The endothelial protein PLVAP in lymphatics controls the entry of lymphocytes and antigens into lymph nodes.

In the lymphatic sinuses of draining lymph nodes, soluble lymph-borne antigens enter the reticular conduits in a size-selective manner and lymphocytes transmigrate to the parenchyma. The molecular mechanisms that control these processes are unknown. Here we unexpectedly found that PLVAP, a prototypic endothelial protein of blood vessels, was synthesized in the sinus-lining lymphatic endothelial cells covering the distal conduits. In PLVAP-deficient mice, both small antigens and large antigens entered the conduit system, and the transmigration of lymphocytes through the sinus floor was augmented. Mechanistically, the filtering function of the lymphatic sinus endothelium was dependent on diaphragms formed by PLVAP fibrils in transendothelial channels. Thus, in the lymphatic sinus, PLVAP forms a physical sieve that regulates the parenchymal entry of lymphocytes and soluble antigens.

Compartmentalization of adenosine metabolism in cancer cells and its modulation during acute hypoxia.

Extracellular adenosine mediates diverse anti-inflammatory, angiogenic and vasoactive effects, and has become an important therapeutic target for cancer, which has been translated into clinical trials. This study was designed to comprehensively assess adenosine metabolism in prostate and breast cancer cells. We identified cellular adenosine turnover as a complex cascade, comprising (1) the ectoenzymatic breakdown of ATP via sequential ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1, officially known as ENPP1), ecto-5'-nucleotidase (CD73, also known as NT5E), and adenosine deaminase reactions, and ATP re-synthesis through a counteracting adenylate kinase and members of the nucleoside diphosphate kinase (NDPK, also known as NME/NM23) family; (2) the uptake of nucleotide-derived adenosine via equilibrative nucleoside transporters; and (3) the intracellular adenosine phosphorylation into ATP by adenosine kinase and other nucleotide kinases. The exposure of cancer cells to 1% O2 for 24 h triggered an ∼2-fold upregulation of CD73, without affecting nucleoside transporters, adenosine kinase activity and cellular ATP content. The ability of adenosine to inhibit the tumor-initiating potential of breast cancer cells via a receptor-independent mechanism was confirmed in vivo using a xenograft mouse model. The existence of redundant pathways controlling extracellular and intracellular adenosine provides a sufficient justification for reexamination of the current concepts of cellular purine homeostasis and signaling in cancer.This article has an associated First Person interview with the first author of the paper.

Subcutaneous B16 melanoma impairs intrinsic pressure generation and relaxation of the heart, which are not restored by short-term voluntary exercise in mice.

The aim of this study was to investigate whether subcutaneous melanoma impairs intrinsic cardiac function and hypoxia tolerance in mice. In addition, it was investigated whether these changes could be prevented by voluntary wheel-running exercise. The roles of different molecular pathways were also analyzed. Male mice (C57Bl/6NCrl) were divided into unexercised tumor-free group, unexercised melanoma group, and exercised melanoma group. The experiment lasted 2.7 ± 0.1 wk (determined by the tumor size) after which the heart function was measured in different oxygen levels ex vivo using Langendorff method. All the melanoma mice had lower pressure amplitude (50.3%), rate of pressure production (54.1%), and decline (52.5%) in hearts ex vivo when compared with tumor-free group. There were no functional differences between the two melanoma groups. All the groups had similar weight changes, heart weights, cardiomyocyte sizes, levels of Ca2+ channels, energy metabolism enzyme activities, lipid peroxidation, and reactive oxygen species in their cardiac tissue homogenates. However, all the melanoma mice had 7.4% lower superoxidase dismutase activity compared with the control animals, which might reduce the ability of the heart to react to changes in oxidative stress. The exercising melanoma group had a 28.6% higher average heart capillary density compared with the unexercised melanoma group. Short-term wheel running did not affect the tumor growth. In conclusion, subcutaneous melanoma seems to impair intrinsic heart function even before cachexia, and these functional alterations were not caused by any of the measured molecular markers. Short-term voluntary wheel-running exercise was insufficient to alleviate the intrinsic cardiac impairments caused by melanoma.NEW & NOTEWORTHY Melanoma has been shown to induce cardiac atrophy and impair cardiac function in vivo, however, it has not been investigated how melanoma affects the intrinsic heart function. Here, we showed that subcutaneous melanoma can impair intrinsic heart function in noncachectic mice, decreasing the heart's pressure production and relaxation. In addition, we investigated whether short-term voluntary wheel-running exercise could attenuate the impairment of intrinsic cardiac function. However, our results do not seem to support this hypothesis.

Vascular adhesion protein-1 defines a unique subpopulation of human hematopoietic stem cells and regulates their proliferation.

Although the development of hematopoietic stem cells (HSC) has been studied in great detail, their heterogeneity and relationships to different cell lineages remain incompletely understood. Moreover, the role of Vascular Adhesion Protein-1 in bone marrow hematopoiesis has remained unknown. Here we show that VAP-1, an adhesin and a primary amine oxidase producing hydrogen peroxide, is expressed on a subset of human HSC and bone marrow vasculature forming a hematogenic niche. Bulk and single-cell RNAseq analyses reveal that VAP-1+ HSC represent a transcriptionally unique small subset of differentiated and proliferating HSC, while VAP-1- HSC are the most primitive HSC. VAP-1 generated hydrogen peroxide acts via the p53 signaling pathway to regulate HSC proliferation. HSC expansion and differentiation into colony-forming units are enhanced by inhibition of VAP-1. Contribution of VAP-1 to HSC proliferation was confirmed with mice deficient of VAP-1, mice expressing mutated VAP-1 and using an enzyme inhibitor. In conclusion, VAP-1 expression allows the characterization and prospective isolation of a new subset of human HSC. Since VAP-1 serves as a check point-like inhibitor in HSC differentiation, the use of VAP-1 inhibitors enables the expansion of HSC.

Gene-expression profiling of different arms of lymphatic vasculature identifies candidates for manipulation of cell traffic.

Afferent lymphatic vessels bring antigens and diverse populations of leukocytes to draining lymph nodes, whereas efferent lymphatics allow only lymphocytes and antigens to leave the nodes. Despite the fundamental importance of afferent vs. efferent lymphatics in immune response and cancer spread, the molecular characteristics of these different arms of the lymphatic vasculature are largely unknown. The objective of this work was to explore molecular differences behind the distinct functions of afferent and efferent lymphatic vessels, and find possible molecules mediating lymphocyte traffic. We used laser-capture microdissection and cell sorting to isolate lymphatic endothelial cells (LECs) from the subcapsular sinus (SS, afferent) and lymphatic sinus (LS, efferent) for transcriptional analyses. The results reveal marked differences between afferent and efferent LECs and identify molecules on lymphatic vessels. Further characterizations of Siglec-1 (CD169) and macrophage scavenger receptor 1 (MSR1/CD204), show that they are discriminatively expressed on lymphatic endothelium of the SS but not on lymphatic vasculature of the LS. In contrast, endomucin (EMCN) is present on the LS endothelium and not on lymphatic endothelium of the SS. Moreover, both murine and human MSR1 on lymphatic endothelium of the SS bind lymphocytes and in in vivo studies MSR1 regulates entrance of lymphocytes from the SS to the lymph node parenchyma. In conclusion, this paper reports surprisingly distinct molecular profiles for afferent and efferent lymphatics and a function for MSR1. These results may open avenues to explore some of the now-identified molecules as targets to manipulate the function of lymphatic vessels.

Stabilin-1 expression defines a subset of macrophages that mediate tissue homeostasis and prevent fibrosis in chronic liver injury.

Macrophages are key regulators of fibrosis development and resolution. Elucidating the mechanisms by which they mediate this process is crucial for establishing their therapeutic potential. Here, we use experimental models of liver fibrosis to show that deficiency of the scavenger receptor, stabilin-1, exacerbates fibrosis and delays resolution during the recovery phase. We detected a subset of stabilin-1(+) macrophages that were induced at sites of cellular injury close to the hepatic scar in mouse models of liver fibrosis and in human liver disease. Stabilin-1 deficiency abrogated malondialdehyde-LDL (MDA-LDL) uptake by hepatic macrophages and was associated with excess collagen III deposition. Mechanistically, the lack of stabilin-1 led to elevated intrahepatic levels of the profibrogenic chemokine CCL3 and an increase in GFAP(+) fibrogenic cells. Stabilin-1(-/-) macrophages demonstrated a proinflammatory phenotype during liver injury and the normal induction of Ly6C(lo) monocytes during resolution was absent in stabilin-1 knockouts leading to persistence of fibrosis. Human stabilin-1(+) monocytes efficiently internalized MDA-LDL and this suppressed their ability to secrete CCL3, suggesting that loss of stabilin-1 removes a brake to CCL3 secretion. Experiments with cell-lineage-specific knockouts revealed that stabilin-1 expression in myeloid cells is required for the induction of this subset of macrophages and that increased fibrosis occurs in their absence. This study demonstrates a previously unidentified regulatory pathway in fibrogenesis in which a macrophage scavenger receptor protects against organ fibrosis by removing fibrogenic products of lipid peroxidation. Thus, stabilin-1(+) macrophages shape the tissue microenvironment during liver injury and healing.

Ecto-5'-nucleotidase/CD73 enhances endothelial barrier function and sprouting in blood but not lymphatic vasculature.

CD73/ecto-5'-nucleotidase is a key enzyme in the regulation of purinergic signaling and inflammatory reactions. It hydrolyzes extracellular AMP into adenosine, which dampens immune cell activation, and reduces leukocyte trafficking. By comparing CD73 expression and function in mononuclear and endothelial cells (ECs) of blood and lymph, we show that extracellular purines and CD73 activity have differential effects in these two vascular systems. We found that CD8-positive T lymphocytes and CD19-positive B lymphocytes in human lymph expressed high levels of CD73 and other purinergic enzymes and adenosine receptors. Soluble CD73 was less abundant in human lymph than in serum, whereas CD73 activity was higher in afferent lymphatic ECs than in blood ECs. Adenosine signaling improved barrier function and induced sprouting of human blood, but not lymphatic, ECs in vitro. Similarly, using CD73-deficient mice we found that CD73 controls only blood vascular permeability at selected lymphoid organs under physiological conditions. Thus, both vascular and lymphatic arms of the immune system synthesize the components of purinergic signaling system, but surprisingly they use CD73 differentially to control endothelial permeability and sprouting.

CD44 binds to macrophage mannose receptor on lymphatic endothelium and supports lymphocyte migration via afferent lymphatics.

RATIONALE: Macrophage mannose receptor (MRC) is one of the few molecules known to be involved in lymphocyte trafficking via the lymphatic vessels. In endothelial cells of efferent lymphatics, it binds L-selectin on lymphocytes. In afferent lymphatics, MRC mediates trafficking of both normal and malignant L-selectin-negative cells to the draining lymph nodes. OBJECTIVE: This work was designed to search for additional lymphocyte ligands of MRC to elucidate how lymphocytes migrate into the draining lymph nodes. METHODS AND RESULTS: Using immunoprecipitation and binding studies with natural and recombinant proteins, we show that MRC and CD44 can interact with each other. Fine mapping revealed that the cysteine-rich domain of MRC binds to the chondroitin sulfate side chains of CD44. In vivo homing experiments with MRC- and CD44-deficient mice verified that MRC and CD44 function as a receptor-ligand pair in supporting lymphocyte migration via the afferent lymphatics into the draining lymph nodes. CONCLUSIONS: These data identify a new counter-receptor for MRC and reveal CD44 as a new molecule involved in the poorly understood process of lymphocyte transit via the lymphatic vasculature.

Consequences of the lack of CD73 and prostatic acid phosphatase in the lymphoid organs.

CD73, ecto-5'-nucleotidase, is the key enzyme catalyzing the conversion of extracellular AMP to adenosine that controls vascular permeability and immunosuppression. Also prostatic acid phosphatase (PAP) possesses ecto-5'-nucleotidase/AMPase activity and is present in leukocytes. However, its role related to immune system is unknown. Therefore, we analyzed enzymatic activities and leukocyte subtypes of CD73 and PAP knockouts and generated CD73/PAP double knockout mice to elucidate the contribution of CD73 and PAP to immunological parameters. Enzymatic assays confirmed the ability of recombinant human PAP to hydrolyze [(3)H]AMP, although at much lower rate than human CD73. Nevertheless, 5'-nucleotidase/AMPase activity in splenocytes and lymphocytes from PAP(-/-) mice tended to be lower than in wild-type controls, suggesting potential contribution of PAP, along with CD73, into lymphoid AMP metabolism ex vivo. Single knockouts had decreased number of CD4(+)/CD25(+)/FoxP3 (+) regulatory T cells in thymus and CD73/PAP double knockouts exhibited reduced percentages of CD4(+) cells in spleen, regulatory T cells in lymph nodes and thymus, and CD4(+) and CD8(+) cells in blood. These findings suggest that PAP has a synergistic role together with CD73 in the immune system by contributing to the balance of leukocyte subpopulations and especially to the number of regulatory T cells in lymph nodes and thymus.

Different role of CD73 in leukocyte trafficking via blood and lymph vessels.

CD73 is involved in the extracellular ATP metabolism by dephosphorylating extracellular AMP to adenosine and thus regulating permeability of the blood vessels and leukocyte traffic into the tissues. It is also present on lymphatic vessels where its distribution and function have not been characterized. We found that CD73 is expressed on a subpopulation of afferent lymph vessels but is absent on efferent lymphatics, unlike LYVE-1 and podoplanin, which are expressed on both types of lymphatics. The extracellular nucleotide metabolism on lymphatic endothelium differs from that on blood vessel endothelium as lymphatic endothelium has lower NTPDase and higher ecto-5'-nucleotidase/CD73 activity than blood vascular endothelium. In knockout mice, the lack of CD73 on lymphocytes decreases migration of lymphocytes to the draining lymph nodes more than 50% while CD73-deficient lymph vessels mediate lymphocyte trafficking as efficiently as the wild-type lymphatics. Thus, although endothelial CD73 is important for permeability and leukocyte extravasation in blood vessels, it does not have a role in these functions on lymphatics. Instead, lymphocyte CD73 is intimately involved in lymphocyte migration via afferent lymphatic vessels.

Altered purinergic signaling in CD73-deficient mice inhibits tumor progression.

CD73/ecto-5'-nucleotidase dephosphorylates extracellular AMP into adenosine, and it is a key enzyme in the regulation of adenosinergic signaling. The contribution of host CD73 to tumor growth and anti-tumor immunity has not been studied. Here, we show that under physiological conditions CD73-deficient mice had significantly elevated ATPase and ADPase activities in LN T cells. In a melanoma model, the growth of primary tumors and formation of metastasis were significantly attenuated in mice lacking CD73. Among tumor-infiltrating leukocytes there were fewer Tregs and mannose receptor-positive macrophages, and increased IFN-γ and NOS2 mRNA production in CD73-deficient mice. Treatment of tumor-bearing animals with soluble apyrase, an enzyme hydrolyzing ATP and ADP, significantly inhibited tumor growth and accumulation of intratumoral Tregs and mannose receptor-positive macrophages in the WT C57BL/6 mice but not in the CD73-deficient mice. Pharmacological inhibition of CD73 with α,ß-methylene-adenosine-5'-diphosphate in WT mice retarded tumor progression similarly to the genetic deletion of CD73. Together these data show that increased pericellular ATP degradation in the absence of CD73 activity in the host cells is a novel mechanism controlling anti-tumor immunity and tumor progression, and that the purinergic balance can be manipulated therapeutically to inhibit tumor growth.

Regulation of mucosal addressin cell adhesion molecule 1 expression in human and mice by vascular adhesion protein 1 amine oxidase activity.

UNLABELLED: Primary sclerosing cholangitis (PSC) and autoimmune hepatitis are hepatic complications associated with inflammatory bowel disease (IBD). The expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) on mucosal endothelium is a prerequisite for the development of IBD, and it is also detected on the hepatic vessels of patients with liver diseases associated with IBD. This aberrant hepatic expression of MAdCAM-1 results in the recruitment of effector cells initially activated in the gut to the liver, in which they drive liver injury. However, the factors responsible for the aberrant hepatic expression of MAdCAM-1 are not known. In this study, we show that deamination of methylamine (MA) by vascular adhesion protein 1 (VAP-1) [a semicarbazide-sensitive amine oxidase (SSAO) expressed in the human liver] in the presence of tumor necrosis factor α induces the expression of functional MAdCAM-1 in hepatic endothelial cells and in intact human liver tissue ex vivo. This is associated with increased adhesion of lymphocytes from patients with PSC to hepatic vessels. Feeding mice MA, a constituent of food and cigarette smoke found in portal blood, led to VAP-1/SSAO-dependent MAdCAM-1 expression in mucosal vessels in vivo. CONCLUSION: Activation of VAP-1/SSAO enzymatic activity by MA, a constituent of food and cigarette smoke, induces the expression of MAdCAM-1 in hepatic vessels and results in the enhanced recruitment of mucosal effector lymphocytes to the liver. This could be an important mechanism underlying the hepatic complications of IBD.

The prototype endothelial marker PAL-E is a leukocyte trafficking molecule.

Pathologische Anatomie Leiden-endothelium antibody has been used for more than 20 years as a marker for vascular endothelium. Despite its widespread use, the target of this antibody was only recently identified as plasmalemma vesicle-associated protein-1 (PV-1). However, no function has been identified for this molecule. Here we report that activation of human umbilical vein endothelial cells with tumor necrosis factor-alpha resulted in a remarkable redistribution of PV-1 toward the peripheral areas of the cells. Furthermore, in vitro endpoint transmigration experiments showed that transcellularly migrating lymphocytes are surrounded by rings containing PV-1 and caveolin-1. Moreover, PV-1 associates physically with vimentin. In addition, administration of anti-PV-1 antibody during capillary flow assays resulted in a significant inhibition of lymphocyte transmigration through the endothelial cell layer, whereas rolling and adhesion were unaffected. In vivo blockage of PV-1 by an antibody in acute peritonitis and air pouch model resulted in a significant decrease in the number of migrating leukocytes. Here we thus define leukocyte transendothelial migration as the first known function for PV-1.

Modeling Rare Human Disorders in Mice: The Finnish Disease Heritage.

The modification of genes in animal models has evidently and comprehensively improved our knowledge on proteins and signaling pathways in human physiology and pathology. In this review, we discuss almost 40 monogenic rare diseases that are enriched in the Finnish population and defined as the Finnish disease heritage (FDH). We will highlight how gene-modified mouse models have greatly facilitated the understanding of the pathological manifestations of these diseases and how some of the diseases still lack proper models. We urge the establishment of subsequent international consortiums to cooperatively plan and carry out future human disease modeling strategies. Detailed information on disease mechanisms brings along broader understanding of the molecular pathways they act along both parallel and transverse to the proteins affected in rare diseases, therefore also aiding understanding of common disease pathologies.

Clever-1/Stabilin-1 regulates lymphocyte migration within lymphatics and leukocyte entrance to sites of inflammation.

Clever-1/Stabilin-1 is a scavenger receptor present on lymphatic and sinusoidal endothelium as well as on a subset of type II macrophages. It is also induced on vasculature at sites of inflammation. However, its in vivo function has remained practically unknown and this work addresses those unknown aspects. We demonstrate using in vivo models that Clever-1/Stabilin-1 mediates migration of T and B lymphocytes to the draining lymph nodes in vivo and identify the adhesive epitope of the Clever-1/Stabilin-1 molecule responsible for the interaction between lymphocytes and lymphatic endothelium. Moreover, we demonstrate that Ab blocking of Clever-1/Stabilin-1 efficiently inhibits peritonitis in mice by decreasing the entrance of granulocytes by 50%, while migration of monocytes and lymphocytes into the inflamed peritoneum is prevented almost completely. Despite efficient anti-inflammatory activity the Ab therapy does not dramatically dampen immune responses against the bacterial and foreign protein Ag tested and bacterial clearance. These results indicate that anti-Clever-1/Stabilin-1 treatment can target two different arms of the vasculature--traffic via lymphatics and inflamed blood vessels.

Macrophage mannose receptor on lymphatics controls cell trafficking.

Macrophage mannose receptor (MR) participates in pathogen recognition, clearance of endogenous serum glycoproteins, and antigen presentation. MR is also present on lymphatic vessels, where its function is unknown. Here we show that migration of lymphocytes from the skin into the draining lymph nodes through the afferent lymphatics is reduced in MR-deficient mice, while the structure of lymphatic vasculature remains normal in these animals. Moreover, in a tumor model the primary tumors grow significantly bigger in MR(-/-) mice than in the wild-type (WT) controls, whereas the regional lymph node metastases are markedly smaller. Adhesion of both normal lymphocytes and tumor cells to lymphatic vessels is significantly decreased in MR-deficient mice. The ability of macrophages to present tumor antigens is indistinguishable between the 2 genotypes. Thus, MR on lymphatic endothelial cells is involved in leukocyte trafficking and contributes to the metastatic behavior of cancer cells. Blocking of MR may provide a new approach to controlling inflammation and cancer metastasis by targeting the lymphatic vasculature.

Clever-1 contributes to lymphocyte entry into the spleen via the red pulp.

Lymphocytes recirculate continuously between the blood and lymphoid organs, a process that is of fundamental importance for proper functioning of the immune system. The molecular mechanisms underlying lymphocyte trafficking to the spleen remain an enigma. Here, we show that lymphocytes enter the spleen preferentially from vessels in the red pulp rather than the marginal sinus or the vasculature in the white pulp. Ex vivo adhesion assays in mice and humans, together with genetic ablation of Clever-1 in mice, indicate that CD8+ T cell and B220+ B cell homing to the spleen via the red pulp is Clever-1 dependent. Moreover, absence of Clever-1 leads to down-regulation of the B cell attractant chemokine, CXCL13, on spleen endothelium. CXCL13 is known to guide B cell trafficking to lymphoid organs, and its lack may contribute to the observed decrease in B cell trafficking into the spleen as well. In summary, this study identifies Clever-1 as an important molecule controlling lymphocyte entry into the spleen, along with a critical role for the splenic red pulp in this regulated trafficking. Furthermore, the results demonstrate that location-specific homing-associated molecules guide lymphocyte entry into the spleen.

Enhanced Antibody Production in Clever-1/Stabilin-1-Deficient Mice.

Clever-1, encoded by the Stab1 gene, is a scavenger and leukocyte trafficking receptor expressed by subsets of vascular and lymphatic endothelial cells and immunosuppressive macrophages. Monocyte Clever-1 also modulates T cell activation. However, nothing is known about the possible links between B cell function and Clever-1. Here, we found that Stab1 knockout mice (Stab1-/-) lacking the Clever-1 protein from all cells present with abnormally high antibody levels under resting conditions and show enhanced humoral immune responses after immunization with protein and carbohydrate antigens. Removal of the spleen does not abolish the augmented basal and post-immunization antibody levels in Clever-1-deficient mice. The increased IgG production is also present in mice in which Clever-1 is selectively ablated from macrophages. When compared to wildtype macrophages, Clever-1-deficient macrophages show increased TNF-α synthesis. In co-culture experiments, monocytes/macrophages deficient of Clever-1 support higher IgM production by B cells, which is blocked by TNF-α depletion. Collectively, our data show that the excessive inflammatory activity of monocytes/macrophages in the absence of Clever-1 results in augmented humoral immune responses in vivo.

Clever-1/stabilin-1 controls cancer growth and metastasis.

PURPOSE: Immunosuppressive leukocytes and vasculature are important host cell components regulating tumor progression. Clever-1/Stabilin-1, a multifunctional scavenger and adhesion receptor, is constitutively present on a subset of type II macrophages and lymphatic endothelium, but its functional role in cancer is unknown. EXPERIMENTAL DESIGN: Here, we generated full Clever-1 knockout mice and cell-specific ones lacking Clever-1 either on macrophages or endothelium. We also used anti-Clever-1 antibody therapy to treat B16 melanoma and EL-4 lymphoma. RESULTS: Clever-1-deficient mice had smaller primary and metastatic tumors than wild-type (WT) controls. Growth of primary tumors, but not of metastases, was attenuated also in mice lacking Clever-1 selectively in macrophages or in vascular endothelium. Anti-Clever-1 antibody treatment inhibited tumor progression in WT mice. Both genetically and therapeutically induced absence of functional Clever-1 led to diminished numbers of immunosuppressive leukocyte types in tumors. Functionally Clever-1 mediated binding of immunosuppressive leukocytes to the intratumoral blood vessels aberrantly expressing Clever-1, and tumor cell traffic via the lymphatics. The antibody therapy did not aggravate autoimmunity. CONCLUSION: This work identifies Clever-1 in type II macrophages and in tumor vasculature as a new immunosuppressive molecule in cancer. Our finding that Clever-1 supports binding of tumor-infiltrating lymphocytes to tumor vasculature increases our understanding of leukocyte immigration to tumors. The ability of anti-Clever-1 antibody treatment to attenuate tumor progression in WT mice in vivo is therapeutically relevant. Thus, Clever-1 may be an emerging new target for modulating immune evasion and lymphatic spread in cancer.