Stepwise Ultra-pH-Sensitive Micelles Overcome a pKa Barrier for Systemic Lymph Node Delivery.

While local nanoparticle delivery to lymph nodes is well studied, there are few design criteria for intravenous delivery to the entire lymph node repertoire. In this study, we investigated the effect of NP pH transition on lymph node targeting by employing a series of ultra-pH-sensitive (UPS) polymeric micelles. The UPS library responds to pH thresholds (pKa 6.9, 6.2, and 5.3) over a range of physiological pH. We observed a dependence of intravenous lymph node targeting on micelle pH transition. UPS6.9 (subscript indicates pKa) shows poor lymph node delivery, while UPS5.3 delivers efficiently to lymph node sets. We investigated targeting mechanisms of UPS5.3, observing an accumulation among lymph node lymphatics and a dependence on lymph node-resident macrophages. To overcome the pH-threshold barrier, which limits UPS6.9, we rationally designed a nanoparticle coassembly of UPS6.9 with UPS5.3, called HyUPS. The HyUPS micelle retains the constitutive pH transitions of each polymer, showing stepwise responses to discrete pH thresholds. We demonstrate that HyUPS improves UPS6.9 delivery to lymph nodes, extending this platform for disease detection of lymph node metastasis.

KRAS-driven model of Gorham-Stout disease effectively treated with trametinib.

Gorham-Stout disease (GSD) is a sporadically occurring lymphatic disorder. Patients with GSD develop ectopic lymphatics in bone, gradually lose bone, and can have life-threatening complications, such as chylothorax. The etiology of GSD is poorly understood, and current treatments for this disease are inadequate for most patients. To explore the pathogenesis of GSD, we performed targeted high-throughput sequencing with samples from a patient with GSD and identified an activating somatic mutation in KRAS (p.G12V). To characterize the effect of hyperactive KRAS signaling on lymphatic development, we expressed an active form of KRAS (p.G12D) in murine lymphatics (iLECKras mice). We found that iLECKras mice developed lymphatics in bone, which is a hallmark of GSD. We also found that lymphatic valve development and maintenance was altered in iLECKras mice. Because most iLECKras mice developed chylothorax and died before they had significant bone disease, we analyzed the effect of trametinib (an FDA-approved MEK1/2 inhibitor) on lymphatic valve regression in iLECKras mice. Notably, we found that trametinib suppressed this phenotype in iLECKras mice. Together, our results demonstrate that somatic activating mutations in KRAS can be associated with GSD and reveal that hyperactive KRAS signaling stimulates the formation of lymphatics in bone and impairs the development of lymphatic valves. These findings provide insight into the pathogenesis of GSD and suggest that trametinib could be an effective treatment for GSD.

The emergence of targetable MEKanisms in sporadic lymphatic disorders.

Sporadic lymphatic diseases are orphans among orphans in the medical community, a diverse collection of disorders at the intersection of cardiac, gastrointestinal, pulmonary, dermatologic, and oncologic disease that receives only passing attention in medical school and that no subspecialty in medicine fully embraces as its own. They often present in a confusing and illusive manner, with a fractured bone, expectoration of blood or a branching airway cast, a swollen limb or a collection of chylous material; protean manifestations that can challenge even the most expert diagnostician. Yet many of these acquired disorders have been discovered to have a targetable genetic basis, and as the case report of Foster et al (2020) demonstrates, the sedulous clinician-patient dyad can be rewarded with an almost miraculous result when the molecular pathogenesis of the disease is pursued and an exquisitely targeted therapy is administered.

Lymphatics in bone arise from pre-existing lymphatics.

Bones do not normally have lymphatics. However, individuals with generalized lymphatic anomaly (GLA) or Gorham-Stout disease (GSD) develop ectopic lymphatics in bone. Despite growing interest in the development of tissue-specific lymphatics, the cellular origin of bone lymphatic endothelial cells (bLECs) is not known and the development of bone lymphatics has not been fully characterized. Here, we describe the development of bone lymphatics in mouse models of GLA and GSD. Through lineage-tracing experiments, we show that bLECs arise from pre-existing Prox1-positive LECs. We show that bone lymphatics develop in a stepwise manner where regional lymphatics grow, breach the periosteum and then invade bone. We also show that the development of bone lymphatics is impaired in mice that lack osteoclasts. Last, we show that rapamycin can suppress the growth of bone lymphatics in our models of GLA and GSD. In summary, we show that bLECs can arise from pre-existing LECs and that rapamycin can prevent the growth of bone lymphatics.

Somatic activating mutations in PIK3CA cause generalized lymphatic anomaly.

Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct somatic PIK3CA variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same PIK3CA variants occur in PIK3CA-related overgrowth spectrum and cause hyperactivation of the PI3K-AKT-mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of PIK3CA (His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.

Cyclooxygenase-2 Inhibition Potentiates the Efficacy of Vascular Endothelial Growth Factor Blockade and Promotes an Immune Stimulatory Microenvironment in Preclinical Models of Pancreatic Cancer.

Resistance to standard therapy remains a major challenge in the treatment of pancreatic ductal adenocarcinoma (PDA). Although anti-VEGF therapy delays PDA progression, therapy-induced hypoxia results in a less differentiated mesenchymal-like tumor cell phenotype, which reinforces the need for effective companion therapies. COX-2 inhibition has been shown to promote tumor cell differentiation and improve standard therapy response in PDA. Here, we evaluate the efficacy of COX-2 inhibition and VEGF blockade in preclinical models of PDA. In vivo, the combination therapy was more effective in limiting tumor growth and metastasis than single-agent therapy. Combination therapy also reversed anti-VEGF-induced epithelial-mesenchymal transition and collagen deposition and altered the immune landscape by increasing tumor-associated CD8+ T cells while reducing FoxP3+ T cells and FasL expression on the tumor endothelium. IMPLICATIONS: Together, these findings demonstrate that COX-2 inhibition enhances the efficacy of anti-VEGF therapy by reducing hypoxia-induced epithelial plasticity and promoting an immune landscape that might facilitate immune activation.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/2/348/F1.large.jpg.

Lymphangiogenesis, lymphatic systemomics, and cancer: context, advances and unanswered questions.

Ever since it was discovered that endothelial cells line lymphatic vessels, investigators have been working on unraveling the mechanisms that control the growth of this distinctive endothelium and its role in normal physiology and human disease. Recent technological advances have ushered in a new era of "omics" research on the lymphatic system. Research on the genome, transcriptome, proteome, and metabolome of lymphatics has increased our understanding of the biology of the lymphatic vasculature. Here, we introduce the context-lymphatic "systemomics," then briefly review some of the latest advances in research on tumor-associated lymphatic vessels highlighting several "omic" studies that have shed light on mechanisms controlling the growth and function of tumor-associated lymphatic vessels. We conclude by returning, with unanswered questions, to the larger context of cancer and the lymphatic system as a vasculature, circulation, route of entry and transport, and control center of the immune network.

Mir-126 is a conserved modulator of lymphatic development.

Organ homeostasis relies upon cellular and molecular processes that restore tissue structure and function in a timely fashion. Lymphatic vessels help maintain fluid equilibrium by returning interstitial fluid that evades venous uptake back to the circulation. Despite its important role in tissue homeostasis, cancer metastasis, and close developmental origins with the blood vasculature, the number of molecular players known to control lymphatic system development is relatively low. Here we show, using genetic approaches in zebrafish and mice, that the endothelial specific microRNA mir-126, previously implicated in vascular integrity, regulates lymphatic development. In zebrafish, in contrast to mir-126 morphants, double mutants (mir-126a-/-; mir-126b-/-, hereafter mir-126-/-) do not exhibit defects in vascular integrity but develop lymphatic hypoplasia; mir-126-/- animals fail to develop complete trunk and facial lymphatics, display severe edema and die as larvae. Notably, following MIR-126 inhibition, human Lymphatic Endothelial Cells (hLECs) respond poorly to VEGFA and VEGFC. In this context, we identify a concomitant reduction in Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) and Vascular Endothelial Growth Factor Receptor-3 (VEGFR3, also known as FLT4) expression upon MIR-126 inhibition. In vivo, we further show that flt4+/- zebrafish embryos exhibit lymphatic defects after mild miR-126 knockdown. Similarly, loss of Mir-126 in Flt4+/- mice results in embryonic edema and lethality. Thus, our results indicate that miR-126 modulation of Vegfr signaling is essential for lymphatic system development in fish and mammals.

Identification of Gene Expression Differences between Lymphangiogenic and Non-Lymphangiogenic Non-Small Cell Lung Cancer Cell Lines.

It is well established that lung tumors induce the formation of lymphatic vessels. However, the molecular mechanisms controlling tumor lymphangiogenesis in lung cancer have not been fully delineated. In the present study, we identify a panel of non-small cell lung cancer (NSCLC) cell lines that induce lymphangiogenesis and use genome-wide mRNA expression to characterize the molecular mechanisms regulating tumor lymphangiogenesis. We show that Calu-1, H1993, HCC461, HCC827, and H2122 NSCLC cell lines form tumors that induce lymphangiogenesis whereas Calu-3, H1155, H1975, and H2073 NSCLC cell lines form tumors that do not induce lymphangiogenesis. By analyzing genome-wide mRNA expression data, we identify a 17-gene expression signature that distinguishes lymphangiogenic from non-lymphangiogenic NSCLC cell lines. Importantly, VEGF-C is the only lymphatic growth factor in this expression signature and is approximately 50-fold higher in the lymphangiogenic group than in the non-lymphangiogenic group. We show that forced expression of VEGF-C by H1975 cells induces lymphangiogenesis and that knockdown of VEGF-C in H1993 cells inhibits lymphangiogenesis. Additionally, we demonstrate that the triple angiokinase inhibitor, nintedanib (small molecule that blocks all FGFRs, PDGFRs, and VEGFRs), suppresses tumor lymphangiogenesis in H1993 tumors. Together, these data suggest that VEGF-C is the dominant driver of tumor lymphangiogenesis in NSCLC and reveal a specific therapy that could potentially block tumor lymphangiogenesis in NSCLC patients.

Warfarin Blocks Gas6-Mediated Axl Activation Required for Pancreatic Cancer Epithelial Plasticity and Metastasis.

Repurposing "old" drugs can facilitate rapid clinical translation but necessitates novel mechanistic insight. Warfarin, a vitamin K "antagonist" used clinically for the prevention of thrombosis for more than 50 years, has been shown to have anticancer effects. We hypothesized that the molecular mechanism underlying its antitumor activity is unrelated to its effect on coagulation, but is due to inhibition of the Axl receptor tyrosine kinase on tumor cells. Activation of Axl by its ligand Gas6, a vitamin K-dependent protein, is inhibited at doses of warfarin that do not affect coagulation. Here, we show that inhibiting Gas6-dependent Axl activation with low-dose warfarin, or with other tumor-specific Axl-targeting agents, blocks the progression and spread of pancreatic cancer. Warfarin also inhibited Axl-dependent tumor cell migration, invasiveness, and proliferation while increasing apoptosis and sensitivity to chemotherapy. We conclude that Gas6-induced Axl signaling is a critical driver of pancreatic cancer progression and its inhibition with low-dose warfarin or other Axl-targeting agents may improve outcome in patients with Axl-expressing tumors.

Viewpoints on vessels and vanishing bones in Gorham-Stout disease.

Gorham-Stout disease (GSD) is a rare disorder characterized by the proliferation of endothelial-lined vessels in bone and the progressive destruction of bone. Although Jackson described the first case of GSD in 1838, the clinical and histological features of GSD were not defined until Gorham and Stout published their report on massive osteolysis in 1955. In the years since Gorham and Stout's groundbreaking publication, more than 300 cases of GSD have been described in the literature. These reports have revealed that the progressive resorption of bone in GSD causes severe physical deformities, disabilities, and life-threatening complications. Unfortunately, the underlying cause of GSD remains unknown and, as a result, the therapeutic options for individuals with GSD are limited. Here we review the latest advances in GSD research and present strategies to address basic and clinical research questions related to GSD.

Collagen signaling enhances tumor progression after anti-VEGF therapy in a murine model of pancreatic ductal adenocarcinoma.

There is growing evidence that antiangiogenic therapy stimulates cancer cell invasion and metastasis. However, the underlying molecular mechanisms responsible for these changes have not been fully defined. Here, we report that anti-VEGF therapy promotes local invasion and metastasis by inducing collagen signaling in cancer cells. We show that chronic VEGF inhibition in a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDA) induces hypoxia, a less differentiated mesenchymal-like tumor cell phenotype, TGF-ß expression, and collagen deposition and signaling. In addition, we show that collagen signaling is critical for protumorigenic activity of TGF-ß in vitro. To further model the impact of collagen signaling in tumors, we evaluated PDA in mice lacking Sparc, a protein that reduces collagen binding to cell surface receptors. Importantly, we show that loss of Sparc increases collagen signaling and tumor progression. Together, these findings suggest that collagen actively promotes PDA spread and that enhanced disease progression associated with anti-VEGF therapy can arise from elevated extracellular matrix-mediated signaling.

RHOA-FAK is a required signaling axis for the maintenance of KRAS-driven lung adenocarcinomas.

UNLABELLED: Non-small cell lung cancer (NSCLC) often expresses mutant KRAS together with tumor-associated mutations of the CDKN2A locus, which are associated with aggressive, therapy-resistant tumors. Here, we unravel specific requirements for the maintenance of NSCLC that carries this genotype. We establish that the extracellular signal-regulated kinase (ERK)/RHOA/focal adhesion kinase (FAK) network is deregulated in high-grade lung tumors. Suppression of RHOA or FAK induces cell death selectively in mutant KRAS;INK4A/ARF-deficient lung cancer cells. Furthermore, pharmacologic inhibition of FAK caused tumor regression specifically in the high-grade lung cancer that developed in mutant Kras;Cdkn2a-null mice. These findings provide a rationale for the rapid implementation of genotype-specific targeted therapies using FAK inhibitors in patients with cancer. SIGNIFICANCE: Targeted therapies are effective for only a small fraction of patients with cancer. We report that FAK inhibitors exert potent antitumor effects in NSCLCs that express mutant KRAS in association with INK4A/ARF deficiency. These results reveal a novel genotype-specific vulnerability of cancer cells that can be exploited for therapeutic purposes.

Stromal platelet-derived growth factor receptor α (PDGFRα) provides a therapeutic target independent of tumor cell PDGFRα expression in lung cancer xenografts.

In lung cancer, platelet-derived growth factor receptor α (PDGFRα) is expressed frequently by tumor-associated stromal cells and by cancer cells in a subset of tumors. We sought to determine the effect of targeting stromal PDGFRα in preclinical lung tumor xenograft models (human tumor, mouse stroma). Effects of anti-human (IMC-3G3) and anti-mouse (1E10) PDGFRα monoclonal antibodies (mAb) on proliferation and PDGFRα signaling were evaluated in lung cancer cell lines and mouse fibroblasts. Therapy studies were conducted using established PDGFRα-positive H1703 cells and PDGFRα-negative Calu-6, H1993, and A549 subcutaneous tumors in immunocompromised mice treated with vehicle, anti-PDGFRα mAbs, chemotherapy, or combination therapy. Tumors were analyzed for growth and levels of growth factors. IMC-3G3 inhibited PDGFRα activation and the growth of H1703 cells in vitro and tumor growth in vivo, but had no effect on PDGFRα-negative cell lines or mouse fibroblasts. 1E10 inhibited growth and PDGFRα activation of mouse fibroblasts, but had no effect on human cancer cell lines in vitro. In vivo, 1E10-targeted inhibition of murine PDGFRα reduced tumor growth as single-agent therapy in Calu-6 cells and enhanced the effect of chemotherapy in xenografts derived from A549 cells. We also identified that low expression cancer cell expression of VEGF-A and elevated expression of PDGF-AA were associated with response to stromal PDGFRα targeting. We conclude that stromal PDGFRα inhibition represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression.

Overlapping biomarkers, pathways, processes and syndromes in lymphatic development, growth and neoplasia.

Recent discoveries in molecular lymphology, developmental biology, and tumor biology in the context of long-standing concepts and observations on development, growth, and neoplasia implicate overlapping pathways, processes, and clinical manifestations in developmental disorders and cancer metastasis. Highlighted in this review are some of what is known (and speculated) about the genes, proteins, and signaling pathways and processes involved in lymphatic/blood vascular development in comparison to those involved in cancer progression and spread. Clues and conundra from clinical disorders that mix these processes and mute them, including embryonic rests, multicentric nests of displaced cells, uncontrolled/invasive "benign" proliferation and lymphogenous/hematogenous "spread", represent a fine line between normal development and growth, dysplasia, benign and malignant neoplasia, and "metastasis". Improved understanding of these normal and pathologic processes and their underlying pathomechanisms, e.g., stem cell origin and bidirectional epithelial-mesenchymal transition, could lead to more successful approaches in classification, treatment, and even prevention of cancer and a whole host of other diseases.

Phosphorylation of Akt and ERK1/2 is required for VEGF-A/VEGFR2-induced proliferation and migration of lymphatic endothelium.

There is growing evidence that vascular endothelial growth factor-A (VEGF-A), a ligand of the receptor tyrosine kinases VEGFR1 and VEGFR2, promotes lymphangiogenesis. However, the underlying mechanisms by which VEGF-A induces the growth of lymphatic vessels remain poorly defined. Here we report that VEGFR2, not VEGFR1, is the primary receptor regulating VEGF-A-induced lymphangiogenesis. We show that specific inhibition of VEGF-A/VEGFR2 signaling with the fully human monoclonal antibody r84 significantly inhibits lymphangiogenesis in MDA-MB-231 tumors. In vitro experiments with primary human dermal lymphatic endothelial cells (LECs) demonstrate that blocking VEGF-A activation of VEGFR2, not VEGFR1, significantly inhibits VEGF-A-induced proliferation and migration of LECs. We show that VEGF-A stimulation of LECs leads to the phosphorylation of VEGFR2 (Tyr 951, 1054, 1059, 1175, and 1214) which subsequently triggers PKC dependent phosphorylation of ERK1/2 and PI3-K dependent phosphorylation of Akt. Additionally, we demonstrate that inhibitors that suppress the phosphorylation of ERK1/2 and Akt significantly block VEGF-A- induced proliferation and migration of LECs. Together, these results shed light on the mechanisms regulating VEGF-A-induced proliferation and migration of LECs, reveal that VEGFR2 is the primary signaling VEGF-A receptor on lymphatic endothelium, and suggest that therapeutic agents targeting the VEGF-A/VEGFR2 axis could be useful in blocking the pathological formation of lymphatic vessels.

Lymphangiogenesis and hemangiogenesis: potential targets for therapy.

This review updates historical background from century-old observations on embryonic lymphatic system development through current understanding of the molecular basis of lymphvasculogenesis/lymphangiogenesis ("molecular lymphology"), highlighting similarities and differences with analogous blood vasculature processes. Topics covered include molecular mechanisms in lymphatic development, structural adaptations of the lymphatic vasculature to particulate and cellular transport and trafficking, lymphogenous route of clinical cancer spread, preservation of delineated lymphatic pathways during cancer operations, and anti-lymphangiogenesis in cancer therapy.

Inhibition of vascular endothelial growth factor reduces angiogenesis and modulates immune cell infiltration of orthotopic breast cancer xenografts.

Vascular endothelial growth factor (VEGF) is a primary stimulant of angiogenesis and is a macrophage chemotactic protein. Inhibition of VEGF is beneficial in combination with chemotherapy for some breast cancer patients. However, the mechanism by which inhibition of VEGF affects tumor growth seems to involve more than its effect on endothelial cells. In general, increased immune cell infiltration into breast tumors confers a worse prognosis. We have shown previously that 2C3, a mouse monoclonal antibody that prevents VEGF from binding to VEGF receptor 2 (VEGFR2), decreases tumor growth, angiogenesis, and macrophage infiltration into pancreatic tumors and therefore hypothesized that r84, a fully human IgG that phenocopies 2C3, would similarly affect breast tumor growth and immune cell infiltration. In this study, we show that anti-VEGF therapy with bevacizumab, 2C3, or r84 inhibits the growth of established orthotopic MDA-MB-231 breast tumors in severe combined immunodeficiency (SCID) mice, reduces tumor microvessel density, limits the infiltration of tumor-associated macrophages, but is associated with elevated numbers of tumor-associated neutrophils. In addition, we found that treatment with r84 reduced the number of CD11b(+)Gr1(+) double-positive cells in the tumor compared with tumors from control-treated animals. These results show that selective inhibition of VEGFR2 with an anti-VEGF antibody is sufficient for effective blockade of the protumorigenic activity of VEGF in breast cancer xenografts. These findings further define the complex molecular interactions in the tumor microenvironment and provide a translational tool that may be relevant to the treatment of breast cancer.