Glioblastomas acquire myeloid-affiliated transcriptional programs via epigenetic immunoediting to elicit immune evasion.

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which current immunotherapy approaches have been unsuccessful. Here, we explore the mechanisms underlying immune evasion in GBM. By serially transplanting GBM stem cells (GSCs) into immunocompetent hosts, we uncover an acquired capability of GSCs to escape immune clearance by establishing an enhanced immunosuppressive tumor microenvironment. Mechanistically, this is not elicited via genetic selection of tumor subclones, but through an epigenetic immunoediting process wherein stable transcriptional and epigenetic changes in GSCs are enforced following immune attack. These changes launch a myeloid-affiliated transcriptional program, which leads to increased recruitment of tumor-associated macrophages. Furthermore, we identify similar epigenetic and transcriptional signatures in human mesenchymal subtype GSCs. We conclude that epigenetic immunoediting may drive an acquired immune evasion program in the most aggressive mesenchymal GBM subtype by reshaping the tumor immune microenvironment.

Redefining macrophage and neutrophil biology in the metastatic cascade.

Tumor cells metastasize to distant organs through a complex series of events that are driven by tumor intrinsic and extrinsic factors. In particular, non-malignant stromal cells, including immune cells, modify tumor metastatic behavior. Of these cells, tumor-associated innate immune cells, particularly macrophages and neutrophils, suppress the cytotoxic activity of innate and adaptive killer cells and interact with tumor cells to promote their growth and malignancy. These findings in mouse cancer models suggest that targeting these sub-populations of immune cells holds therapeutic promise in treating metastatic disease. In this review, we describe the origin and role of the macrophages, neutrophils, and their progenitors in the metastatic cascade and suggest strategies that might enhance cancer therapy.

Transcellular Pathways in Lymphatic Endothelial Cells Regulate Changes in Solute Transport by Fluid Stress.

RATIONALE: The transport of interstitial fluid and solutes into lymphatic vessels is important for maintaining interstitial homeostasis and delivering antigens and soluble factors to the lymph node for immune surveillance. Transendothelial transport across lymphatic endothelial cells (LECs) is commonly considered to occur paracellularly, or between cell-cell junctions, and driven by local pressure and concentration gradients. However, emerging evidence suggests that LECs also play active roles in regulating interstitial solute balance and can scavenge and store antigens, raising the possibility that vesicular or transcellular pathways may be important in lymphatic solute transport. OBJECTIVE: The aim of this study was to determine the relative importance of transcellular (vesicular) versus paracellular transport pathways by LECs and how mechanical stress (ie, fluid flow conditioning) alters either pathway. METHODS AND RESULTS: We demonstrate that transcellular transport mechanisms substantially contribute to lymphatic solute transport and that solute uptake occurs in both caveolae- and clathrin-coated vesicles. In vivo, intracelluar uptake of fluorescently labeled albumin after intradermal injection by LECs was similar to that of dermal dendritic cells. In vitro, we developed a method to differentially quantify intracellular solute uptake versus transendothelial transport by LECs. LECs preconditioned to 1 µm/s transmural flow demonstrated increased uptake and basal-to-apical solute transport, which could be substantially reversed by blocking dynamin-dependent vesicle formation. CONCLUSIONS: These findings reveal the importance of intracellular transport in steady-state lymph formation and suggest that LECs use transcellular mechanisms in parallel to the well-described paracellular route to modulate solute transport from the interstitium according to biomechanical cues.

Perivascular Macrophages Limit Permeability.

OBJECTIVE: Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions. APPROACH AND RESULTS: We combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents. CONCLUSIONS: This study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.

Fatty acid based hyperbranched polymeric nanoparticles for hydrophobic drug delivery.

BACKGROUND: In recent years nano-sized dendrimer/hyperbranched polymers gained importance in drug delivery applications. OBJECTIVE: In this study, a novel fatty acid-based hyperbranched resin (HBR) was synthesized and used for tamoxifen (TAM) and idarubicin (IDA) delivery. METHODS: The core of the HBR was dipentaerythritol, and the branching was provided by dimethylolpropionic acid. The molecule was terminated by ricinoleic acid. Chemical and structural characterization of the resin was carried out and then drug-loading experiments were performed. CONCLUSION: The loading efficiencies were found to be 73.3% for TAM and 74% for IDA. The Fourier transform infrared spectroscopy analysis showed that TAM physically bounded onto the resin whereas IDA interacted chemically. Controlled release in phosphate buffer was improved by Pseudomonas sp. lipase and sodium dodecyl sulfate. The release rates decreased with the increase of loading concentrations. The cytotoxicity analyses were carried out on MCF-7 breast cancer cells for both drug-free and drug-loaded HBR. Drug-free particles did not have significant toxicity. Drug-loaded nanoparticles caused higher levels of cell death than pure drugs.

Generation of mouse bone marrow-derived macrophages using tumor coculture assays to mimic the tumor microenvironment.

Macrophages are one of the key immune cells within the tumor microenvironment that encourage the growth of tumors at the primary site as well as contributing to all parts of the metastatic cascade. Although it is possible to isolate macrophages directly from the tumor, this can be a laborious process and due to their plasticity, it is not possible to maintain their in vivo phenotype in vitro. For this reason, differentiating macrophages from bone marrow is an attractive alternative. Here we present robust methods to study in vitro derived macrophages including (i) the isolation and generation of macrophages from bone marrow, (ii) differentiation/characterization of classically activated, alternatively activated and tumor-conditioned macrophages, as well as (iii) in vitro co-culturing assays for tumor cell-macrophage interaction/transmigration.

Adjuvant-free immunization with infective filarial larvae as lymphatic homing antigen carriers.

Controlled infection with intestinal nematodes has therapeutic potential for preventing the symptoms of allergic and autoimmune diseases. Here, we engineered larvae of the filarial nematode Litomosoides sigmodontis as a vaccine strategy to induce adaptive immunity against a foreign, crosslinked protein, chicken egg ovalbumin (OVA), in the absence of an external adjuvant. The acylation of filarial proteins with fluorescent probes or biotin was not immediately detrimental to larval movement and survival, which died 3 to 5 days later. At least some of the labeled and skin-inoculated filariae migrated through lymphatic vessels to draining lymph nodes. The immunization potential of OVA-biotin-filariae was compared to that of an OVA-bound nanoparticulate carrier co-delivered with a CpG adjuvant in a typical vaccination scheme. Production of IFNγ and TNFα by restimulated CD4+ cells but not CD8+ confirmed the specific ability of filariae to stimulate CD4+ T cells. This alternative method of immunization exploits the intrinsic adjuvancy of the attenuated nematode carrier and has the potential to shift the vaccination immune response towards cellular immunity.

Growth factors with enhanced syndecan binding generate tonic signalling and promote tissue healing.

Growth factors can stimulate tissue regeneration, but the side effects and low effectiveness associated with suboptimal delivery systems have impeded their use in translational regenerative medicine. Physiologically, growth factor interactions with the extracellular matrix control their bioavailability and spatiotemporal cellular signalling. Growth factor signalling is also controlled at the cell surface level via binding to heparan sulfate proteoglycans, such as syndecans. Here we show that vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) that were engineered to have a syndecan-binding sequence trigger sustained low-intensity signalling (tonic signalling) and reduce the desensitization of growth factor receptors. We also show in mouse models that tonic signalling leads to superior morphogenetic activity, with syndecan-binding growth factors inducing greater bone regeneration and wound repair than wild-type growth factors, as well as reduced tumour growth (associated with PDGF-BB delivery) and vascular permeability (triggered by VEGF-A). Tonic signalling via syndecan binding may also enhance the regenerative capacity of other growth factors.

Dampening the fire to prevent surgery- and chemotherapy-induced metastasis.

In this issue of the JCI, Panigrahy et al. demonstrate that preoperative administration of the antiinflammatory drug ketorolac or specialized proresolving mediators (SPM) called resolvins increases disease-free survival rates and prevents metastasis after surgery and chemotherapy in mouse models of cancer. The antitumor response was partially mediated by tumor-specific T cell immunity and immunological memory.

Chemotherapy elicits pro-metastatic extracellular vesicles in breast cancer models.

Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy.

Dorsal Ear Skin Window for Intravital Imaging and Functional Analysis of Lymphangiogenesis.

Postdevelopmental lymphangiogenesis occurs in chronic inflammation and wound healing, and here we describe a window preparation in the mouse ear in which lymphangiogenesis can be observed and manipulated. This model has many advantages, including access for intravital immunostaining and imaging to assess morphological features and regeneration kinetics, as well as functional assays such as lymphatic clearance. We describe five procedures: (1) the creation of a collagen-fibrin-filled window in the mouse ear as a model for regenerative lymphangiogenesis, (2) intravital immunostaining for live analysis of morphology and structure, (3) lymphatic clearance assay for functional quantification, (4) whole-mount imaging with tissue clearing for confocal imaging, and (5) postmortem lymphangiography. These procedures allow for identification of morphological and functional abnormalities in both preexisting and newly formed lymphatic vessels.

Characterization of a B16-F10 melanoma model locally implanted into the ear pinnae of C57BL/6 mice.

The common experimental use of B16-F10 melanoma cells focuses on exploring their metastatic potential following intravenous injection into mice. In this study, B16-F10 cells are used to develop a primary tumor model by implanting them directly into the ears of C57BL/6J mice. The model represents a reproducible and easily traceable tool for local tumor growth and for making additional in vivo observations, due to the localization of the tumors. This model is relatively simple and involves (i) surgical opening of the ear skin, (ii) removal of a square-piece of cartilage followed by (iii) the implantation of tumor cells with fibrin gel. The remodeling of the fibrin gel within the cartilage chamber, accompanying tumor proliferation, results in the formation of blood vessels, lymphatics and tissue matrix that can be readily distinguished from the pre-existing skin structures. Moreover, this method avoids the injection-enforced artificial spread of cells into the pre-existing lymphatic vessels. The tumors have a highly reproducible exponential growth pattern with a tumor doubling time of around 1.8 days, reaching an average volume of 85mm3 16 days after implantation. The melanomas are densely cellular with proliferative indices of between 60 and 80%. The induced angiogenesis and lymphangiogenesis resulted in the development of well-vascularized tumors. Different populations of immunologically active cells were also present in the tumor; the population of macrophages decreases with time while the population of T cells remained quasi constant. The B16-F10 tumors in the ear frequently metastasized to the cervical lymph nodes, reaching an incidence of 75% by day 16. This newly introduced B16-F10 melanoma model in the ear is a powerful tool that provides a new opportunity to study the local tumor growth and metastasis, the associated angiogenesis, lymphangiogenesis and tumor immune responses. It could potentially be used to test different treatment strategies.

Local induction of lymphangiogenesis with engineered fibrin-binding VEGF-C promotes wound healing by increasing immune cell trafficking and matrix remodeling.

Lymphangiogenesis occurs in inflammation and wound healing, yet its functional roles in these processes are not fully understood. Consequently, clinically relevant strategies for therapeutic lymphangiogenesis remain underdeveloped, particularly using growth factors. To achieve controlled, local capillary lymphangiogenesis with protein engineering and determine its effects on fluid clearance, leukocyte trafficking, and wound healing, we developed a fibrin-binding variant of vascular endothelial growth factor C (FB-VEGF-C) that is slowly released upon demand from infiltrating cells. Using a novel wound healing model, we show that implanted fibrin containing FB-VEGF-C, but not free VEGF-C, could stimulate local lymphangiogenesis in a dose-dependent manner. Importantly, the effects of FB-VEGF-C were restricted to lymphatic capillaries, with no apparent changes to blood vessels and downstream collecting vessels. Leukocyte intravasation and trafficking to lymph nodes were increased in hyperplastic lymphatics, while fluid clearance was maintained at physiological levels. In diabetic wounds, FB-VEGF-C-induced lymphangiogenesis increased extracellular matrix deposition and granulation tissue thickening, indicators of improved wound healing. Together, these results indicate that FB-VEGF-C is a promising strategy for inducing lymphangiogenesis locally, and that such lymphangiogenesis can promote wound healing by enhancing leukocyte trafficking without affecting downstream lymphatic collecting vessels.

The dual role of tumor lymphatic vessels in dissemination of metastases and immune response development.

Lymphatic vasculature plays a crucial role in the immune response, enabling transport of dendritic cells (DCs) and antigens (Ags) into the lymph nodes. Unfortunately, the lymphatic system has also a negative role in the progression of cancer diseases, by facilitating the metastatic spread of many carcinomas to the draining lymph nodes. The lymphatics can promote antitumor immune response as well as tumor tolerance. Here, we review the role of lymphatic endothelial cells (LECs) in tumor progression and immunity and mechanism of action in the newest anti-lymphatic therapies, including photodynamic therapy (PDT).

Long-term intravital immunofluorescence imaging of tissue matrix components with epifluorescence and two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

Growth factors engineered for super-affinity to the extracellular matrix enhance tissue healing.

Growth factors (GFs) are critical in tissue repair, but their translation to clinical use has been modest. Physiologically, GF interactions with extracellular matrix (ECM) components facilitate localized and spatially regulated signaling; therefore, we reasoned that the lack of ECM binding in their clinically used forms could underlie the limited translation. We discovered that a domain in placenta growth factor-2 (PlGF-2(123-144)) binds exceptionally strongly and promiscuously to ECM proteins. By fusing this domain to the GFs vascular endothelial growth factor-A, platelet-derived growth factor-BB, and bone morphogenetic protein-2, we generated engineered GF variants with super-affinity to the ECM. These ECM super-affinity GFs induced repair in rodent models of chronic wounds and bone defects that was greatly enhanced as compared to treatment with the wild-type GFs, demonstrating that this approach may be useful in several regenerative medicine applications.

Intravital immunofluorescence for visualizing the microcirculatory and immune microenvironments in the mouse ear dermis.

Visualizing the dynamic behaviors of immune cells in living tissue has dramatically increased our understanding of how cells interact with their surroundings, contributing important insights into mechanisms of leukocyte trafficking, tumor cell invasion, and T cell education by dendritic cells, among others. Despite substantial advances with various intravital imaging techniques including two-photon microscopy and the generation of multitudes of reporter mice, there is a growing need to assess cell interactions in the context of specific extracellular matrix composition and microvascular functions, and as well, simpler and more widely accessible methods are needed to image cell behaviors in the context of living tissue physiology. Here we present an antibody-based method for intravital imaging of cell interactions with the blood, lymphatic, and the extracellular matrix compartments of the living dermis while simultaneously assessing capillary permeability and lymphatic drainage function. Using the exposed dorsal ear of the anesthetized mouse and a fluorescence stereomicroscope, such events can be imaged in the context of specific extracellular matrix proteins, or matrix-bound chemokine stores. We developed and optimized the method to minimize tissue damage to the ear, rapidly immunostain for multiple extracellular or cell surface receptors of interest, minimize immunotoxicity with pre-blocking Fcγ receptors and phototoxicity with extracellular antioxidants, and highlight the major dermal tissue structures with basement membrane markers. We demonstrate differential migration behaviors of bone marrow-derived dendritic cells, blood-circulating leukocytes, and dermal dendritic cells, with the latter entering sparse CCL21-positive areas of pre-collecting lymphatic vessels. This new method allows simultaneous imaging of cells and tissue structures, microvascular function, and extracellular microenvironment in multiple skin locations for 12 hours or more, with the flexibility of immunolabeling in addition to genetic-based fluorescent reporters.