Lymphangiogenic Phenotype Research Articles

Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through an NGFR-dependent mechanism.

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.

Angiogenic and lymphangiogenic profiles in histological variants of papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is a well‑differentiated tumor that occurs in several histological variants whose biological behaviors remain unclear. Angiogenesis and lymphangiogenesis are critical processes that enable tumor progression. The aim of this study was to evaluate the angiogenic and lymphangiogenic phenotypes of PTC, considering the differences between histological variants. Angiogenic and lymphangiogenic profiles were analyzed by determining microvascular density (MVD) and lymphatic vessel density (LVD) in 73 cases of PTC, using immunohistochemistry. To assess the biological markers involved in blood and lymph vessel formation, the expression of vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX‑2), and p27kip1 (p27) was determined. MVD was significantly higher in patients with high‑risk PTC and in those with local extrathyroidal and vascular invasion. Positive VEGF expression was strongly associated with high MVD and age‑related tumor enlargement. The presence of lymph vessel invasion was associated with the expression of either VEGF or COX‑2. The analysis of angiogenesis and lymphangiogenesis in different histological variants of PTC revealed elevated LVD rather than MVD in the follicular variant of PTC (FV‑PTC).Lower MVD was observed in FV‑PTC relative to the classic variant of PTC (CV‑PTC). The frequency of VEGF‑positive tumors was higher in CV‑PTC than in FV‑PTC. A significant association between COX‑2 and p27 expression was observed in FV‑PTC but not in CV‑PTC. These results suggest that VEGF, COX‑2, and p27 may be important biological markers that determine the angiogenic and lymphangiogenic potentials of PTC, particularly between the follicular and classic variants.

Lymphangiogenesis in rat asthma model.

Although bronchial angiogenesis has been well documented in allergic asthma, lymphangiogenesis has not been widely studied. Therefore, we evaluated changes in lung lymphatics in a rat model of allergen-induced asthma using house dust mite (Der p 1; 100μg/challenge). Additionally, properties of isolated lung lymphatic endothelial cells (CD45-, CD141+, LYVE-1+, Prox-1+) were studied in vitro. Threeweeks after the onset of intranasal allergen exposure (twice-weekly), an increase in the number of lung lymphatic vessels was measured (34% increase) by lung morphometry. New lymphatic structures were seen predominantly in the peribronchial and periarterial interstitial space but also surrounding large airways. Isolated lymphatic endothelial cells from sensitized lungs showed enhanced proliferation (% Ki67+), chemotaxis, and tube formation (number and length) compared to lymphatic endothelial cells isolated from naive rat lungs. This hyper-proliferative lymphangiogenic phenotype was preserved through multiple cell passages (2-8). Lymphatic endothelial cells isolated from naive and HDM-sensitized rats produced similar in vitro levels of VEGF-C, VEGF-D, and VEGFR3 protein, each recognized as critical lymphangiogenic factors. Inhibition with anti-VEGFR (axitinib, 0.1μM) blocked proliferation and chemotaxis. Results suggest that in vivo sensitization causes fundamental changes to lymphatic endothelium, which are retained in vitro, and may relate to VEGFR downstream signaling.

MT1-MMP sheds LYVE-1 on lymphatic endothelial cells and suppresses VEGF-C production to inhibit lymphangiogenesis.

Lymphangiogensis is involved in various pathological conditions, such as arthritis and cancer metastasis. Although many factors have been identified to stimulate lymphatic vessel growth, little is known about lymphangiogenesis inhibitors. Here we report that membrane type 1-matrix metalloproteinase (MT1-MMP) is an endogenous suppressor of lymphatic vessel growth. MT1-MMP-deficient mice exhibit spontaneous corneal lymphangiogenesis without concomitant changes in angiogenesis. Mice lacking MT1-MMP in either lymphatic endothelial cells or macrophages recapitulate corneal lymphangiogenic phenotypes observed in Mmp14−/− mice, suggesting that the spontaneous lymphangiogenesis is both lymphatic endothelial cells autonomous and macrophage associated. Mechanistically, MT1-MMP directly cleaves LYVE-1 on lymphatic endothelial cells to inhibit LYVE-1-mediated lymphangiogenic responses. In addition, MT1-MMP-mediated PI3Kδ signalling restrains the production of VEGF-C from prolymphangiogenic macrophages through repressing the activation of NF-κB signalling. Thus, we identify MT1-MMP as an endogenous inhibitor of physiological lymphangiogenesis.

Increased lymphangiogenesis in lung metastases from colorectal cancer is associated with early lymph node recurrence and decreased overall survival.

Pulmonary metastasectomy (PM) is an accepted treatment modality in colorectal cancer (CRC) patients with pulmonary tumor spread. Positive intrathoracic lymph nodes at the time of PM are associated with a poor prognosis and 5-year survival rates of <20%. Increased lymphangiogenesis in pulmonary metastases might represent an initial step for a subsequent lymphangiogenic spreading. We aimed to evaluate the presence of lymphangiogenesis in clinically lymph node negative patients undergoing PM and its impact on outcome parameters. 71 patients who underwent PM for CRC metastases were included in this dual-center study. Tissue specimens of pulmonary metastases and available corresponding primary tumors were assessed by immunohistochemistry for lymphatic microvessel density (LMVD) and lymphovascular invasion (LVI). Results were correlated with clinical outcome parameters. LMVD was 13.9±8.1 and 13.3±8.5 microvessels/field (mean±SD) in metastases and corresponding primary CRC; LVI was evident in 46.5 and 58.6% of metastases and corresponding primary CRC, respectively. Samples with high LMVD had a higher likelihood of LVI. LVI was associated with early tumor recurrence in intrathoracic lymph nodes and a decreased overall survival (p<0.001 and p=0.029). Herein, we present first evidence in a well-defined patient collective that increased lymphangiogenesis is already present in a subtype of pulmonary metastases of patients staged as N0 at the time of PM. This lymphangiogenic phenotype has a strong impact on patients' prognosis. Our findings may have impact on the post-surgical therapeutic management of CRC patients with pulmonary spreading.

The Naïve Murine Cornea as a Model System to Identify Novel Endogenous Regulators of Lymphangiogenesis: TRAIL and rtPA

In the murine cornea, which is an established model for analyzing pathologic lymphatic vessel growth, phenotypic heterogeneity of the endogenous lymphatic vessels in the limbus of the cornea was previously described. In this study, the cornea of BALB/c, C57BL/6, and FVB mice with different limbal lymphangiogenic phenotypes was analyzed to identify novel candidates potentially influencing lymphatic vessel growth. Pathway specific expression analysis of the cornea was performed to identify novel candidate genes. Corneal protein expression of the respective candidates was analyzed by fluorescent immunohistochemistry. The effect of the candidates on proliferation of human dermal lymphatic endothelial cells (HDLECs) was analyzed by BrdU proliferation ELISA. Thirteen genes were differentially regulated in corneas of mouse strains with more endogenous limbal lymphatic vessels (high-lymphangiogenic) (C57BL/6) compared to mouse strains with less endogenous limbal lymphatic vessels (low-lymphangiogenic) (BALB/c, FVB). Two candidates, Tumor necrosis factor (ligand) superfamily member 10 (Tnfsf10/Trail) and Plasminogen activator, tissue (Plat/tPA) were expressed in the cornea of BALB/c and C57BL/6 mice on the protein level. In vitro, Trail and recombinant tPA inhibited the proliferation of human dermal lymphatic endothelial cells. Molecular analysis of the naive cornea in mouse strains with different limbal lymphatic phenotypes is a valuable model to identify novel endogenous regulators of lymphangiogenesis.

HIF-2α attenuates lymphangiogenesis by up-regulating IGFBP1 in hepatocellular carcinoma.

Tumour-associated lymphangiogenesis was identified as an important clinical determinant for the prognosis of hepatocellular carcinoma (HCC) and significantly influences patient survival. However, in this context, little is known about regulation of lymphangiogenesis by hypoxia-inducible factors (HIF). In HCC, mainly HIF-1α was positively correlated with lymphatic invasion and metastasis, whereas a defined role of HIF-2α is missing. We created a stable knockdown (k/d) of HIF-1α and HIF-2α in HepG2 cells and generated co-cultures of HepG2 spheroids with embryonic bodies. This constitutes an in vitro tumour model mimicking the cancer microenvironment and allows addressing the role of distinct HIF isoforms in regulating HCC lymphangiogenesis. In co-cultures with a HIF-2α k/d, lymphangiogenesis was significantly increased, whereas the k/d of HIF-1α showed no effect. The HIF-2α-dependent lymphangiogenic phenotype was confirmed in vivo using matrigel plug assays with supernatants of HIF-2α k/d HepG2 cells. We identified and verified insulin-like growth factor binding protein 1 (IGFBP1) as a HIF-2α target gene. The potential of HepG2 cells to induce lymphangiogenesis in two independent functional assays was significantly enhanced either by a k/d of HIF-2α or by silencing IGFBP1. Moreover, we confirmed IGF as a potent pro-lymphatic growth factor with IGFBP1 being its negative modulator. We propose that HIF-2α acts as an important negative regulator of hepatic lymphangiogenesis in vitro and in vivo by inducing IGFBP1 and thus, interfering with IGF signalling. Therefore, HIF-2α may constitute a critical target in HCC therapy.

The Role of Podoplanin in the Lymphangiogenesis of Oral Squamous Carcinomas.

Oral squamous cells carcinoma (OSCC) mortality rate ranges between 29-40/100,000 inhabitants. Regional lymph node metastases occur in 30-40% cases and are associated with unfavorable prognosis and decreased survival. Recentely it was suggested that the tumor-associated lymphatic vessels formation plays an active role in metastasis proces of several human malignancies, including OSCC. In the present study we investigated comparatively podoplanin immunoexpression in OSCC in both the tumor cells and lymphatic vessels reported to clinicopathological prognostic parameters. Material and methods The study included a total of 25 cases of OSCC. We investigated clinico-pathological parameters as age, gender, tumor site, and degree of differentiation, size and depth of invasion (pT), lymph node status (pN). Histologic classification was done according to the WHO criteria. For immunohistochemical (IHC) analysis we used podoplanin (Dako, clone D2-40). As visualization system it was used LSAB2 (Dako, Redox, Romania, code K0675) and chromogen DAB (Dako, Redox, Romania, code K3468). Negative controls were obtained by omitting the primary antibodies. IHC quantification was done intensity score and by lymphatic microdensity (LVD), intratumoral and on the advancing edge. For the statistical analysis we used Student's t-tests, ANOVA, chi square and Pearson, using SPSS 10 software. Results Podoplanin immunoexpression in tumor parenchyma presented with an average of 43%, varying intensity. We found a higher intensity in weak and moderately differentiated SCC then in well differentiated ones and no difference intratumoral aand advancing edge. In relation to the degree of tumor differentiation the mean LVD D2-40 was higher in the advancing edge. SCC who presented lymph node metastasis mean values for LVD D2-4 was higher then at advancing edge and higher than those of non-metastatic carcinomas. Conclusions Podoplanin immunoexpresion suggests the involvement both in tumor growth and the acquisition of an lymphangiogenic phenotype invasive by autocrine mechanisms.

Activating PIK3CA alleles and lymphangiogenic phenotype of lymphatic endothelial cells isolated from lymphatic malformations.

Lymphatic malformations (LMs) are developmental anomalies of the lymphatic system associated with the dysmorphogenesis of vascular channels lined by lymphatic endothelial cells (LECs). Seeking to identify intrinsic defects in affected LECs, cells were isolated from malformation tissue or fluid on the basis of CD31 and podoplanin (PDPN) expression. LECs from five unrelated LM lesions were characterized, including cells derived from one patient previously diagnosed with CLOVES. CLOVES-related LECs carried a known, activating mutation in PIK3CA (p.H1047L), confirmed by direct sequencing. Activating PIK3CA mutations (p.E542K and p.E545A) were identified in lesion-derived cells from the other four patients, also by direct sequencing. The five LM-LEC cultures shared a lymphangiogenic phenotype distinguished by PI3K/AKT activation, enhanced sprouting efficiency, elevated VEGF-C expression and COX2 expression, shorter doubling times and reduced expression of angiopoietin 2 and CXCR4. Nine additional LM-LEC populations and 12 of 15 archived LM tissue samples were shown to bear common PIK3CA variants by allele-specific PCR. The activation of a central growth/survival pathway (PI3K/AKT) represents a feasible target for the non-invasive treatment of LMs bearing in mind that background genetics may individualize lesions and influence treatments.

MET expression in melanoma correlates with a lymphangiogenic phenotype

Melanomas contain high frequencies of tumorigenic cells and their tumorigenic capacity resides in several distinct subpopulations within melanoma. Since their metastatic potential is linked to their ability to recruit lymphatic vessels, we aimed at identifying lymphangiogenic subpopulations by comparative in vitro analysis of single cell clones derived from a melanoma of a single patient. Selected lymphangiogenic clones were then grafted into severe combined immunodeficient mice, where they induced lymphangiogenesis and metastasized into sentinel nodes, whereas non-lymphangiogenic clones from the same patient did not metastasize. Transcriptome analysis revealed high expression of vascular endothelial growth factor C (VEGF-C) and platelet derived growth factor C (PDGF-C) as well as of the met proto-oncogene (MET) and its targets to be associated with this lymphangiogenic phenotype. Screening of a set of independently isolated melanoma cell lines from other patients confirmed this association between expression of high levels of MET and of VEGF-C and PDGF-C. Hence, we provide a model to screen for the lymphangiogenic potential of tumor cells. We show that the lymphangiogenic potential is heterogeneously distributed among melanoma cells within one given tumor and is associated with activation of MET signaling.

KSHV Induction of Angiogenic and Lymphangiogenic Phenotypes

Kaposi’s sarcoma (KS) is a highly vascularized tumor supporting large amounts of neo-angiogenesis. The major cell type in KS tumors is the spindle cell, a cell that expresses markers of lymphatic endothelium. KSHV, the etiologic agent of KS, is found in the spindle cells of all KS tumors. Considering the extreme extent of angiogenesis in KS tumors at all stages it has been proposed that KSHV directly induces angiogenesis in a paracrine fashion. In accordance with this theory, KSHV infection of endothelial cells in culture induces a number of host pathways involved in activation of angiogenesis and a number of KSHV genes themselves can induce pathways involved in angiogenesis. Spindle cells are phenotypically endothelial in nature, and therefore, activation through the induction of angiogenic and/or lymphangiogenic phenotypes by the virus may also be directly involved in spindle cell growth and tumor induction. Accordingly, KSHV infection of endothelial cells induces cell autonomous angiogenic phenotypes to activate host cells. KSHV infection can also reprogram blood endothelial cells to lymphatic endothelium. However, KSHV induces some blood endothelial specific genes upon infection of lymphatic endothelial cells creating a phenotypic intermediate between blood and lymphatic endothelium. Induction of pathways involved in angiogenesis and lymphangiogenesis are likely to be critical for tumor cell growth and spread. Thus, induction of both cell autonomous and non-autonomous changes in angiogenic and lymphangiogenic pathways by KSHV likely plays a key role in the formation of KS tumors.

Transgenic Induction of Vascular Endothelial Growth Factor-C Is Strongly Angiogenic in Mouse Embryos but Leads to Persistent Lymphatic Hyperplasia in Adult Tissues

Vascular endothelial growth factor-C (VEGF-C) is the quintessential lymphangiogenic growth factor that is required for the development of the lymphatic system and is capable of stimulating lymphangiogenesis in adults by activating its receptor, VEGFR-3. Although VEGF-C is a major candidate molecule for the development of prolymphangiogenic therapy for defective lymphatic vessels in lymphedema, the stability of lymph vessels generated by exogenous VEGF-C administration is not currently known. We studied VEGF-C-stimulated lymphangiogenesis in inducible transgenic mouse models in which growth factor expression can be spatially and temporally controlled without side effects, such as inflammation. VEGF-C induction in adult mouse skin for 1 to 2 weeks caused robust lymphatic hyperplasia that persisted for at least 6 months. VEGF-C induced lymphangiogenesis in numerous tissues and organs when expressed in the vascular endothelium in either neonates or adult mice. Very few or no effects were observed in either blood vessels or collecting lymph vessels. Additionally, VEGF-C stimulated lymphangiogenesis in embryos after the onset of lymphatic vessel development. Strikingly, a strong angiogenic effect was observed after VEGF-C induction in vascular endothelium at any point before embryonic day 16.5. Our results indicate that blood vessels can undergo VEGF-C-induced angiogenesis even after down-regulation of VEGFR-3 in embryos; however, transient VEGF-C expression in adults can induce long-lasting lymphatic hyperplasia with no obvious side effects on the blood vasculature.

Adenovirus-Mediated Gene Transfer of Human Vascular Endothelial Growth Factor-D Induces Transient Angiogenic Effects in Mouse Hind Limb Muscle

We evaluated the therapeutic potential of adenovirus (Ad)-mediated human vascular endothelial growth factor-D (hVEGF-D) gene delivery in mice. Hind limbs of hypercholesterolemic mice ( n = 120) were injected with AdhVEGF-D, AdhVEGF-A, control AdLacZ (all at 1x10(11)viral particles) or saline. Animals were killed at 4, 7, 14, 28, and 42 days. Newly formed vessels were characterized for their quantity, sprouting, angiogenic versus lymphangiogenic phenotype, and arterial versus venous phenotype by endothelial enzymes markers, pericyte coverage, and electron microscopy. Perfusion was measured by power Doppler ultrasound and edema by magnetic resonance imaging (MRI). AdhVEGF-D induced significant formation of new blood vessels, which featured lumenal enlargement, branching, and sprouting. Branching originated mainly from arterioles. The highest vessel density was present on days 4-7 and the effect lasted up to 28 days. Endothelial marker enzyme activity indicated the predominance of arterial capillaries and arterioles. Forty percent of the neovessels were positive for desmin, indicating that VEGF-D increased pericyte coverage. However, branching vessels were highly positive for smooth muscle actin pericyte marker but negative for desmin. Maximal perfusion was measured during the first week after AdhVEGF-D gene transfer. Ultrastructural analysis showed endothelial cells enriched with vesiculo-vacuolar organelles and cytoplasmic protrusions. Modest lymphangiogenic activity was also detected, which could contribute to the relatively low level of edema detected by MRI. In conclusions, AdhVEGF-D has a strong angiogenic effect and a modest lymphangiogenic effect in mouse skeletal muscle. VEGF-D also increases the presence of pericytes/smooth muscle cells in neovessels. AdhVEGF-D is a potential new agent for the induction of therapeutic vascular growth in skeletal muscle.

Angiogenesis and lymphangiogenesis in thyroid proliferative lesions: relationship to type and tumour behaviour

The role of angiogenesis and lymphangiogenesis in thyroid cancer pathogenesis has not been elucidated. Patterns for tumour behaviour and metastasic spread vary according to tumour type and whether differences in the angiogenic or lymphangiogenic phenotype influence the route for tumour metastases or determine a more aggressive behaviour has not been fully explored. The angiogenic and lymphangiogenic phenotypes of a large cohort of thyroid proliferative lesions (n=191) were studied. Using immunohistochemistry for CD34, lymphatic vessel endothelial receptor-1 (LYVE-1) (specific markers for vascular and lymphatic endothelium respectively), vascular endothelial growth factor (VEGF-A), VEGF-C and fibroblast growth factor-2 (FGF-2), this study analyses microvascular density (MVD), lymphatic vascular density (LVD), and expression of angiogenic and lymphangiogenic factors in normal thyroid (NT; n=19), multinodular goitre (n=25), toxic multinodular goitre (n=8), Graves' hyperplasia (n=22), follicular adenoma (n=54), papillary carcinoma (PC; n=27), incidental papillary microcarcinoma (PMC; n=8), follicular carcinoma (FC; n=20) and medullary carcinoma (MC; n=8). MVD was decreased in proliferative lesions, benign and malignant, compared with NT (P<0.0001). In contrast, VEGF-A expression was increased in thyroid carcinomas (PC, FC and MC) when compared with PMC, benign lesions and NT (P<0.0001). LVD was higher in PC and PMC (P=0.001), and VEGF-C expression was increased in PC (P<0.0001). Despite higher LVD and increased expression of VEGF-A and VEGF-C in thyroid cancers, these markers were not related to poor prognosis in terms of tumour size, multifocality and/or presence of lymphatic or distant metastases. In conclusion, angiogenesis is reduced in thyroid proliferative lesions compared with NT tissue. However, VEGF-A expression is upregulated in thyroid cancers. Lymphangiogenesis and VEGF-C expression are increased in thyroid tumours prone to lymphatic metastases. This may be an important mechanism underlying the differences in metastatic behaviour between papillary and follicular thyroid cancer.

Analysis of the VEGF family and their receptors in serum/plasma of patients with pre-invasive and invasive cervical cancer

5016 Background: The vascular endothelial growth factor (VEGF) family and their receptors are essential regulators of angiogenesis and lymphangiogenesis. This study examined the significance of circulating VEGF, VEGF-C, VEGF-D and their receptors VEGFR-1 and VEGFR-2 in patients with preinvasive and invasive cancer in relation to conventional prognostic parameters. Methods: Blood samples were obtained from 125 women before initial treatment (CIN I-III n = 50; FIGO stage I-IV n = 51; relapse n = 24). Plasma (p) and serum(s) levels of pVEGF, sVEGF-D, sVEGFR-1, sVEGFR-2 (R&amp;D Systems, USA) and sVEGF-C (IBL, Japan) were determined by using a quantitative sandwich enzyme-linked immunosorbent assay (ELISA). Results: The highest level of pVEGF (p = 0.007) and sVEGFR-2 (p = 0.014) were detected in patients with recurrent disease, whereas the highest level of sVEGF-D (p = 0.046) were measured in patients with preinvasive lesions (CIN I-III). Furthermore, significantly elevated levels of sVEGF-C (p = 0.021) were detected in early stages (FIGO I-II) in comparison with advanced stages (FIGO III-IV) of cervical cancer. No significant difference in concentration was observed between the various grades of cervical intraepithelial neoplasia (CIN I-III). Correlations between conventional prognostic markers such as lymph node status (N0/N1), lymphangioinvasion (L0/L1), angioinvasion (V0/V1) and grading (G1/G2/G3) were investigated. Increased plasma levels of VEGF (p = 0.036) correlate with the dissemination of tumor cells to regional lymph nodes (N1),whereas serum VEGF-D levels were significantly decreased in women with lymph-node involvement (N1) (p = 0.045) and vascular invasion (L1) (p = 0.019). None of these members of the VEGF family were found to correlate significantly with histological subtype (squamous cell carcinoma, adenocarcinoma, adeno-squamous cell carcinoma) or grading of the tumor cells (G1/G2/G3). Conclusions: These results show that circulating concentrations of VEGF are associated with the stage of disease. In an early stage of cervical cancer a switch to a lymphangiogenic phenotype (VEGF-D, VEGF-C) might be possible. No significant financial relationships to disclose.

Expression of vascular endothelial growth factor (VEGF)-C and VEGF-D, and their receptor VEGFR-3, during different stages of cervical carcinogenesis.

Cervical carcinogenesis has well-defined stages of disease progression including three grades of pre-invasive lesions--cervical intraepithelial neoplasia grades 1-3 (CIN 1-3)--and invasive cervical cancer. However, the biological properties of CIN lesions prone to develop invasive disease are not well defined. Recent observations suggest that early invasive disease spreads to regional lymph nodes in several tumour types and that growth factors (VEGF-C and VEGF-D) involved in new lymphatic vessel formation may play a crucial role in this process. The present study has assessed the expression of VEGF-C and VEGF-D, and their receptor VEGFR-3, in 152 cervical lesions (33 CIN 1, 33 CIN 2, 37 CIN 3, and 49 squamous cell carcinomas) to determine whether expression of lymphangiogenic factors occurs prior to invasion. The presence of lymphatic vessels was determined using LYVE-1 and podoplanin staining, as well as double immunostaining for LYVE-1/CD34 and podoplanin/CD34. In situ hybridization was performed to determine VEGFR-3 mRNA expression. A significant positive correlation was found between VEGF-C, VEGF-D, and VEGFR-3 expression through the different stages of cervical carcinogenesis. Significant differences in protein expression for VEGF-C, VEGF-D, and VEGFR-3 were found between CIN 1-2 and CIN 3 (p<0.001 for all), but not between CIN 3 and cervical cancer. More than 50% of the CIN 3 lesions showed moderate to strong staining for VEGF-C and VEGF-D, whereas most of the early pre-cancerous lesions (CIN 1 and 2) were negative. In cervical cancer, similar observations to those in CIN 3 were found. VEGFR-3 mRNA expression was found in the cytoplasm of epithelial neoplastic cells and VEGFR3 protein expression was found in more than 50% of CIN 3 lesions and cervical cancers, compared with 15% in CIN 1 and 2. These findings suggest an autocrine growth stimulation pattern via VEGFR-3. Adjacent CIN 3 was present in nine cervical cancers and displayed strong expression for VEGF-C, VEGF-D, and VEGFR-3. These results suggest that in cervical carcinogenesis a switch to the lymphangiogenic phenotype may occur at the stage of CIN 3.

C-erbB receptors in squamous cell carcinomas of the head and neck: clinical significance and correlation with matrix metalloproteinases and vascular endothelial growth factors.

We studied the profile of four c-erbB receptors in head and neck squamous cell carcinomas (HNSCC) and to determine whether their expression was associated with clinicopathological features and key molecules involved in angiogenesis and metastasis. We also assessed the impact of expression on survival. This study included 54 cases of primary HNSCC, of which 27 cases showed lymph node metastasis. The expression of c-erbB receptors, matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) family members was analysed in the same tissue homogenates by semi-quantitative RT-PCR. HNSCC frequently co-expressed multiple c-erbB receptors and showed significant correlations amongst their levels. High expression of epidermal growth factor receptor (EGFR), c-erbB-2 or c-erbB-3 was associated with an infiltrating mode of invasion, nodal metastases and advanced pathological stages. EGFR and c-erbB-2 levels were strongly correlated (P=0.0004-0.029) with the expression of MMP-2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, VEGF-A and VEGF-C whereas the levels of c-erbB-3 and B-4 showed a weaker correlation (P=0.049-0.01) with some MMPs and VEGF-C. Only nodal metastasis and EGFR levels were significantly associated with poor outcome in uni- and multi-variate analysis. We conclude that co-operative signalling of all four c-erbB receptors may play a significant role in the pathogenesis of HNSCC. Amongst these, EGFR appears to be the dominant component controlling the invasive and angio-/lymphangiogenic phenotype in HNSCC via upregulation of multiple MMPs and VEGFs.