Simona Podgrabinska

Molecular characterization of lymphatic endothelial cells

The lymphatic microvasculature is uniquely adapted for the continuous removal of interstitial fluid and proteins and is an important entry point for leukocytes and tumor cells. Specialized functions of lymphatics suggest differences in the molecular composition of the lymphatic and blood vascular endothelium. However, the extent to which the two cell types differ is still unclear, and few molecules that are truly specific to lymphatic endothelial cells have been identified to date. We have isolated primary lymphatic and blood microvascular endothelial cells from human skin by immunoselection with the lymphatic marker LYVE-1 and demonstrate that the two cell lineages express distinct sets of vascular markers and respond differently to growth factors and extracellular matrix. Comparative microarray analysis of gene-expression profiles revealed a number of unique molecular properties that distinguish lymphatic and blood vascular endothelium. The molecular profile of lymphatic endothelium seems to reflect characteristic functional and structural features of the lymphatic capillaries. Classification of the differentially expressed genes into functional groups revealed particularly high levels of genes implicated in protein sorting and trafficking, indicating a more active role of lymphatic endothelium in uptake and transport of molecules than previously anticipated. The identification of a large number of genes selectively expressed by lymphatic endothelium should facilitate the discovery of hitherto unknown lymphatic vessel markers and provide a basis for the analysis of the molecular mechanisms accounting for the characteristic functions of lymphatic capillaries.

Inhibition of VEGFR-3 Activation with the Antagonistic Antibody More Potently Suppresses Lymph Node and Distant Metastases than Inactivation of VEGFR-2

Lymph nodes are the first site of metastases for most types of cancer, and lymph node status is a key indicator of patient prognosis. Induction of tumor lymphangiogenesis by vascular endothelial growth factor-C (VEGF-C) has been shown to play an important role in promoting tumor metastases to lymph nodes. Here, we employed receptor-specific antagonist antibodies in an orthotopic spontaneous breast cancer metastasis model to provide direct evidence for the key role of VEGFR-3 activation in metastasis. Inhibition of VEGFR-3 activation more potently suppressed regional and distant metastases than inactivation of VEGFR-2, although VEGFR-2 blockade was more effective in inhibiting angiogenesis and tumor growth. Despite prominent proliferation, metastases were not vascularized in any of the control and treatment groups, indicating that the growth of metastases was not dependent on angiogenesis at the secondary site for the duration of the experiment. Systemic treatment with either VEGFR-2 or VEGFR-3 antagonistic antibodies suppressed tumor lymphangiogenesis, indicating that VEGFR-3 signaling affects the rate of tumor cell entry into lymphatic vessels through both lymphangiogenesis-dependent and independent mechanisms. Combination treatment with the anti-VEGFR-2 and anti-VEGFR-3 antibodies more potently decreased lymph node and lung metastases than each antibody alone. These results validate the concept of targeting the lymphatic dissemination and thereby very early steps of the metastatic process for metastasis control and suggest that a combination therapy with antiangiogenic agents may be a particularly promising approach for controlling metastases.

Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

The Notch family of cell surface receptors and its ligands are highly conserved proteins that regulate cell fate determination, including those involved in mammalian vascular development. We report that Notch induces VEGFR-3 expression in vitro in human endothelial cells and in vivo in mice. In vitro, Notch in complex with the DNA-binding protein CBF-1/suppressor of hairless/Lag1 (CSL) bound the VEGFR-3 promoter and transactivated VEGFR-3 specifically in endothelial cells. Through induction of VEGFR-3, Notch increased endothelial cell responsiveness to VEGF-C, promoting endothelial cell survival and morphological changes. In vivo, VEGFR-3 was upregulated in endothelial cells with active Notch signaling. Mice heterozygous for null alleles of both Notch1 and VEGFR-3 had significantly reduced viability and displayed midgestational vascular patterning defects analogous to Notch1 nullizygous embryos. We found that Notch1 and Notch4 were expressed in normal and tumor lymphatic endothelial cells and that Notch1 was activated in lymphatic endothelium of invasive mammary micropapillary carcinomas. These results demonstrate that Notch1 and VEGFR-3 interact genetically, that Notch directly induces VEGFR-3 in blood endothelial cells to regulate vascular development, and that Notch may function in tumor lymphangiogenesis.

Inflamed lymphatic endothelium suppresses dendritic cell maturation and function via Mac-1/ICAM-1-dependent mechanism.

The lymphatic system is essential for the generation of immune responses by facilitating immune cell trafficking to lymph nodes. Dendritic cells (DCs), the most potent APCs, exit tissues via lymphatic vessels, but the mechanisms of interaction between DCs and the lymphatic endothelium and the potential implications of these interactions for immune responses are poorly understood. In this study, we demonstrate that lymphatic endothelial cells (LECs) modulate the maturation and function of DCs. Direct contact of human monocyte-derived DCs with an inflamed, TNF-α-stimulated lymphatic endothelium reduced expression of the costimulatory molecule CD86 by DCs and suppressed the ability of DCs to induce T cell proliferation. These effects were dependent on adhesive interactions between DCs and LECs that were mediated by the binding of Mac-1 on DCs to ICAM-1 on LECs. Importantly, the suppressive effects of the lymphatic endothelium on DCs were observed only in the absence of pathogen-derived signals. In vivo, DCs that migrated to the draining lymph nodes upon inflammatory stimuli, but in the absence of a pathogen, showed increased levels of CD86 expression in ICAM-1-deficient mice. Together, these data demonstrate a direct role of LECs in the modulation of immune response and suggest a function of the lymphatic endothelium in preventing undesired immune reactions in inflammatory conditions.

Activation of NF-κB by the Latent vFLIP Gene of Kaposi's Sarcoma-Associated Herpesvirus Is Required for the Spindle Shape of Virus-Infected Endothelial Cells and Contributes to Their Proinflammatory Phenotype

ABSTRACT Kaposis sarcoma (KS) is an inflammatory angioproliferative lesion induced by the infection of endothelial cells with the KS-associated herpesvirus (KSHV). Infected endothelial cells assume an elongated (spindle) shape that is one of the histologic signatures of KS. In vitro, latent viral infection of primary endothelial cells (but no other cell type) strikingly recapitulates these morphological findings. Here we report that the spindling phenotype involves major rearrangement of the actin cytoskeleton and can be attributed to the expression of a single viral protein, vFLIP, a known activator of NF-κB. Consistent with this, the inhibition of NF-κB activation blocks vFLIP-induced spindling in cultured endothelial cells. vFLIP expression in spindle cells also induces the production of a variety of proinflammatory cytokines and cell surface adhesion proteins that likely contribute to the inflammatory component of KS lesions.

Tumor cell entry into the lymph node is controlled by CCL1 chemokine expressed by lymph node lymphatic sinuses

Blocking CCR8 inhibits entry of metastases from the collecting lymphatic vessel into the lymph node.

Role of lymphatic vasculature in regional and distant metastases

In cancer, lymphatic vasculature has been traditionally viewed only as a transportation system for metastatic cells. It has now become clear that lymphatics perform many additional functions which could influence cancer progression. Lymphangiogenesis, induced at the primary tumor site and at distant sites, potently augments metastasis. Lymphatic endothelial cells (LECs) control tumor cell entry and exit from the lymphatic vessels. LECs also control immune cell traffic and directly modulate adaptive immune responses. This review highlights advances in our understanding of the mechanisms by which lymphatic vessels, and in particular lymphatic endothelium, impact metastasis.

Vascular Endothelial Growth Factor-C Induces Lymphangitic Carcinomatosis, an Extremely Aggressive Form of Lung Metastases

The lymphatic system is an important pathway for tumor dissemination to the lymph nodes, but to which extent it contributes to the formation of distant metastases remains unknown. We report that induction of lymphangiogenesis by vascular endothelial growth factor-C (VEGF-C) at the secondary site, in the lung, facilitates expansion of already disseminated cancer cells throughout the lung tissue. By using orthotopic spontaneous metastasis models in nude mice, we show that VEGF-C expression by tumor cells altered the pattern of pulmonary metastases from nodular to diffuse and facilitated disease progression. Metastases expressing VEGF-C were tightly associated with the airways, in contrast to the control cells that were scattered in the lung parenchyma, throughout the alveolar region. VEGF-C induced lung lymphangiogenesis and promoted intralymphatic spread of metastases in the lung and formation of tumor emboli in the pulmonary arteries. This pattern of metastasis corresponds to lymphangitic carcinomatosis metastatic phenotype in human cancer patients, an extremely aggressive pattern of pulmonary metastases. In accordance, pulmonary breast cancer metastases from patients which were classified as lymphangitic carcinomatosis showed high levels of VEGF-C expression in cancer cells. These data show that VEGF-C promotes late steps of the metastatic process and identify the VEGF-C/VEGF receptor-3 pathway as the target not only for prevention of metastases, but also for treatment of established metastatic disease.

Abstract LB-333: Lymphatic endothelium increases antioxidant capacity of triple-negative breast cancer cells and protects from cell death

Involvement of lymphatic system with cancer and the extent of lymph node metastases are directly correlated with the poor patient outcome. However, it is not understood whether the presence of lymphatic metastases is only indicative of an aggressive cancer or if the lymphatic vessel microenvironment directly contributes to the metastatic progression. We demonstrate that soluble factors produced by lymphatic endothelial cells (LECs) protect triple negative breast cancer cells from cell death in vitro. Co-culture with LECs or LEC-conditioned medium (LEC-CM) protected cancer cells from death induced by the loss of homotypic cell adhesion, nutrient deprivation, or loss of matrix attachment. High levels of reactive oxygen species (ROS) preceded cell death, and were significantly decreased in tumor cells upon treatment with LEC-CM. Furthermore, LEC-CM protected tumor cells from death induced by exogenous oxidative stress (H2O2), while treatment with the anti-oxidant N-acetyl-cysteine (NAC) recapitulated the cytoprotective effect of LEC-CM. RNA-Seq analysis revealed Nrf2 pathway as the most upregulated stress-pathway induced in tumor cells upon loss of adhesion. Nrf2 and other stress signaling pathways were significantly diminished in the presence of LEC-CM. Pharmacological inhibition of the pentose phosphate pathway (PPP) and the components of thioredoxin and glutathione scavanging systems increased ROS and cell death in LEC-CM indicating that the maintenance of redox homeostasis and cell viability by LEC-CM is dependent on the PPP pathway and in particular thioredoxin system. Furthermore, LEC-CM preserved integrity and function of mitochondria. These results demonstrate that soluble factors produced by lymphatic endothelium promote survival of triple-negative breast cancer cells under stress by regulating tumor cell redox homeostasis and promoting mitochondrial function. Citation Format: Mirela Berisa, Simona Podgrabinska, Jerry Chipuk, Mihaela Skobe. Lymphatic endothelium increases antioxidant capacity of triple-negative breast cancer cells and protects from cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-333. doi:10.1158/1538-7445.AM2017-LB-333

Abstract 1931: Mathematical modeling predicts exponential growth kinetics for metastases in the lymphatic vessels in the absence of vascularization

The objective of this study was to examine how the biophysical characteristics of different metastatic niches influence survival and growth of metastatic cells using mathematical modeling. In the lung, metastases can form in the lung parenchyma or in the lymphatic vessels. The presence of cancer metastases in the lung lymphatics is associated with rapid disease progression and extremely poor prognosis for patients. Remarkably, metastases in pulmonary lymphatics are avascular, yet grow larger than metastases in the lung parenchyma. To explain rapid growth of metastases in the lymphatics in the absence of angiogenesis, we have developed a 3D mathematical model of intralymphatic tumor growth. This is a deterministic continuum model based on partial differential equations used to describe avascular tumor growth and adapted to reflect the unique architecture of the lymphatic vasculature. Our model predicts that the cylindrical shape of the lymphatic vessel, which constrains growth of the tumor in two dimensions yet allows indefinite growth along the vessel, enables oxygen levels to remain high throughout the tumor. The greater diffusion coefficient of oxygen in lymph further improves oxygenation of intralymphatic metastases, which rarely become hypoxic. Improved tumor oxygenation leads to decreased tumor cell death and a rapid increase of metastatic burden in the lymphatics. Importantly, our model predicts that the kinetics of growth of intralymphatic metastases is exponential. This contrasts the established view that all tumors follow Gompertzian growth kinetics, i.e., that tumor growth rate decreases as tumor size increases. Furthermore, these findings indicate that the lymphatic niche is a favorable environment for metastatic growth, and that angiogenesis is not always required for the progressive growth of metastases. Finally, our predictions of different growth kinetics of primary tumors and their metastases suggest that the treatment schedules for metastatic disease could be further optimized to achieve better efficacy. Citation Format: Ruth E. Griswold, Simona Podgrabinska, Suvendu Das, Boyce Griffith, Charles S. Peskin, Mihaela Skobe. Mathematical modeling predicts exponential growth kinetics for metastases in the lymphatic vessels in the absence of vascularization. [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 1931. doi:10.1158/1538-7445.AM2015-1931