Allison S. Harney

Real-Time Imaging Reveals Local, Transient Vascular Permeability, and Tumor Cell Intravasation Stimulated by TIE2hi Macrophage–Derived VEGFA

UNLABELLED Dissemination of tumor cells is an essential step in metastasis. Direct contact between a macrophage, mammalian-enabled (MENA)-overexpressing tumor cell, and endothelial cell [Tumor MicroEnvironment of Metastasis (TMEM)] correlates with metastasis in breast cancer patients. Here we show, using intravital high-resolution two-photon microscopy, that transient vascular permeability and tumor cell intravasation occur simultaneously and exclusively at TMEM. The hyperpermeable nature of tumor vasculature is described as spatially and temporally heterogeneous. Using real-time imaging, we observed that vascular permeability is transient, restricted to the TMEM, and required for tumor cell dissemination. VEGFA signaling from TIE2(hi) TMEM macrophages causes local loss of vascular junctions, transient vascular permeability, and tumor cell intravasation, demonstrating a role for the TMEM within the primary mammary tumor. These data provide insight into the mechanism of tumor cell intravasation and vascular permeability in breast cancer, explaining the value of TMEM density as a predictor of distant metastatic recurrence in patients. SIGNIFICANCE Tumor vasculature is abnormal with increased permeability. Here, we show that VEGFA signaling from TIE2(hi) TMEM macrophages results in local, transient vascular permeability and tumor cell intravasation. These data provide evidence for the mechanism underlying the association of TMEM with distant metastatic recurrence, offering a rationale for therapies targeting TMEM.

The Multifaceted Role of Perivascular Macrophages in Tumors

Evidence has emerged for macrophages in the perivascular niche of tumors regulating important processes like angiogenesis, various steps in the metastatic cascade, the recruitment and activity of other tumor-promoting leukocytes, and tumor responses to frontline therapies like irradiation and chemotherapy. Understanding the mechanisms controlling the recruitment, retention, and function of these cells could identify important targets for anti-cancer therapeutics.

Brightness-equalized quantum dots.

As molecular labels for cells and tissues, fluorescent probes have shaped our understanding of biological structures and processes. However, their capacity for quantitative analysis is limited because photon emission rates from multicolour fluorophores are dissimilar, unstable and often unpredictable, which obscures correlations between measured fluorescence and molecular concentration. Here we introduce a new class of light-emitting quantum dots with tunable and equalized fluorescence brightness across a broad range of colours. The key feature is independent tunability of emission wavelength, extinction coefficient and quantum yield through distinct structural domains in the nanocrystal. Precise tuning eliminates a 100-fold red-to-green brightness mismatch of size-tuned quantum dots at the ensemble and single-particle levels, which substantially improves quantitative imaging accuracy in biological tissue. We anticipate that these materials engineering principles will vastly expand the optical engineering landscape of fluorescent probes, facilitate quantitative multicolour imaging in living tissue and improve colour tuning in light-emitting devices.

Neoadjuvant chemotherapy induces breast cancer metastasis through a TMEM-mediated mechanism

Chemotherapy induces prometastatic changes in breast cancer, reversible by TIE2 or MENA inhibition. Closing the door to cancer cells Breast cancer is one of the most common tumor types, and metastasis greatly increases the risk of death from this disease. By studying the process of intravasation or entry of cells into the vasculature, Karagiannis et al. discovered that, in addition to killing tumor cells, chemotherapy treatment can also increase intravasation. Groups of cells collectively known as tumor microenvironment of metastasis (TMEM) can serve as gateways for tumor cells entering the vasculature, and the authors discovered that several types of chemotherapy can increase the amounts of TMEM complexes and circulating tumor cells in the bloodstream. The researchers also determined that a drug called rebastinib can interfere with TMEM activity and help overcome the increased risk of cancer cell dissemination. Breast cancer cells disseminate through TIE2/MENACalc/MENAINV-dependent cancer cell intravasation sites, called tumor microenvironment of metastasis (TMEM), which are clinically validated as prognostic markers of metastasis in breast cancer patients. Using fixed tissue and intravital imaging of a PyMT murine model and patient-derived xenografts, we show that chemotherapy increases the density and activity of TMEM sites and Mena expression and promotes distant metastasis. Moreover, in the residual breast cancers of patients treated with neoadjuvant paclitaxel after doxorubicin plus cyclophosphamide, TMEM score and its mechanistically connected MENAINV isoform expression pattern were both increased, suggesting that chemotherapy, despite decreasing tumor size, increases the risk of metastatic dissemination. Chemotherapy-induced TMEM activity and cancer cell dissemination were reversed by either administration of the TIE2 inhibitor rebastinib or knockdown of the MENA gene. Our results indicate that TMEM score increases and MENA isoform expression pattern changes with chemotherapy and can be used in predicting prometastatic changes in response to chemotherapy. Furthermore, inhibitors of TMEM function may improve clinical benefits of chemotherapy in the neoadjuvant setting or in metastatic disease.

Imaging interactions between macrophages and tumour cells that are involved in metastasis in vivo and in vitro

Tumour‐associated macrophages participate in several protumour functions including tumour growth and angiogenesis, and facilitate almost every step of the metastatic cascade. Interfering with macrophage functions may therefore provide an important strategy in the clinical management of cancer and metastatic disease. Our understanding of macrophage functions has been greatly expanded by direct observations of macrophage–carcinoma cell interactions using light microscopy. Imaging approaches include intravital microscopy of tumours in mouse models of cancer and visualization of macrophage–carcinoma cell interactions in in vitro assays; whether atop 2D substrates, embedded in 3D matrices or in more complex assemblies of multiple cell types that mimic specific topologies of the tumour microenvironment. Such imaging and reconstitution approaches have provided us with a wealth of information on the motile behaviour and physical associations between macrophages and carcinoma cells and the role of the tumour microenvironment in influencing the movement of these cells. Finally, high‐resolution imaging techniques have permitted researchers to correlate motility patterns with specific gene signatures and biochemical pathways in cells, pointing to potential targets for intervention. Here, we review experimental approaches employed in the study of macrophage interactions with carcinoma cells with an emphasis on imaging invasive and metastatic cell motility in breast carcinomas.

The Selective Tie2 Inhibitor Rebastinib Blocks Recruitment and Function of Tie2Hi Macrophages in Breast Cancer and Pancreatic Neuroendocrine Tumors

Tumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2+ myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2Hi/Vegf-AHi macrophages in the tumor microenvironment of metastasis (TMEM). The antitumor effects of rebastinib enhance the efficacy of microtubule inhibiting chemotherapeutic agents, either eribulin or paclitaxel, by reducing tumor volume, metastasis, and improving overall survival. Rebastinib inhibition of angiopoietin/Tie2 signaling impairs multiple pathways in tumor progression mediated by protumoral Tie2+ macrophages, including TMEM-dependent dissemination and angiopoietin/Tie2-dependent angiogenesis. Rebastinib is a promising therapy for achieving Tie2 inhibition in cancer patients. Mol Cancer Ther; 16(11); 2486–501. ©2017 AACR.

A Unidirectional Transition from Migratory to Perivascular Macrophage Is Required for Tumor Cell Intravasation

Summary Tumor-associated macrophages (TAMs) are critical for tumor metastasis. Two TAM subsets support cancer cell intravasation: migratory macrophages guide cancer cells toward blood vessels, where sessile perivascular macrophages assist their entry into the blood. However, little is known about the inter-relationship between these functionally distinct TAMs or their possible inter-conversion. We show that motile, streaming TAMs are newly arrived monocytes, recruited via CCR2 signaling, that then differentiate into the sessile perivascular macrophages. This unidirectional process is regulated by CXCL12 and CXCR4. Cancer cells induce TGF-β-dependent upregulation of CXCR4 in monocytes, while CXCL12 expressed by perivascular fibroblasts attracts these motile TAMs toward the blood vessels, bringing motile cancer cells with them. Once on the blood vessel, the migratory TAMs differentiate into perivascular macrophages, promoting vascular leakiness and intravasation.

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

Abstract 397: Rebastinib potently inhibits function of perivascular TIE2 expressing macrophagesin vitroandin vivo

Introduction Significant cross-talk between tumor cells and the surrounding stromal tissue are essential for tumor vascularization, survival, immunotolerance, invasion, and metastasis. The angiopoietin (ANG)/TIE2 kinase signaling pathway is a pivotal cross talk axis in the tumor microenvironment. It has been demonstrated that TIE2-expressing macrophages (TEMs) mediate invasion and metastasis in the PyMT syngeneic breast cancer model, and that TIE2 expression correlates with poor overall survival and high risk of metastasis in breast cancer patients. In this study, rebastinib was evaluated as a TIE2 inhibitor in in vitro and in vivo models, and was shown to have profound effects on the structure and function of perivascular TEMs. Procedures TIE2 kinase assays used a standard PK/LDH coupled spectrophotometric continuous assay. CHO cells were transiently transfected to express human TIE2 for cellular studies. CHOs, HUVECs and EA.hy926 cells were used to evaluate rebastinib for inhibition of ANG1-stimulated TIE2 phosphorylation. In vitro intravasation studies were performed in a transwell transendothelial migration chamber wherein TIE2HI macrophages interact with breast tumor cells to cause transendothelial migration of tumor cells across a sealed HUVEC endothelial monolayer. In vivo evaluations of rebastinib were performed using intravital high-resolution two-photon microscopy in the murine PyMT breast cancer model to evaluate effects on tumor vascular permeability and tumor cell intravasation. Results Rebastinib is a potent inhibitor of TIE2 kinase (IC50 = 0.63 nM). Rebastinib slowly dissociated from TIE2 (koff = 0.0012 minutes−1; T1/2 = 10 hr). In HUVECs or EA.hy926 cells, rebastinib inhibited ANG1-stimulated TIE2 kinase activity (IC50s of 0.018 and 0.091 nM, respectively). In TIE2 CHO cells, rebastinib inhibited TIE2 phosphorylation (IC50 2.0 nM), and demonstrated a prolonged off-rate (> 24 hr) against TIE2 after inhibitor washout. Rebastinib exhibited an IC50 Rebastinib was evaluated in vivo in the PyMT syngeneic breast cancer model. Rebastinib dosed at 10 mg/kg orally twice weekly impaired tumoral perivascular TEMs, resulting in a significant reduction in vascular permeability and in tumor cell intravasation as quantified by CTCs. Conclusion Rebastinib is a potent inhibitor of TIE2 kinase and exhibits durable cellular inhibition in endothelial cells and in TIE2 macrophages. Oral dosing of rebastinib resulted in a significant reduction in TIE2-macrophage mediated tumor vascular permeability and in the intravasation of tumor cells into the circulation. Rebastinib is currently in Phase 1 clinical evaluation in solid tumors. Citation Format: Allison Harney, Jeanine Pignatelli, Edison Leung, Maja Oktay, Yarong Wang, Bryan D. Smith, Daniel L. Flynn, John S. Condeelis. Rebastinib potently inhibits function of perivascular TIE2 expressing macrophages in vitro and in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 397. doi:10.1158/1538-7445.AM2015-397

The Tumor Microenvironment as a Metastasis Biomarker in Breast Cancer

Distant metastasis is the primary cause of death in breast cancer, and metastasis often occurs despite potentially curative local therapy of the primary tumor. Prognostic factors for distant recurrence after local therapy are largely “tumor-centric”. Defining the interactions between tumor cells and their microenvironment, and identifying the molecular mechanisms that define their interactions, provides a basis for development of metastasis biomarkers, and the ability to therapeutically target individual steps in the metastatic cascade. In vivo imaging modalities and other techniques have facilitated identification of the steps required for metastasis. These initial steps include streaming of tumor cells toward endothelial cells in collaboration with tumor-associated macrophages, formation of microanatomic structures consisting of tumor cells, macrophages, and endothelial cells, and transendothelial migration of tumor cells at these sites resulting in intravasation and dissemination to distant sites. Metastasis biomarkers that have been associated with distant recurrence in humans, and are based on observations of the tumor microenvironment, include a multiplex immunofluorescence assay that measures invasive isoforms of the actin regulatory protein Mena (a marker which we call “Menacalc”) that enable tumor cell streaming. Multiplex immunohistochemical assays that identify where Mena-expressing tumor cells, endothelial cells, and macrophages form microanatomic structures can serve as platforms for transendothelial migration, intravasation, and metastasis (which we call “TMEM” for tumor microenvironment of metastasis). An understanding of the signaling molecules that drive these interactions may provide a foundation for developing therapeutic strategies to prevent metastasis. There is potential for these biomarkers to both exhibit clinical utility by more accurately characterizing prognosis and thus the potential to benefit from standard therapies. They could also to predict benefit from novel interventions that prevent metastasis.