Adipose tissue dysfunction and cardiometabolic risk. Ex vitro, in vivo and clinical studies

While the obesity epidemic develops at an alarming rate, scientifically we are still far behind with regard to diagnostic and therapeutic actions. In this thesis, we aimed to explore current and novel pathways in adipose tissue dysfunction, as a result of obesity, and investigated how they might contribute to metabolic and cardiovascular disease. In chapter 2, current knowledge of pathophysiological mechanisms linking abdominal adipose tissue to obesity-related metabolic dysfunction is reviewed, with a special focus on distinct adipose tissue depots and the role of adipose tissue-derived extracellular vesicles. Adipose tissue consists of different depots which are anatomically linked to distinct organs. Evidence suggests that intrinsic differences as well as interactions of adipose tissue with surrounding organs accounts for different contributions to cardiometabolic disease. In chapter 3 we explored the inflammatory profile of four different abdominal adipose tissue depots and their relation with parameters of metabolic dysfunction in a clinical study considering abdominally lean versus abdominally obese male patients. The most striking finding was the differential contribution of distinct visceral fat depots with metabolic dysfunction. Mesenteric fat morphology was mainly related to insulin resistance, whereas omental fat morphology was more strongly related to higher triglyceride and lower HDL-cholesterol levels. Extracellular vesicles (EVs) are active signaling vesicles important for communication between cells. In obesity, communication between adipocytes and immune cells such as macrophages is a key mechanism in adipose tissue inflammation, leading to metabolic complications like insulin resistance. In chapter 4 we have shown that EVs released by human in vitro differentiated adipocytes or ex vivo human adipose tissue explants can stimulate macrophages which subsequently induced insulin resistance in in vitro differentiated adipocytes. Furthermore, EVs of visceral but not subcutaneous adipose tissue were also quantitatively related to systemic insulin resistance. In chapter 5 we have shown that adiponectin-positive EVs were isolated from human plasma suggesting secretion of EVs into the circulation enabling endocrine signaling of fat EVs. Adipose tissue EVs, derived from ex vivo human fat explants, could inhibit insulin signaling in liver cells in vitro, which was related to pro-inflammatory adipokines present in EVs of visceral adipose tissue. In chapter 6, we have explored the potential of EV-associated markers, associated with cardiovascular disease, to serve as biomarkers for obesity-induced metabolic disease in patients with manifest cardiovascular disease. EV-cystatin C levels were positively related to metabolic complications of obesity. In contrast, EV-CD14 levels were inversely related to visceral obesity in males and associated with a relative risk reduction for the development of type 2 diabetes, indicating that EV-CD14 might be a protective biomarker for obesity-induced metabolic disease. In chapter 7, the potential prognostic value of adiponectin, an adipocyte specific marker with insulin sensitizing properties, as biomarker for cardiovascular disease is studied in a meta-analysis, which showed no relation between circulating levels of adiponectin and incident CHD or stroke. Therefore, despite all metabolic beneficial properties of adiponectin observed in vivo or in vitro, systemic levels of adiponectin do not seem to cover enough of the underlying pathological activity to be used as a biomarker for obesity-induced cardiovascular disease. Exploiting the composition of adipose tissue EVs would enable multi-parameter biomarker development which seems crucial for complex diseases such as obesity-induced cardiometabolic disease. Furthermore, manipulation of the contents and binding specificity of adipose tissue-EVs hold true therapeutic potential for targeted delivery in vivo

Tumor-specific uptake of fluorescent bevacizumab-IRDye800CW microdosing in patients with primary breast cancer: A phase I feasibility study.

Purpose: to provide proof of principle of safety, breast tumor-specific uptake and positive tumor margin assessment of the systemically administered near-infrared fluorescent (NIRF) tracer bevacizumab-IRDye800CW targeting vascular endothelial growth factor (VEGF)-A in breast cancer patients. Experimental Design: Twenty patients with primary invasive breast cancer eligible for primary surgery received 4.5 mg bevacizumab-IRDye800CW as intravenous bolus injection. Safety aspects were assessed as well as tracer uptake and tumor delineation during surgery and ex vivo in surgical specimens using an optical imaging system. Ex vivo multiplexed histopathology analyses were performed for evaluation of biodistribution of tracer uptake and co-registration of tumor tissue and healthy tissue. Results: None of the patients experienced adverse events. Tracer levels in primary tumor tissue were higher compared to those in the tumor margin (P < 0.05) and healthy tissue (P < 0.0001). VEGF-A tumor levels also correlated with tracer levels (r = 0.63, P < 0.0002). All but one tumor showed specific tracer uptake. Two out of 20 surgically excised lumps contained microscopic positive margins detected ex vivo by fluorescent macro- and microscopy and confirmed at the cellular level. Conclusions: Our study shows that systemic administration of the bevacizumab-IRDye800CW tracer is safe for breast cancer guidance and confirms tumor and tumor-margin uptake as evaluated by a systematic validation methodology. The findings are a step towards a phase II dose-finding study aimed at in vivo margin assessment and point to a novel drug assessment tool that provides a detailed picture of drug distribution in tumor tissue

Threshold analysis and biodistribution of fluorescently labeled bevacizumab in human breast cancer.

In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and non-malignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybrid multi-scale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized threshold that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labelled bevacizumab to outline the tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging

Fluorescently labeled bevacizumab in human breast cancer: Defining the classification threshold.

In-vivo fluorescently labelled drug (bevacizumab) breast cancer specimen where obtained from patients. We propose a new structured method to determine the optimal classification threshold in targeted fluorescence intra-operative imaging

PATZ1 fusions define a novel molecularly distinct neuroepithelial tumor entity with a broad histological spectrum

Large-scale molecular profiling studies in recent years have shown that central nervous system (CNS) tumors display a much greater heterogeneity in terms of molecularly distinct entities, cellular origins and genetic drivers than anticipated from histological assessment. DNA methylation profiling has emerged as a useful tool for robust tumor classification, providing new insights into these heterogeneous molecular classes. This is particularly true for rare CNS tumors with a broad morphological spectrum, which are not possible to assign as separate entities based on histological similarity alone. Here, we describe a molecularly distinct subset of predominantly pediatric CNS neoplasms (n = 60) that harbor PATZ1 fusions. The original histological diagnoses of these tumors covered a wide spectrum of tumor types and malignancy grades. While the single most common diagnosis was glioblastoma (GBM), clinical data of the PATZ1-fused tumors showed a better prognosis than typical GBM, despite frequent relapses. RNA sequencing revealed recurrent MN1:PATZ1 or EWSR1:PATZ1 fusions related to (often extensive) copy number variations on chromosome 22, where PATZ1 and the two fusion partners are located. These fusions have individually been reported in a number of glial/glioneuronal tumors, as well as extracranial sarcomas. We show here that they are more common than previously acknowledged, and together define a biologically distinct CNS tumor type with high expression of neural development markers such as PAX2, GATA2 and IGF2. Drug screening performed on the MN1:PATZ1 fusion-bearing KS-1 brain tumor cell line revealed preliminary candidates for further study. In summary, PATZ1 fusions define a molecular class of histologically polyphenotypic neuroepithelial tumors, which show an intermediate prognosis under current treatment regimens. © 2021, The Author(s)