Unbiased metastatic niche-labeling identifies estrogen receptor-positive macrophages as a barrier of T cell infiltration during bone colonization.

Microenvironment niches determine cellular fates of metastatic cancer cells. However, robust and unbiased approaches to identify niche components and their molecular profiles are lacking. We established Sortase A-Based Microenvironment Niche Tagging (SAMENT), which selectively labels cells encountered by cancer cells during metastatic colonization. SAMENT was applied to multiple cancer models colonizing the same organ and the same cancer to different organs. Common metastatic niche features include macrophage enrichment and T cell depletion. Macrophage niches are phenotypically diverse between different organs. In bone, macrophages express the estrogen receptor alpha (ERα) and exhibit active ERα signaling in male and female hosts. Conditional knockout of Esr1 in macrophages significantly retarded bone colonization by allowing T cell infiltration. ERα expression was also discovered in human bone metastases of both genders. Collectively, we identified a unique population of ERα+ macrophages in the metastatic niche and functionally tied ERα signaling in macrophages to T cell exclusion during metastatic colonization. HIGHLIGHTS: SAMENT is a robust metastatic niche-labeling approach amenable to single-cell omics.Metastatic niches are typically enriched with macrophages and depleted of T cells.Direct interaction with cancer cells induces ERα expression in niche macrophages. Knockout of Esr1 in macrophages allows T cell infiltration and retards bone colonization.

Osteoprogenitor-GMP crosstalk underpins solid tumor-induced systemic immunosuppression and persists after tumor removal.

Remote tumors disrupt the bone marrow (BM) ecosystem (BME), eliciting the overproduction of BM-derived immunosuppressive cells. However, the underlying mechanisms remain poorly understood. Herein, we characterized breast and lung cancer-induced BME shifts pre- and post-tumor removal. Remote tumors progressively lead to osteoprogenitor (OP) expansion, hematopoietic stem cell dislocation, and CD41- granulocyte-monocyte progenitor (GMP) aggregation. The tumor-entrained BME is characterized by co-localization between CD41- GMPs and OPs. OP ablation abolishes this effect and diminishes abnormal myeloid overproduction. Mechanistically, HTRA1 carried by tumor-derived small extracellular vesicles upregulates MMP-13 in OPs, which in turn induces the alterations in the hematopoietic program. Importantly, these effects persist post-surgery and continue to impair anti-tumor immunity. Conditional knockout or inhibition of MMP-13 accelerates immune reinstatement and restores the efficacies of immunotherapies. Therefore, tumor-induced systemic effects are initiated by OP-GMP crosstalk that outlasts tumor burden, and additional treatment is required to reverse these effects for optimal therapeutic efficacy.

Bone Metastasis Initiation Is Coupled with Bone Remodeling through Osteogenic Differentiation of NG2+ Cells.

The bone microenvironment is dynamic and undergoes remodeling in normal and pathologic conditions. Whether such remodeling affects disseminated tumor cells (DTC) and bone metastasis remains poorly understood. Here, we demonstrated that pathologic fractures increase metastatic colonization around the injury. NG2+ cells are a common participant in bone metastasis initiation and bone remodeling in both homeostatic and fractured conditions. NG2+ bone mesenchymal stem/stromal cells (BMSC) often colocalize with DTCs in the perivascular niche. Both DTCs and NG2+ BMSCs are recruited to remodeling sites. Ablation of NG2+ lineage impaired bone remodeling and concurrently diminished metastatic colonization. In cocultures, NG2+ BMSCs, especially when undergoing osteodifferentiation, enhanced cancer cell proliferation and migration. Knockout of N-cadherin in NG2+ cells abolished these effects in vitro and phenocopied NG2+ lineage depletion in vivo. These findings uncover dual roles of NG2+ cells in metastasis and remodeling and indicate that osteodifferentiation of BMSCs promotes metastasis initiation via N-cadherin-mediated cell-cell interaction. SIGNIFICANCE: The bone colonization of cancer cells occurs in an environment that undergoes constant remodeling. Our study provides mechanistic insights into how bone homeostasis and pathologic repair lead to the outgrowth of disseminated cancer cells, thereby opening new directions for further etiologic and epidemiologic studies of tumor recurrences. This article is highlighted in the In This Issue feature, p. 247.

Review old bone, new tricks.

Despite the significant progress made over the past decade with combination of molecular profiling data and the development of new clinical strategies, our understanding of metastasis remains elusive. Bone metastasis is a complex process and a major cause of mortality in breast and prostate cancer patients, for which there is no effective treatment to-date. The current review summarizes the routes taken by the metastatic cells and the interactions between them and the bone microenvironment. We emphasize the role of the specified niches and cues that promote cellular adhesion, colonization, prolonged dormancy, and reactivation. Understanding these mechanisms will provide better insights for future studies and treatment strategies for bone metastatic conditions.

Improving the odds together: a framework for breast cancer research scientists to include patient advocates in their research.

Including patient advocates in basic cancer research ensures that breast cancer research is intentional, supports effective communication with broader audiences, and directly connects researchers with those who they are striving to help. Despite this utility, many cancer research scientists do not work with patient advocates. To understand barriers to engagement and build a framework for enhanced interactions in the future, we hosted a workshop with patient advocates and researchers who do engage, then discussed findings at an international metastatic breast cancer conference to solicit additional feedback and suggestions. Findings demonstrate that researchers are uncertain about how to initiate and maintain relationships with advocates. We offer actionable steps to support researchers working with patient advocates to improve cancer research and accomplish our collective goal of improving lives of those who have been diagnosed with breast cancer. We hope that this initiative will facilitate such collaborative efforts.

Chemotherapy Coupled to Macrophage Inhibition Induces T-cell and B-cell Infiltration and Durable Regression in Triple-Negative Breast Cancer.

Immunosuppressive elements within the tumor microenvironment, such as tumor-associated macrophages (TAM), can present a barrier to successful antitumor responses by cytolytic T cells. Here we employed preclinical syngeneic p53 null mouse models of triple-negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small-molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was effective in treating several highly aggressive TNBC murine mammary tumor and lung metastasis models. Single-cell RNA sequencing characterized tumor-infiltrating lymphocytes including Th cells and antigen-presenting B cells that were highly enriched in responders to combination therapy. In one model that exhibited long-term posttreatment tumor regression, high-dimensional imaging techniques identified the close spatial localization of B220+/CD86+-activated B cells and CD4+ T cells in tertiary lymphoid structures that were present up to 6 weeks posttreatment. The transcriptional and metabolic heterogeneity of TAMs was also characterized in two closely related claudin-low/mesenchymal subtype tumor models with differential treatment responses. A murine TAM signature derived from the T12 model was highly conserved in human claudin-low breast cancers, and high expression of the TAM signature correlated with reduced overall survival in patients with breast cancer. This TAM signature may help identify human patients with claudin-low breast cancer that will benefit from the combination of cyclophosphamide and anti-CSF1R therapy. These studies illustrate the complexity of the tumor immune microenvironment and highlight different immune responses that result from rational immunotherapy combinations. SIGNIFICANCE: Immunostimulatory chemotherapy combined with pharmacologic inhibition of TAMs results in durable treatment responses elicited by Th cells and B cells in claudin-low TNBC models.

Harnessing the power of antibodies to fight bone metastasis.

Antibody-based therapies have proved to be of great value in cancer treatment. Despite the clinical success of these biopharmaceuticals, reaching targets in the bone microenvironment has proved to be difficult due to the relatively low vascularization of bone tissue and the presence of physical barriers. Here, we have used an innovative bone-targeting (BonTarg) technology to generate a first-in-class bone-targeting antibody. Our strategy involves the use of pClick antibody conjugation technology to chemically couple the bone-targeting moiety bisphosphonate to therapeutic antibodies. Bisphosphonate modification of these antibodies results in the delivery of higher conjugate concentrations to the bone metastatic niche, relative to other tissues. In xenograft mice models, this strategy provides enhanced inhibition of bone metastases and multiorgan secondary metastases that arise from bone lesions. Specific delivery of therapeutic antibodies to the bone, therefore, represents a promising strategy for the treatment of bone metastatic cancers and other bone diseases.

The bone microenvironment invigorates metastatic seeds for further dissemination.

Metastasis has been considered as the terminal step of tumor progression. However, recent genomic studies suggest that many metastases are initiated by further spread of other metastases. Nevertheless, the corresponding pre-clinical models are lacking, and underlying mechanisms are elusive. Using several approaches, including parabiosis and an evolving barcode system, we demonstrated that the bone microenvironment facilitates breast and prostate cancer cells to further metastasize and establish multi-organ secondary metastases. We uncovered that this metastasis-promoting effect is driven by epigenetic reprogramming that confers stem cell-like properties on cancer cells disseminated from bone lesions. Furthermore, we discovered that enhanced EZH2 activity mediates the increased stemness and metastasis capacity. The same findings also apply to single cell-derived populations, indicating mechanisms distinct from clonal selection. Taken together, our work revealed an unappreciated role of the bone microenvironment in metastasis evolution and elucidated an epigenomic reprogramming process driving terminal-stage, multi-organ metastases.

The bone microenvironment increases phenotypic plasticity of ER+ breast cancer cells.

Estrogen receptor-positive (ER+) breast cancer exhibits a strong bone tropism in metastasis. How the bone microenvironment (BME) impacts ER signaling and endocrine therapy remains poorly understood. Here, we discover that the osteogenic niche transiently and reversibly reduces ER expression and activities specifically in bone micrometastases (BMMs), leading to endocrine resistance. As BMMs progress, the ER reduction and endocrine resistance may partially recover in cancer cells away from the osteogenic niche, creating phenotypic heterogeneity in macrometastases. Using multiple approaches, including an evolving barcoding strategy, we demonstrated that this process is independent of clonal selection, and represents an EZH2-mediated epigenomic reprogramming. EZH2 drives ER+ BMMs toward a basal and stem-like state. EZH2 inhibition reverses endocrine resistance. These data exemplify how epigenomic adaptation to BME promotes phenotypic plasticity of metastatic seeds, fosters intra-metastatic heterogeneity, and alters therapeutic responses. Our study provides insights into the clinical enigma of ER+ metastatic recurrences despite endocrine therapies.

Senesce to Survive: YAP-Mediated Dormancy Escapes EGFR/MEK Inhibition.

Therapeutic resistance is a major challenge in cancer treatment. In this issue of Cancer Cell, Kurppa et al. demonstrated that a senescence-like state enables lung cancer cells to survive dual inhibition of EGFR and MEK. This was mediated by the YAP/TEAD pathway, which drives epigenomic reprogramming and EMT to counteract apoptosis.

Bone Tropism in Cancer Metastases.

Bone is a frequent site of metastases in many cancers. Both bone properties and the tumor-intrinsic traits are associated with the metastatic propensity to bone (i.e., the bone tropism). Whereas an increasing body of mechanistic studies expanded our understanding on bone tropism, they also revealed complexity across the bone lesions originated from different cancer types. In this review, we will discuss the physical, chemical, and biological properties of bone microenvironment, identify potential players in every stage of bone metastases, and introduce some of the known mechanisms regulating the bone colonization. Our objectives are to integrate the knowledge established in different biological contexts and highlight the determinants of bone tropism.

Immuno-subtyping of breast cancer reveals distinct myeloid cell profiles and immunotherapy resistance mechanisms.

Cancer-induced immune responses affect tumour progression and therapeutic response. In multiple murine models and clinical datasets, we identified large variations of neutrophils and macrophages that define 'immune subtypes' of triple-negative breast cancer (TNBC), including neutrophil-enriched (NES) and macrophage-enriched subtypes (MES). Different tumour-intrinsic pathways and mutual regulation between macrophages (or monocytes) and neutrophils contribute to the development of a dichotomous myeloid compartment. MES contains predominantly macrophages that are CCR2-dependent and exhibit variable responses to immune checkpoint blockade (ICB). NES exhibits systemic and local accumulation of immunosuppressive neutrophils (or granulocytic myeloid-derived suppressor cells), is resistant to ICB, and contains a minority of macrophages that seem to be unaffected by CCR2 knockout. A MES-to-NES conversion mediated acquired ICB resistance of initially sensitive MES models. Our results demonstrate diverse myeloid cell frequencies, functionality and potential roles in immunotherapies, and highlight the need to better understand the inter-patient heterogeneity of the myeloid compartment.

Metastasis Organotropism: Redefining the Congenial Soil.

Metastasis is the most devastating stage of cancer progression and causes the majority of cancer-related deaths. Clinical observations suggest that most cancers metastasize to specific organs, a process known as "organotropism." Elucidating the underlying mechanisms may help identify targets and treatment strategies to benefit patients. This review summarizes recent findings on tumor-intrinsic properties and their interaction with unique features of host organs, which together determine organ-specific metastatic behaviors. Emerging insights related to the roles of metabolic changes, the immune landscapes of target organs, and variation in epithelial-mesenchymal transitions open avenues for future studies of metastasis organotropism.

Bone Metastasis: Find Your Niche and Fit in.

Metastasis to bones is determined by both intrinsic traits of metastatic tumor cells and properties appertaining to the bone microenvironment. Bone marrow niches are critical for all major steps of metastasis, including the seeding of disseminated tumor cells (DTCs) to bone, the survival of DTCs and microscopic metastases under dormancy, and the eventual outgrowth of overt metastases. In this review, we discuss the role of bone marrow niches in bone colonization. The emphasis is on complicated and dynamic nature of cancer cells-niche interaction, which may underpin the long-standing mystery of metastasis dormancy, and represent a therapeutic target for elimination of minimal residue diseases and prevention of life-taking, overt metastases.

The Osteogenic Niche Is a Calcium Reservoir of Bone Micrometastases and Confers Unexpected Therapeutic Vulnerability.

The fate of disseminated tumor cells is largely determined by microenvironment (ME) niche. The osteogenic niche promotes cancer cell proliferation and bone metastasis progression. We investigated the underlying mechanisms using pre-clinical models and analyses of clinical data. We discovered that the osteogenic niche serves as a calcium (Ca) reservoir for cancer cells through gap junctions. Cancer cells cannot efficiently absorb Ca from ME, but depend on osteogenic cells to increase intracellular Ca concentration. The Ca signaling, together with previously identified mammalian target of rapamycin signaling, promotes bone metastasis progression. Interestingly, effective inhibition of these pathways can be achieved by danusertib, or a combination of everolimus and arsenic trioxide, which provide possibilities of eliminating bone micrometastases using clinically established drugs.

ERß alters the chemosensitivity of luminal breast cancer cells by regulating p53 function.

Estrogen receptor α (ERα)-positive breast cancers tend to develop resistance to both endocrine therapy and chemotherapy. Despite recent progress in defining molecular pathways that confer endocrine resistance, the mechanisms that regulate chemotherapy response in luminal tumors remain largely elusive. Luminal tumors often express wild-type p53 that is a major determinant of the cellular DNA damage response. Similar to p53, the second ER subtype, ERß, has been reported to inhibit breast tumorigenesis by acting alone or in collaboration with p53. However, a synergistic mechanism of action has not been described. Here, we suggest that ERß relies on p53 to elicit its tumor repressive actions in ERα-positive breast cancer cells. Upregulation of ERß and treatment with ERß agonists potentiates the tumor suppressor function of p53 resulting in decreased survival. This effect requires molecular interaction between the two proteins that disrupts the inhibitory action of ERα on p53 leading to increased transcriptional activity of p53. In addition, we show that the same interaction alters the chemosensitivity of endocrine-resistant cells including their response to tamoxifen therapy. Our results suggest a collaboration of ERß and p53 tumor suppressor activity in breast cancer cells that indicates the importance of ligand-regulated ERß as a tool to target p53 activity and improve the clinical management of resistant disease.

ERß Sensitizes NSCLC to Chemotherapy by Regulating DNA Damage Response.

The expression of wild-type estrogen receptor ß (ESR2/ERß1) correlates with clinical outcome in patients with non-small cell lung cancer (NSCLC). However, the molecular mechanism that accounts for this association is currently poorly understood. ERß1 was previously linked to chemotherapy response in patients with breast cancer and in breast cancer cells. The effect of the receptor in NSCLC cells after chemotherapy treatment, a common remedy for advanced NSCLC, has not been studied. Here, upregulation of ERß1 increases the sensitivity of NSCLC cells to treatment with doxorubicin and etoposide. This effect was primarily observed in p53-defecient NSCLC cells. In these cells, ERß1 either enhanced G2-M cell-cycle arrest by activating the checkpoint kinase 1 (Chk1) and altering downstream signaling or induced apoptosis. The expression of p63 target genes that control G2-M checkpoint activation was altered by ERß1 suggesting an ERß1-p63 transcriptional cooperation in lung cancer cells that affects DNA damage response (DDR). These results suggest involvement of ERß1 in the mechanism that regulates DNA damage response in NSCLC cells and support the potential predictive and therapeutic value of the receptor in clinical management of the disease.Implications: This study demonstrating the impact of ERß1 on chemosensitivity of NSCLC cells suggests the predictive value of the receptor for successful response of tumors to chemotherapy and the potential benefit of chemotherapy-treated patients from the use of ER ligands. Mol Cancer Res; 16(2); 233-42. ©2017 AACR.

Somatic loss of estrogen receptor beta and p53 synergize to induce breast tumorigenesis.

BACKGROUND: Upregulation of estrogen receptor beta (ERß) in breast cancer cells is associated with epithelial maintenance, decreased proliferation and invasion, and a reduction in the expression of the receptor has been observed in invasive breast tumors. However, proof of an association between loss of ERß and breast carcinogenesis is still missing. METHODS: To study the role of ERß in breast oncogenesis, we generated mouse conditional mutants with specific inactivation of ERß and p53 in the mammary gland epithelium. For epithelium-specific knockout of ERß and p53, ERß F/F and p53 F/F mice were crossed to transgenic mice that express the Cre recombinase under the control of the human keratin 14 promoter. RESULTS: Somatic loss of ERß significantly accelerated formation of p53-deficient mammary tumors. Loss of the receptor also resulted in the development of less differentiated carcinomas with stronger spindle cell morphology and decreased expression of luminal epithelial markers. CONCLUSIONS: Our results show that synergism between ERß and p53 inactivation functions to determine important aspects of breast oncogenesis and cancer progression.

ERß decreases the invasiveness of triple-negative breast cancer cells by regulating mutant p53 oncogenic function.

Most (80%) of the triple-negative breast cancers (TNBCs) express mutant p53 proteins that acquire oncogenic activities including promoting metastasis. We previously showed that wild-type ERß (ERß1) impedes epithelial to mesenchymal transition (EMT) and decreases the invasiveness of TNBC cells. In the present study we searched for signaling pathways that ERß1 uses to inhibit EMT and invasion in TNBC cells. We show that ERß1 binds to and opposes the transcriptional activity of mutant p53 at the promoters of genes that regulate metastasis. p63 that transcriptionally cooperates with mutant p53 also binds to ERß1. Downregulation of p63 represses the epithelial phenotype of ERß1-expressing cells and alters the expression of mutant p53 target genes. These results describe a novel mechanism through which ERß1 can disturb oncogenic signals to inhibit aggressiveness in TNBCs.