Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

Lung cancer is the leading cause of cancer-related deaths in the world, and non-small cell lung cancer (NSCLC) is the most common subset. We previously found that infiltration of tumor inflammatory monocytes (TIMs) into lung squamous carcinoma (LUSC) tumors is associated with increased metastases and poor survival. To further understand how TIMs promote metastases, we compared RNA-Seq profiles of TIMs from several LUSC metastatic models with inflammatory monocytes (IMs) of non-tumor-bearing controls. We identified Spon1 as upregulated in TIMs and found that Spon1 expression in LUSC tumors corresponded with poor survival and enrichment of collagen extracellular matrix signatures. We observed SPON1+ TIMs mediate their effects directly through LRP8 on NSCLC cells, which resulted in TGF-ß1 activation and robust production of fibrillar collagens. Using several orthogonal approaches, we demonstrated that SPON1+ TIMs were sufficient to promote NSCLC metastases. Additionally, we found that Spon1 loss in the host, or Lrp8 loss in cancer cells, resulted in a significant decrease of both high-density collagen matrices and metastases. Finally, we confirmed the relevance of the SPON1/LRP8/TGF-ß1 axis with collagen production and survival in patients with NSCLC. Taken together, our study describes how SPON1+ TIMs promote collagen remodeling and NSCLC metastases through an LRP8/TGF-ß1 signaling axis.

PTEN-restoration abrogates brain colonisation and perivascular niche invasion by melanoma cells.

BACKGROUND: Melanoma brain metastases (MBM) continue to be a significant clinical problem with limited treatment options. Highly invasive melanoma cells migrate along the vasculature and perivascular cells may contribute to residual disease and recurrence. PTEN loss and hyperactivation of AKT occur in MBM; however, a role for PTEN/AKT in perivascular invasion has not been described. METHODS: We used in vivo intracranial injections of murine melanoma and bulk RNA sequencing of melanoma cells co-cultured with brain endothelial cells (brECs) to investigate brain colonisation and perivascular invasion. RESULTS: We found that PTEN-null melanoma cells were highly efficient at colonising the perivascular niche relative to PTEN-expressing counterparts. PTEN re-expression (PTEN-RE) in melanoma cells significantly reduced brain colonisation and migration along the vasculature. We hypothesised this phenotype was mediated through vascular-induced TGFß secretion, which drives AKT phosphorylation. Disabling TGFß signalling in melanoma cells reduced colonisation and perivascular invasion; however, the introduction of constitutively active myristolated-AKT (myrAKT) restored overall tumour size but not perivascular invasion. CONCLUSIONS: PTEN loss facilitates perivascular brain colonisation and invasion of melanoma. TGFß-AKT signalling partially contributes to this phenotype, but further studies are needed to determine the complementary mechanisms that enable melanoma cells to both survive and spread along the brain vasculature.

The modes of angiogenesis: an updated perspective.

Following the process of vasculogenesis during development, angiogenesis generates new vascular structures through a variety of different mechanisms or modes. These different modes of angiogenesis involve, for example, increasing microvasculature density by sprouting of endothelial cells, splitting of vessels to increase vascular surface area by intussusceptive angiogenesis, fusion of capillaries to increase blood flow by coalescent angiogenesis, and the recruitment of non-endothelial cells by vasculogenic mimicry. The recent reporting on coalescent angiogenesis as a new mode of vessel formation warrants a brief overview of angiogenesis mechanisms to provide a more complete picture. The journal Angiogenesis is devoted to the delineation of the different modes and mechanisms that collectively dictate blood vessel formation, inhibition, and function in health and disease.

Periostin+ Stromal Cells Guide Lymphovascular Invasion by Cancer Cells.

Cancer cell dissemination to sentinel lymph nodes is associated with poor patient outcomes, particularly in breast cancer. The process by which cancer cells egress from the primary tumor upon interfacing with the lymphatic vasculature is complex and driven by dynamic interactions between cancer cells and stromal cells, including cancer-associated fibroblasts (CAF). The matricellular protein periostin can distinguish CAF subtypes in breast cancer and is associated with increased desmoplasia and disease recurrence in patients. However, as periostin is secreted, periostin-expressing CAFs are difficult to characterize in situ, limiting our understanding of their specific contribution to cancer progression. Here, we used in vivo genetic labeling and ablation to lineage trace periostin+ cells and characterize their functions during tumor growth and metastasis. Periostin-expressing CAFs were spatially found at periductal and perivascular margins, were enriched at lymphatic vessel peripheries, and were differentially activated by highly metastatic cancer cells versus poorly metastatic counterparts. Surprisingly, genetically depleting periostin+ CAFs slightly accelerated primary tumor growth but impaired intratumoral collagen organization and inhibited lymphatic, but not lung, metastases. Periostin ablation in CAFs impaired their ability to deposit aligned collagen matrices and inhibited cancer cell invasion through collagen and across lymphatic endothelial cell monolayers. Thus, highly metastatic cancer cells mobilize periostin-expressing CAFs in the primary tumor site that promote collagen remodeling and collective cell invasion within lymphatic vessels and ultimately to sentinel lymph nodes. SIGNIFICANCE: Highly metastatic breast cancer cells activate a population of periostin-expressing CAFs that remodel the extracellular matrix to promote escape of cancer cells into lymphatic vessels and drive colonization of proximal lymph nodes.

Pathological angiogenesis: mechanisms and therapeutic strategies.

In multicellular organisms, angiogenesis, the formation of new blood vessels from pre-existing ones, is an essential process for growth and development. Different mechanisms such as vasculogenesis, sprouting, intussusceptive, and coalescent angiogenesis, as well as vessel co-option, vasculogenic mimicry and lymphangiogenesis, underlie the formation of new vasculature. In many pathological conditions, such as cancer, atherosclerosis, arthritis, psoriasis, endometriosis, obesity and SARS-CoV-2(COVID-19), developmental angiogenic processes are recapitulated, but are often done so without the normal feedback mechanisms that regulate the ordinary spatial and temporal patterns of blood vessel formation. Thus, pathological angiogenesis presents new challenges yet new opportunities for the design of vascular-directed therapies. Here, we provide an overview of recent insights into blood vessel development and highlight novel therapeutic strategies that promote or inhibit the process of angiogenesis to stabilize, reverse, or even halt disease progression. In our review, we will also explore several additional aspects (the angiogenic switch, hypoxia, angiocrine signals, endothelial plasticity, vessel normalization, and endothelial cell anergy) that operate in parallel to canonical angiogenesis mechanisms and speculate how these processes may also be targeted with anti-angiogenic or vascular-directed therapies.

Priming a vascular-selective cytokine response permits CD8+ T-cell entry into tumors.

Targeting DNA methyltransferase 1 (DNMT1) has immunomodulatory and anti-neoplastic activity, especially when paired with cancer immunotherapies. Here we explore the immunoregulatory functions of DNMT1 in the tumor vasculature of female mice. Dnmt1 deletion in endothelial cells (ECs) impairs tumor growth while priming expression of cytokine-driven cell adhesion molecules and chemokines important for CD8+ T-cell trafficking across the vasculature; consequently, the efficacy of immune checkpoint blockade (ICB) is enhanced. We find that the proangiogenic factor FGF2 promotes ERK-mediated DNMT1 phosphorylation and nuclear translocation to repress transcription of the chemokines Cxcl9/Cxcl10 in ECs. Targeting Dnmt1 in ECs reduces proliferation but augments Th1 chemokine production and extravasation of CD8+ T-cells, suggesting DNMT1 programs immunologically anergic tumor vasculature. Our study is in good accord with preclinical observations that pharmacologically disrupting DNMT1 enhances the activity of ICB but suggests an epigenetic pathway presumed to be targeted in cancer cells is also operative in the tumor vasculature.

The rising impact of angiogenesis research.

While inhibiting pathological angiogenesis has been long associated with the field of oncology, recent advances in angiogenesis research have impacted the progress of disease treatment for additional non-malignant diseases or chronic conditions in the fields of ophthalmology, cardiology, and gynecology. Moreover, stimulators of angiogenesis find application in ischemic diseases, while inhibitors of angiogenesis are being used to limit blood vessel formation, but in judicious ways that modify or "reprogram" the vasculature as a reinforcement for immunotherapy. We have noticed an increasing impact, as evidenced by increases in the total number of citations, in the literature surrounding the angiogenesis field suggesting that targeting angiogenesis per se is well established as a tractable approach for therapy in diverse conditions.

Reporter mice for isolating and auditing cell type-specific extracellular vesicles in vivo.

Extracellular vesicles (EVs) are abundant, lipid-enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type-specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR-Cas9-mediated genome editing to generate mice bearing a CD63-emGFPloxP/stop/loxP knock-in cassette that enables the specific labeling of circulating CD63+ vesicles from any cell type when crossed with lineage-specific Cre recombinase driver mice. As proof-of-principle, we have crossed these mice with Cdh5-CreERT2 mice to generate CD63emGFP+ vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma-purified EVs contain a subpopulation of emGFP+ vesicles that are derived from the endothelium, co-express additional EV (e.g., CD9 and CD81) and endothelial cell (e.g., CD105) markers, and they harbor specific miRNAs (e.g., miR-126, miR-30c, and miR-125b). This new mouse strain should be a useful genetic tool for generating cell type-specific, CD63+ EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.

Models and molecular mechanisms of blood vessel co-option by cancer cells.

Cancer cells have diverse mechanisms for utilizing the vasculature; they can initiate the formation of new blood vessels from preexisting ones (sprouting angiogenesis) or they can form cohesive interactions with the abluminal surface of preexisting vasculature in the absence of sprouting (co-option). The later process has received renewed attention due to the suggested role of blood vessel co-option in resistance to antiangiogenic therapies and the reported perivascular positioning and migratory patterns of cancer cells during tumor dormancy and invasion, respectively. However, only a few molecular mechanisms have been identified that contribute to the process of co-option and there has not been a formal survey of cell lines and laboratory models that can be used to study co-option in different organ microenvironments; thus, we have carried out a comprehensive literature review on this topic and have identified cell lines and described the laboratory models that are used to study blood vessel co-option in cancer. Put into practice, these models may help to shed new light on the molecular mechanisms that drive blood vessel co-option during tumor dormancy, invasion, and responses to different therapies.

A miRNA signature in endothelial cell-derived extracellular vesicles in tumor-bearing mice.

Extracellular vesicles (EVs) play important roles in tumor progression by altering immune surveillance, promoting vascular dysfunction, and priming distant sites for organotropic metastases. The miRNA expression patterns in circulating EVs are important diagnostic tools in cancer. However, multiple cell types within the tumor microenvironment (TME) including cancer cells and stromal cells (e.g. immune cells, fibroblasts, and endothelial cells, ECs) contribute to the pool of circulating EVs. Because EVs of different cellular origins have different functional properties, auditing the cargo derived from cell type-specific EVs in the TME is essential. Here, we demonstrate that a murine EC lineage-tracing model (Cdh5-CreERT2:ZSGreenl/s/l mice) can be used to isolate EC-derived extracellular vesicles (EC-EVs). We further show that purified ZSGreen+ EVs express expected EV markers, they are transferable to multiple recipient cells, and circulating EC-EVs from tumor-bearing mice harbor elevated levels of specific miRNAs (e.g. miR-30c, miR-126, miR-146a, and miR-125b) compared to non tumor-bearing counterparts. These results suggest that, in the tumor setting, ECs may systemically direct the function of heterotypic cell types either in the circulation or in different organ micro-environments via the cargo contained within their EVs.

Quaking orchestrates a post-transcriptional regulatory network of endothelial cell cycle progression critical to angiogenesis and metastasis.

Angiogenesis is critical to cancer development and metastasis. However, anti-angiogenic agents have only had modest therapeutic success, partly due to an incomplete understanding of tumor endothelial cell (EC) biology. We previously reported that the microRNA (miR)-200 family inhibits metastasis through regulation of tumor angiogenesis, but the underlying molecular mechanisms are poorly characterized. Here, using integrated bioinformatics approaches, we identified the RNA-binding protein (RBP) quaking (QKI) as a leading miR-200b endothelial target with previously unappreciated roles in the tumor microenvironment in lung cancer. In lung cancer samples, both miR-200b suppression and QKI overexpression corresponded with tumor ECs relative to normal ECs, and QKI silencing phenocopied miR-200b-mediated inhibition of sprouting. Additionally, both cancer cell and endothelial QKI expression in patient samples significantly corresponded with poor survival and correlated with angiogenic indices. QKI supported EC function by stabilizing cyclin D1 (CCND1) mRNA to promote EC G1/S cell cycle transition and proliferation. Both nanoparticle-mediated RNA interference of endothelial QKI expression and palbociclib blockade of CCND1 function potently inhibited metastasis in concert with significant effects on tumor vasculature. Altogether, this work demonstrates the clinical relevance and therapeutic potential of a novel, actionable miR/RBP axis in tumor angiogenesis and metastasis.

Endothelial miR-30c suppresses tumor growth via inhibition of TGF-ß-induced Serpine1.

In tumors, extravascular fibrin forms provisional scaffolds for endothelial cell (EC) growth and motility during angiogenesis. We report that fibrin-mediated angiogenesis was inhibited and tumor growth delayed following postnatal deletion of Tgfbr2 in the endothelium of Cdh5-CreERT2 Tgfbr2fl/fl mice (Tgfbr2iECKO mice). ECs from Tgfbr2iECKO mice failed to upregulate the fibrinolysis inhibitor plasminogen activator inhibitor 1 (Serpine1, also known as PAI-1), due in part to uncoupled TGF-ß-mediated suppression of miR-30c. Bypassing TGF-ß signaling with vascular tropic nanoparticles that deliver miR-30c antagomiRs promoted PAI-1-dependent tumor growth and increased fibrin abundance, whereas miR-30c mimics inhibited tumor growth and promoted vascular-directed fibrinolysis in vivo. Using single-cell RNA-Seq and a NanoString miRNA array, we also found that subtypes of ECs in tumors showed spectrums of Serpine1 and miR-30c expression levels, suggesting functional diversity in ECs at the level of individual cells; indeed, fresh EC isolates from lung and mammary tumor models had differential abilities to degrade fibrin and launch new vessel sprouts, a finding that was linked to their inverse expression patterns of miR-30c and Serpine1 (i.e., miR-30chi Serpine1lo ECs were poorly angiogenic and miR-30clo Serpine1hi ECs were highly angiogenic). Thus, by balancing Serpine1 expression in ECs downstream of TGF-ß, miR-30c functions as a tumor suppressor in the tumor microenvironment through its ability to promote fibrin degradation and inhibit blood vessel formation.

Deadly DAaRTS destroy cancer cells via a tumor microenvironment-mediated trigger.

Stromal cells within the tumor microenvironment play a supportive role in tumor growth, progression, and treatment resistance; therefore, these nonmalignant cells are potential therapeutic targets. In this issue of the JCI, Szot et al. devised a strategy to exploit the cell-surface marker TEM8 (also known as ANTXR1), which is expressed by cancer-associated stromal cells, as a zip code to deliver an antibody-drug conjugate (ADC) linked to the potent cancer-killing drug monomethyl auristatin E (MMAE). In preclinical tumor and experimental metastasis models of multiple cancer types, TEM8-ADC targeted TEM8-expressing cancer-associated stromal cells, which processed and liberated membrane-permeable MMAE and released this drug via the P-glycoprotein (P-gp) drug transporter. Released MMAE killed cancer cells through a bystander mechanism that did minimal damage to the stromal cells themselves. P-gp-expressing tumor cells displayed MMAE resistance, suggesting that P-gp expression status may identify patients who might benefit the most from TEM8-ADC. This strategy, termed DAaRTS (drug activation and release through stroma), represents an elegant example of how selective expression of a cell-surface molecule on cancer-associated stroma can be exploited to facilitate drug delivery and shrink solid tumors.

Consensus guidelines for the use and interpretation of angiogenesis assays.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Suppression of TGFß-mediated conversion of endothelial cells and fibroblasts into cancer associated (myo)fibroblasts via HDAC inhibition.

BACKGROUND: Cancer-associated fibroblasts (CAFs) support tumour progression and invasion, and they secrete abundant extracellular matrix (ECM) that may shield tumour cells from immune checkpoint or kinase inhibitors. Targeting CAFs using drugs that revert their differentiation, or inhibit their tumour-supportive functions, has been considered as an anti-cancer strategy. METHODS: We have used human and murine cell culture models, atomic force microscopy (AFM), microarray analyses, CAF/tumour cell spheroid co-cultures and transgenic fibroblast reporter mice to study how targeting HDACs using small molecule inhibitors or siRNAs re-directs CAF differentiation and function in vitro and in vivo. RESULTS: From a small molecule screen, we identified Scriptaid, a selective inhibitor of HDACs 1/3/8, as a repressor of TGFß-mediated CAF differentiation. Scriptaid inhibits ECM secretion, reduces cellular contraction and stiffness, and impairs collective cell invasion in CAF/tumour cell spheroid co-cultures. Scriptaid also reduces CAF abundance and delays tumour growth in vivo. CONCLUSIONS: Scriptaid is a well-tolerated and effective HDACi that reverses many of the functional and phenotypic properties of CAFs. Impeding or reversing CAF activation/function by altering the cellular epigenetic regulatory machinery could control tumour growth and invasion, and be beneficial in combination with additional therapies that target cancer cells or immune cells directly.

Fine-tuning vascular fate during endothelial-mesenchymal transition.

In the heart and other organs, endothelial-mesenchymal transition (EndMT) has emerged as an important developmental process that involves coordinated migration, differentiation, and proliferation of the endothelium. In multiple disease states including cancer angiogenesis and cardiovascular disease, the processes that regulate EndMT are recapitulated, albeit in an uncoordinated and dysregulated manner. Members of the transforming growth factor beta (TGFß) superfamily are well known to impart cellular plasticity during EndMT by the timely activation (or repression) of transcription factors and miRNAs in addition to epigenetic regulation of gene expression. On the other hand, fibroblast growth factors (FGFs) are reported to augment or oppose TGFß-driven EndMT in specific contexts. Here, we have synthesized the currently understood roles of TGFß and FGF signalling during EndMT and have provided a new, comprehensive paradigm that delineates how an autocrine and paracrine TGFß/FGF axis coordinates endothelial cell specification and plasticity. We also provide new guidelines and nomenclature that considers factors such as endothelial cell heterogeneity to better define EndMT across different vascular beds. This perspective should therefore help to clarify why TGFß and FGF can both cooperate with or oppose one another during the complex process of EndMT in both health and disease. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Variants of Rab GTPase-Effector Binding Protein-2 Cause Variation in the Collateral Circulation and Severity of Stroke.

BACKGROUND AND PURPOSE: The extent (number and diameter) of collateral vessels varies widely and is a major determinant, along with arteriogenesis (collateral remodeling), of variation in severity of tissue injury after large artery occlusion. Differences in genetic background underlie the majority of the variation in collateral extent in mice, through alterations in collaterogenesis (embryonic collateral formation). In brain and other tissues, ≈80% of the variation in collateral extent among different mouse strains has been linked to a region on chromosome 7. We recently used congenic (CNG) fine mapping of C57BL/6 (B6, high extent) and BALB/cByJ (BC, low extent) mice to narrow the region to a 737 Kb locus, Dce1. Herein, we report the causal gene. METHODS: We used additional CNG mapping and knockout mice to narrow the number of candidate genes. Subsequent inspection identified a nonsynonymous single nucleotide polymorphism between B6 and BC within Rabep2 (rs33080487). We then created B6 mice with the BC single nucleotide polymorphism at this locus plus 3 other lines for predicted alteration or knockout of Rabep2 using gene editing. RESULTS: The single amino acid change caused by rs33080487 accounted for the difference in collateral extent and infarct volume between B6 and BC mice attributable to Dce1. Mechanistically, variants of Rabep2 altered collaterogenesis during embryogenesis but had no effect on angiogenesis examined in vivo and in vitro. Rabep2 deficiency altered endosome trafficking known to be involved in VEGF-A→VEGFR2 signaling required for collaterogenesis. CONCLUSIONS: Naturally occurring variants of Rabep2 are major determinants of variation in collateral extent and stroke severity in mice.