Lymphatic Endothelial Cell Markers Research Articles

The Roles of Podoplanin-Positive/Podoplanin-Negative Cells from Adipose-Derived Stem Cells in Lymphatic Regeneration.

Secondary lymphedema is a refractory disease, for which adipose-derived stem cells have shown some therapeutic potential. However, the mechanism of this action remains poorly understood. The authors identified podoplanin-expressing adipose-derived stem cells, which allowed them to divide adipose-derived stem cells into podoplanin-positive and podoplanin-negative groups that they characterized in vitro. The authors then used a mouse hindlimb model for lymphedema to trace the fate of podoplanin-positive, podoplanin-negative, and unsorted adipose-derived stem cells in vivo. When induced in culture, podoplanin-positive cells were noted to up-regulate the expression of lymphatic endothelial cell markers, including LYVE-1, and assumed a cobblestone morphology. In addition, a substantial increase in lymphangiogenic cytokines was detected in the podoplanin-positive supernatant. The above findings were largely absent from the podoplanin-negative and unsorted groups. In the mouse model, the implanted cells relieved the limb lymphedema by promoting lymphangiogenesis, with the podoplanin-positive group showing the most significant effect. Immunocolocalization further revealed that the podoplanin-positive cells incorporated into lymphatic vessels were positive for LYVE-1. These data demonstrated that actions by means of both paracrine and differentiation pathways were involved in the adipose-derived stem cell-mediated therapeutic effects. The podoplanin-positive cells possessed lymphatic paracrine and differentiation abilities and may represent lymphatic endothelial cell precursor cells. The podoplanin-negative cells, which constitute a considerable proportion of the adipose-derived stem cells, may play an important paracrine role by secreting mesenchymal stem cell-related factors.

PMab-241 Specifically Detects Bear Podoplanin of Lymphatic Endothelial Cells in the Lung of Brown Bear.

Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses to detect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel anti-bPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic endothelial cells specifically in the bear lung tissues.

Abstract 711: Therapeutic Effects of Engineered Human Pluripotent Stem Cell-derived Lymphatic Endothelial Cells on Experimental Lymphedema

Current systems generating lymphatic endothelial cell (LEC) from human pluripotent stem cells (hPSCs) have limited value due to low purity, the use of undefined components for differentiation, and poor cell survival in vivo . Here, we developed a fully defined system to differentiate hPSCs into LECs and evaluated their therapeutic and engraftment potential when encapsulated in a nanomatrix gel. hPSCs were cultured with GSK3-β inhibitor for 3 days to induce differentiation into the mesodermal lineage. The mesodermal cells were cultured for another 6 days and double-sorted by PDPN and FLT4. These hPSC-PDPN + FLT4 + cells showed highly purified and fully functional LEC characteristics in vitro. These hPSC-LECs express LEC markers such as PDPN, LYVE1, PROX1, and FLT4 at the mRNA level and the protein level, and formed tube-like structures in Matrigel. We next determined the lymphatic vascular reparative effects of engineered hPSC-LECs. After inducing lymphedema in the tail of mouse, hPSC-LECs, hPSC-LECs/PA-RGDS, human dermal lymphatic endothelial cells (hdLEC), PA-RGDS, or PBS were injected into the tail of mouse. Tail thickness significantly decreased in the groups injected with hPSC-LECs with or without PA-RGDS compared to the other groups at day 28. At day 45, mice injected with PA-RGDS encapsulated hPSC-LECs showed significant decrease in the tail diameter compared to all other groups including those injected with hPSC-LECs. This study demonstrated for the first time that PA-RGDS encapsulation can substantially improve lymphedema repair in mouse tail through enhancement of cell survival and lymphatic neovascularizationThis engineered hPSC-LEC therapy represents a novel option for treating lymphedema.

PMab-235: A monoclonal antibody for immunohistochemical analysis against goat podoplanin

Sensitive and specific monoclonal antibodies (mAbs) against not only human but also mouse, rat, rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. However, anti-goat PDPN (gPDPN) has not been established yet. PDPN has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses; therefore, mAbs for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues are needed. Although we recently demonstrated that an anti-bovine PDPN mAb, PMab-44 cross-reacted with gPDPN, PMab-44 did not detect lymphatic endothelial cells in immunohistochemistry. In this study, we immunized mice with gPDPN-overexpressing Chinese hamster ovary (CHO)–K1 (CHO/gPDPN) cells, and screened mAbs against gPDPN using flow cytometry. One of the mAbs, PMab-235 (IgG1, kappa), specifically detected CHO/gPDPN cells by flow cytometry. Furthermore, PMab-235 strongly detected lung type I alveolar cells, renal podocytes, and lymphatic endothelial cells of colon by immunohistochemistry. These findings suggest that PMab-235 may be useful as a lymphatic endothelial cell marker for goat tissues.

Immunohistochemical Analysis of the Harbor Porpoise Using Antipodoplanin Antibody PMab-237.

Podoplanin (PDPN)/T1 alpha is known as a specific marker of lymphatic endothelial cells and type I alveolar cells. Sensitive and specific monoclonal antibodies (mAbs) for PDPN are needed for immunohistochemical analyses. Recently, we developed an anticetacean PDPN mAb, PMab-237. Herein, immunohistochemical analyses showed that PMab-237 strongly detected pulmonary type I alveolar cells, renal podocytes, and lymphatic endothelial cells of the harbor porpoise. These findings suggest that PMab-237 may be useful for immunohistochemical analyses for cetacean tissues.

Establishment of an Anticetacean Podoplanin Monoclonal Antibody PMab-237 for Immunohistochemical Analysis.

Podoplanin (PDPN) has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses. Therefore, sensitive and specific monoclonal antibodies (mAbs) targeting PDPN are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues. Recently, anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse were established in our studies. However, anticetacean (whale) PDPN (wPDPN) has not been established yet. In this study, we immunized mice with wPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/wPDPN) cells, and screened mAbs against wPDPN using flow cytometry. One of the mAbs, PMab-237 (IgG1, kappa), specifically detected CHO/wPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-237 for the functional analyses of wPDPN.

Establishment of a monoclonal antibody PMab-225 against alpaca podoplanin for immunohistochemical analyses

Podoplanin (PDPN) is known as a lymphatic endothelial cell marker. Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse PDPN have been established in our previous studies. However, mAbs against alpaca PDPN (aPDPN), required for immunohistochemical analysis, remain to be developed. In the present study, we employed the Cell-Based Immunization and Screening (CBIS) method for producing anti-aPDPN mAbs. We immunized mice with aPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/aPDPN), and hybridomas producing mAbs against aPDPN were screened using flow cytometry. One of the mAbs, PMab-225 (IgG2b, kappa), specifically detected CHO/aPDPN cells via flow cytometry and recognized the aPDPN protein on Western blotting. Further, PMab-225 strongly stained lung type I alveolar cells, colon lymphatic endothelial cells, and kidney podocytes via immunohistochemistry. These findings demonstrate that PMab-225 antibody is useful to investigate the function of aPDPN via different techniques.

Establishment of a monoclonal antibody PMab-233 for immunohistochemical analysis against Tasmanian devil podoplanin

Monoclonal antibodies (mAbs) against not only human, mouse, and rat but also rabbit, dog, cat, bovine, pig, and horse podoplanins (PDPNs) have been established in our previous studies. PDPN is used as a lymphatic endothelial cell marker in pathological diagnoses. However, mAbs against Tasmanian devil PDPN (tasPDPN), which are useful for immunohistochemical analysis, remain to be developed. Herein, mice were immunized with tasPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/tasPDPN) cells, and hybridomas producing mAbs against tasPDPN were screened using flow cytometry. One of the mAbs, PMab-233 (IgG1, kappa), specifically detected CHO/tasPDPN cells by flow cytometry and recognized tasPDPN protein by western blotting. Furthermore, PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These findings suggest that PMab-233 may be useful as a lymphatic endothelial cell marker of the Tasmanian devil.

IL-7 enhances the differentiation of adipose-derived stem cells toward lymphatic endothelial cells through AKT signaling.

Our study was designed to investigate the effects of IL-7 during the differentiation process of adipose-derived stem cells (ADSCs) toward lymphatic endothelial cells (LECs). IL-7 was added to the traditional induced medium, which was called the IL-7 (+) group, while the group that used traditional induced medium was called the IL-7 (-) group. After 7 days of induction of ADSCs, a comprehensive analysis was conducted between these two groups. We examined the changes in Prox1, LYVE-1, Podoplanin and VEGFR-3 on the RNA and protein level and found that the expression of LEC markers in the IL-7 (+) group was higher than in the IL-7 (-) group. The characteristics of differentiated cells were confirmed by flow cytometry and immunofluorescence. At the same time, we detected the MAPK/ERK and PI3K/AKT pathway involved in the differentiation process, and we found that the phosphorylation of AKT increased, however the expression of ERK was not significantly changed. In conclusion, our study found that IL-7 could improve the differentiation efficiency of ADSCs toward LECs through AKT signaling pathways.

PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.

Generating Vegfr3 reporter transgenic mouse expressing membrane-tagged Venus for visualization of VEGFR3 expression in vascular and lymphatic endothelial cells.

Vascular endothelial growth factor receptor 3 (Vegfr3) has been widely used as a marker for lymphatic and vascular endothelial cells during mouse embryonic development and in adult mouse, making it valuable for studying angiogenesis and lymphangiogenesis under normal and pathological conditions. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a membrane-localized fluorescent reporter protein, Gap43-Venus, under the control of the Vegfr3 regulatory sequence. Vegfr3-Gap43-Venus BAC Tg recapitulated endogenous Vegfr3 expression in vascular and lymphatic endothelial cells during embryonic development and tumor development. Thus, this Tg mouse line contributes a valuable model to study angiogenesis and lymphangiogenesis in physiological and pathological contexts.

Platelet-derived growth factor-D enables liver myofibroblasts to promote tumor lymphangiogenesis in cholangiocarcinoma

In cholangiocarcinoma, early metastatic spread via lymphatic vessels often precludes curative therapies. Cholangiocarcinoma invasiveness is fostered by an extensive stromal reaction, enriched in cancer-associated fibroblasts (CAFs) and lymphatic endothelial cells (LECs). Cholangiocarcinoma cells recruit and activate CAFs by secreting PDGF-D. Herein, we investigated the role of PDGF-D and liver myofibroblasts in promoting lymphangiogenesis in cholangiocarcinoma. Human cholangiocarcinoma specimens were immunostained for podoplanin (LEC marker), α-SMA (CAF marker), VEGF-A, VEGF-C, and their cognate receptors (VEGFR2, VEGFR3). VEGF-A and VEGF-C secretion was evaluated in human fibroblasts obtained from primary sclerosing cholangitis explants. Using human LECs incubated with conditioned medium from PDGF-D-stimulated fibroblasts we assessed migration, 3D vascular assembly, transendothelial electric resistance and transendothelial migration of cholangiocarcinoma cells (EGI-1). We then studied the effects of selective CAF depletion induced by the BH3 mimetic navitoclax on LEC density and lymph node metastases in vivo. In cholangiocarcinoma specimens, CAFs and LECs were closely adjacent. CAFs expressed VEGF-A and VEGF-C, while LECs expressed VEGFR2 and VEGFR3. Upon PDGF-D stimulation, fibroblasts secreted increased levels of VEGF-C and VEGF-A. Fibroblasts, stimulated by PDGF-D induced LEC recruitment and 3D assembly, increased LEC monolayer permeability, and promoted transendothelial EGI-1 migration. These effects were all suppressed by the PDGFRβ inhibitor, imatinib. In the rat model of cholangiocarcinoma, navitoclax-induced CAF depletion, markedly reduced lymphatic vascularization and reduced lymph node metastases. PDGF-D stimulates VEGF-C and VEGF-A production by fibroblasts, resulting in expansion of the lymphatic vasculature and tumor cell intravasation. This critical process in the early metastasis of cholangiocarcinoma may be blocked by inducing CAF apoptosis or by inhibiting the PDGF-D-induced axis. Cholangiocarcinoma is a highly malignant cancer affecting the biliary tree, which is characterized by a rich stromal reaction involving a dense population of cancer-associated fibroblasts that promote early metastatic spread. Herein, we show that cholangiocarcinoma-derived PDGF-D stimulates fibroblasts to secrete vascular growth factors. Thus, targeting fibroblasts or PDGF-D-induced signals may represent an effective tool to block tumor-associated lymphangiogenesis and reduce the invasiveness of cholangiocarcinoma.

Detection of Tiger Podoplanin Using the Anti-Cat Podoplanin Monoclonal Antibody PMab-52.

Podoplanin (PDPN) is expressed in type I alveolar cells of lung but not in type II alveolar cells. PDPN is also known as a specific lymphatic endothelial cell marker because PDPN is not expressed in vascular endothelial cells. PDPNs of several animals have been characterized using specific anti-PDPN monoclonal antibodies (mAbs): PMab-1, PMab-2, PMab-32, PMab-38, PMab-44, and PMab-52 for mouse, rat, rabbit, dog, bovine, and cat PDPNs, respectively. In this study, we investigated the possible crossreaction between these anti-PDPN mAbs and tiger PDPN. Flow cytometry and western blot analyses revealed that the anti-cat PDPN mAb PMab-52 (IgM, kappa) reacted with tiger PDPN, which is overexpressed in Chinese hamster ovary-K1 cells. Using immunohistochemical analysis, type I alveolar cells of the tiger lung were strongly detected by PMab-52. These results indicate that PMab-52 may be useful for the detection of tiger PDPN.

Elucidation of Critical Epitope of Anti-Rat Podoplanin Monoclonal Antibody PMab-2.

Rat podoplanin (rPDPN) is a recognized lymphatic endothelial cell marker and is expressed on the podocytes of kidney and type I lung alveolar cells. rPDPN is a type I transmembrane sialoglycoprotein and induces platelet aggregation via the C-type lectin-like receptor-2 of platelets. It comprises four platelet aggregation-stimulating (PLAG) domains: PLAG1–3, present in the N-terminus, and PLAG4, in the center of the PDPN protein. Previously, we developed a mouse anti-rPDPN monoclonal antibody clone, PMab-2, by immunizing the PLAG2 and PLAG3 domains of rPDPN. PMab-2 has applications in Western blot, flow cytometry, and immunohistochemical analyses for detection of both normal and cancer cells. However, the binding epitope of PMab-2 remains to be determined. Herein, we investigated the epitope of PMab-2 using enzyme-linked immunosorbent assay, immunohistochemical analysis, and flow cytometry. The results revealed that the critical epitope of PMab-2 is Leu46 and Glu47 of rPDPN.

Steroid receptor coactivator-1 interacts with NF-κB to increase VEGFC levels in human thyroid cancer.

Thyroid cancer is the most common endocrine cancer, and has a high incidence of lymphatic metastasis. Vascular endothelial growth factor C (VEGFC) is essential for development of lymphatic vessels and lymphatic metastases during carcinogenesis. Steroid receptor coactivator-1 (SRC-1) interacts with nuclear receptors and transcription factors to promote tumor proliferation and metastasis. However, the correlation between SRC-1 and VEGFC levels in the lymphatic metastases of thyroid cancer remains unclear. We analyzed 20-paired specimens of thyroid cancer tissue and normal thyroid tissue and found increased levels of SRC-1 and VEGFC proteins in 13/20 and 15/20 thyroid cancer specimens, respectively, when compared with those levels in specimens of normal thyroid tissue. A high level of SRC-1 expression was positively correlated with VEGFC and lymphatic endothelial cell marker LYVE-1 expression. Papillary thyroid carcinoma cell line TPC-1 displayed high levels of SRC-1 and VEGFC expression and was selected for stable knockdown of SRC-1 in vitro. Inhibition of SRC-1 significantly reduced the VEGFC levels in TPC-1 cells. We found that SRC-1 binds to transcription factor NF-kB (p50/p65), and that this coactivation complex directly promoted VEGFC transcription, which could be abrogated by SRC-1 knockdown. Up-regulated NF-kB signaling was also confirmed in thyroid cancer tissues. In vivo studies showed that SRC-1 knockdown restricted tumor growth, reduced the numbers of LYVE-1-positive lymphatic vessels, and decreased the levels of VEGFC in tumor tissues. These results suggest a tumorigenic role for SRC-1 in thyroid cancer via its ability to regulate VEGFC expression.

What podoplanin tells us about cells with telopodes

Telocytes (TCs) are stromal cells with telopodes, which represent long, thin, moniliform cell processes; however, this morphological feature alone is insufficient to define a cell type. Specific markers of lymphatic endothelial cells (LECs), such as Prox-1, podoplanin (D2-40) or LYVE-1, are not usually tested in TCs. We thus aimed at performing a study in light microscopy to evaluate whether or not LECs could be mistaken for TCs. Therefore we used CD34, α-smooth muscle actin and D2-40 for an immunohistochemical study on archived paraffin-embedded samples of uterine leiomyoma. Lymphatic vessels were identified by the expression of D2-40, but on the microscopic slides, false spindle-shaped TCs appearances either corresponded to collapsed lymphatic lumina or were determined by grazing longitudinal cuts of lymphatics. It is therefore mandatory to check the expression of lymphatic markers in telocyte-like cells and, moreover, to carefully examine the bidimensional cuts in order to avoid false results.

Expression of Genes in Primo Vasculature Floating in Lymphatic Endothelium Under Lipopolysaccharide and Acupuncture Electric Stimulation

It is known that the primo vascular system (PVS) includes the primo nodes and vessels. However, the relevant genes in the PVS system for both pathologic and physiologic condition are poorly understood. Here, we first examined the gene expression in primo vessels (PVs) floating in lymphatic endothelium by isolation of PVS and lymphatic vessels (LVs) containing PVS. To investigate therapeutic effects, both PVs and LVs containing PVS were isolated after lipopolysaccharide injection and acupuncture electric stimulation at two acupoints Joksamni (ST36) and Hapgok (LI04) following lipopolysaccharide injection. We used reverse transcriptase–polymerase chain reaction to examine expression of lymphatic endothelial cell markers and inflammatory related genes. We found that lymphatic endothelial cell markers such as fms-related tyrosine kinase 4 (Flt4), lymphatic vessel endothelial receptor (Lyve-1), prospero homeobox protein 1 (Prox-1), and podoplanin (Pdpn) were highly expressed in PV compared to that of lymphatic endothelium, suggesting pivotal roles of PV in LV under inflammation. Furthermore, lymphatic-related genes including metal-response element-binding transcription factor 2 (Mtf2), hypoxia inducible factor (Hif1a), angiotensin II type 1 receptor (Agtr1), and angiotensin II type 2 receptor (Agtr2) were also overall increased in PV, and remarkably increased and these genes except peroxisome proliferator–activated receptor gamma (Pparg) after acupuncture electric stimulation in two acupoints implying central role of PV by gene activation.

Rediscovery of forgotten structures inside mucosa of uterine tubes ‐ lymphatic lacunae‐ and their possible role in reproduction.

Human uterine tubes are paired, muscular tubes of great mobility that measure about 12 cm in length. The uterine tube wall is composed of a mucosa, a muscular layer, and a serosa. Mucosa forms longitudinal mucosal folds and is lined with a simple columnar epithelium. In this poster presentation, we described in detail a neglected structure of uterine tubes, central lymphatic lacunae of mucosal folds and fimbriae. These lymphatic lacunae were first time described by German gynecologist Paul Kroemer in 1904. Dr. Kroemer, as a part of his habilitation, injected ink to visualize lymphatic spaces in the pregnant woman's ovarian tube, which he named as “Lymphbahnen”, that is “lymphatic channels”. In recent textbooks, we found only one note about these lymphatic lacunae inside fimbriae, and also we found no mention about these structures in Terminologia Histologica. The specimens from the uterine tubes were taken from 48 women (age from 21 to75, mean age 48.8 years) who underwent trans-abdominal or laparoscopic surgery - salpingectomy with or without hysterectomy. The study protocol was approved by the ethical committee of local hospital, and informed consent was obtained from all patients. Tissue samples from anatomically different parts of the uterine tubes (infundibulum with fimbriae, ampulla and isthmus) were fixed in formalin, embedded in paraffin, and 5 μm thick sections were used for hematoxylin and eosin staining and for immunohistochemistry. Primary antibodies were used to label and detect podoplanin D2–40, a relatively new and selective marker of lymphatic endothelia, and CD34 antigen, a universal marker of blood endothelial cells. For visualization of the histological sections by means of light microscopy, the LEICA DM2500 microscope was used. In the histological slides of uterine tubes, stained with hematoxylin and eosin, there are noticeable slits or gaps within the loose connective tissue of lamina propria of the mucosal folds. They are lined with one layer of squamous cells resembling endothelial cells and are devoid of content. These “empty spaces” are most prominent in fimbriae, but are still well recognized in mucosal folds of ampullary region. In the isthmus, they are least visible. They always run thru the central part of the fold, reminding with their histotopographical localization lacteals in the center of intestinal villi. Squamous endothelial cells that line these gaps in the connective tissue do not express CD34 antigen, what can be regarded as a proof, that they are not blood vessels nor blood sinusoids. However, they are positive on podoplanin (D2–40) – selective marker of lymphatic endothelial cells. As a result, we confirmed that in the center of every mucosal fold and in fimbriae of the uterine tubes are situated dilated lymphatic spaces, which are lined with simple layer of endothelial cells. As there is no mention on them in current Terminologia Histologica (2008), we propose the term “central lymphatic lacunae of tubal mucosal folds and fimbriae”. Remains a question, why these lacunae are only rarely (and unsatisfactory) described in the literature, as the visibility of these large spaces even with the routine hematoxylin and eosin staining is excellent. Many times, the structures we had been looking for, had been well seen on microphotographs, however, without any description. This confirms, that lymphatic lacunae are physiologically present in the mucosa of uterine tubes and that until now, they had been just neglected and overlooked. Ampullary region of human uterine tube in hematoxylin and eosin staining (lymphatic lacunae are visualised by red color) and visualisation of lymphatic endothelia with podoplanin-antibodies (and diaminobenzidine as brown chromogen) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FOXF2 deficiency permits basal-like breast cancer cells to form lymphangiogenic mimicry by enhancing the response of VEGF-C/VEGFR3 signaling pathway

Lymphatic metastasis is the main route of breast cancer metastasis. It is known that lymphangiogenesis facilitates lymphatic metastasis through vascular endothelial growth factor-C (VEGF-C)/VEGF receptor 3 (VEGFR3) pathway-linked interactions between the tumor and its microenvironment. Here, we report a novel mechanism of lymphatic metastasis by which aggressive basal-like breast cancer (BLBC) cells form lymphatic vessel-like structures that are identified by the positive expression of lymphatic endothelial cell markers lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), podoplanin, and VEGFR3, and termed as lymphangiogenic mimicry (LM), for the first time. Our clinical evidence and experimental data in vivo and in vitro revealed that forkhead box F2 (FOXF2) deficiency promotes the lymphatic metastasis of BLBC by conferring a lymphangiogenic mimetic feature upon cancer cells through directly activating VEGFR3 transcription. The fact that FOXF2 controls the activation of the VEGF-C/VEGFR3 signaling pathway in BLBC cells provides potential molecular diagnostic and therapeutic strategies for lymphatic metastasis in BLBC patients.

Migration of lymphatic endothelial cells and lymphatic vascular development in the craniofacial region of embryonic mice.

Lymphatic development in mice is initiated in the trunk at embryonic day (E) 9.5. This study aimed to examine the origin of craniofacial lymphatic endothelial cells (LECs) and the developmental process of lymphatic vessels in the mouse craniofacial region. Serial sections from ICR mouse embryos at E9.5-E14.5 were immunolabeled with LEC and venous endothelial cell (VEC) markers. These markers included prospero homeobox protein 1 (Prox1), vascular endothelial growth factor receptor 3 (Vegfr3), lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), and C-C motif chemokine 2 (Ccl21) for LEC, and COUP transcription factor 2 (CoupTF2) and endomucin (Emcn) for VEC. LECs were monitored as an index in Prox1/Vegfr3 double-positive cells using three-dimensional analysis because LECs express Prox1 and Vegfr3 ab initio during lymphatic vascular development. LECs appeared in VECs of the lateral walls of cardinal veins (CVs) at E9.5. These LECs were dichotomized into LEC populations that formed lymph sacs close to CVs and were scattered in the surrounding CVs. The scattered LECs formed cellular streams and extended from the trunk to the mandibular arches at E10.5 - E11.5. In the mandibular arches, individual LECs aggregated, and formed lymph sacs and tubular lymphatic vessels at E11.5-E14.5. Expression of the LEC marker proteins Lyve1 and Ccl21 in LECs changed during craniofacial lymphatic vascular development. Collectively, these findings suggest that craniofacial LECs originate from CVs of the trunk and migrate into the mandibular arches. Additionally, we found that craniofacial lymphatic vessels are formed according to morphogenesis of individual LECs that migrate from CVs.