Mobilization of innate and adaptive antitumor immune responses by the RNP-targeting antibody ATRC-101.

Immunotherapy approaches focusing on T cells have provided breakthroughs in treating solid tumors. However, there remains an opportunity to drive anticancer immune responses via other cell types, particularly myeloid cells. ATRC-101 was identified via a target-agnostic process evaluating antibodies produced by the plasmablast population of B cells in a patient with non-small cell lung cancer experiencing an antitumor immune response during treatment with checkpoint inhibitor therapy. Here, we describe the target, antitumor activity in preclinical models, and data supporting a mechanism of action of ATRC-101. Immunohistochemistry studies demonstrated tumor-selective binding of ATRC-101 to multiple nonautologous tumor tissues. In biochemical analyses, ATRC-101 appears to target an extracellular, tumor-specific ribonucleoprotein (RNP) complex. In syngeneic murine models, ATRC-101 demonstrated robust antitumor activity and evidence of immune memory following rechallenge of cured mice with fresh tumor cells. ATRC-101 increased the relative abundance of conventional dendritic cell (cDC) type 1 cells in the blood within 24 h of dosing, increased CD8+ T cells and natural killer cells in blood and tumor over time, decreased cDC type 2 cells in the blood, and decreased monocytic myeloid-derived suppressor cells in the tumor. Cellular stress, including that induced by chemotherapy, increased the amount of ATRC-101 target in tumor cells, and ATRC-101 combined with doxorubicin enhanced efficacy compared with either agent alone. Taken together, these data demonstrate that ATRC-101 drives tumor destruction in preclinical models by targeting a tumor-specific RNP complex leading to activation of innate and adaptive immune responses.

Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1.

An aged circulatory environment can activate microglia, reduce neural precursor cell activity and impair cognition in mice. We hypothesized that brain endothelial cells (BECs) mediate at least some of these effects. We observe that BECs in the aged mouse hippocampus express an inflammatory transcriptional profile with focal upregulation of vascular cell adhesion molecule 1 (VCAM1), a protein that facilitates vascular-immune cell interactions. Concomitantly, levels of the shed, soluble form of VCAM1 are prominently increased in the plasma of aged humans and mice, and their plasma is sufficient to increase VCAM1 expression in cultured BECs and the hippocampi of young mice. Systemic administration of anti-VCAM1 antibody or genetic ablation of Vcam1 in BECs counteracts the detrimental effects of plasma from aged individuals on young brains and reverses aging aspects, including microglial reactivity and cognitive deficits, in the brains of aged mice. Together, these findings establish brain endothelial VCAM1 at the blood-brain barrier as a possible target to treat age-related neurodegeneration.

Collagenase-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry.

The brain endothelium is a highly specialized vascular structure that maintains the activity and integrity of the central nervous system (CNS). Previous studies have reported that the integrity of the brain endothelium is compromised in a plethora of neuropathologies. Therefore, it is of particular interest to establish a method that enables researchers to investigate and understand the molecular changes in CNS endothelial cells and underlying mechanisms in conjunction with murine models of disease. In the past, approaches to isolate endothelial cells have either involved the use of transgenic reporter mice or suffered from insufficiently pure cell populations and poor yield. This protocol here is based on well-established protocols that were modified and combined to allow single cell isolation of highly pure brain endothelial cell populations using fluorescence activated cell sorting (FACS). Briefly, after careful removal of the meninges and dissection of the cortex/hippocampus, the brain tissue is mechanically homogenized and enzymatically digested in two steps resulting in a single cell suspension. Cells are stained with a cocktail of fluorochrome-conjugated antibodies identifying not only brain endothelial cells, but also potentially contaminating cell types such as pericytes, astrocytes, and lineage cells. Using flow cytometry, cell populations are separated and sorted directly into either RNA lysis buffer for bulk RNA analyses (e.g., RNA microarray and RNA-Seq) or in pure fetal bovine serum to preserve viability for other downstream applications such as single cell RNA-Seq and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-Seq). The protocol does not require the expression of a transgene to label brain endothelial cells and thus, may be applied to any mouse model. In our hands, the protocol has been highly reproducible with an average yield of 3 × 105 cells from a pool of four adult mice.

Papain-based Single Cell Isolation of Primary Murine Brain Endothelial Cells Using Flow Cytometry.

Brain endothelial cells (BECs) form the integral component of the blood-brain barrier (BBB) which separates the systemic milieu from the brain parenchyma and protects the brain from pathogens and circulating factors. In order to study BEC biology, it was of particular interest to establish a method that enables researchers to investigate and understand the underlying molecular mechanisms regulating their function during homeostasis, aging and disease. Furthermore, due to the heterogeneity of the cerebrovasculature and different vessel types that comprise the BBB, it is of particular interest to isolate primary BECs for single cell analysis from various subregions of the brain, such as the neurogenic and highly vascularized hippocampus and to enrich for specific vessel types. In the past, approaches to isolate endothelial cells were dependent on transgenic mice and often resulted in insufficiently pure cell populations and poor yield. This protocol describes a technique that allows single-cell isolation of highly pure brain endothelial cell populations using fluorescence activated cell sorting (FACS). Briefly, after perfusion and careful removal of the meninges, and dissection of the cortex/hippocampus, the brain tissue is mechanically homogenized and enzymatically digested resulting in a single cell suspension. Cells are stained with fluorochrome-conjugated antibodies identifying CD31+ brain endothelial cells, as well as CD45+CD11b+ myeloid cells for exclusion. Using flow cytometry, cell populations are separated and CD31+BECs are sorted in bulk into RNA later or as single cells directly into either RNA lysis buffer for single or bulk RNA-Seq analyses. The protocol does not require the expression of a transgene to label brain endothelial cells and thus, may be applied to any mouse model. In our hands, the protocol has been highly reproducible with an average yield of 1 × 105 cells isolated from an adult mouse cortex/hippocampus.

Patterns of expression of factor VIII and von Willebrand factor by endothelial cell subsets in vivo.

UNLABELLED: Circulating factor VIII (FVIII) is derived from liver and from extrahepatic sources probably of endothelial origin, but the vascular sites of FVIII production remain unclear. Among organs profiled, only liver and lymph nodes (LNs) show abundant expression of F8 messenger RNA (mRNA). Transcriptomic profiling of subsets of stromal cells, including endothelial cells (ECs) from mouse LNs and other tissues, showed that F8 mRNA is expressed by lymphatic ECs (LECs) but not by capillary ECs (capECs), fibroblastic reticular cells, or hematopoietic cells. Among blood ECs profiled, F8 expression was seen only in fenestrated ECs (liver sinusoidal and renal glomerular ECs) and some high endothelial venules. In contrast, von Willebrand factor mRNA was expressed in capECs but not in LECs; it was coexpressed with F8 mRNA in postcapillary high endothelial venules. Purified LECs and liver sinusoidal ECs but not capECs from LNs secrete active FVIII in culture, and human and mouse lymph contained substantial FVIII: C activity. Our results revealed localized vascular expression of FVIII and von Willebrand factor and identified LECs as a major cellular source of FVIII in extrahepatic tissues.

Tumour necrosis factor receptor superfamily member 9 (TNFRSF9) is up-regulated in reactive astrocytes in human gliomas.

AIMS: The prognosis of patients with malignant gliomas is still dismal despite maximum treatment. Novel therapeutic alternatives targeting tumorigenic pathways are, therefore, demanded. In murine glioma models, targeting of tumour necrosis factor receptor superfamily (TNFRSF) 9 led to complete tumour eradication. Thus, TNFRSF9 might also constitute a promising target in human diffuse gliomas. As there is a lack of data, we aimed to define the expression pattern and cellular source of TNFRSF9 in human gliomas. METHODS: We investigated TNFRSF9 expression in normal human central nervous system (CNS) tissue and glioma specimens using immunohistochemistry, immunofluorescence and Western blotting techniques. RESULTS: Our results show that TNFRSF9 is considerably up-regulated in human gliomas when compared with normal brain tissue. In addition, our data provides evidence for an immune cell-independent de novo expression pattern of TNFRSF9 in mainly non-neoplastic reactive astrocytes and excludes classic immunological cell types, namely lymphocytes and microglia as the source of TNFRSF9. Moreover, TNFRSF9 is predominantly expressed in a perivascular and peritumoural distribution with significantly higher expression in IDH-1 mutant gliomas. CONCLUSIONS: Our findings provide a novel, TNFRSF9-positive, reactive astrocytic phenotype and challenge the therapeutic suitability of TNFRSF9 as a promising target for human gliomas.

In vitro models of the blood-brain barrier.

The blood-brain barrier (BBB) proper is composed of endothelial cells (ECs) of the cerebral microvasculature, which are interconnected by tight junctions (TJs) that in turn form a physical barrier restricting paracellular flux. Tight control of vascular permeability is essential for the homeostasis and functionality of the central nervous system (CNS). In vitro BBB models have been in use for decades and have been of great benefit in the process of investigating and understanding the cellular and molecular mechanisms underlying BBB establishment. BBB integrity changes can be addressed in vitro by determining cell monolayer permeability (Pe) to different solutes and measuring trans-endothelial electrical resistance (TEER).This chapter describes procedures that can be utilized for both freshly isolated mouse brain microvascular ECs (MBMECs) and murine or human brain EC lines (bEnd5 or hCMEC/D3), cultivated either as a single monolayer or in cocultivation with primary mouse astrocytes (ACs). It starts with detailed information on how to perform transwell cell culture, including coating of inserts and seeding of the ECs and ACs. Moreover, it encompasses instructions for electrical assessment of the in vitro BBB using the more recent cellZscope(®) device, which was traditionally performed with chopstick electrodes of voltohmmeter type (EVOM). From continuous impedance measurements, the cellZscope(®) device provides TEER (paracellular resistance) and cell membrane capacitance (Ccl-transcellular resistance), two independent measures of monolayer integrity. Additionally, this chapter provides guidance through subsequent experiments such as permeability analysis (Pe, flux), expression analysis (qRT-PCR and Western blotting), and localization analysis of BBB junction proteins (immunocytochemistry) using the same inserts subjected earlier to impedance analysis.As numerous diseases are associated with BBB breakdown, researchers aim to continuously improve and refine in vitro BBB models to mimic in vivo conditions as closely as possible. This chapter summarizes protocols with the intention to provide a collection of BBB in vitro assays that generate reproducible results not only with primary brain ECs but also with EC lines to open up the field for a broader spectrum of researchers who intend to investigate the BBB in vitro particularly aiming at therapeutic aspects.

Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro.

Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize ß-catenin, or they are infected with a transcriptionally active form of ß-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

Endothelial Wnt/ß-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression.

Endothelial Wnt/ß-catenin signaling is necessary for angiogenesis of the central nervous system and blood-brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/ß-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active ß-catenin specifically in the endothelium. Enforced endothelial ß-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/ß-catenin-Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that ß-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/ß-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy.

Current concepts of blood-brain barrier development.

Homeostasis of the central nervous system (CNS) microenvironment is essential for its normal function and is maintained by the blood-brain barrier (BBB). The BBB proper is made up of endothelial cells (ECs) interconnected by tight junctions (TJs) that reveal a unique morphology and biochemical composition of the body's vasculature. In this article, we focus on developmental aspects of the BBB and describe morphological as well as molecular special features of the neuro-vascular unit (NVU) involved in barrier induction. Recently, we and others identified the Wnt/b-catenin pathway as crucial for brain angiogenesis, TJ and BBB formation. Based on these findings we discuss other pathways and molecular interactions for BBB establishment and maintenance. At the morphological level, our concept favors a major role for polarized astrocytes (ACs) therein. Orthogonal arrays of particles (OAPs) that are the morphological correlate of the water channel protein aquaporin-4 (AQP4) are specifically formed in the membrane of the AC endfoot. The polarized AC endfoot and hence OAPs are dependent on agrin and dystroglycan, of which agrin is a developmentally regulated extracellular matrix (ECM) component. Understanding the mechanisms leading to BBB development will be key to the understanding of barrier maintenance that is crucial for, but frequently disturbed, in the diseased adult brain.

Sonic hedgehog acts as a negative regulator of {beta}-catenin signaling in the adult tongue epithelium.

Wnt/beta-catenin signaling has been implicated in taste papilla development; however, its role in epithelial maintenance and tumor progression in the adult tongue remains elusive. We show Wnt/beta-catenin pathway activation in reporter mice and by nuclear beta-catenin staining in the epithelium and taste papilla of adult mouse and human tongues. beta-Catenin activation in APC(min/+) mice, which carry a mutation in adenomatous poliposis coli (APC), up-regulates Sonic hedgehog (Shh) and Jagged-2 (JAG2) in the tongue epithelium without formation of squamous cell carcinoma (SCC). We demonstrate that Shh suppresses beta-catenin transcriptional activity in a signaling-dependent manner in vitro and in vivo. A similar regulation and function was observed for JAG2, suggesting that both pathways negatively regulate beta-catenin, thereby preventing SCC formation in the tongue. This was supported by reduced nuclear beta-catenin in the tongue epithelium of Patched(+/-) mice, exhibiting dominant active Shh signaling. At the invasive front of human tongue cancer, nuclear beta-catenin and Shh were increased, suggesting their participation in tumor progression. Interestingly, Shh but not JAG2 was able to reduce beta-catenin signaling in SCC cells, arguing for a partial loss of negative feedback on beta-catenin transcription in tongue cancer. We show for the first time that the putative Wnt/beta-catenin targets Shh and JAG2 control beta-catenin signaling in the adult tongue epithelium, a function that is partially lost in lingual SCC.

Wnt/beta-catenin signaling controls development of the blood-brain barrier.

The blood-brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/beta-catenin (beta-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of beta-cat in vivo enhances barrier maturation, whereas inactivation of beta-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of beta-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of beta-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of beta-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.