Esther N. Arwert

Epithelial stem cells, wound healing and cancer

It is well established that tissue repair depends on stem cells and that chronic wounds predispose to tumour formation. However, the association between stem cells, wound healing and cancer is poorly understood. Lineage tracing has now shown how stem cells are mobilized to repair skin wounds and how they contribute to skin tumour development. The signalling pathways, including WNT and Hedgehog, that control stem cell behaviour during wound healing are also implicated in tumour formation. Furthermore, tumorigenesis and wound repair both depend on communication between epithelial cells, mesenchymal cells and bone marrow-derived cells. These studies suggest ways to harness stem cells for wound repair while minimizing cancer risk.

Stem cells are dispensable for lung homeostasis but restore airways after injury

Local tissue stem cells have been described in airways of the lung but their contribution to normal epithelial maintenance is currently unknown. We therefore developed aggregation chimera mice and a whole-lung imaging method to determine the relative contributions of progenitor (Clara) and bronchiolar stem cells to epithelial maintenance and repair. In normal and moderately injured airways chimeric patches were small in size and not associated with previously described stem cell niches. This finding suggested that single, randomly distributed progenitor cells maintain normal epithelial homeostasis. In contrast we found that repair following severe lung injury resulted in the generation of rare, large clonal cell patches that were associated with stem cell niches. This study provides evidence that epithelial stem cells are dispensable for normal airway homeostasis. We also demonstrate that stem cell activation and robust clonal cellular expansion occur only during repair from severe lung injury.

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.

Tumor formation initiated by nondividing epidermal cells via an inflammatory infiltrate

In mammalian epidermis, integrin expression is normally confined to the basal proliferative layer that contains stem cells. However, in epidermal hyperproliferative disorders and tumors, integrins are also expressed by suprabasal cells, with concomitant up-regulation of Erk mitogen-activated protein kinase (MAPK) signaling. In transgenic mice, expression of activated MAPK kinase 1 (MEK1) in the suprabasal, nondividing, differentiated cell layers (InvEE transgenics) results in epidermal hyperproliferation and skin inflammation. We now demonstrate that wounding induces benign tumors (papillomas and keratoacanthomas) in InvEE mice. By generating chimeras between InvEE mice and mice that lack the MEK1 transgene, we demonstrate that differentiating, nondividing cells that express MEK1 stimulate adjacent transgene-negative cells to divide and become incorporated into the tumor mass. Dexamethasone treatment inhibits tumor formation, suggesting that inflammation is involved. InvEE skin and tumors express high levels of IL1α; treatment with an IL1 receptor antagonist delays tumor onset and reduces incidence. Depletion of γδ T cells and macrophages also reduces tumor incidence. Because a hallmark of cancer is uncontrolled proliferation, it is widely assumed that tumors arise only from dividing cells. In contrast, our studies show that differentiated epidermal cells can initiate tumor formation without reacquiring the ability to divide and that they do so by triggering an inflammatory infiltrate.

Innate sensing of microbial products promotes wound-induced skin cancer

The association between tissue damage, chronic inflammation and cancer is well known. However, the underlying mechanisms are unclear. Here we characterize a mouse model in which constitutive epidermal extracellular-signal-regulated kinase-MAP-kinase signalling results in epidermal inflammation, and skin wounding induces tumours. We show that tumour incidence correlates with wound size and inflammatory infiltrate. Ablation of tumour necrosis factor receptor (TNFR)-1/-2, Myeloid Differentiation primary response gene 88 or Toll-like receptor (TLR)-5, the bacterial flagellin receptor, but not other innate immune sensors, in radiosensitive leukocytes protects against tumour formation. Antibiotic treatment inhibits, whereas injection of flagellin induces, tumours in a TLR-5-dependent manner. TLR-5 is also involved in chemical-induced skin carcinogenesis in wild-type mice. Leukocytic TLR-5 signalling mediates upregulation of the alarmin HMGB1 (High Mobility Group Box 1) in wound-induced papillomas. HMGB1 is elevated in tumours of patients with Recessive Dystrophic Epidermolysis Bullosa, a disease characterized by chronic skin damage. We conclude that in our experimental model the combination of bacteria, chronic inflammation and wounding cooperate to trigger skin cancer.

Upregulation of CD26 expression in epithelial cells and stromal cells during wound-induced skin tumour formation.

We have previously described InvEE transgenic mice in which non-dividing, differentiating epidermal cells express oncogenically activated MAPK kinase 1 (MEK1). Skin wounding triggers tumour formation in InvEE mice via a mechanism that involves epidermal release of IL-1α and attraction of a pro-tumorigenic inflammatory infiltrate. To look for potential effects on the underlying connective tissue, we screened InvEE and wild-type epidermis for differential expression of cytokines and immune modulators. We identified a single protein, CD26 (dipeptidyl peptidase-4). CD26 serum levels were not increased in InvEE mice. In contrast, CD26 was upregulated in keratinocytes expressing mutant MEK1 and in the epithelial compartment of InvEE tumours, where it accumulated at cell–cell borders. CD26 expression was increased in dermal fibroblasts following skin wounding but was downregulated in tumour stroma. CD26 activity was stimulated by calcium-induced intercellular adhesion in keratinocytes, suggesting that the upregulation of CD26 in InvEE epidermis is due to expansion of the differentiated cell layers. IL-1α treatment of dermal fibroblasts stimulated CD26 activity, and therefore epidermal IL-1α release may contribute to the upregulation of CD26 expression in wounded dermis. Pharmacological blockade of CD26, via Sitagliptin, reduced growth of InvEE tumours, while combined inhibition of IL-1α and CD26 delayed tumour onset and reduced tumour incidence. Our results demonstrate that inappropriate activation of MEK1 in the epidermis leads to changes in dermal fibroblasts that, like the skin inflammatory infiltrate, contribute to tumour formation.

Macrophage Infiltration and Alternative Activation during Wound Healing Promote MEK1-Induced Skin Carcinogenesis.

Macrophages are essential for the progression and maintenance of many cancers, but their role during the earliest stages of tumor formation is unclear. To test this, we used a previously described transgenic mouse model of wound-induced skin tumorigenesis, in which expression of constitutively active MEK1 in differentiating epidermal cells results in chronic inflammation (InvEE mice). Upon wounding, the number of epidermal and dermal monocytes and macrophages increased in wild-type and InvEE skin, but the increase was greater, more rapid, and more sustained in InvEE skin. Macrophage ablation reduced tumor incidence. Furthermore, bioluminescent imaging in live mice to monitor macrophage flux at wound sites revealed that macrophage accumulation was predictive of tumor formation; wounds with the greatest number of macrophages at day 5 went on to develop tumors. Gene expression profiling of flow-sorted monocytes, macrophages, and T cells from InvEE and wild-type skin showed that as wound healing progressed, InvEE macrophages altered their phenotype. Throughout wound healing and after wound closure, InvEE macrophages demonstrated sustained upregulation of several markers implicated in alternative macrophage activation including arginase-1 (ARG1) and mannose receptor (CD206). Notably, inhibition of ARG1 activity significantly reduced tumor formation and epidermal proliferation in vivo, whereas addition of L-arginase to cultured keratinocytes stimulated proliferation. We conclude that macrophages play a key role in early, inflammation-mediated skin tumorigenesis, with mechanistic evidence suggesting that ARG1 secretion drives tumor development by stimulating epidermal cell proliferation. These findings highlight the importance of cancer immunotherapies aiming to polarize tumor-associated macrophages toward an antitumor phenotype.

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.

Abstract 4940: Perivascular macrophages induce localized, transient blood vessel permeability and tumor cell intravasation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Metastasis is a multistep process involving tumor and stromal cells. The microanatomical site consisting of a perivascular macrophage interacting with a Mena over-expressing tumor cell has been named the “tumor microenvironment of metastasis” (TMEM). TMEM density predicts distant metastatic recurrence in breast cancer patients making the study of TMEM function essential. In spontaneous orthotopic mouse mammary tumors (MMTV-PyMT), as the tumor progresses to malignancy tumor cells have increased Mena expression and assemble TMEM. TMEM assembly is correlated with elevated levels of circulating tumor cells and lung metastases, indicating a functional role for TMEM in tumor cell dissemination. High-resolution multiphoton-based microscopy at single cell resolution has revealed transient blood vessel permeability events at TMEM, marked by the extravasation of otherwise impermeable serum markers such as Qdots and fluorescent 155 kDa dextran. In proximity to sites of permeability there is increased tumor cell and macrophage motility towards the blood vessel, and local intravasation of tumor cells. Blocking macrophage function results in decreased blood vessel permeability and decreased numbers of circulating tumor cells. TMEM-associated macrophages produce VEGF-A, and blocking VEGF-A reduces blood vessel permeability and circulating tumor cells. The observation that TMEM mediates tumor cell intravasation through localized blood vessel permeability in live animals demonstrates that TMEM is a key metastatic microenvironment in the primary tumor, explains the prognostic value of TMEM density in predicting distant metastatic recurrence in breast cancer patients and suggests a potentially valuable re-tasking of existing anti-angiogenesis drugs. This research is supported by the Department of Defense [Breast Cancer Research Program)] under award number BC120227 (ASH). Note: This abstract was not presented at the meeting. Citation Format: Allison S. Harney, Esther N. Arwert, David Entenberg, Yarong Wang, Joan G. Jones, John S. Condeelis. Perivascular macrophages induce localized, transient blood vessel permeability and tumor cell intravasation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4940. doi:10.1158/1538-7445.AM2014-4940

Abstract 5125: Imaging the tumor microenvironment of metastasis reveals the mechanism of transient blood vessel permeability and tumor cell intravasation

Sites of direct contact between a macrophage, a tumor cell and endothelial cell [Tumor MicroEnvironment of Metastasis (TMEM)], correlates with metastasis in breast cancer patients independently of other clinical prognostic indicators suggesting a direct role for TMEM function in hematogenous dissemination. Here we show, using intravital high-resolution two-photon microscopy, that tumor cell intravasation occurs only at TMEM. Tumor cell intravasation occurs concurrently with transient, local vascular permeability at TMEM in an autochthonous mouse mammary carcinoma model and a human patient-derived xenograft model. Ablation of the presence or activity of the TMEM associated macrophages blocks tumor cell intravasation at TMEM demonstrating an essential role of perivascular macrophages in TMEM function. A subset of TMEM macrophages are identified as Tie2-expressing macrophages that are characterized by F4/80+/CD11b+/CD68+/MRC1+/Tie2 Hi /VEGFA Hi /CD11c-. VEGFA signaling from Tie2 Hi TMEM-associated macrophages causes the local loss of vascular junctions resulting in transient vascular permeability and tumor cell intravasation at TMEM. Macrophage-specific ablation of VEGFA results in increased vascular junction stability and inhibition of intravasation, demonstrating that vascular junction dissolution at VEGFA Hi /Tie2 Hi TMEM-associated macrophages leads to vascular permeability and tumor cell intravasation. Inhibition of Tie2 with the first in class small molecular inhibitor rebastinib impairs TMEM function leading to a reduction in vascular permeability, tumor cell dissemination and metastasis. Further, rebastinib inhibition of Tie2 blocks tumor cell extravasation and metastatic growth in the lungs. Together, the findings that TMEM macrophages mediate vascular permeability and tumor cell intravasation demonstrate an essential role for TMEM within the primary mammary tumor as sites of tumor cell dissemination. These data reveal the mechanism of tumor cell intravasation in breast cancer, explain the prognostic value of TMEM density in predicting distant metastatic recurrence in breast cancer patients and document a strategy for inhibition of dissemination. This research is supported by the Department of Defense Breast Cancer Research Program under award number BC120227 (ASH), NIH CA100324 (JSC) and the Integrated Imaging Program. Citation Format: Allison S. Harney, Esther N. Arwert, David Entenberg, Yarong Wang, Peng Guo, Bin-Zhi Qian, Bryan D. Smith, Jeffrey W. Pollard, Joan G. Jones, Daniel L. Flynn, John S. Condeelis. Imaging the tumor microenvironment of metastasis reveals the mechanism of transient blood vessel permeability and tumor cell intravasation. [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 5125. doi:10.1158/1538-7445.AM2015-5125