context dependent. Here, we isolate epithelial and mesenchymal populations from human breast cancer metastatic biopsies and assess their functional potential in vivo. Strikingly, progressively decreasing epithelial cell adhesion molecule (EPCAM) levels correlate with declining disease propagation. Mechanistically, we find that persistent EPCAM expression marks epithelial clones that resist EMT induction and propagate competitively. In contrast, loss of EPCAM defines clones arrested in a mesenchymal state, with concomitant suppression of tumorigenicity and metastatic potential. This dichotomy results from distinct clonal trajectories impacting global epigenetic programs that are determined by the interplay between human ZEB1 and its target GRHL2. Collectively, our results indicate that susceptibility to irreversible EMT restrains clonal propagation, whereas resistance to mesenchymal reprogramming sustains disease spread in multiple models of human metastatic breast cancer, including patient-derived cells in vivo.

ICAM-1 on Breast Cancer Cells Suppresses Lung Metastasis but Is Dispensable for Tumor Growth and Killing by Cytotoxic T Cells.

Breast tumors and their derived circulating cancer cells express the leukocyte ß2 integrin ligand Intercellular adhesion molecule 1 (ICAM-1). We found that elevated ICAM-1 expression in breast cancer cells results in a favorable outcome and prolonged survival of breast cancer patients. We therefore assessed the direct in vivo contribution of ICAM-1 expressed by breast cancer cells to breast tumorigenesis and lung metastasis in syngeneic immunocompetent mice hosts using spontaneous and experimental models of the lung metastasis of the C57BL/6-derived E0771 cell line, a luminal B breast cancer subtype. Notably, the presence of ICAM-1 on E0771 did not alter tumor growth or the leukocyte composition in the tumor microenvironment. Interestingly, the elimination of Tregs led to the rapid killing of primary tumor cells independently of tumor ICAM-1 expression. The in vivo elimination of a primary E0771 tumor expressing the ovalbumin (OVA) model neoantigen by the OVA-specific OVA-tcr-I mice (OT-I) transgenic cytotoxic T lymphocytes (CTLs) also took place normally in the absence of ICAM-1 expression by E0771 breast cancer target cells. The whole lung imaging of these cells by light sheet microscopy (LSM) revealed that both Wild type (WT)- and ICAM-1-deficient E0771 cells were equally disseminated from resected tumors and accumulated inside the lung vasculature at similar magnitudes. ICAM-1-deficient breast cancer cells developed, however, much larger metastatic lesions than their control counterparts. Strikingly, the vast majority of these cells gave rise to intravascular tumor colonies both in spontaneous and experimental metastasis models. In the latter model, ICAM-1 expressing E0771- but not their ICAM-1-deficient counterparts were highly susceptible to elimination by neutrophils adoptively transferred from E0771 tumor-bearing donor mice. Ex vivo, neutrophils derived from tumor-bearing mice also killed cultured E0771 cells via ICAM-1-dependent interactions. Collectively, our results are a first indication that ICAM-1 expressed by metastatic breast cancer cells that expand inside the lung vasculature is involved in innate rather than in adaptive cancer cell killing. This is also a first indication that the breast tumor expression of ICAM-1 is not required for CTL-mediated killing but can function as a suppressor of intravascular breast cancer metastasis to lungs.

The metastatic spread of breast cancer accelerates during sleep.

The metastatic spread of cancer is achieved by the haematogenous dissemination of circulating tumour cells (CTCs). Generally, however, the temporal dynamics that dictate the generation of metastasis-competent CTCs are largely uncharacterized, and it is often assumed that CTCs are constantly shed from growing tumours or are shed as a consequence of mechanical insults1. Here we observe a striking and unexpected pattern of CTC generation dynamics in both patients with breast cancer and mouse models, highlighting that most spontaneous CTC intravasation events occur during sleep. Further, we demonstrate that rest-phase CTCs are highly prone to metastasize, whereas CTCs generated during the active phase are devoid of metastatic ability. Mechanistically, single-cell RNA sequencing analysis of CTCs reveals a marked upregulation of mitotic genes exclusively during the rest phase in both patients and mouse models, enabling metastasis proficiency. Systemically, we find that key circadian rhythm hormones such as melatonin, testosterone and glucocorticoids dictate CTC generation dynamics, and as a consequence, that insulin directly promotes tumour cell proliferation in vivo, yet in a time-dependent manner. Thus, the spontaneous generation of CTCs with a high proclivity to metastasize does not occur continuously, but it is concentrated within the rest phase of the affected individual, providing a new rationale for time-controlled interrogation and treatment of metastasis-prone cancers.

Cooperation between melanoma cell states promotes metastasis through heterotypic cluster formation.

Melanomas can have multiple coexisting cell states, including proliferative (PRO) versus invasive (INV) subpopulations that represent a "go or grow" trade-off; however, how these populations interact is poorly understood. Using a combination of zebrafish modeling and analysis of patient samples, we show that INV and PRO cells form spatially structured heterotypic clusters and cooperate in the seeding of metastasis, maintaining cell state heterogeneity. INV cells adhere tightly to each other and form clusters with a rim of PRO cells. Intravital imaging demonstrated cooperation in which INV cells facilitate dissemination of less metastatic PRO cells. We identified the TFAP2 neural crest transcription factor as a master regulator of clustering and PRO/INV states. Isolation of clusters from patients with metastatic melanoma revealed a subset with heterotypic PRO-INV clusters. Our data suggest a framework for the co-existence of these two divergent cell populations, in which heterotypic clusters promote metastasis via cell-cell cooperation.

Circulating Tumor Cell-Neutrophil Tango along the Metastatic Process.

The crosstalk between cancer cells and the immune system is crucial for disease progression and its therapeutic targeting is providing exciting results, in particular with newly developed immune checkpoint inhibitors. Current approaches primarily focus on cellular interactions occurring between tumor cells and T lymphocytes; however, recent data highlight a crucial role of neutrophils in support of tumor progression and suggest yet unexplored treatment opportunities. In this review, we summarize the current understanding of those interactions that occur between neutrophils and cancer cells, focusing on both protumor and antitumor activities of neutrophils at different stages of cancer progression. These include infiltration of neutrophils into the primary tumor, their interactions with circulating tumor cells (CTC) within the bloodstream, and their involvement in the establishment of a metastatic niche. Additionally, we discuss how further investigation of CTCs and their interacting immune cell partners may point towards novel immune checkpoint inhibition strategies and provide new insights on the efficacy of already existing immunotherapies.

Sustained prognostic impact of circulating tumor cell status and kinetics upon further progression of metastatic breast cancer.

PURPOSE: Serial longitudinal enumeration of circulating tumor cells (CTCs) has shown its prognostic value on progression-free survival and overall survival (OS) in patients with stage IV breast cancer. This study prospectively evaluated the role of CTCs as a prognostic marker during further progression of metastatic breast cancer (MBC). METHODS: Among 476 MBC patients recruited between 2010 and 2015, the 103 patients with a known CTC status at baseline (CTCBL) and within 4 weeks of tumor progression (CTCPD) were included. Progressive disease (PD) was defined according to the Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1). Using the CellSearch method, < 5 and ≥ 5 CTCs per 7.5 ml blood were determined as negative and positive, respectively. A shift in CTC status from baseline to progression ([Formula: see text] to [Formula: see text] and vice versa) was considered as alternating KineticsBL-PD. RESULTS: Median follow-up was 29.9 [21.2, 40.0] months. CTCPD positivity (37%, n = 38) was associated with a significantly shorter OS than CTCPD negativity (8.0 [5.1, 10.9] vs 22.6 [15.3, 39.8] months; P < 0.001). Alternating KineticsBL-PD was observed in 24% of the patients. This significantly changed the OS prediction of [Formula: see text] patients ([Formula: see text] vs [Formula: see text], 11.4 [9.7, not available (NA)] vs. 7.6 [4.4, 11.5] months; P = 0.044) and [Formula: see text] patients ([Formula: see text] vs. [Formula: see text], 8.4 [4.0, NA] vs. 22.6 [18.9, NA] months, respectively; P < 0.001). Prediction of survival was significantly improved (P = 0.002) by adding CTCPD status to clinicopathological characteristics and CTCBL status. CONCLUSIONS: CTC status upon further disease progression is a prognostic factor that could significantly improve well-established models. Thus, it represents a potential additional instrument supporting treatment decision.

Single cell polarity in liquid phase facilitates tumour metastasis.

Dynamic polarisation of tumour cells is essential for metastasis. While the role of polarisation during dedifferentiation and migration is well established, polarisation of metastasising tumour cells during phases of detachment has not been investigated. Here we identify and characterise a type of polarisation maintained by single cells in liquid phase termed single-cell (sc) polarity and investigate its role during metastasis. We demonstrate that sc polarity is an inherent feature of cells from different tumour entities that is observed in circulating tumour cells in patients. Functionally, we propose that the sc pole is directly involved in early attachment, thereby affecting adhesion, transmigration and metastasis. In vivo, the metastatic capacity of cell lines correlates with the extent of sc polarisation. By manipulating sc polarity regulators and by generic depolarisation, we show that sc polarity prior to migration affects transmigration and metastasis in vitro and in vivo.

Acquired CYP19A1 amplification is an early specific mechanism of aromatase inhibitor resistance in ERα metastatic breast cancer.

Tumor evolution is shaped by many variables, potentially involving external selective pressures induced by therapies. After surgery, patients with estrogen receptor (ERα)-positive breast cancer are treated with adjuvant endocrine therapy, including selective estrogen receptor modulators (SERMs) and/or aromatase inhibitors (AIs). However, more than 20% of patients relapse within 10 years and eventually progress to incurable metastatic disease. Here we demonstrate that the choice of therapy has a fundamental influence on the genetic landscape of relapsed diseases. We found that 21.5% of AI-treated, relapsed patients had acquired CYP19A1 (encoding aromatase) amplification (CYP19A1amp). Relapsed patients also developed numerous mutations targeting key breast cancer-associated genes, including ESR1 and CYP19A1. Notably, CYP19A1amp cells also emerged in vitro, but only in AI-resistant models. CYP19A1 amplification caused increased aromatase activity and estrogen-independent ERα binding to target genes, resulting in CYP19A1amp cells showing decreased sensitivity to AI treatment. These data suggest that AI treatment itself selects for acquired CYP19A1amp and promotes local autocrine estrogen signaling in AI-resistant metastatic patients.

The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features.

INTRODUCTION: Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells. METHODS: We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations. RESULTS: High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial-mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-ß-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy. CONCLUSIONS: In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.

Hematopoietic stem cell quiescence and function are controlled by the CYLD-TRAF2-p38MAPK pathway.

The status of long-term quiescence and dormancy guarantees the integrity of hematopoietic stem cells (HSCs) during adult homeostasis. However the molecular mechanisms regulating HSC dormancy remain poorly understood. Here we show that cylindromatosis (CYLD), a tumor suppressor gene and negative regulator of NF-κB signaling with deubiquitinase activity, is highly expressed in label-retaining dormant HSCs (dHSCs). Moreover, Cre-mediated conditional elimination of the catalytic domain of CYLD induced dHSCs to exit quiescence and abrogated their repopulation and self-renewal potential. This phenotype is dependent on the interactions between CYLD and its substrate TRAF2 (tumor necrosis factor-associated factor 2). HSCs expressing a mutant CYLD with an intact catalytic domain, but unable to bind TRAF2, showed the same HSC phenotype. Unexpectedly, the robust cycling of HSCs lacking functional CYLD-TRAF2 interactions was not elicited by increased NF-κB signaling, but instead by increased activation of the p38MAPK pathway. Pharmacological inhibition of p38MAPK rescued the phenotype of CYLD loss, identifying the CYLD-TRAF2-p38MAPK pathway as a novel important regulator of HSC function restricting HSC cycling and promoting dormancy.

Co-expression of MET and CD47 is a novel prognosticator for survival of luminal breast cancer patients.

Although luminal-type primary breast cancer can be efficiently treated, development of metastatic disease remains a significant clinical problem. We have previously shown that luminal-type circulating tumor cells (CTCs) co-expressing the tyrosine-kinase MET and CD47, a ligand involved in cancer cell evasion from macrophage scavenging, are able to initiate metastasis in xenografts. Here, we investigated the clinical relevance of MET-CD47 co-expression in 255 hormone receptor positive breast tumors by immunohistochemistry and found a 10.3- year mean overall-survival difference between MET-CD47 double-positive and double-negative patients (p<0.001) MET-CD47 co-expression defined a novel independent prognosticator for overall-survival by multivariate analysis (Cox proportional hazards model: HR: 4.1, p<0.002) and CD47 expression alone or in combination with MET was strongly associated with lymph node metastasis. Furthermore, flow cytometric analysis of metastatic patient blood revealed consistent presence of MET+CD47+ CTCs (range 0.8 - 33.3% of CTCs) and their frequency was associated with increased metastatic spread. Finally, primary uncultured CTCs with high MET+CD47+ content showed an enhanced capacity to initiate metastasis in mice. Detection and targeting of MET and CD47 may thus provide a rational basis for risk stratification and treatment of patients with luminal-type breast cancer.

Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay.

It has been hypothesized that carcinoma metastasis is initiated by a subpopulation of circulating tumor cells (CTCs) found in the blood of patients. However, although the presence of CTCs is an indicator of poor prognosis in several carcinoma entities, the existence and phenotype of metastasis-initiating cells (MICs) among CTCs has not been experimentally demonstrated. Here we developed a xenograft assay and used it to show that primary human luminal breast cancer CTCs contain MICs that give rise to bone, lung and liver metastases in mice. These MIC-containing CTC populations expressed EPCAM, CD44, CD47 and MET. In a small cohort of patients with metastases, the number of EPCAM(+)CD44(+)CD47(+)MET(+) CTCs, but not of bulk EPCAM(+) CTCs, correlated with lower overall survival and increased number of metastasic sites. These data describe functional circulating MICs and associated markers, which may aid the design of better tools to diagnose and treat metastatic breast cancer.

A role for miR-155 in enabling tumor-infiltrating innate immune cells to mount effective antitumor responses in mice.

A productive immune response requires transient upregulation of the microRNA miR-155 in hematopoietic cells mediating innate and adaptive immunity. In order to investigate miR-155 in the context of tumor-associated immune responses, we stably knocked down (KD) miR-155 in the myeloid compartment of MMTV-PyMT mice, a mouse model of spontaneous breast carcinogenesis that closely mimics tumor-host interactions seen in humans. Notably, miR-155/KD significantly accelerated tumor growth by impairing classic activation of tumor-associated macrophages (TAMs). This created an imbalance toward a protumoral microenvironment as evidenced by a lower proportion of CD11c(+) TAMs, reduced expression of activation markers, and the skewing of immune cells within the tumor toward an macrophage type 2/T helper 2 response. This study highlights the importance of tumor-infiltrating hematopoietic cells in constraining carcinogenesis and establishes an antitumoral function of a prototypical oncomiR.

Attenuation of miR-126 activity expands HSC in vivo without exhaustion.

Lifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3ß pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.