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.

Functional annotation of human long noncoding RNAs via molecular phenotyping.

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.

Epigenetic regulation of the lineage specificity of primary human dermal lymphatic and blood vascular endothelial cells.

Lymphatic and blood vascular endothelial cells (ECs) share several molecular and developmental features. However, these two cell types possess distinct phenotypic signatures, reflecting their different biological functions. Despite significant advances in elucidating how the specification of lymphatic and blood vascular ECs is regulated at the transcriptional level during development, the key molecular mechanisms governing their lineage identity under physiological or pathological conditions remain poorly understood. To explore the epigenomic signatures in the maintenance of EC lineage specificity, we compared the transcriptomic landscapes, histone composition (H3K4me3 and H3K27me3) and DNA methylomes of cultured matched human primary dermal lymphatic and blood vascular ECs. Our findings reveal that blood vascular lineage genes manifest a more 'repressed' histone composition in lymphatic ECs, whereas DNA methylation at promoters is less linked to the differential transcriptomes of lymphatic versus blood vascular ECs. Meta-analyses identified two transcriptional regulators, BCL6 and MEF2C, which potentially govern endothelial lineage specificity. Notably, the blood vascular endothelial lineage markers CD34, ESAM and FLT1 and the lymphatic endothelial lineage markers PROX1, PDPN and FLT4 exhibited highly differential epigenetic profiles and responded in distinct manners to epigenetic drug treatments. The perturbation of histone and DNA methylation selectively promoted the expression of blood vascular endothelial markers in lymphatic endothelial cells, but not vice versa. Overall, our study reveals that the fine regulation of lymphatic and blood vascular endothelial transcriptomes is maintained via several epigenetic mechanisms, which are crucial to the maintenance of endothelial cell identity.

In vivo CRISPRi screen identified lncRNA portfolio crucial for cutaneous squamous cell carcinoma tumor growth.

Cutaneous squamous cell carcinoma (cSCC) accounts for 20% of all skin cancer deaths globally, making it the second-highest subtype of skin cancer. The prevalence of cSCC in humans, as well as the poor capacity for an efficient prognosis, highlights the need to uncover alternative actors and mechanisms at the foundation of skin cancer development. Significant advances have been made to better understand some key factors in cSCC progression. However, little is known about the role of noncoding RNAs, particularly of a specific category called long noncoding RNA (lncRNA). By performing pseudobulk analysis of single-cell sequencing data from normal and cSCC human skin tissues, we determined a global portfolio of lncRNAs specifically expressed in keratinocyte subpopulations. Integration of CRISPR interference screens in vitro and the xenograft model identified several lncRNAs impacting the growth of cSCC cancer lines both in vitro and in vivo. Among these, we further validated LINC00704 and LINC01116 as proliferation-regulating lncRNAs in cSCC lines and potential biomarkers of cSCC progression. Taken together, our study provides a comprehensive signature of lncRNAs with roles in regulating cSCC progression.

Mitochondrial Raf1 Regulates Glutamine Catabolism.

Raf kinases play vital roles in normal mitogenic signaling and cancer, however, the identities of functionally important Raf-proximal proteins throughout the cell are not fully known. Raf1 proximity proteomics/BioID in Raf1-dependent cancer cells unexpectedly identified Raf1-adjacent proteins known to reside in the mitochondrial matrix. Inner-mitochondrial localization of Raf1 was confirmed by mitochondrial purification and super-resolution microscopy. Inside mitochondria, Raf1 associated with glutaminase (GLS) in diverse human cancers and enabled glutaminolysis, an important source of biosynthetic precursors in cancer. These impacts required Raf1 kinase activity and were independent of canonical MAP kinase pathway signaling. Kinase-dead mitochondrial matrix-localized Raf1 impaired glutaminolysis and tumorigenesis in vivo. These data indicate that Raf1 localizes inside mitochondria where it interacts with GLS to engage glutamine catabolism and support tumorigenesis.

irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein associations on RNA.

RNA binding proteins ( RBPs ) control varied processes, including RNA splicing, stability, transport, and translation 1-3 . Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease 1,4,5 , however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation 6 was combined with mass spectrometry ( irCLIP-RNP ) to identify RNA-dependent associated proteins ( RDAPs ) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor ( EGF ), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP ( Re-CLIP ) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and detected simultaneous HNRNPC and UPF1 co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.

Analyzing RNA-Protein Interactions by Cross-Link Rates and CLIP-seq Libraries.

UV cross-linking-based methods are the most common tool to explore in vivo RNA-protein interactions. UV cross-linking enables the freezing of direct interactions in the cell, which can then be mapped by high-throughput sequencing through a family of methods termed CLIP-seq. CLIP-seq measures the distribution of cross-link events by purifying a protein of interest and sequencing the covalently bound RNA fragments. However, there are disagreements and ambiguities as to which proteins are RNA-binding proteins and what interactions are significant as all proteins contact all RNAs at some frequency. Here we describe a protocol for both determining RNA-protein interactions through a combination of RNA library preparation and the measurement of absolute cross-link rates, which helps determine what proteins are RNA-binding proteins and what interactions are significant. This protocol, comprising an updated form of the easyCLIP protocol, describes guidelines for RNA library preparation, oligo and protein standard construction, and the measurement of cross-link rates. These methods are easily visualizable through their fluorescent labels and can be adapted to study RNA-binding properties of both functional, high affinity RNA-binding proteins, and the accidental RNA interactions of non-RNA-binding proteins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: RNA library construction Basic Protocol 2: Determining UV cross-link rates Support Protocol 1: Cross-linking and lysing cells Support Protocol 2: Adapter preparation Support Protocol 3: Preparation of cross-linked RBP standard.

The p300/CBP Inhibitor A485 Normalizes Psoriatic Fibroblast Gene Expression In Vitro and Reduces Psoriasis-Like Skin Inflammation In Vivo.

Psoriasis is a chronic inflammatory skin disease that often recurs at the same locations, indicating potential epigenetic changes in lesional skin cells. In this study, we discovered that fibroblasts isolated from psoriatic skin lesions retain an abnormal phenotype even after several passages in culture. Transcriptomic profiling revealed the upregulation of several genes, including the extra domain A splice variant of fibronectin and ITGA4 in psoriatic fibroblasts. A phenotypic library screening of small-molecule epigenetic modifier drugs revealed that selective CBP/p300 inhibitors were able to rescue the psoriatic fibroblast phenotype, reducing the expression levels of extra domain A splice variant of fibronectin and ITGA4. In the imiquimod-induced mouse model of psoriasis-like skin inflammation, systemic treatment with A485, a potent CBP/p300 blocker, significantly reduced skin inflammation, immune cell recruitment, and inflammatory cytokine production. Our findings indicate that epigenetic reprogramming might represent a new approach for the treatment and/or prevention of relapses of psoriasis.

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.

Beyond PROX1: transcriptional, epigenetic, and noncoding RNA regulation of lymphatic identity and function.

The lymphatic vascular system acts as the major transportation highway of tissue fluids, and its activation or impairment is associated with a wide range of diseases. There has been increasing interest in understanding the mechanisms that control lymphatic vessel formation (lymphangiogenesis) and function in development and disease. Here, we discuss recent insights into new players whose identification has contributed to deciphering the lymphatic regulatory code. We reveal how lymphatic endothelial cells, the building blocks of lymphatic vessels, utilize their transcriptional, post-transcriptional, and epigenetic portfolio to commit to and maintain their vascular lineage identity and function, with a particular focus on development.

Single-Cell Transcriptional Heterogeneity of Lymphatic Endothelial Cells in Normal and Inflamed Murine Lymph Nodes.

The lymphatic system plays a crucial role in immunity and lymph nodes (LNs) undergo drastic remodeling during inflammation. Here, we used single-cell RNA sequencing to investigate transcriptional changes in lymphatic endothelial cells (LECs) in LNs draining naïve and inflamed skin. We found that subsets of LECs lining the different LN sinuses responded individually to skin inflammation, suggesting that they exert distinct functions under pathological conditions. Among the genes dysregulated during inflammation, we confirmed an up-regulation of CD200 in the LECs lining the subcapsular sinus floor with a possible function in immune regulation. Furthermore, by in silico analysis, we predicted numerous possible interactions of LECs with diverse immune cells in the LNs and found similarities in the transcriptional changes of LN LECs in different skin inflammation settings. In summary, we provide an in-depth analysis of the transcriptional landscape of LN LECs in the naïve state and in skin inflammation.

The choice of negative control antisense oligonucleotides dramatically impacts downstream analysis depending on the cellular background.

BACKGROUND: The lymphatic and the blood vasculature are closely related systems that collaborate to ensure the organism's physiological function. Despite their common developmental origin, they present distinct functional fates in adulthood that rely on robust lineage-specific regulatory programs. The recent technological boost in sequencing approaches unveiled long noncoding RNAs (lncRNAs) as prominent regulatory players of various gene expression levels in a cell-type-specific manner. RESULTS: To investigate the potential roles of lncRNAs in vascular biology, we performed antisense oligonucleotide (ASO) knockdowns of lncRNA candidates specifically expressed either in human lymphatic or blood vascular endothelial cells (LECs or BECs) followed by Cap Analysis of Gene Expression (CAGE-Seq). Here, we describe the quality control steps adopted in our analysis pipeline before determining the knockdown effects of three ASOs per lncRNA target on the LEC or BEC transcriptomes. In this regard, we especially observed that the choice of negative control ASOs can dramatically impact the conclusions drawn from the analysis depending on the cellular background. CONCLUSION: In conclusion, the comparison of negative control ASO effects on the targeted cell type transcriptomes highlights the essential need to select a proper control set of multiple negative control ASO based on the investigated cell types.

LETR1 is a lymphatic endothelial-specific lncRNA governing cell proliferation and migration through KLF4 and SEMA3C.

Recent studies have revealed the importance of long noncoding RNAs (lncRNAs) as tissue-specific regulators of gene expression. There is ample evidence that distinct types of vasculature undergo tight transcriptional control to preserve their structure, identity, and functions. We determine a comprehensive map of lineage-specific lncRNAs in human dermal lymphatic and blood vascular endothelial cells (LECs and BECs), combining RNA-Seq and CAGE-Seq. Subsequent antisense oligonucleotide-knockdown transcriptomic profiling of two LEC- and two BEC-specific lncRNAs identifies LETR1 as a critical gatekeeper of the global LEC transcriptome. Deep RNA-DNA, RNA-protein interaction studies, and phenotype rescue analyses reveal that LETR1 is a nuclear trans-acting lncRNA modulating, via key epigenetic factors, the expression of essential target genes, including KLF4 and SEMA3C, governing the growth and migratory ability of LECs. Together, our study provides several lines of evidence supporting the intriguing concept that every cell type expresses precise lncRNA signatures to control lineage-specific regulatory programs.

MINA-1 and WAGO-4 are part of regulatory network coordinating germ cell death and RNAi in C. elegans.

Post-transcriptional control of mRNAs by RNA-binding proteins (RBPs) has a prominent role in the regulation of gene expression. RBPs interact with mRNAs to control their biogenesis, splicing, transport, localization, translation, and stability. Defects in such regulation can lead to a wide range of human diseases from neurological disorders to cancer. Many RBPs are conserved between Caenorhabditis elegans and humans, and several are known to regulate apoptosis in the adult C. elegans germ line. How these RBPs control apoptosis is, however, largely unknown. Here, we identify mina-1(C41G7.3) in a RNA interference-based screen as a novel regulator of apoptosis, which is exclusively expressed in the adult germ line. The absence of MINA-1 causes a dramatic increase in germ cell apoptosis, a reduction in brood size, and an impaired P granules organization and structure. In vivo crosslinking immunoprecipitation experiments revealed that MINA-1 binds a set of mRNAs coding for RBPs associated with germ cell development. Additionally, a system-wide analysis of a mina-1 deletion mutant compared with wild type, including quantitative proteome and transcriptome data, hints to a post-transcriptional regulatory RBP network driven by MINA-1 during germ cell development in C. elegans. In particular, we found that the germline-specific Argonaute WAGO-4 protein levels are increased in mina-1 mutant background. Phenotypic analysis of double mutant mina-1;wago-4 revealed that contemporary loss of MINA-1 and WAGO-4 strongly rescues the phenotypes observed in mina-1 mutant background. To strengthen this functional interaction, we found that upregulation of WAGO-4 in mina-1 mutant animals causes hypersensitivity to exogenous RNAi. Our comprehensive experimental approach allowed us to describe a phenocritical interaction between two RBPs controlling germ cell apoptosis and exogenous RNAi. These findings broaden our understanding of how RBPs can orchestrate different cellular events such as differentiation and death in C. elegans.

Distinct transcriptional responses of lymphatic endothelial cells to VEGFR-3 and VEGFR-2 stimulation.

Vascular endothelial growth factors (VEGFs) and their receptors play crucial roles in the formation of blood and lymphatic vessels during embryogenesis, and also under pathologic conditions in the adult. Despite intensive efforts over the last decades to elucidate the precise functions of VEGFs, transcriptional responses to VEGF receptor stimulation are still not fully characterized. To investigate the specific transcriptional effects of VEGFR-2 and VEGFR-3 activation, we performed a correlation analysis of previously published CAGE sequencing and microarray data of human lymphatic endothelial cells (LECs) stimulated with distinct VEGFs acting through either VEGFR-2 or VEGFR-3. We identified that specific activation of VEGFR-3 by VEGF-C156S results in the downregulation of many genes involved in immune regulation and inflammation, suggesting that VEGFR-3 stimulation has direct anti-inflammatory effects. Comparing CAGE and microarray data sets, we furthermore identified a small number of genes that showed a receptor-dependent response in LECs, demonstrating that these receptors, despite activating very similar signaling pathways, fulfill overlapping but not identical functions within the same cell type (LECs).