Integrin-Mediated Macrophage Adhesion Promotes Lymphovascular Dissemination in Breast Cancer

Lymphatic vasculature is crucial for metastasis in triple-negative breast cancer (TNBC); however, cellular and molecular drivers controlling lymphovascular metastasis are poorly understood. We define a macrophage-dependent signaling cascade that facilitates metastasis through lymphovascular remodeling. TNBC cells instigate mRNA changes in macrophages, resulting in beta 4 integrin-dependent adhesion to the lymphovasculature. beta 4 integrin retains macrophages proximal to lymphatic endothelial cells (LECs), where release of TGF-beta 1 drives LEC contraction via RhoA activation. Macrophages promote gross architectural changes to lymphovasculature by increasing dilation, hyperpermeability, and disorganization. TGF-beta 1 drives beta 4 integrin clustering at the macrophage plasma membrane, further promoting macrophage adhesion and demonstrating the dual functionality of TGF-beta 1 signaling in this context. beta 4 integrin-expressing macrophages were identified in human breast tumors, and a combination of vascular-remodeling macrophage gene signature and TGF-beta signaling scores correlates with metastasis. We postulate that future clinical strategies for patients with TNBC should target crosstalk between beta 4 integrin and TGF-beta 1.

The interplay between the VEGF-C/VEGFR-3 axis and CCL21/CCR7 axis in tumor cell invasion

Amine Issa

There are two main routes for tumor spread and metastasis, the blood vasculature and the lymphatic system. A wide range of human carcinomas, including lung, colon and breast cancer, spread to distant sites via the lymphatic system. Preclinical and clinical studies have demonstrated a positive correlation between the incidence of lymph node metastasis and the secretion of vascular endothelial growth factor-C (VEGF-C) by tumor cells. Indeed, VEGF-C is critical for the formation of new lymphatic vessels in a process called lymphangiogenesis, suggesting this mechanism as the main "escape route" for tumor cells. However, many studies have failed to identify functional lymphatic vessels inside tumors, and much research has demonstrated that metastasis can occur in the absence of lymphangiogenesis. Furthermore, there is growing evidence that many tumor cells may express vascular endothelial growth factor receptor-3 (VEGFR-3), the primary receptor for VEGF-C, suggesting that autologous VEGFR-3 signaling loops may enhance the invasive and metastatic potential of tumor cells. Other than lymphangiogenic factors, the lymphatic homing chemokine receptor CCR7 is strongly correlated to lymph node metastasis, suggesting that tumor cells mimic dendritic cells in sensing and homing to lymphatic vessels and lymph nodes. Potential mechanisms underlying these observations have not been elucidated. Using several in vitro 3D models, we examined the interactions between tumor and lymphatic endothelial cells (LECs) to gain insight into the mechanistic roles of VEGF-C and CCR7-mediated tumor cell invasion towards lymphatics as well as elucidate their potential interplay. All the designed experimental setups utilized a 3D matrix for a more physiologically relevant chemokine and growth factor signaling environment. For example, we designed one culture model where tumor cells are seeded in one gel compartment and lymphatic endothelial cells in a horizontally adjacent one, allowing both cell types to establish contact through soluble mediators without being initially mixed while simultaneously allowing direct visualization of their interactions. We first demonstrated that receptor expression patterns by cells are strongly influenced by the extracellular matrix (ECM). Indeed, while we failed to detect tumor expression of VEGFR-3 in 2D conditions, we found both expression and phosphorylation of VEGFR-3 in three of four tumor cell lines tested. Furthermore, in the three VEGFR-3 positive cell lines, invasive capacity and chemoattraction towards lymphatics was demonstrated to be dependent on both VEGF-C secretion and CCR7 expression by the tumor cells. We found that VEGF-C could act in both an autocrine manner, by enhancing the migratory and invasive capacity of tumor cells, and in a paracrine manner where it enhances CCL21 secretion by LECs to promote the chemoinvasion of tumor cells via CCL21 secretion. The same mechanism was also tested in vivo by injecting VEGF-C into mouse skin: VEGF-C, but not saline nor VEGF-A, caused marked upregulation of CCL21 associated with lymphatic vessels. In this way, VEGF-C and CCR7 act synergistically to promote the invasive phenotype of tumor cells. Additionally, we uncovered a new and critical role for VEGFR-3 on mammary epithelial cells. We demonstrated that the receptor is involved in epithelial tubulogenesis in a collagen matrix, and furthermore, it helps to preserve the integrity of a mammary cell layer (since blocking the receptor leads to increased epithelial permeability). In conclusion, this work highlights the importance of the VEGFR-3/VEGF-C and CCR7/CCL21 axis in promoting tumor cell migration and invasion. It also shows that VEGFR-3 may play many roles in the process of tumorigenesis and metastasis.

EPFL2009

TIE-2-expressing monocytes are lymphangiogenic and associate specifically with lymphatics of human breast cancer

George Coukos, Nicolas Jean Philippe Guex, Luc Henry, Emanuela Marina Iancu, Hans-Anton Lehr, Nathalie Rufer, Ioannis Xenarios

In experimental mouse models of cancer, increasingly compelling evidence point toward a contribution of tumor associated macrophages (TAM) to tumor lymphangiogenesis. Corresponding experimental observations in human cancer remain scarce although lymphatic metastasis is widely recognized as a predominant route for tumor spread. We previously showed that, in malignant tumors of untreated breast cancer (BC) patients, TIE-2-expressing monocytes (TEM) are highly proangiogenic immunosuppressive cells and that TIE-2 and VEGFR signaling pathways drive TEM immunosuppressive function. We report here that, in human BC, TEM express the canonical lymphatic markers LYVE-1, Podoplanin, VEGFR-3 and PROX-1. Critically, both TEM acquisition of lymphatic markers and insertion into lymphatic vessels were observed in tumors but not in adjacent non-neoplastic tissues, suggesting that the tumor microenvironment shapes both TEM phenotype and spatial distribution. We assessed the lymphangiogenic activity of TEM isolated from dissociated primary breast tumors in vitro and in vivo using endothelial cells (EC) sprouting assay and corneal vascularization assay, respectively. We show that, in addition to their known hemangiogenic function, TEM isolated from breast tumor display a lymphangiogenic activity. Importantly, TIE-2 and VEGFR pathways display variable contributions to TEM angiogenic and lymphangiogenic activities across BC patients; however, combination of TIE-2 and VEGFR kinase inhibitors abrogated these activities and overcame inter-patient variability. These results highlight the direct contribution of tumor TEM to the breast tumor lymphatic network and suggest a combined use of TIE-2 and VEGFR kinase inhibitors as a therapeutic approach to block hem-and lymphangiogenesis in BC.

Taylor & Francis Inc2016

Elucidating the role of lymphatic vessels within the tumor microenvironment of breast cancer

Ingrid Maria van Mier

Breast cancer is the most common cancer in women worldwide, with an estimated 1.7 million newly diagnosed cases in 2012. Despite the many clinical manifestations, metastatic disease is the main cause of death, and is responsible for 90% of all breast cancer-related deaths. Aggressive carcinomas of the breast preferentially spread via the lymphogenous route, and often co-opt and actively remodel the lymphatic vasculature in order to invade and metastasize to their draining lymph nodes (LNs) and, ultimately, to distant organs. Indeed, expression of vascular endothelial growth factor (VEGF)-C, the principal lymphangiogenic growth factor, by the tumor or recruited ancillary cells has been shown to correlate both with LN and distant metastasis, and shorter overall survival (OS), and LN status represents an important prognostic factor for patients diagnosed with breast cancer. Although traditionally considered as passive conduits that solely mediate tumor cell escape, more recent research aimed at discerning the interactions between the developing tumor and its associated lymphatic vessels has revealed that the lymphatic vasculature actively promotes lymphogenous metastasis of invasive tumor cells. Likewise, collaborative interactions between tumor cells and a multitude of other tissue-resident and recruited ancillary cells, which together constitute the tumor microenvironment, not only coercively contribute to tumor growth, progression and metastasis, but also suggest that interactions among primed stromal cells themselves may be important in the process of metastatic dissemination as well. Notably, expansion of tumor-associated lymphatic vessels in the tumor periphery leads to increased lymphatic drainage from the tumor to the tumor-draining LN, and interstitial flow-induced mechanical changes may alter tensional homeostasis within the tumor through stimulation of local fibroblasts that remodel and stiffen the extracellular matrix (ECM). Furthermore, VEGF-C-induced remodeling of the tumor-associated lymphatic vasculature has been shown to directly suppress the anti-tumor immune response, thereby promoting tolerance to the nascent tumor. Thus, to explore whether tumor-associated lymphatic vessels promote tumor metastasis not only via interactions with the incipient tumor, but also through cross-talk with the surrounding stromal cells, the different components of the tumor microenvironment, specifically tensional homeostasis and anti-tumor immunity, were evaluated upon modulation of lymphatic vessel growth in various experimental models of breast cancer. Accordingly, inhibition of VEGFR-3-mediated signaling upon administration of an antagonistic antibody in a genetically engineered preclinical mouse model specifically reduced the number of lymphatic endothelial cells (LECs) in the tumor, whereas conversely, orthotopic inoculation of a stable murine breast cancer cell line with enhanced expression of VEGF-C induced an expansion of the lymphatic endothelium. Although either experimental approach affected either regional or distant metastatic dissemination of the primary tumor to LN or the lungs, respectively, these effects did not seem to be mediated by interactions of the tumor-associated lymphatic vasculature with the tumor microenvironment in these particular experimental models.

EPFL2017