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Lymphatic endothelium of metastatic tumours has a distinct transcription profile.

ABSTRACT: Invasion of lymphatic vessels is a key step in the metastasis of primary tumour cells to draining lymph nodes. Recent evidence indicates that such metastasis can be facilitated by tumour lymphangiogenesis, although it remains unclear whether this is a consequence of increased lymphatic vessel numbers or alteration in the properties of the vessels themselves. Here we have addressed this important question by comparing the RNA profile of normal dermal lymphatic endothelial cells (LEC) with those isolated from tumours of murine T-241/VEGF-C metastatic fibrosarcoma. Our findings reveal significant changes in the expression of some 792 genes in tumour lymphatics (≥ 2 fold up/downregulation, p ≤ 0.05), involving particularly transcripts associated with junctional adhesion, immunomodulation, extracellular matrix and vessel growth/patterning, several of which we have confirmed by RT-PCR and/or immunohistochemistry. Interestingly, this altered phenotype could not be attributed solely to VEGF-C induced lymphoproliferation, as no similar change in gene expression was reported when human LEC were cultured with VEGF-C in vitro. Moreover, we show that a key protein upregulated in the mouse model, namely the tight junction protein Endothelial Cell Specific Adhesion Molecule (ESAM), is similarly upregulated in tumour lymphatic vessels from 2/2 patients with head and neck squamous cell carcinoma and 4/4 patients with aggressive bladder carcinoma. These findings demonstrate a previously unrecognized influence of tumour environment on lymphatic gene expression and identify candidate tumour specific vessel markers that may prove valuable for either prognosis or therapy. Keywords: Tumor, Metastasis, Lymphatic endothelium, comparative transcriptional profiling

ORGANISM(S): Mus musculus

PROVIDER: GSE6255 | GEO | 2008/09/22

SECONDARY ACCESSION(S): PRJNA99687

REPOSITORIES: GEO

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Project description:Invasion of lymphatic vessels is a key step in the metastasis of primary tumour cells to draining lymph nodes. Recent evidence indicates that such metastasis can be facilitated by tumour lymphangiogenesis, although it remains unclear whether this is a consequence of increased lymphatic vessel numbers or alteration in the properties of the vessels themselves. Here we have addressed this important question by comparing the RNA profile of normal dermal lymphatic endothelial cells (LEC) with those isolated from tumours of murine T-241/VEGF-C metastatic fibrosarcoma. Our findings reveal significant changes in the expression of some 792 genes in tumour lymphatics (â?¥ 2 fold up/downregulation, p â?¤ 0.05), involving particularly transcripts associated with junctional adhesion, immunomodulation, extracellular matrix and vessel growth/patterning, several of which we have confirmed by RT-PCR and/or immunohistochemistry. Interestingly, this altered phenotype could not be attributed solely to VEGF-C induced lymphoproliferation, as no similar change in gene expression was reported when human LEC were cultured with VEGF-C in vitro. Moreover, we show that a key protein upregulated in the mouse model, namely the tight junction protein Endothelial Cell Specific Adhesion Molecule (ESAM), is similarly upregulated in tumour lymphatic vessels from 2/2 patients with head and neck squamous cell carcinoma and 4/4 patients with aggressive bladder carcinoma. These findings demonstrate a previously unrecognized influence of tumour environment on lymphatic gene expression and identify candidate tumour specific vessel markers that may prove valuable for either prognosis or therapy. Experiment Overall Design: Here we have investigated the invasion of lymphatic vessels as a key step in the metastasis of primary tumour cells to draining lymph nodes by comparing the gene expression profile of normal dermal lymphatic endothelial cells (LEC) with those isolated from tumours of murine T241/VEGF-C/GFP metastatic fibrosarcoma. Three biological replicates were analyzed from each group.

Project description:The lymphatic vascular system plays important roles in the maintenance of interstitial fluid pressure, the afferent immune response and the absorption of dietary lipids. However, the molecular mechanisms that control lymphatic vessel network maturation and function remain largely unknown. To identify novel players in lymphatic vessel function, we isolated pure populations of lymphatic and blood vascular endothelial cells from mouse intestine using fluorescence-activated high-speed cell sorting and performed transcriptional profiling. We found that the axonal guidance molecules semaphorin 3A (Sema3A) and Sema3D were specifically expressed by lymphatic vessels. Quantitative PCR of ex vivo isolated cells and immunohistochemical analysis confirmed these results. Importantly, we found that the semaphorin receptor neuropilin-1 (Nrp-1) is expressed on the valves of collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1, but not with an antibody that blocks VEGFA binding to Nrp-1, resulted in abnormal development of collecting lymphatic vessels and valves, and aberrant smooth muscle cell coverage. Conversely, Sema3A-deficient mice displayed branching defects of collecting lymphatic vessels as well as impaired valve development. Together, these results reveal an unanticipated role of Sema3A/Nrp-1 signaling in the maturation of the lymphatic vascular network. Colon single-cell suspensions were prepared by a fast protocol that minimizes the RNA degradation. Fluorescence-activated cell sorting (FACS) was used to sort blood vascular endothelial cells (BEC) and lymphatic endothelial cells (LEC). 4 animal-matched pairs of LEC and BEC were chosen based on the quality of extracted and amplified material to provide homogenous groups of biological replicates. This gave 8 samples to analyze. Samples present LEC and BEC isolated from 4 healthy normal mice. The 4 mice used present the 4 biological replicates.

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Lymphatic endothelium of metastatic tumours has a distinct transcription profile.

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