Lymphatic vessels in cancer.

The lymphatic system, composed of initial and collecting lymphatic vessels as well as lymph nodes that are present in almost every tissue of the human body, acts as an essential transport system for fluids, biomolecules, and cells between peripheral tissues and the central circulation. Consequently, it is required for normal body physiology but is also involved in the pathogenesis of various diseases, most notably cancer. The important role of tumor-associated lymphatic vessels and lymphangiogenesis in the formation of lymph node metastasis has been elucidated during the last two decades, whereas the underlying mechanisms and the relation between lymphatic and peripheral organ dissemination of cancer cells are incompletely understood. Lymphatic vessels are also important for tumor-host communication, relaying molecular information from a primary or metastatic tumor to regional lymph nodes and the circulatory system. Beyond antigen transport, lymphatic endothelial cells, particularly those residing in lymph node sinuses, have recently been recognized as direct regulators of tumor immunity and immunotherapy responsiveness, presenting tumor antigens and expressing several immune-modulatory signals including PD-L1. In this review, we summarize recent discoveries in this rapidly evolving field and highlight strategies and challenges of therapeutic targeting of lymphatic vessels or specific lymphatic functions in cancer patients.

Single-cell mapping reveals new markers and functions of lymphatic endothelial cells in lymph nodes.

Lymph nodes (LNs) are highly organized secondary lymphoid organs that mediate adaptive immune responses to antigens delivered via afferent lymphatic vessels. Lymphatic endothelial cells (LECs) line intranodal lymphatic sinuses and organize lymph and antigen distribution. LECs also directly regulate T cells, mediating peripheral tolerance to self-antigens, and play a major role in many diseases, including cancer metastasis. However, little is known about the phenotypic and functional heterogeneity of LN LECs. Using single-cell RNA sequencing, we comprehensively defined the transcriptome of LECs in murine skin-draining LNs and identified new markers and functions of distinct LEC subpopulations. We found that LECs residing in the subcapsular sinus (SCS) have an unanticipated function in scavenging of modified low-density lipoprotein (LDL) and also identified a specific cortical LEC subtype implicated in rapid lymphocyte egress from LNs. Our data provide new, to our knowledge, insights into the diversity of LECs in murine LNs and a rich resource for future studies into the regulation of immune responses by LN LECs.

The tumor organismal environment: Role in tumor development and cancer immunotherapy.

Tumor immunotherapy has resulted in dramatic effects in some cancer types, including curing of previously untreatable patients. However, the response rates are typically very heterogenous, with some patients showing dramatic responses whereas others do not or only barely respond. Consequently, there has been an ever-increasing research effort to better understand the factors that govern immunotherapy responsiveness and efficiency in order to identify predictive biomarkers and novel therapeutic targets. Clearly, traits of the tumor cells as well as aspects of the tumor microenvironment (TME) play an important role in this regard. However, a growing tumor not only interacts with cells in its immediate vicinity, but also reciprocally communicates with the entire host organism (and its microbiota). Thus, systemic influences on tumor growth and progression are likely to be similarly important as the microenvironment. In this review, we focus on various aspects of the "tumor organismal environment" (TOE), namely the lymphatic, the hematopoietic, the microbial, the neurogenic and the metabolic environment, and discuss their impact on tumor growth and immunotherapy.

Lymphatic MAFB regulates vascular patterning during developmental and pathological lymphangiogenesis.

MAFB is a transcription factor involved in the terminal differentiation of several cell types, including macrophages and keratinocytes. MAFB is also expressed in lymphatic endothelial cells (LECs) and is upregulated by VEGF-C/VEGFR-3 signaling. Recent studies have revealed that MAFB regulates several genes involved in lymphatic differentiation and that global Mafb knockout mice show defects in patterning of lymphatic vessels during embryogenesis. However, it has remained unknown whether this effect is LEC-intrinsic and whether MAFB might also be involved in postnatal lymphangiogenesis. We established conditional, lymphatic-specific Mafb knockout mice and found comparable lymphatic patterning defects during embryogenesis as in the global MAFB knockout. Lymphatic MAFB deficiency resulted in increased lymphatic branching in the diaphragm at P7, but had no major effect on lymphatic patterning or function in healthy adult mice. By contrast, tumor-induced lymphangiogenesis was enhanced in mice lacking lymphatic MAFB. Together, these data reveal that LEC-expressed MAFB is involved in lymphatic vascular morphogenesis during embryonic and postnatal development as well as in pathological conditions. Therefore, MAFB could represent a target for therapeutic modulation of lymphangiogenesis.

Transcriptional profiling of breast cancer-associated lymphatic vessels reveals VCAM-1 as regulator of lymphatic invasion and permeability.

Tumor-associated lymphangiogenesis and lymphatic invasion of tumor cells correlate with poor outcome in many tumor types, including breast cancer. Various explanations for this correlation have been suggested in the past, including the promotion of lymphatic metastasis and an immune-inhibitory function of lymphatic endothelial cells (LECs). However, the molecular features of tumor-associated lymphatic vessels and their implications for tumor progression have been poorly characterized. Here, we report the first transcriptional analysis of tumor-associated LECs directly isolated from the primary tumor in an orthotopic mouse model of triple negative breast cancer (4T1). Gene expression analysis showed a strong upregulation of inflammation-associated genes, including endothelial adhesion molecules such as VCAM-1, in comparison to LECs derived from control tissue. In vitro experiments demonstrated that VCAM-1 is not involved in the adhesion of tumor cells to LECs but unexpectedly promoted lymphatic permeability by weakening of lymphatic junctions, most likely through a mechanism triggered by interactions with integrin α4 which was also induced in tumor-associated LECs. In line with this, in vivo blockade of VCAM-1 reduced lymphatic invasion of 4T1 cells. Taken together, our findings suggest that disruption of lymphatic junctions and increased permeability via tumor-induced lymphatic VCAM-1 expression may represent a new target to block lymphatic invasion and metastasis.

DeepCAGE transcriptomics identify HOXD10 as a transcription factor regulating lymphatic endothelial responses to VEGF-C.

Lymphangiogenesis plays a crucial role during development, in cancer metastasis and in inflammation. Activation of VEGFR-3 (also known as FLT4) by VEGF-C is one of the main drivers of lymphangiogenesis, but the transcriptional events downstream of VEGFR-3 activation are largely unknown. Recently, we identified a wave of immediate early transcription factors that are upregulated in human lymphatic endothelial cells (LECs) within the first 30 to 80 min after VEGFR-3 activation. Expression of these transcription factors must be regulated by additional pre-existing transcription factors that are rapidly activated by VEGFR-3 signaling. Using transcription factor activity analysis, we identified the homeobox transcription factor HOXD10 to be specifically activated at early time points after VEGFR-3 stimulation, and to regulate expression of immediate early transcription factors, including NR4A1. Gain- and loss-of-function studies revealed that HOXD10 is involved in LECs migration and formation of cord-like structures. Furthermore, HOXD10 regulates expression of VE-cadherin, claudin-5 and NOS3 (also known as e-NOS), and promotes lymphatic endothelial permeability. Taken together, these results reveal an important and unanticipated role of HOXD10 in the regulation of VEGFR-3 signaling in lymphatic endothelial cells, and in the control of lymphangiogenesis and permeability.

High expression of insulin receptor on tumour-associated blood vessels in invasive bladder cancer predicts poor overall and progression-free survival.

Bladder cancer is a frequently recurring disease with a very poor prognosis once progressed to invasive stages, and tumour-associated blood vessels play a crucial role in this process. In order to identify novel biomarkers associated with progression, we isolated blood vascular endothelial cells (BECs) from human invasive bladder cancers and matched normal bladder tissue, and found that tumour-associated BECs greatly up-regulated the expression of insulin receptor (INSR). High expression of INSR on BECs of invasive bladder cancers was significantly associated with shorter progression-free and overall survival. Furthermore, increased expression of the INSR ligand IGF-2 in invasive bladder cancers was associated with reduced overall survival. INSR may therefore represent a novel biomarker to predict cancer progression. Mechanistically, we observed pronounced hypoxia in human bladder cancer tissue, and found a positive correlation between the expression of the hypoxia marker gene GLUT1 and vascular INSR expression, indicating that hypoxia drives INSR expression in tumour-associated blood vessels. In line with this, exposure of cultured BECs and human bladder cancer cell lines to hypoxia led to increased expression of INSR and IGF-2, respectively, and IGF-2 increased BEC migration through the activation of INSR in vitro. Taken together, we identified vascular INSR expression as a potential biomarker for progression in bladder cancer. Furthermore, our data suggest that IGF-2/INSR mediated paracrine crosstalk between bladder cancer cells and endothelial cells is functionally involved in tumour angiogenesis and may thus represent a new therapeutic target. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

VEGF suppresses T-lymphocyte infiltration in the tumor microenvironment through inhibition of NF-κB-induced endothelial activation.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) signaling pathway is in clinical use, but its effect on vascular function and the tumor microenvironment is poorly understood. Here, we investigate cross-talk between VEGF and proinflammatory TNF-α signaling in endothelial cells and its impact on leukocyte recruitment. We found that cotreatment with VEGF decreased TNF-α-induced Jurkat cell adhesion to human microvascular endothelial cells by 40%. This was associated with inhibition of TNF-α-mediated regulation of 86 genes, including 2 T-lymphocyte-attracting chemokines, CXCL10 and CXCL11 [TNF-α concentration 1 ng/ml; 50% inhibition/inhibitory concentration (IC50) VEGF, 3 ng/ml]. Notably, VEGF directly suppressed TNF-α-induced gene expression through negative cross-talk with the NF-κB-signaling pathway, leading to an early decrease in IFN regulatory factor 1 (IRF-1) expression and reduced phosphorylation of signal transducer and activator of transcription 1 (p-Stat1) at later times. Inhibition of VEGF signaling in B16 melanoma tumor-bearing mice by sunitinib treatment resulted in up-regulation of CXCL10 and CXCL11 in tumor vessels, accompanied by up to 18-fold increased infiltration of CD3(+) T-lymphocytes in B16 tumors. Our results demonstrate a novel role of VEGF in negative regulation of NF-κB signaling and endothelial activation in the tumor microenvironment and provide evidence that pharmacological inhibition of VEGF signaling enhances T-lymphocyte recruitment through up-regulation of chemokines CXCL10 and CXCL11.

Lymphatic vessels: new targets for the treatment of inflammatory diseases.

The lymphatic system plays an important role in the physiological control of the tissue fluid balance and in the initiation of immune responses. Recent studies have shown that lymphangiogenesis, the growth of new lymphatic vessels and/or the expansion of existing lymphatic vessels, is a characteristic feature of acute inflammatory reactions and of chronic inflammatory diseases. In these conditions, lymphatic vessel expansion occurs at the tissue level but also within the draining lymph nodes. Surprisingly, activation of lymphatic vessel function by delivery of vascular endothelial growth factor-C exerts anti-inflammatory effects in several models of cutaneous and joint inflammation. These effects are likely mediated by enhanced drainage of extravasated fluid and inflammatory cells, but also by lymphatic vessel-mediated modulation of immune responses. Although some of the underlying mechanisms are just beginning to be identified, lymphatic vessels have emerged as important targets for the development of new therapeutic strategies to treat inflammatory conditions. In this context, it is of great interest that some of the currently used anti-inflammatory drugs also potently activate lymphatic vessels.

αB-Crystallin regulates expansion of CD11b⁺Gr-1⁺ immature myeloid cells during tumor progression.

The molecular chaperone αB-crystallin has emerged as a target for cancer therapy due to its expression in human tumors and its role in regulating tumor angiogenesis. αB-crystallin also reduces neuroinflammation, but its role in other inflammatory conditions has not been investigated. Here, we examined whether αB-crystallin regulates inflammation associated with tumors and ischemia. We found that CD45(+) leukocyte infiltration is 3-fold increased in tumors and ischemic myocardium in αB-crystallin-deficient mice. Notably, αB-crystallin is prominently expressed in CD11b(+) Gr-1(+) immature myeloid cells (IMCs), known as regulators of angiogenesis and immune responses, while lymphocytes and mature granulocytes show low αB-crystallin expression. αB-Crystallin deficiency results in a 3-fold higher accumulation of CD11b(+) Gr-1(+) IMCs in tumors and a significant rise in CD11b(+) Gr-1(+) IMCs in spleen and bone marrow. Similarly, we noted a 2-fold increase in CD11b(+) Gr-1(+) IMCs in chronically inflamed livers in αB-crystallin-deficient mice. The effect of αB-crystallin on IMC accumulation is limited to pathological conditions, as CD11b(+) Gr-1(+) IMCs are not elevated in naive mice. Through ex vivo differentiation of CD11b(+) Gr-1(+) cells, we provide evidence that αB-crystallin regulates systemic expansion of IMCs through a cell-intrinsic mechanism. Our study suggests a key role of αB-crystallin in limiting expansion of CD11b(+) Gr-1(+) IMCs in diverse pathological conditions.

αB-crystallin/HspB5 regulates endothelial-leukocyte interactions by enhancing NF-κB-induced up-regulation of adhesion molecules ICAM-1, VCAM-1 and E-selectin.

αB-crystallin is a small heat shock protein, which has pro-angiogenic properties by increasing survival of endothelial cells and secretion of vascular endothelial growth factor A. Here we demonstrate an additional role of αB-crystallin in regulating vascular function, through enhancing tumor necrosis factor α (TNF-α) induced expression of endothelial adhesion molecules involved in leukocyte recruitment. Ectopic expression of αB-crystallin in endothelial cells increases the level of E-selectin expression in response to TNF-α, and enhances leukocyte-endothelial interaction in vitro. Conversely, TNF-α-induced expression of intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and E-selectin is markedly inhibited in endothelial cells isolated from αB-crystallin-deficient mice. This is associated with elevated levels of IκB in αB-crystallin deficient cells and incomplete degradation upon TNF-α stimulation. Consistent with this, endothelial adhesion molecule expression is reduced in inflamed vessels of αB-crystallin deficient mice, and leukocyte rolling velocity is increased. Our data identify αB-crystallin as a new regulator of leukocyte recruitment, by enhancing pro-inflammatory nuclear factor κ B-signaling and endothelial adhesion molecule expression during endothelial activation.

Transcriptional profiling of human glioblastoma vessels indicates a key role of VEGF-A and TGFß2 in vascular abnormalization.

Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGFß2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGFß signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGFß2 may represent a new target for vascular normalization therapy.

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development.

BACKGROUND: Angiogenesis is implicated in many pathological conditions. The role of the proteins involved remains largely unknown, and few vascular-specific drug targets have been discovered. Previously, in a screen for angiogenesis regulators, we identified Paladin (mouse: X99384, human: KIAA1274), a protein containing predicted S/T/Y phosphatase domains. RESULTS: We present a mouse knockout allele for Paladin with a ß-galactosidase reporter, which in combination with Paladin antibodies demonstrate that Paladin is expressed in the vasculature. During mouse embryogenesis, Paladin is primarily expressed in capillary and venous endothelial cells. In adult mice Paladin is predominantly expressed in arterial pericytes and vascular smooth muscle cells. Paladin also displays vascular-restricted expression in human brain, astrocytomas, and glioblastomas. CONCLUSIONS: Paladin, a novel putative phosphatase, displays a dynamic expression pattern in the vasculature. During embryonic stages it is broadly expressed in endothelial cells, while in the adult it is selectively expressed in arterial smooth muscle cells.

Associations amongst genes, molecules, cells, and organs in breast cancer metastasis.

This paper is a cross fertilization of ideas about the importance of molecular aspects of breast cancer metastasis by basic scientists, a pathologist, and clinical oncologists at the Henry Ford Health symposium. We address four major topics: (i) the complex roles of lymphatic endothelial cells and the molecules that stimulate them to enhance lymph node and systemic metastasis and influence the anti-tumor immunity that might inhibit metastasis; (ii) the interaction of molecules and cells when breast cancer spreads to bone, and how bone metastases may themselves spread to internal viscera; (iii) how molecular expression and morphologic subtypes of breast cancer assist clinicians in determining which patients to treat with more or less aggressive therapies; (iv) how the outcomes of patients with oligometastases in breast cancer are different from those with multiple metastases and how that could justify the aggressive treatment of these patients with the hope of cure.

Mechanisms of extracellular vesicle-mediated immune evasion in melanoma.

Melanoma-derived extracellular vesicles (EVs) have been found to promote tumor growth and progression, and to predict patient responsiveness to immunotherapy. Consequently, EVs have been implicated in tumor immune evasion, and multiple studies reported immune-regulatory activities of melanoma EVs in vitro and in vivo. This review highlights mechanistic insights in EV-mediated regulation of various immune cell types, including effects on inflammatory, apoptotic, stress-sensing and immune checkpoint pathways as well as antigen-dependent responses. Additionally, current challenges in the field are discussed that need to be overcome to determine the clinical relevance of these various mechanisms and to develop corresponding therapeutic approaches to promote tumor immunity and immunotherapy responsiveness in melanoma patients in the future.

Isolation and Fluorescent Labeling of Extracellular Vesicles from Cultured Tumor Cells.

Extracellular vesicles (EVs), comprising exosomes, ectosomes, and apoptotic bodies, are an important component of molecular cell-to-cell communication, and are critically involved in the pathophysiology of various diseases, including tumors. In order to study the interaction of tumor cell-derived EVs with their target cells and to investigate their biological functions in comparison to other tumor cell-released factors, efficient isolation of EVs from cultured tumor cells, as well as fluorescent labeling of these EVs, is often necessary. In addition, EVs and EV-like particles are emerging as versatile vehicles for the delivery of therapeutic substances. Here, we describe an easy size exclusion chromatography-based method to isolate EVs from the mouse melanoma cell line B16F10 that yields highly enriched EV samples for subsequent applications such as molecular and functional studies. Our protocol also includes an optional labeling step with the lipophilic dye DiD, which allows tracking of EV uptake by recipient cells in vitro and in vivo.

Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling.

Ample evidence pinpoints the phenotypic diversity of blood vessels (BVs) and site-specific functions of their lining endothelial cells (ECs). We harnessed single-cell RNA sequencing (scRNA-seq) to dissect the molecular heterogeneity of blood vascular endothelial cells (BECs) in healthy adult human skin and identified six different subpopulations, signifying arterioles, post-arterial capillaries, pre-venular capillaries, post-capillary venules, venules and collecting venules. Individual BEC subtypes exhibited distinctive transcriptomic landscapes associated with diverse biological pathways. These functionally distinct dermal BV segments were characterized by their unique compositions of conventional and novel markers (e.g., arteriole marker GJA5; arteriole capillary markers ASS1 and S100A4; pre-venular capillary markers SOX17 and PLAUR; venular markers EGR2 and LRG1), many of which have been implicated in vascular remodeling upon inflammatory responses. Immunofluorescence staining of human skin sections and whole-mount skin blocks confirmed the discrete expression of these markers along the blood vascular tree in situ, further corroborating BEC heterogeneity in human skin. Overall, our study molecularly refines individual BV compartments, whilst the identification of novel subtype-specific signatures provides more insights for future studies dissecting the responses of distinct vessel segments under pathological conditions.

Immunomodulatory Responses of Subcapsular Sinus Floor Lymphatic Endothelial Cells in Tumor-Draining Lymph Nodes.

Tumor-draining lymph nodes (LNs), composed of lymphocytes, antigen-presenting cells, and stromal cells, are highly relevant for tumor immunity and the efficacy of immunotherapies. Lymphatic endothelial cells (LECs) represent an important stromal cell type within LNs, and several distinct subsets of LECs that interact with various immune cells and regulate immune responses have been identified. In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize LECs from LNs draining B16F10 melanomas compared to non-tumor-draining LNs. Several upregulated genes with immune-regulatory potential, especially in LECs lining the subcapsular sinus floor (fLECs), were identified and validated. Interestingly, some of these genes, namely, podoplanin, CD200, and BST2, affected the adhesion of macrophages to LN LECs in vitro. Congruently, lymphatic-specific podoplanin deletion led to a decrease in medullary sinus macrophages in tumor-draining LNs in vivo. In summary, our data show that tumor-derived factors induce transcriptional changes in LECs of the draining LNs, especially the fLECs, and that these changes may affect tumor immunity. We also identified a new function of podoplanin, which is expressed on all LECs, in mediating macrophage adhesion to LECs and their correct localization in LN sinuses.

Antibody-Mediated Delivery of VEGF-C Promotes Long-Lasting Lymphatic Expansion That Reduces Recurrent Inflammation.

The lymphatic vascular system plays a fundamental role in inflammation by draining interstitial fluid, immune cells, antigens, and inflammatory mediators from peripheral tissues. Site-specific delivery of the lymphangiogenic growth factor VEGF-C alleviates acute inflammation in mouse models of psoriasis and chronic colitis by enhancing local drainage. However, it is unclear whether therapeutically induced lymphangiogenesis is transient or long-lasting and whether it might prevent relapses of inflammation. Here, we investigated the long-term effects of targeted VEGF-C delivery in a chronic dermatitis model in mice. Congruent with our previous results, intravenous injection with a VEGF-C fusion protein targeted to the EDA domain of fibronectin initially resulted in reduced inflammation. Importantly, we found that targeted VEGF-C-mediated expansion of lymphatic vessels in the skin persisted for more than 170 days, long after primary inflammation had resolved. Furthermore, the treatment markedly decreased tissue swelling upon inflammatory re-challenge at the same site. Simultaneously, infiltration of leukocytes, including CD4+ T cells, macrophages, and dendritic cells, was significantly reduced in the previously treated group. In conclusion, our data show that targeted delivery of VEGF-C leads to long-lasting lymphatic expansion and long-term protection against repeated inflammatory challenge, suggesting that it is a promising new approach for the treatment of chronic, recurrent inflammatory diseases.

Mediators of Capillary-to-Venule Conversion in the Chronic Inflammatory Skin Disease Psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperplasia and hyperkeratosis, immune cell infiltration and vascular remodeling. Despite the emerging recognition of vascular normalization as a potential strategy for managing psoriasis, an in-depth delineation of the remodeled dermal vasculature has been missing. In this study, we exploited 5' single-cell RNA sequencing to investigate the transcriptomic alterations in different subpopulations of blood vascular and lymphatic endothelial cells directly isolated from psoriatic and healthy human skin. Individual subtypes of endothelial cells underwent specific molecular repatterning associated with cell adhesion and extracellular matrix organization. Blood capillaries, in particular, showed upregulation of the melanoma cell adhesion molecule as well as its binding partners and adopted postcapillary venule‒like characteristics during chronic inflammation that are more permissive to leukocyte transmigration. We also identified psoriasis-specific interactions between cis-regulatory enhancers and promoters for each endothelial cell subtype, revealing the dysregulated gene regulatory networks in psoriasis. Together, our results provide more insights into the specific transcriptional responses and epigenetic signatures of endothelial cells lining different vessel compartments in chronic skin inflammation.