You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation}
Article:
Abstract:
Lymphatic invasion and lymph node metastasis correlate with poor clinical outcome in melanoma. However, the mechanisms of lymphatic dissemination in distant metastasis remain incompletely understood. We show here that exposure of expansively growing human WM852 melanoma cells, but not singly invasive Bowes cells, to lymphatic endothelial cells( LEC) in 3D co-culture facilitates melanoma distant organ metastasis in mice. To dissect the underlying molecular mechanisms, we established LEC co-cultures with different melanoma cells originating from primary tumors or metastases. Notably, the expansively growing metastatic melanoma cells adopted an invasively sprouting phenotype in 3D matrix that was dependent on MMP14, Notch3 and β1-integrin. Unexpectedly, MMP14 was necessary for LEC-induced Notch3 induction and coincident β1-integrin activation. Moreover, MMP14 and Notch3 were required for LEC-mediated metastasis of zebrafish xenografts. This study uncovers a unique mechanism whereby LEC contact promotes melanoma metastasis by inducing a reversible switch from 3D growth to invasively sprouting cell phenotype.
Introduction:
Distant organ metastasis requires that the tumor cells gain access into the hematogenous circulation( Lambert et al., 2017). The metastatic dissemination of cancer cells is expedited by molecular changes promoting the ability of cancer cells to invade across the surrounding extracellular matrices( ECM) and into the lumen of lymphatic or blood vessels. In order to survive in circulation as well as extravasate and colonize the distant organ sites the cancer cells need to survive a variety of stresses such as hemodynamic shear forces, trapping to vascular beds and ROS, and gain the ability to invasively grow at the new tissue microenvironment( Piskounova et al., 2015; Strilic and Offermanns, 2017). Many of these activities have been found to be enhanced in tumor cell clusters relative to singly invading tumor cells. The local microenvironment including the ECM and cell-non-autonomous interactions between cancer cells and stromal cells play a vital role in metastasis. In addition to providing a direct route for dissemination, the tumor lymphatics have been proposed to directly modulate the metastatic cascade through mechanisms that have remained elusive( Alitalo and Detmar, 2012). Clinical observations of satellite melanoma tumors growing between the primary tumor and draining lymph nodes have suggested that the surrounding lymphatic endothelium serves as a protective microenvironment for the survival of incipient metastatic cells( Meier et al., 2002). In support of this hypothesis, over the last few years it has become increasingly clear that lymphatic endothelial cells( LECs) in fact actively interact with the surrounding cells in the tissue, thus regulating both physiological and pathological processes including tumor progression and metastasis. Both paracrine communication and direct cell-cell interactions between tumor cells and the associated lymphatics have been shown to drive tumor progression and dissemination. For example, chemokine receptor-ligand interactions between melanoma and LECs drive chemotaxis of tumor cells towards the lymphatics( Cabioglu et al., 2005; Das et al., 2013; Shields et al., 2007). Alternatively, tumor cells secrete factors like lipoxygenase, which can induce downregulation of the endothelial surface molecules and loosening of the LEC junctions in vitro( Kerjaschki et al., 2011). In this study, we set to investigate how the LECs in the tumor microenvironment affect the metastatic melanoma cell phenotype. To this end, we implemented 2D- and 3D melanoma-LEC co-culture models, which enable a systematic analysis of the molecular crosstalk between the tumor cells and the lymphatic endothelium. We found that the interaction of melanoma cells with LECs induced matrix-metalloproteinase-14( MMP14, also known as MT1-MMP)-dependent Notch3 and β1-integrin activation in the expansively growing metastatic melanoma cells, leading to invasive sprouting of cells in 3D matrices. Importantly, the interaction of these melanoma cells with LECs led to significantly increased metastasis of melanoma xenografts in vivo, which was dependent on MMP14 and Notch3. Thus, the crosstalk with LECs promotes melanoma metastasis by inducing a reversible switch to invasively sprouting melanoma cells.
Discussion:
Melanoma cells have been shown to be in close contact with the lymphatic vessels in human specimens and often metastasize via the lymphatic system,( Niakosari et al., 2008), implying that the melanoma cell-LEC interactions are common events in human melanomas. In addition, clinical data correlating metastatic spread with lymphatic infiltration suggest that melanoma-LEC interactions could contribute to the tumor progression and metastasis. Here we investigated the consequences of such interactions by coupling unique 2D and 3D LEC-melanoma co-culture models to in vivo mouse tumor model, transcriptome profiling, in vitro functional studies and a zebrafish xenograft/metastasis assay. In the xenograft mouse model, dissemination of the metastatic WM852 cell line to distant organs was increased by LEC priming. Furthermore, in vitro molecular and functional studies revealed that the direct contact of LECs with metastatic melanoma cell lines triggered changes particularly in the localization of MMP14, an important protease for cancer-cell invasion( Tatti et al., 2015; Turunen et al., 2017), which in turn induced expression and activation of Notch3 and activation of β1-integrin leading to a significant change in the 3D growth phenotype of the melanoma cells. The interaction with LECs did not, however, induce metastasis or change the 3D growth phenotype of non-metastatic Bowes cells. Although Bowes have active MMP14 and an invasive, single cell 3D sprouting phenotype in vitro( Tatti et al., 2011), these intrinsic properties were not sufficient to support distant organ metastatic colonization in vivo. Moreover, ectopic expression of MMP14 or NICD3 did not induce any change in their 3D growth phenotype. Similar to Bowes, LEC priming did not markedly alter the 3D phenotype of another non-metastatic melanoma cell line WM793, that, despite of MMP14 expression( Tatti et al., 2011), continued to grow expansively in 3D as indicated by the sphere-like growth in 3D fibrin. However, these cells switched to the invasively sprouting growth upon introduction of NICD3, and the sprouting was dependent on β1-integrin. Another possible contributor to this change could be theβ1-integrin processing and trafficking, which appears quite different in Bowes as compared to WM793 and WM852. Based on our results, it is plausible that the capacity for expansive growth is a property required for the NICD3 dependent change in the 3D growth. Fittingly, WM852 cells with strong intrinsic capacity to grow expansively, switched to the invasively sprouting growth phenotype after the LEC-mediated transient activation of the MMP14-Notch3-β1-integrin axis, which resulted in increased MMP14 and Notch3 dependent metastasis in vivo. The LEC contact likewise switched the metastatic, expansively growing WM165 cells to invasive sprouting via activation of MMP14-Notch3-β1-integrin axis. Supporting the importance of this signaling axis, neither Bowes nor WM793 responded to the LEC contact by activating these key effectors needed for the metastatic phenotype. Melanoma cell invasion into the deeper layers of the skin and distant sites is associated with molecular changes such as increased activation of the tissue degrading proteases including the MMPs( Moro et al., 2014; Villanueva and Herlyn, 2008). Our results demonstrate that the LEC contact increased MMP14 on the melanoma cell plasma membrane and cell-cell contacts in the metastatic melanoma cell lines. This was accompanied with an MMP14-dependent increase in Notch3 expression by a yet unidentified mechanism. MMP14 was recently shown to interact with and activate Notch1 at the cell membrane of melanoma cells, which supported melanoma cell growth( Ma et al., 2014). Although Notch receptors 1 and 3 are highly activated in melanoma with clear pro-tumorigenic functions, no Notch-activating mutations have been identified in melanoma. Therefore, the herein identified MMP14 dependent Notch3 upregulation may prove important in metastatic melanoma progression. Our results also revealed that LEC contact induced β1-integrin activation in the metastatic WM852 and WM165 cells, which was required for the 3D sprouting of WM852* cells. β1-integrin activation was dependent on MMP14 as its depletion by siRNA abolished the β1-integrin activation and slightly, but non-significantly, reduced total β1-integrin protein levels These findings concur with previous studies demonstrating MMP14 incorporation into β1-integrin rich adhesion complexes( Woskowicz et al., 2013) and MMP14 binding to β1-integrin upon mammary branching morphogenesis( Mori et al., 2013) as well as its role in controlling the 3D cell-shape of stem cells through activation of β1-integrin signaling( Tang et al., 2013). Moreover, melanoma metastases express more activated β1-integrin than the primary tumors( Kato et al., 2012). Our current results add to this concept by demonstrating that endothelial cell contact activates integrins in melanoma cells, thus providing a putative mechanism for the contribution of the stromal lymphatic microenvironment to melanoma metastasis. Negative regulator of β1-integrin, ICAP-1, has been shown to act as a potent inducer of Notch1-signaling by transcriptionally upregulating Notch ligands DLL1 and DLL4 and the downstream transcription factors HEY1, HEY2, and HES5 in blood endothelial cells and thereby restrict the sprouting angiogenesis of the ECs( Brütsch et al., 2010). However, as our transcriptomic data of the LEC-primed melanoma cells did not reveal changes in ICAP-1 expression upon LEC-priming it probably does not play a significant role in the Notch3-dependent invasive sprouting of metastatic melanoma cells. Furthermore, our results indicate that β1-integrin functions downstream of Notch3 in the LEC-primed melanomas. Although melanoma cells at the primary tumor site predominantly disseminate through the lymphatic vascular route, the distant organ metastasis requires cells entering blood circulation and colonization in the distant organ tissue. MMP14, Notch3 and β1-integrin are all proteins linked to epithelial-to-mesenchymal transition in carcinoma cell metastasis( Cao et al., 2008; Kato et al., 2012; Liu et al., 2014), supporting the idea that the metastatic WM852* and WM165* melanoma cells have also undergone a transient transition into a more mesenchymally invasive state that can promote hematogenous dissemination. However, since the Bowes cells with high MMP14 levels did not metastasize, MMP14 activity by itself is apparently not enough for efficient distant organ metastasis. In the absence of strong cancer cell-cell interactions, constitutively high MMP14 activity can facilitate single cell invasion, thereby compromising the adhesive and expansive growth potential essential for efficient tissue colonization of the tumor cells. We therefore propose that the capacity for transiently induced 3D invasive sprouting coupled to expansive growth behavior are required for the most aggressive melanoma cells to enter and survive in the blood circulation, as well as to efficiently colonize the distant organs. Melanoma progression is suggested to be driven by reversible and functional reprogramming of different signaling routes, known as reversible phenotypic plasticity of cell( Vandamme and Berx, 2014). Changes and interactions in the tumor microenvironment are believed to direct this phenotype-switching, but the detailed mechanisms are largely unknown. Our current results support the role of reversible phenotype-switching in melanoma progression and identifies the stromal lymphatic endothelium as one of the key triggers for the process to support both hematogenous dissemination and tissue colonization at the distant organs.
The death rates for many types of cancer have dropped , but melanoma remains a serious concern . This type of skin cancer is especially aggressive because it can spread to distant organs . Melanoma often spreads via the lymphatic system , a network of vessels that extends throughout the body to drain fluid from the body’s tissues . The lymphatic system also includes structures – called lymph nodes – that filter bacteria from this fluid; this helps to defend against infection . When melanoma spreads to lymph nodes and distant organs , clinicians diagnose it as Stage IV melanoma . For patients at this stage , the outcome is often poor . It is clear that melanoma exploits lymph vessels to spread throughout the body . But researchers also suspect that vessel cells interact with the cancer cells , helping the melanoma invade distant organs . Understanding exactly how lymph vessels promote the spread of melanoma will lead to better options for treating this aggressive cancer . Pekkonen , Alve et al . investigated whether exposing human melanoma cells to cells from the walls of human lymph vessels would make the cancer cells more aggressive . Indeed , after growing the two cell types together in the laboratory , the melanoma cells became more invasive . When transplanted into mice , these cancer cells spread to and invaded the rodents’ distant organs . Pekkonen , Alve et al . conducted a series of experiments to identify specific proteins in the melanoma cellsthat were responsible for making the cancer more invasive after it interacted with the lymph vessel cells . These experiments identified proteins called MMP14 , Notch3 , and β1-integrin as critical to the invasive spread of melanoma cells . When melanoma cells with less MMP14 or Notch3 were implanted into zebrafish , the cancer cells spread less efficiently . These findings may represent new leads that clinicians can test to see if they are markers of cancers that are most likely to spread and that the pharmaceutical industry can pursue to treat melanoma patients .