Whole-Mount Imaging of Adipose Tissue Macrophages.

The adipose tissue comprises highly heterogeneous macrophage populations, which play critical roles in the regulation of adipose tissue function and dysfunction during health and disease. Whole-amount staining is a powerful technique for macrophage characterization within the 3D environment of the adipose tissue, enabling the visualization of different macrophage populations and their interaction with other cells within their in vivo niche. Due to the high-fat content and softness, freezing and sectioning of adipose tissue is difficult, and distortion of tissue morphology typically occurs, especially in the case of white adipose tissue. We describe here a whole-mount staining alternative for adipose tissue imaging that preserves all structures and allows high-resolution image acquisition. We address in a step-by-step manner how to perform immunofluorescence staining of different fat pads and how to optimally visualize cellular and acellular (extracellular matrix) constituents of the adipose tissue and its vasculature, as well as resident and infiltrating macrophage populations.

The Impact of Adipose Tissue-Derived miRNAs in Metabolic Syndrome, Obesity, and Cancer.

Obesity is a multifactorial and complex condition that is characterized by abnormal and excessive white adipose tissue accumulation, which can lead to the development of metabolic diseases, such as type 2 diabetes mellitus, nonalcoholic fatty liver disease, cardiovascular diseases, and several types of cancer. Obesity is characterized by excessive adipose tissue accumulation and associated with alterations in immunity, displaying a chronic low-grade inflammation profile. Adipose tissue is a dynamic and complex endocrine organ composed not only by adipocytes, but several immunological cells, which can secrete hormones, cytokines and many other factors capable of regulating metabolic homeostasis and several critical biological pathways. Remarkably, adipose tissue is a major source of circulating microRNAs (miRNAs), recently described as a novel form of adipokines. Several adipose tissue-derived miRNAs are deeply associated with adipocytes differentiation and have been identified with an essential role in obesity-associated inflammation, insulin resistance, and tumor microenvironment. During obesity, adipose tissue can completely change the profile of the secreted miRNAs, influencing circulating miRNAs and impacting the development of different pathological conditions, such as obesity, metabolic syndrome, and cancer. In this review, we discuss how miRNAs can act as epigenetic regulators affecting adipogenesis, adipocyte differentiation, lipid metabolism, browning of the white adipose tissue, glucose homeostasis, and insulin resistance, impacting deeply obesity and metabolic diseases. Moreover, we characterize how miRNAs can often act as oncogenic and tumor suppressor molecules, significantly modulating cancer establishment and progression. Furthermore, we highlight in this manuscript how adipose tissue-derived miRNAs can function as important new therapeutic targets.

Obesity and Breast Cancer: The Role of Crown-Like Structures in Breast Adipose Tissue in Tumor Progression, Prognosis, and Therapy.

Obesity is associated with increased risk and aggressiveness of many types of cancer. Women with obesity and breast cancer are more likely to be diagnosed with larger and higher-grade tumors and have higher incidence of metastases than lean individuals. Increasing evidence indicates that obesity includes systemic, chronic low-grade inflammation, and that adipose tissue can act as an important endocrine site, secreting a variety of substances that may regulate inflammation, immune response, and cancer predisposition. Obesity-associated inflammation appears to be initially mediated by macrophage infiltration into adipose tissue. Macrophages can surround damaged or necrotic adipocytes, forming "crown-like" structures (CLS). CLS are increased in breast adipose tissue from breast cancer patients and are more abundant in patients with obesity conditions. Moreover, the CLS index-ratio from individuals with obesity seems to influence breast cancer recurrence rates and survival. In this review, we discuss the most recent cellular and molecular mechanisms involved in CLS establishment in the white adipose tissue of women with obesity and their implications for breast cancer biology. We also explain how CLS influence the tumor microenvironment and affect breast cancer behavior. Targeting breast adipose tissue CLS can be a crucial therapeutic tool in cancer treatment, especially in patients with obesity.

The Impact of the Adipose Organ Plasticity on Inflammation and Cancer Progression.

Obesity is characterized by chronic and low-grade systemic inflammation, an increase of adipose tissue, hypertrophy, and hyperplasia of adipocytes. Adipose tissues can be classified into white, brown, beige and pink adipose tissues, which display different regulatory, morphological and functional characteristics of their adipocyte and immune cells. Brown and white adipocytes can play a key role not only in the control of energy homeostasis, or through the balance between energy storage and expenditure, but also by the modulation of immune and inflammatory responses. Therefore, brown and white adipocytes can orchestrate important immunological crosstalk that may deeply impact the tumor microenvironment and be crucial for cancer establishment and progression. Recent works have indicated that white adipose tissues can undergo a process called browning, in which an inducible brown adipocyte develops. In this review, we depict the mechanisms involved in the differential role of brown, white and pink adipocytes, highlighting their structural, morphological, regulatory and functional characteristics and correlation with cancer predisposition, establishment, and progression. We also discuss the impact of the increased adiposity in the inflammatory and immunological modulation. Moreover, we focused on the plasticity of adipocytes, describing the molecules produced and secreted by those cells, the modulation of the signaling pathways involved in the browning phenomena of white adipose tissue and its impact on inflammation and cancer.

Lysophosphatidylcholine Induces NLRP3 Inflammasome-Mediated Foam Cell Formation and Pyroptosis in Human Monocytes and Endothelial Cells.

Foam cells are specialized lipid-loaded macrophages derived from monocytes and are a key pathological feature of atherosclerotic lesions. Lysophosphatidylcholine (LPC) is a major lipid component of the plasma membrane with a broad spectrum of proinflammatory activities and plays a key role in atherosclerosis. However, the role of LPC in lipid droplet (LD) biogenesis and the modulation of inflammasome activation is still poorly understood. In the present study, we investigated whether LPC can induce foam cell formation through an analysis of LD biogenesis and determined whether the cell signaling involved in this process is mediated by the inflammasome activation pathway in human endothelial cells and monocytes. Our results showed that LPC induced foam cell formation in both types of cells by increasing LD biogenesis via a NLRP3 inflammasome-dependent pathway. Furthermore, LPC induced pyroptosis in both cells and the activation of the inflammasome with IL-1ß secretion, which was dependent on potassium efflux and lysosomal damage in human monocytes. The present study described the IL-1ß secretion and foam cell formation triggered by LPC via an inflammasome-mediated pathway in human monocytes and endothelial cells. Our results will help improve our understanding of the relationships among LPC, LD biogenesis, and NLRP3 inflammasome activation in the pathogenesis of atherosclerosis.

The influence of female mice age on biodistribution and biocompatibility of citrate-coated magnetic nanoparticles.

BACKGROUND: Magnetic nanoparticles (MNPs) have been successfully tested for several purposes in medical applications. However, knowledge concerning the effects of nanostructures on elderly organisms is remarkably scarce. PURPOSE: To fill part of this gap, this work aimed to investigate biocompatibility and bio-distribution aspects of magnetic nanoparticles coated with citrate (NpCit) in both elderly and young healthy mice. METHODS: NpCit (2.4 mg iron) was administered intraperitoneally, and its toxicity was evaluated for 28 days through clinical, biochemical, hematological, and histopathological examinations. In addition, its biodistribution was evaluated by spectrometric (inductively coupled plasma optical emission spectrometry) and histological methods. RESULTS: NpCit presented age-dependent effects, inducing very slight and temporary biochemical and hematological changes in young animals. These changes were even weaker than the effects of the aging process, especially those related to the hematological data, tumor necrosis factor alpha, and nitric oxide levels. On the other hand, NpCit showed a distinct set of results in the elderly group, sometimes reinforcing (decrease of lymphocytes and increase of monocytes) and sometimes opposing (erythrocyte parameters and cytokine levels) the aging changes. Leukocyte changes were still observed on the 28th day after treatment in the elderly group. Slight evidence of a decrease in liver and immune functions was detected in elderly mice treated or not treated with NpCit. It was noted that tissue damage or clinical changes related to aging or to the NpCit treatment were not observed. As detected for aging, the pattern of iron biodistribution was significantly different after NpCit administration: extra iron was detected until the 28th day, but in different organs of elderly (liver and kidneys) and young (spleen, liver, and lungs) mice. CONCLUSION: Taken together, the data show NpCit to be a stable and reasonably biocompatible sample, especially for young mice, and thus appropriate for biomedical applications. The data showed important differences after NpCit treatment related to the animals' age, and this emphasizes the need for further studies in older animals to appropriately extend the benefits of nanotechnology to the elderly population.

Omega 3-DHA and Delta-Tocotrienol Modulate Lipid Droplet Biogenesis and Lipophagy in Breast Cancer Cells: the Impact in Cancer Aggressiveness.

Omega 3-docosahexaenoic acid (DHA) and vitamin E Delta-tocotrienol (Delta-T3) are extensively studied as protective nutrients against cancer development. Little is known about the biological mechanisms targeted by these bioactive molecules on lipid droplet (LD) biogenesis, an important breast cancer aggressiveness marker, and the occurrence of lipophagy in breast cancer cells. The aim of this study was to investigate the effect of DHA, Delta-T3 and DHA plus Delta-T3 co-treatment in LD biogenesis and lipophagy process in triple negative breast cancer cell line MDA-MB-231. Cells were treated with 50 µM DHA and/or 5 µM Delta-T3. Our results demonstrated that DHA can trigger an increase in LD biogenesis and co-treatment with Delta-T3 was able to reduce this LD biogenesis. In addition, we showed that a higher cytoplasmic LD content is associated with a higher breast cancer cells malignance and proliferation. Reduction of cytoplasmic LD content by silencing ADRP (adipose differentiation-related protein), a structural LD protein, also decreased cell proliferation in MDA-MB-231 cells. Treatment with DHA and Delta-T3 alone or co-treatment did not reduce cell viability. Moreover, we showed here that DHA can trigger lipophagy in MDA-MB-231 cells and DHA plus Delta-T3 co-treatment was able to enhance this lipophagy process. Our findings demonstrated that co-treatment with DHA plus Delta-T3 in MDA-MB-231 cells could reduce LD biogenesis and potentiate lipophagy in these cells, possibly having a positive impact to inhibit breast cancer malignancy. Therefore, suitable doses of DHA and Delta-T3 vitamin E isoform supplementation can be a prominent tool in therapeutic treatments against breast cancer.

Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells.

The implication of inflammation in pathophysiology of several type of cancers has been under intense investigation. Omega-3 fatty acids can modulate inflammation and present anticancer effects, promoting cancer cell death. Pyroptosis is an inflammation related cell death and so far, the function of docosahexaenoic acid (DHA) in pyroptosis cell death has not been described. This study investigated the role of DHA in triggering pyroptosis activation in breast cancer cells. MDA-MB-231 breast cancer cells were supplemented with DHA and inflammation cell death was analyzed. DHA-treated breast cancer cells triggered increased caspase-1and gasdermin D activation, enhanced IL-1ß secretion, translocated HMGB1 towards the cytoplasm, and membrane pore formation when compared to untreated cells, suggesting DHA induces pyroptosis programmed cell death in breast cancer cells. Moreover, caspase-1 inhibitor (YVAD) could protect breast cancer cells from DHA-induced pyroptotic cell death. In addition, membrane pore formation showed to be a lysosomal damage and ROS formation-depended event in breast cancer cells. DHA triggered pyroptosis cell death in MDA-MB-231by activating several pyroptosis markers in these cells. This is the first study that shows the effect of DHA triggering pyroptosis programmed cell death in breast cancer cells and it could improve the understanding of the omega-3 supplementation during breast cancer treatment.

Author Correction: Omega-3 docosahexaenoic acid induces pyroptosis cell death in triple-negative breast cancer cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Adipocytes and Macrophages Interplay in the Orchestration of Tumor Microenvironment: New Implications in Cancer Progression.

Inflammation has been known as one of the main keys to the establishment and progression of cancers. Chronic low-grade inflammation is also a strategic condition that underlies the causes and development of metabolic syndrome and obesity. Moreover, obesity has been largely related to poor prognosis of tumors by modulating tumor microenvironment with secretion of several inflammatory mediators by tumor-associated adipocytes (TAAs), which can modulate and recruit tumor-associated macrophages. Thus, the understanding of cellular and molecular mechanisms that underlay and link inflammation, obesity, and cancer is crucial to identify potential targets that interfere with this important route. Knowledge about the exact role of each component of the tumor microenvironment is not yet fully understood, but the new insights in literature highlight the essential role of adipocytes and macrophages interplay as key factor to determine the fate of cancer progression. In this review article, we focus on the functions of adipocytes and macrophages orchestrating cellular and molecular mechanisms that lead to inflammatory modulation in tumor microenvironment, which will be crucial to cancer establishment. We also emphasized the mechanisms by which the tumor promotes itself by recruiting and polarizing macrophages, discussing the role of adipocytes in this process. In addition, we discuss here the newest possible anticancer therapeutic treatments aiming to retard the development of the tumor based on what is known about cancer, adipocyte, and macrophage polarization.