The Arteria Lymphatica and Lymphatic Microperforators: A Dedicated Blood Supply to Collecting Lymphatics and Their Potential Implications in Lymphedema: Anatomical Description.

Background: Lymphedema is common after lymphatic damage in cancer treatment, with negative impacts on function and quality of life. Evidence suggests that blood vessel microvasculature is sensitive to irradiation and trauma; however, despite knowledge regarding dedicated mural blood supply to arteries and veins (vasa vasorum), equivalent blood vessels supplying lymphatics have not been characterized. We studied collecting lymphatics for dedicated mural blood vessels in our series of 500 lymphaticovenous anastomosis procedures for lymphedema, and equivalent controls. Methods: Microscopic images of lymphatics from lymphedema and control patients were analyzed for lymphatic wall vascular density. Collecting lymphatics from 20 patients with lymphedema and 10 control patients were sampled for more detailed analysis (podoplanin immunostaining, light/confocal microscopy, microcomputed tomography, and transmission electron microscopy) to assess lymphatic wall ultrastructure and blood supply. Results: Analysis revealed elaborate, dense blood microvessel networks associating with lymphatic walls in lymphedema patients and smaller equivalent vessels in controls. These vasa vasora or "arteria lymphatica" were supplied by regular axial blood vessels, parallel to lymphatic microperforators linking dermal and collecting lymphatics. Lymphatic walls were thicker in lymphedema patients than controls, with immunohistochemistry, computed tomography, transmission electron microscopy, and confocal microscopy characterizing abnormal blood vessels (altered appearance, thickened walls, elastin loss, narrow lumina, and fewer red blood cells) on these lymphatic walls. Conclusions: Dedicated blood vessels on lymphatics are significantly altered in lymphedema. A better understanding of the role of these vessels may reveal mechanistic clues into lymphedema pathophysiology and technical aspects of lymphedema microsurgery, and suggest potential novel therapeutic targets.

Radiation therapy attenuates lymphatic vessel repair by reducing VEGFR-3 signalling.

Introduction: Surgery and radiotherapy are key cancer treatments and the leading causes of damage to the lymphatics, a vascular network critical to fluid homeostasis and immunity. The clinical manifestation of this damage constitutes a devastating side-effect of cancer treatment, known as lymphoedema. Lymphoedema is a chronic condition evolving from the accumulation of interstitial fluid due to impaired drainage via the lymphatics and is recognised to contribute significant morbidity to patients who survive their cancer. Nevertheless, the molecular mechanisms underlying the damage inflicted on lymphatic vessels, and particularly the lymphatic endothelial cells (LEC) that constitute them, by these treatment modalities, remain poorly understood. Methods: We used a combination of cell based assays, biochemistry and animal models of lymphatic injury to examine the molecular mechanisms behind LEC injury and the subsequent effects on lymphatic vessels, particularly the role of the VEGF-C/VEGF-D/VEGFR-3 lymphangiogenic signalling pathway, in lymphatic injury underpinning the development of lymphoedema. Results: We demonstrate that radiotherapy selectively impairs key LEC functions needed for new lymphatic vessel growth (lymphangiogenesis). This effect is mediated by attenuation of VEGFR-3 signalling and downstream signalling cascades. VEGFR-3 protein levels were downregulated in LEC that were exposed to radiation, and LEC were therefore selectively less responsive to VEGF-C and VEGF-D. These findings were validated in our animal models of radiation and surgical injury. Discussion: Our data provide mechanistic insight into injury sustained by LEC and lymphatics during surgical and radiotherapy cancer treatments and underscore the need for alternative non-VEGF-C/VEGFR-3-based therapies to treat lymphoedema.

KSHV infection of endothelial precursor cells with lymphatic characteristics as a novel model for translational Kaposi's sarcoma studies.

Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), a hyperplasia consisting of enlarged malformed vasculature and spindle-shaped cells, the main proliferative component of KS. While spindle cells express markers of lymphatic and blood endothelium, the origin of spindle cells is unknown. Endothelial precursor cells have been proposed as the source of spindle cells. We previously identified two types of circulating endothelial colony forming cells (ECFCs), ones that expressed markers of blood endothelium and ones that expressed markers of lymphatic endothelium. Here we examined both blood and lymphatic ECFCs infected with KSHV. Lymphatic ECFCs are significantly more susceptible to KSHV infection than the blood ECFCs and maintain the viral episomes during passage in culture while the blood ECFCs lose the viral episome. Only the KSHV-infected lymphatic ECFCs (K-ECFCLY) grew to small multicellular colonies in soft agar whereas the infected blood ECFCs and all uninfected ECFCs failed to proliferate. The K-ECFCLYs express high levels of SOX18, which supported the maintenance of high copy number of KSHV genomes. When implanted subcutaneously into NSG mice, the K-ECFCLYs persisted in vivo and recapitulated the phenotype of KS tumor cells with high number of viral genome copies and spindling morphology. These spindle cell hallmarks were significantly reduced when mice were treated with SOX18 inhibitor, SM4. These data suggest that KSHV-infected lymphatic ECFCs can be utilized as a KSHV infection model for in vivo translational studies to test novel inhibitors representing potential treatment modalities for KS.

Corrigendum to 'A transcriptomic dataset evaluating the effect of radiotherapy injury on cells of skin and soft tissue' Data in Brief 41 (2022) 107828.

[This corrects the article DOI: 10.1016/j.dib.2022.107828.].

A transcriptomic dataset evaluating the effect of radiotherapy injury on cells of skin and soft tissue.

Radiotherapy injury to cells of the skin and subcutaneous tissue is an inevitable consequence of external beam radiation for treatment of cancer. This sublethal injury to normal tissues plays a significant role in the development of fibrosis, lymphedema, impaired wound healing, and recurrent infections. To elucidate the transcriptional changes that occur in cells of the skin and soft tissues after radiotherapy injury, we performed genome-wide RNA-sequencing comparing irradiated cells (10Gy) with non-irradiated (0Gy) controls in normal human dermal fibroblasts, normal human keratinocytes, human microvascular endothelial cells, human dermal lymphatic endothelial cells, pericytes and adipose derived stem cell populations. These data are publicly available from the Gene Expression Omnibus database (accession number GSE184119). Further insights can be gained by comparing the mRNA signatures arising from radiation injury derived from these data to publicly available signatures from other studies involving similar or different tissue types. These global targets hold potential for manipulation to mitigate radiotherapy soft tissue injury.

Non-beta blocker enantiomers of propranolol and atenolol inhibit vasculogenesis in infantile hemangioma.

Propranolol and atenolol, current therapies for problematic infantile hemangioma (IH), are composed of R(+) and S(-) enantiomers: the R(+) enantiomer is largely devoid of beta blocker activity. We investigated the effect of R(+) enantiomers of propranolol and atenolol on the formation of IH-like blood vessels from hemangioma stem cells (HemSCs) in a murine xenograft model. Both R(+) enantiomers inhibited HemSC vessel formation in vivo. In vitro, similar to R(+) propranolol, both atenolol and its R(+) enantiomer inhibited HemSC to endothelial cell differentiation. As our previous work implicated the transcription factor sex-determining region Y (SRY) box transcription factor 18 (SOX18) in propranolol-mediated inhibition of HemSC to endothelial differentiation, we tested in parallel a known SOX18 small-molecule inhibitor (Sm4) and show that this compound inhibited HemSC vessel formation in vivo with efficacy similar to that seen with the R(+) enantiomers. We next examined how R(+) propranolol alters SOX18 transcriptional activity. Using a suite of biochemical, biophysical, and quantitative molecular imaging assays, we show that R(+) propranolol directly interfered with SOX18 target gene trans-activation, disrupted SOX18-chromatin binding dynamics, and reduced SOX18 dimer formation. We propose that the R(+) enantiomers of widely used beta blockers could be repurposed to increase the efficiency of current IH treatment and lower adverse associated side effects.

Therapeutic Reversal of Radiotherapy Injury to Pro-fibrotic Dysfunctional Fibroblasts In Vitro Using Adipose-derived Stem Cells.

Cancer patients often require radiotherapy (RTx) to enhance their survival. Unfortunately, RTx also damages nearby healthy non-cancer tissues, leading to progressive fibrotic soft-tissue injury, consisting of pain, contracture, tissue-breakdown, infection, and lymphoedema. Mechanisms underlying the clinically observed ability of fat grafting to ameliorate some of these effects, however, are poorly understood. It was hypothesized that RTx significantly alters fibroblast cell function and the paracrine secretome of adipose-derived stem cells (ADSC) may mitigate these changes. METHODS: To investigate cellular changes resulting in the fibrotic side-effects of RTx, cultured normal human dermal fibroblasts (NHDF) were irradiated (10Gy), then studied using functional assays that reflect key fibroblast functions, and compared with unirradiated controls. RNA-Seq and targeted microarrays (with specific examination of TGFß) were performed to elucidate altered gene pathways. Finally, conditioned-media from ADSC was used to treat irradiated fibroblasts and model fat graft surgery. RESULTS: RTx altered NHDF morphology, with cellular functional changes reflecting transition into a more invasive phenotype: increased migration, adhesion, contractility, and disordered invasion. Changes in genes regulating collagen and MMP homeostasis and cell-cycle progression were also detected. However, TGFß was not identified as a key intracellular regulator of the fibroblast response. Finally, treatment with ADSC-conditioned media reversed the RTx-induced hypermigratory state of NHDF. CONCLUSIONS: Our findings regarding cellular and molecular changes in irradiated fibroblasts help explain clinical manifestations of debilitating RTx-induced fibrosis. ADSC-secretome-mediated reversal indicated that these constituents may be used to combat the devastating side-effects of excessive unwanted fibrosis in RTx and other human fibrotic diseases.

Fat Therapeutics: The Clinical Capacity of Adipose-Derived Stem Cells and Exosomes for Human Disease and Tissue Regeneration.

Fat grafting is a well-established surgical technique used in plastic surgery to restore deficient tissue, and more recently, for its putative regenerative properties. Despite more frequent use of fat grafting, however, a scientific understanding of the mechanisms underlying either survival or remedial benefits of grafted fat remain lacking. Clinical use of fat grafts for breast reconstruction in tissues damaged by radiotherapy first provided clues regarding the clinical potential of stem cells to drive tissue regeneration. Healthy fat introduced into irradiated tissues appeared to reverse radiation injury (fibrosis, scarring, contracture and pain) clinically; a phenomenon since validated in several animal studies. In the quest to explain and enhance these therapeutic effects, adipose-derived stem cells (ADSCs) were suggested as playing a key role and techniques to enrich ADSCs in fat, in turn, followed. Stem cells - the body's rapid response 'road repair crew' - are on standby to combat tissue insults. ADSCs may exert influences either by releasing paracrine-signalling factors alone or as cell-free extracellular vesicles (EVs, exosomes). Alternatively, ADSCs may augment vital immune/inflammatory processes; or themselves differentiate into mature adipose cells to provide the 'building-blocks' for engineered tissue. Regardless, adipose tissue constitutes an ideal source for mesenchymal stem cells for therapeutic application, due to ease of harvest and processing; and a relative abundance of adipose tissue in most patients. Here, we review the clinical applications of fat grafting, ADSC-enhanced fat graft, fat stem cell therapy; and the latest evolution of EVs and nanoparticles in healing, cancer and neurodegenerative and multiorgan disease.

CCL27/CCL28-CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells.

Metastasis via the lymphatic vasculature is an important step in cancer progression. The formation of new lymphatic vessels (lymphangiogenesis), or remodeling of existing lymphatics, is thought to facilitate the entry and transport of tumor cells into lymphatic vessels and on to distant organs. The migration of lymphatic endothelial cells (LEC) toward guidance cues is critical for lymphangiogenesis. While chemokines are known to provide directional navigation for migrating immune cells, their role in mediating LEC migration during tumor-associated lymphangiogenesis is not well defined. Here, we undertook gene profiling studies to identify chemokine-chemokine receptor pairs that are involved in tumor lymphangiogenesis associated with lymph node metastasis. CCL27 and CCL28 were expressed in tumor cells with metastatic potential, while their cognate receptor, CCR10, was expressed by LECs and upregulated by the lymphangiogenic growth factor VEGFD and the proinflammatory cytokine TNFα. Migration assays demonstrated that LECs are attracted to both CCL27 and CCL28 in a CCR10-dependent manner, while abnormal lymphatic vessel patterning in CCR10-deficient mice confirmed the significant role of CCR10 in lymphatic patterning. In vivo analyses showed that LECs are recruited to a CCL27 or CCL28 source, while VEGFD was required in combination with these chemokines to enable formation of coherent lymphatic vessels. Moreover, tumor xenograft experiments demonstrated that even though CCL27 expression by tumors enhanced LEC recruitment, the ability to metastasize was dependent on the expression of VEGFD. These studies demonstrate that CCL27 and CCL28 signaling through CCR10 may cooperate with inflammatory mediators and VEGFD during tumor lymphangiogenesis. SIGNIFICANCE: The study shows that the remodeling of lymphatic vessels in cancer is influenced by CCL27 and CCL28 chemokines, which may provide a future target to modulate metastatic spread.

Genome-wide functional analysis reveals central signaling regulators of lymphatic endothelial cell migration and remodeling.

Lymphatic vessels constitute a specialized vasculature that is involved in development, cancer, obesity, and immune regulation. The migration of lymphatic endothelial cells (LECs) is critical for vessel growth (lymphangiogenesis) and vessel remodeling, processes that modify the lymphatic network in response to developmental or pathological demands. Using the publicly accessible results of our genome-wide siRNA screen, we characterized the migratome of primary human LECs and identified individual genes and signaling pathways that regulate LEC migration. We compared our data set with mRNA differential expression data from endothelial and stromal cells derived from two in vivo models of lymphatic vessel remodeling, viral infection and contact hypersensitivity-induced inflammation, which identified genes selectively involved in regulating LEC migration and remodeling. We also characterized the top candidates in the LEC migratome in primary blood vascular endothelial cells to identify genes with functions common to lymphatic and blood vascular endothelium. On the basis of these analyses, we showed that LGALS1, which encodes the glycan-binding protein Galectin-1, promoted lymphatic vascular growth in vitro and in vivo and contributed to maintenance of the lymphatic endothelial phenotype. Our results provide insight into the signaling networks that control lymphangiogenesis and lymphatic remodeling and potentially identify therapeutic targets and biomarkers in disease specific to lymphatic or blood vessels.

Systematic high-content genome-wide RNAi screens of endothelial cell migration and morphology.

Many cell types undergo migration during embryogenesis and disease. Endothelial cells line blood vessels and lymphatics, which migrate during development as part of angiogenesis, lymphangiogenesis and other types of vessel remodelling. These processes are also important in wound healing, cancer metastasis and cardiovascular conditions. However, the molecular control of endothelial cell migration is poorly understood. Here, we present a dataset containing siRNA screens that identify known and novel components of signalling pathways regulating migration of lymphatic endothelial cells. These components are compared to signalling in blood vascular endothelial cells. Further, using high-content microscopy, we captured a dataset of images of migrating cells following transfection with a genome-wide siRNA library. These datasets are suitable for the identification and analysis of genes involved in endothelial cell migration and morphology, and for computational approaches to identify signalling networks controlling the migratory response and integration of cell morphology, gene function and cell signaling. This may facilitate identification of protein targets for therapeutically modulating angiogenesis and lymphangiogenesis in the context of human disease.

A Three-Dimensional Lymphatic Endothelial Cell Tube Formation Assay to Identify Novel Kinases Involved in Lymphatic Vessel Remodeling.

The lymphatic system is a series of vessels that transport cells and excess fluid from tissues to the blood vascular system. Normally quiescent, the lymphatics can grow or remodel in response to developmental, immunological, or cells pathological stimuli. Lymphatic vessels comprise lymphatic endothelial cells (LECs) that can respond to external growth factors by undergoing proliferation, migration, adhesion, and tube and lumen formation into new vessel structures, a process known as lymphangiogenesis. To understand the key gene and signaling pathways necessary for lymphangiogenesis and lymphatic vessel remodeling, we have developed a three-dimensional LEC tube formation assay to explore the role of kinase signaling in these processes. The collagen-overlay-based assay was used with primary human adult dermal LECs to investigate a library of 60 tyrosine kinase (TK) and TK-like genes by siRNA knockdown. Nine candidate genes were identified and characterized for their ability to modify key parameters of lymphatic tube formation, including tube length, area, thickness, branching, and number of blind-ended sacs. Four genes-ZAP70, IRAK4, RIPK1, and RIPK2-were identified as high-confidence hits after tertiary deconvolution screens and demonstrate the utility of the assay to define LEC genes critical for the formation of tube structures. This assay facilitates the identification of potential molecular targets for novel drugs designed to modulate the remodeling of lymphatics that is important for the metastatic spread of cancer and other pathologies.

Differential Receptor Binding and Regulatory Mechanisms for the Lymphangiogenic Growth Factors Vascular Endothelial Growth Factor (VEGF)-C and -D.

VEGF-C and VEGF-D are secreted glycoproteins that induce angiogenesis and lymphangiogenesis in cancer, thereby promoting tumor growth and spread. They exhibit structural homology and activate VEGFR-2 and VEGFR-3, receptors on endothelial cells that signal for growth of blood vessels and lymphatics. VEGF-C and VEGF-D were thought to exhibit similar bioactivities, yet recent studies indicated distinct signaling mechanisms (e.g. tumor-derived VEGF-C promoted expression of the prostaglandin biosynthetic enzyme COX-2 in lymphatics, a response thought to facilitate metastasis via the lymphatic vasculature, whereas VEGF-D did not). Here we explore the basis of the distinct bioactivities of VEGF-D using a neutralizing antibody, peptide mapping, and mutagenesis to demonstrate that the N-terminal α-helix of mature VEGF-D (Phe93-Arg108) is critical for binding VEGFR-2 and VEGFR-3. Importantly, the N-terminal part of this α-helix, from Phe93 to Thr98, is required for binding VEGFR-3 but not VEGFR-2. Surprisingly, the corresponding part of the α-helix in mature VEGF-C did not influence binding to either VEGFR-2 or VEGFR-3, indicating distinct determinants of receptor binding by these growth factors. A variant of mature VEGF-D harboring a mutation in the N-terminal α-helix, D103A, exhibited enhanced potency for activating VEGFR-3, was able to promote increased COX-2 mRNA levels in lymphatic endothelial cells, and had enhanced capacity to induce lymphatic sprouting in vivo This mutant may be useful for developing protein-based therapeutics to drive lymphangiogenesis in clinical settings, such as lymphedema. Our studies shed light on the VEGF-D structure/function relationship and provide a basis for understanding functional differences compared with VEGF-C.

Chronic stress in mice remodels lymph vasculature to promote tumour cell dissemination.

Chronic stress induces signalling from the sympathetic nervous system (SNS) and drives cancer progression, although the pathways of tumour cell dissemination are unclear. Here we show that chronic stress restructures lymphatic networks within and around tumours to provide pathways for tumour cell escape. We show that VEGFC derived from tumour cells is required for stress to induce lymphatic remodelling and that this depends on COX2 inflammatory signalling from macrophages. Pharmacological inhibition of SNS signalling blocks the effect of chronic stress on lymphatic remodelling in vivo and reduces lymphatic metastasis in preclinical cancer models and in patients with breast cancer. These findings reveal unanticipated communication between stress-induced neural signalling and inflammation, which regulates tumour lymphatic architecture and lymphogenous tumour cell dissemination. These findings suggest that limiting the effects of SNS signalling to prevent tumour cell dissemination through lymphatic routes may provide a strategy to improve cancer outcomes.

Lymphangiogenesis and lymphatic vessel remodelling in cancer.

The generation of new lymphatic vessels through lymphangiogenesis and the remodelling of existing lymphatics are thought to be important steps in cancer metastasis. The past decade has been exciting in terms of research into the molecular and cellular biology of lymphatic vessels in cancer, and it has been shown that the molecular control of tumour lymphangiogenesis has similarities to that of tumour angiogenesis. Nevertheless, there are significant mechanistic differences between these biological processes. We are now developing a greater understanding of the specific roles of distinct lymphatic vessel subtypes in cancer, and this provides opportunities to improve diagnostic and therapeutic approaches that aim to restrict the progression of cancer.

Lymphovascular and neural regulation of metastasis: shared tumour signalling pathways and novel therapeutic approaches.

The progression of cancer is supported by a wide variety of non-neoplastic cell types which make up the tumour stroma, including immune cells, endothelial cells, cancer-associated fibroblasts and nerve fibres. These host cells contribute molecular signals that enhance primary tumour growth and provide physical avenues for metastatic dissemination. This article provides an overview of the role of blood vessels, lymphatic vessels and nerve fibres in the tumour microenvironment and highlights the interconnected molecular signalling pathways that control their development and activation in cancer. Further, this article highlights the known pharmacological agents which target these pathways and discusses the potential therapeutic uses of drugs that target angiogenesis, lymphangiogenesis and stress-response pathways in the different stages of cancer care.

Signaling for lymphangiogenesis via VEGFR-3 is required for the early events of metastasis.

Metastasis to regional lymph nodes is an important and early event in many tumors. Vascular endothelial growth factor-C (VEGF-C), VEGF-D and their receptor VEGFR-3, play a role in tumor spread via the lymphatics, although the timing of their involvement is not understood. In contrast, VEGFR-2, activated by VEGF-A, VEGF-C and VEGF-D, is a mediator of angiogenesis and drives primary tumor growth. We demonstrate the critical role for VEGFR-3, but not VEGFR-2, in the early events of metastasis. In a tumor model exhibiting both VEGF-D-dependent angiogenesis and lymphangiogenesis, an antibody to VEGFR-2 (DC101) was capable of inhibiting angiogenesis (79 % reduction in PECAM + blood vessels) and growth (93 % reduction in tumor volume). However, unlike an anti-VEGFR-3 Mab (mF4-31C1), DC101 was not capable of eliminating either tumor lymphangiogenesis or lymphogenous metastasis (60 % reduction of lymph node metastasis by DC101 vs 95 % by mF4-31C1). Early excision of the primary tumors demonstrated that VEGF-D-mediated tumor spread precedes angiogenesis-induced growth. Small but highly metastatic primary human breast cancers had significantly higher lymphatic vessel density (23.1 vessels/mm(2)) than size-matched (11.7) or larger non-metastatic tumors (12.4) thus supporting the importance of lymphatic vessels, as opposed to angiogenesis-mediated primary tumor growth, for nodal metastasis. These results suggest that lymphangiogenesis via VEGF-D is more critical than angiogenesis for nodal metastasis.

The propeptides of VEGF-D determine heparin binding, receptor heterodimerization, and effects on tumor biology.

VEGF-D is an angiogenic and lymphangiogenic glycoprotein that can be proteolytically processed generating various forms differing in subunit composition due to the presence or absence of N- and C-terminal propeptides. These propeptides flank the central VEGF homology domain, that contains the binding sites for VEGF receptors (VEGFRs), but their biological functions were unclear. Characterization of propeptide function will be important to clarify which forms of VEGF-D are biologically active and therefore clinically relevant. Here we use VEGF-D mutants deficient in either propeptide, and in the capacity to process the remaining propeptide, to monitor the functions of these domains. We report for the first time that VEGF-D binds heparin, and that the C-terminal propeptide significantly enhances this interaction (removal of this propeptide from full-length VEGF-D completely prevents heparin binding). We also show that removal of either the N- or C-terminal propeptide is required for VEGF-D to drive formation of VEGFR-2/VEGFR-3 heterodimers which have recently been shown to positively regulate angiogenic sprouting. The mature form of VEGF-D, lacking both propeptides, can also promote formation of these receptor heterodimers. In a mouse tumor model, removal of only the C-terminal propeptide from full-length VEGF-D was sufficient to enhance angiogenesis and tumor growth. In contrast, removal of both propeptides is required for high rates of lymph node metastasis. The findings reported here show that the propeptides profoundly influence molecular interactions of VEGF-D with VEGF receptors, co-receptors, and heparin, and its effects on tumor biology.

Tumor location and nature of lymphatic vessels are key determinants of cancer metastasis.

Tumor metastasis to lymph nodes is a key indicator of patient survival, and is enhanced by the neo-lymphatics induced by tumor-secreted VEGF-C or VEGF-D, acting via VEGFR-3 signalling. These targets constitute important avenues for anti-metastatic treatment. Despite this new understanding, clinical observations linking metastasis with tumor depth or location suggest that lymphangiogenic growth factors are not the sole determinants of metastasis. Here we explored the influence of tumor proximity to lymphatics capable of responding to growth factors on nodal metastasis in a murine VEGF-D over-expression tumor model. We found that primary tumor location profoundly influenced VEGF-D-mediated lymph node metastasis: 89 % of tumors associated with the flank skin metastasised, in contrast with only 19 % of tumors located more deeply on the body wall (p < 0.01). Lymphatics in metastatic tumors arose from small lymphatics, and displayed distinct molecular and morphological profiles compared with those found in normal lymphatics. Smaller lymphatic subtypes were more abundant in skin (2.5-fold, p < 0.01) than in body wall, providing a richer source of lymphatics for VEGF-D(+) skin tumors, a phenomenon also confirmed in human samples. This study shows that the proximity of a VEGF-D(+) primary tumor to small lymphatics is an important determinant of metastasis. These observations may explain why tumor location relative to the lymphatic network is prognostically important for some human cancers.

The connection between lymphangiogenic signalling and prostaglandin biology: a missing link in the metastatic pathway.

Substantial evidence supports important independent roles for lymphangiogenic growth factor signaling and prostaglandins in the metastatic spread of cancer. The significance of the lymphangiogenic growth factors, vascular endothelial growth factor (VEGF)-C and VEGF-D, is well established in animal models of metastasis, and a strong correlation exits between an increase in expression of VEGF-C and VEGF-D, and metastatic spread in various solid human cancers. Similarly, key enzymes that control the production of prostaglandins, cyclooxygenases (COX-1 and COX-2, prototypic targets of Non-steroidal anti-inflammatory drugs (NSAIDs)), are frequently over-expressed or de-regulated in the progression of cancer. Recent data have suggested an intersection of lymphangiogenic growth factor signaling and the prostaglandin pathways in the control of metastatic spread via the lymphatic vasculature. Furthermore, this correlates with current clinical data showing that some NSAIDs enhance the survival of cancer patients through reducing metastasis. Here, we discuss the potential biochemical and cellular basis for such anti-cancer effects of NSAIDs through the prostaglandin and VEGF signaling pathways.