Project description:Secreted extracellular vesicles are known to influence the tumor microenvironment and promote metastasis. In this work, we have analyzed the involvement of extracellular vesicles in the establishment of lymph node pre-metastatic niches by melanoma cells. We found that small extracellular vesicles (sEVs) derived from highly metastatic melanoma cell lines spread broadly through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Melanoma-derived sEVs induce lymphangiogenesis, a hallmark of pre-metastatic niche formation, in vitro and in lymphoreporter mice in vivo. We found that neural growth factor receptor (NGFR) is secreted in melanoma-derived small extracellular vesicles and shuttled to lymphatic endothelial cells, inducing lymphangiogenesis and tumor cell adhesion through the activation of ERK and NF-B pathways and ICAM1 expression. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased melanoma lymph node metastasis and extended the survival. Importantly, analysis of NGFR expression in lymph node metastases and matched primary tumors shows that levels of MITF+NGFR+ lymph node metastatic cells are correlated with disease outcome. Our data support the idea that NGFR secreted in sEVs favors lymph node pre-metastatic niche formation and lymph node metastasis in melanoma
Project description:Secreted extracellular vesicles are known to influence the tumor microenvironment and promote metastasis. In this work, we have analyzed the involvement of extracellular vesicles in establishing the lymph node pre-metastatic niche by melanoma cells. We found that small extracellular vesicles (sEVs) derived from highly metastatic melanoma cell lines spread broadly through the lymphatic system and are taken up by lymphatic endothelial cells reinforcing lymph node metastasis. Melanoma-derived sEVs induce lymphangiogenesis, a hallmark of pre-metastatic niche formation, in vitro and in lymphoreporter mice in vivo. Analysis of involved factors demonstrated that the neural growth factor receptor (NGFR) is secreted in melanoma-derived small extracellular vesicles and shuttled to lymphatic endothelial cells inducing lymphangiogenesis and tumor cell adhesion through the activation of ERK and NF-B pathways and ICAM1 expression. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased melanoma lymph node metastasis and extended mice survival. Importantly, analysis of NGFR expression in lymph node metastases and matched primary tumors shows that levels of MITF+NGFR+ lymph node metastatic cells are correlated with disease outcome. Our data support that NGFR is secreted in sEVs favoring lymph node pre-metastatic niche formation and lymph node metastasis in melanoma.
Project description:Lymphatic endothelial cells (LEC) residing in lymph nodes (LN) have been shown to express genes normally restricted to one or a few tissues, termed peripheral tissue antigens (PTA). The expression of one of these PTA, tyrosinase, by LN-resident LEC has been shown to mediate peripheral T cell tolerance. We used a microarray approach to determine the gene expression profile of LN-resident LEC and blood endothelial cells as a comparison with the objective of determining the global PTA repertoire in these LN stromal populations. Skin draining and mesenteric lymph nodes were pooled from 6 week old adult C57BL/6 mice, minced, and enzymatically digested yielding single cell suspensions. Lymph node stromal cells were purified via CD45 magnetic bead negative selection and pure populations of lymphatic endothelial cells (LEC) and blood endothelial cells (BEC) were obtained via electronic cell sorting according to their expression of gp38 and CD31 (LEC: gp38+ CD31+, BEC: gp38- CD31+). Total RNA was extracted, amplified, and hybridized to Affymetrix microarrays. 3 paired independent samples of purified lymph node LEC and BEC were analyzed.
Project description:Intra- and extracellular metabolomics dataset of human dermal blood endothelial cells (HDBECs), human umbilical vein endothelial cells (HUVECs), human dermal lymphatic endothelial cells (HDLECs) and intestinal lymphatic endothelial cells (iLECs) in proliferation and quiescence.
Project description:Purpose: The objective was to identify genes regulted by RANK signalling in lymph node lymphatic endothelial cells Method: Lymphatic endothelial cells were FACS sorted from peripheral lymph nodes of mice deficient for stromal RANKL (Ccl19 Cre Rankl flox/flox) versus littermate controls (Rankl flox/flox) and processed for bulk RNA sequencing Results: Upregulated and downregulated genes were identified
Project description:We examined transcriptional remodeling of lymph node lymphatic endothelial cells in response to imiquimod-induced inflammation using single-cell RNA sequencing.
Project description:Using single-cell RNA sequencing, we provide a comprehensive map of lymph node resident lymphatic endothelial cells, identifying subpopulations, new markers and functions.
Project description:Metastasis to lymph nodes is an early and prognostically important event in the progression of many human cancers, and is associated with expression of vascular endothelial growth factor-D (VEGF-D). Changes to lymph node vasculature occur during metastasis, and may establish a metastatic niche capable of attracting and supporting tumor cells. We used microarrays to characterise the molecular profiles of endothelial cells from lymph nodes draining metastatic (VEGF-D-overexpressing) and non-metastatic tumors, and to identify differentially-expressed genes that might have therapeutic or prognostic potential. Draining lymph nodes of metastatic (VEGF-D-overexpressing) or non-metastatic tumors were pooled from 1-5 mice and enzymatically digested. Lymph nodes draining metastatic tumors were included for the analysis only if macroscopically enlarged, indicating the presence of metastatic cells. After digestion, tumor cells and leukocytes were depleted via immunomagnetic selection, and the resulting lymph node stromal cells were cultured briefly. Podoplanin was then used as a positive immunomagnetic selection marker to enrich for lymphatic and other endothelial cells in the lymph node. RNA was isolated from biological duplicate lymph node endothelial cell (LN EC) preparations and analysed by microarray.
Project description:The exit of antigen-presenting cells (APC) and lymphocytes from inflamed skin to afferent lymph is vital for the initiation and maintenance of dermal immune responses. How such exit is achieved and how cells transmigrate the distinct endothelium of lymphatic vessels is however unknown. Here we show that inflammatory cytokines trigger activation of dermal lymphatic endothelial cells (LEC) leading to expression of the key leukocyte adhesion receptors ICAM-1, VCAM-1 and E-selectin, as well as a discrete panel of chemokines and other potential regulators of leukocyte transmigration. Furthermore, we show that both ICAM-1 and VCAM-1 are induced in the dermal lymphatic vessels of mice exposed to skin contact hypersensitivity where they mediate lymph node trafficking of DC via afferent lymphatics. Lastly, we show that TNF_-stimulates both DC adhesion and transmigration of dermal LEC monolayers in vitro and that the process is efficiently inhibited by ICAM-1 and VCAM-1 adhesion-blocking mAbs. These results reveal a CAM-mediated mechanism for recruiting leukocytes to the lymph nodes in inflammation and highlight the process of lymphatic transmigration as a potential new target for anti-inflammatory therapy. Experiment Overall Design: Global gene expression profile of normal dermal lymphatic endothelial cells cultured in media alone (no TNF) compared to that of normal dermal lymphatic endothelial cells stimulated with TNFalpha, 1 ng/ml for 48h.Triplicate biological samples were analyzed from human lymphatic endothelial cells (3 x controls; 3 x TNF treated) and a single sample analyzed from mouse lymphatic endothelial cells (1 x controls; 1 x TNF treated).