A practical and sensitive method of quantitating lymphangiogenesis in vivo

Majumder, Mousumi; Xin, Xiping; Lala, Peeyush K.

doi:10.1038/labinvest.2013.72

Cited by 12 publications

(19 citation statements)

References 36 publications

(54 reference statements)

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“…In the present study we validated the roles of PGE2 and EP4 in lymphangiogenesis in vitro and in vivo studies in nude mice using directed in vivo lymphangiogenesis assay (DIVLA) previously developed in our lab [29]. Inclusion of PGE2 and two EP4 agonists in the angioreactors significantly increased lymphangiogenesis and to a minor extent angiogenesis, identified by three approaches: expression of lymphatic endothelial marker molecules Prox-1 and Lyve-1 and vascular endothelial marker molecule CD31 at the mRNA and protein levels; and a dual immunostaining of lymphatics for Lyve-1 or prox-1 and blood vessels for CD31.…”

Section: Discussionmentioning

confidence: 86%

“…Directed in-vivo angiogenesis and lymphangiogenesis assays in mice [29] was performed by implanting the angioreactors with VEGF-C, PGE2, and EP4 agonists PGE1OH or L-902,688 mixed in BME. After ten days, the angioreactors were removed to visualize their gross appearances (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This assay was conducted as reported by us [29]. In brief 6 weeks old athymic nude female mice were allowed to acclimatize for 2 weeks, maintained on standard mouse chow and tap water on a 12 h light/dark cycle.…”

Section: Methodsmentioning

confidence: 99%

“…Four angioreactors containing the same agents were implanted per mouse, two on each side of the dorsal flank region, and 2 mice were used per condition. After a period of 10 days, mice were euthanized and the angioreactors were retrieved and used for three purposes as reported earlier [29]. All the angioreactors were retrieved without severing the ingrowing vessels and excising the rest of the tissues with fine scissors.…”

Section: Methodsmentioning

confidence: 99%

“…We addressed whether: (a) the native tube forming capacity of the LEC on Matrigel is dependent on COX-2 or EP4 activity; (b) soluble products of COX-2-expressing breast cancer cells stimulate tube formation by the LEC; (c) proliferation, migration and tube forming capacity of the LEC are stimulated by exogenous PGE2 or selective EP4 agonists and if so, what are the underlying signaling mechanisms. In additional experiments we utilized a directed in vivo lymphangiogenesis assay (DIVLA) devised in our laboratory [28, 29] to examine the roles of exogenous PGE2 and EP4 agonists in promoting lymphatic vessel outgrowth in nude mice. Results revealed that tumor or host-derived PGE2 in the tumor micro-environment or exogenous PGE2 or EP4 agonists can directly stimulate lymphangiogenesis by activation of EP4 receptors on the LEC via PI3K/Akt and Erk signaling pathways and VEGFR-3 stimulation, so that EP4 antagonists may be useful in the prevention and intervention of lymphatic metastasis in breast cancer.…”

Section: Introductionmentioning

confidence: 99%

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PGE2 promotes breast cancer-associated lymphangiogenesis by activation of EP4 receptor on lymphatic endothelial cells

et al. 2017

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BackgroundLymphatic metastasis, facilitated by lymphangiogenesis is a common occurrence in breast cancer, the molecular mechanisms remaining incompletely understood. We had earlier shown that cyclooxygenase (COX)-2 expression by human or murine breast cancer cells promoted lymphangiogenesis and lymphatic metastasis by upregulating VEGF-C/D production by tumor cells or tumor-associated macrophages primarily due to activation of the prostaglandin receptor EP4 by endogenous PGE2. It is not clear whether tumor or host-derived PGE2 has any direct effect on lymphangiogenesis, and if so, whether EP4 receptors on lymphatic endothelial cells (LEC) play any role.MethodsHere, we address these questions employing in vitro studies with a COX-2-expressing and VEGF-C/D-producing murine breast cancer cell line C3L5 and a rat mesenteric (RM) LEC line and in vivo studies in nude mice.ResultsRMLEC responded to PGE2, an EP4 agonist PGE1OH, or C3L5 cell-conditioned media (C3L5-CM) by increased proliferation, migration and accelerated tube formation on growth factor reduced Matrigel. Native tube formation by RMLEC on Matrigel was abrogated in the presence of a selective COX-2 inhibitor or an EP4 antagonist. Addition of PGE2 or EP4 agonist, or C3L5-CM individually in the presence of COX-2 inhibitor, or EP4 antagonist, restored tube formation, reinforcing the role of EP4 on RMLEC in tubulogenesis. These results were partially duplicated with a human dermal LEC (HMVEC-dLyAd) and a COX-2 expressing human breast cancer cell line MDA-MB-231. Knocking down EP4 with shRNA in RMLEC abrogated their tube forming capacity on Matrigel in the absence or presence of PGE2, EP4 agonist, or C3L5-CM. RMLEC tubulogenesis following EP4 activation by agonist treatment was dependent on PI3K/Akt and Erk signaling pathways and VEGFR-3 stimulation. Finally in a directed in vivo lymphangiogenesis assay (DIVLA) we demonstrated the lymphangiogenic as well as angiogenic capacity of PGE2 and EP4 agonist in vivo.Discussion/conclusionsThese results demonstrate the roles of tumor as well as host-derived PGE2 in inducing lymphangiogenesis, at least in part, by activating EP4 and VEGFR-3 on LEC. EP4 being a common target on both tumor and host cells contributing to tumor-associated lymphangiogenesis reaffirms the therapeutic value of EP4 antagonists in the intervention of lymphatic metastasis in breast cancer.

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Section: Discussionmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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PGE2 promotes breast cancer-associated lymphangiogenesis by activation of EP4 receptor on lymphatic endothelial cells

et al. 2017

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Fluorescent reporter transgenic mice for in vivo live imaging of angiogenesis and lymphangiogenesis

et al. 2018

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The study of lymphangiogenesis is an emerging science that has revealed the lymphatic system as a central player in many pathological conditions including cancer metastasis, lymphedema, and organ graft rejection. A thorough understanding of the mechanisms of lymphatic growth will play a key role in the development of therapeutic strategies against these conditions. Despite the known potential of this field, the study of lymphatics has historically lagged behind that of hemangiogenesis. Until recently, significant strides in lymphatic studies were impeded by a lack of lymphatic-specific markers and suitable experimental models compared to those of the more immediately visible blood vasculature. Lymphangiogenesis has also been shown to be a key phenomenon in developmental biological processes, such as cell proliferation, guided migration, differentiation, and cell-to-cell communication, making lymphatic-specific visualization techniques highly desirable and desperately needed. Imaging modalities including immunohistochemistry and in situ hybridization are limited by the need to sacrifice animal models for tissue harvesting at every experimental time point. Moreover, the processes of mounting and staining harvested tissues may introduce artifacts that can confound results. These traditional methods for investigating lymphatic and blood vasculature are associated with several problems including animal variability (e.g., between mice) when replicating lymphatic growth environments and the cost concerns of prolonged, labor-intensive studies, all of which complicate the study of dynamic lymphatic processes. With the discovery of lymphatic-specific markers, researchers have been able to develop several lymphatic and blood vessel-specific, promoter-driven, fluorescent-reporter transgenic mice for visualization of lymphatics in vivo and in vitro. For instance, GFP, mOrange, tdTomato, and other fluorescent proteins can be expressed under control of a lymphatic-specific marker like Prospero-related homeobox 1 (Prox1), which is a highly conserved transcription factor for determining embryonic organogenesis in vertebrates that is implicated in lymphangiogenesis as well as several human cancers. Importantly, Prox1-null mouse embryos develop without lymphatic vessels. In human adults, Prox1 maintains lymphatic endothelial cells and upregulates proteins associated with lymphangiogenesis (e.g., VEGFR-3) and downregulates angiogenesis-associated gene expression (e.g., STAT6). To visualize lymphatic development in the context of angiogenesis, dual fluorescent-transgenic reporters, like Prox1-GFP/Flt1-DsRed mice, have been bred to characterize lymphatic and blood vessels simultaneously in vivo. In this review, we discuss the trends in lymphatic visualization and the potential usage of transgenic breeds in hemangiogenesis and lymphangiogenesis research to understand spatial and temporal correlations between vascular development and pathological progression.

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Roles of prostaglandins in tumor-associated lymphangiogenesis with special reference to breast cancer

Lala

Nandi

Majumder

2018

Cancer Metastasis Rev

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Lymphangiogenesis (formation of new lymphatic vessels), unlike angiogenesis, has been a lesser-focused field in cancer biology, because of earlier controversy regarding whether lymphatic metastasis occurs via pre-existing or newly formed lymphatics. Recent evidence reveals that peri-tumoral or intra-tumoral lymphangiogenesis is a precursor for lymphatic metastasis in most carcinomas and melanomas. Two major lymphangiogenic factors, vascular endothelial growth factor (VEGF)-C and VEGF-D, are produced by cancer cells or immune cells such as macrophages in the tumor-stroma to promote sprouting of lymphatics from lymphatic endothelial cells (LEC) or LEC precursors (LECP) by binding to their primary (high affinity) receptor VEGF-R3 or secondary receptors VEGF-R2, neuropilin (NRP)2 and α9/β1 integrin. Many other growth factors/receptors such as VEGF-A/VEGF-R2, fibroblast growth factor (FGF)2/FGF-R, platelet-derived growth factor (PDGF)/PDGF-R, hepatocyte growth factor (HGF)/C-Met, angiopoietins (Ang)1, 2/Tie2, and chemokines/ chemokine receptors (CCL21/CCR7, CCL12/CCR4) can also stimulate LEC sprouting directly or indirectly. This review deals with the roles of prostaglandins (PG), in particular PGE2, in cancer-associated lymphangiogenesis, with special emphasis on breast cancer. We show that cyclooxygenase (COX)-2 expression by breast cancer cells or tumor stroma leading to high PGE2 levels in the tumor milieu promotes lymphangiogenesis and lymphatic metastases, resulting from binding of PGE2 to PGE receptors (EP, in particular EP4) on multiple cell types: tumor cells, tumor-infiltrating immune cells, and LEC. EP4 activation on cancer cells and macrophages upregulated VEGF-C/D production to stimulate LEC sprouting. Furthermore, ligation of EP4 with PGE2 on cancer or host cells can initiate a new cascade of molecular events leading to cross-talk between cancer cells and LEC, facilitating lymphangiogenesis and lympho-vascular transport of cancer cells. We make a case for EP4 as a potential therapeutic target for breast cancer.

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A practical and sensitive method of quantitating lymphangiogenesis in vivo

Cited by 12 publications

References 36 publications

PGE2 promotes breast cancer-associated lymphangiogenesis by activation of EP4 receptor on lymphatic endothelial cells

PGE2 promotes breast cancer-associated lymphangiogenesis by activation of EP4 receptor on lymphatic endothelial cells

Fluorescent reporter transgenic mice for in vivo live imaging of angiogenesis and lymphangiogenesis

Roles of prostaglandins in tumor-associated lymphangiogenesis with special reference to breast cancer

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