Endothelial TrkA coordinates vascularization and innervation in thermogenic adipose tissue and can be targeted to control metabolism.

OBJECTIVE: Brown adipogenesis and thermogenesis in brown and beige adipose tissue (AT) involve vascular remodeling and sympathetic neuronal guidance. Here, we investigated the molecular mechanism coordinating these processes. METHODS: We used mouse models to identify the molecular target of a peptide CPATAERPC homing to the endothelium of brown and beige AT. RESULTS: We demonstrate that CPATAERPC mimics nerve growth factor (NGF) and identify a low molecular weight isoform of NGF receptor, TrkA, as the CPATAERPC cell surface target. We show that the expression of truncated endothelial TrkA is selective for brown and subcutaneous AT. Analysis of mice with endothelium-specific TrkA knockout revealed the role of TrkA in neuro-vascular coordination supporting the thermogenic function of brown adipocytes. A hunter-killer peptide D-BAT, composed of CPATAERPC and a pro-apoptotic domain, induced cell death in the endothelium and adipocytes. This resulted in thermogenesis impairment, and predisposed mice to obesity and glucose intolerance. We also tested if this treatment can inhibit the tumor recruitment of lipids mobilized from adipocytes from adjacent AT. Indeed, in a mouse model of breast cancer D-BAT suppressed tumor-associated AT lipolysis, which resulted in reduced fatty acid utilization by cancer cells. CONCLUSION: Our study demonstrates that TrkA signaling in the endothelium supports neuro-vascular coordination enabling beige adipogenesis.

Endothelial Prohibitin Mediates Bidirectional Long-Chain Fatty Acid Transport in White and Brown Adipose Tissues.

The function of prohibitin-1 (PHB1) in adipocyte mitochondrial respiration, adaptive thermogenesis, and long-chain fatty acid (LCFA) metabolism has been reported. While intracellular PHB1 expression is ubiquitous, cell surface PHB1 localization is selective for adipocytes and endothelial cells of adipose tissue. The importance of PHB1 in adipose endothelium has not been investigated, and its vascular cell surface function has remained unclear. Here, we generated and analyzed mice with PHB1 knock-out specifically in endothelial cells (PHB1 EC-KO). Despite the lack of endothelial PHB1, mice developed normally and had normal vascularization in both white adipose tissue and brown adipose tissue (BAT). Tumor and ex vivo explant angiogenesis assays also have not detected a functional defect in PHB1 KO endothelium. No metabolic phenotype was observed in PHB1 EC-KO mice raised on a regular diet. We show that both male and female PHB1 EC-KO mice have normal body composition and adaptive thermogenesis. However, PHB1 EC-KO mice displayed higher insulin sensitivity and increased glucose clearance when fed a high-fat diet. We demonstrate that the efficacy of LCFA deposition by adipocytes is decreased by PHB1 EC-KO, in particular in BAT. Consistent with that, EC-KO mice have a defect in clearing triglycerides from systemic circulation. Free fatty acid release upon lipolysis induction was also found to be reduced in PHB1 EC-KO mice. Our results demonstrate that PHB1 in endothelial cells regulates bidirectional LCFA transport and thereby suppresses glucose utilization.

Constitutive loss of DNMT3A causes morbid obesity through misregulation of adipogenesis.

DNA Methyltransferase 3 A (DNMT3A) is an important facilitator of differentiation of both embryonic and hematopoietic stem cells. Heterozygous germline mutations in DNMT3A lead to Tatton-Brown-Rahman Syndrome (TBRS), characterized by obesity and excessive height. While DNMT3A is known to impact feeding behavior via the hypothalamus, here we investigated a role in adipocyte progenitors utilizing heterozygous knockout mice that recapitulate cardinal TBRS phenotypes. These mice become morbidly obese due to adipocyte enlargement and tissue expansion. Adipose tissue in these mice exhibited defects in preadipocyte maturation and precocious activation of inflammatory gene networks, including interleukin-6 signaling. Adipocyte progenitor cell lines lacking DNMT3A exhibited aberrant differentiation. Furthermore, mice in which Dnmt3a was specifically ablated in adipocyte progenitors showed enlarged fat depots and increased progenitor numbers, partly recapitulating the TBRS obesity phenotypes. Loss of DNMT3A led to constitutive DNA hypomethylation, such that the DNA methylation landscape of young adipocyte progenitors resemble that of older wild-type mice. Together, our results demonstrate that DNMT3A coordinates both the central and local control of energy storage required to maintain normal weight and prevent inflammatory obesity.

Characterization of Peptides Targeting Metastatic Tumor Cells as Probes for Cancer Detection and Vehicles for Therapy Delivery.

Metastasis is the leading cause of cancer-related deaths, and metastatic cancers remain largely incurable due to chemoresistance. Biomarkers of metastatic cells are lacking, and probes that could be used to detect and target metastases would be highly valuable. Here we hypothesize that metastatic cancer cells express cell-surface receptors that can be harnessed for identification of molecules homing to metastases. Screening a combinatorial library in a mouse mammary tumor model of spontaneous metastasis identified cyclic peptides with tropism for cancer cells disseminated to the lungs. Two lead peptides, CLRHSSKIC and CRAGVGRGC, bound murine and human cells derived from breast carcinoma and melanoma in culture and were selective for metastatic cells in vivo. In mice, peptide CRAGVGRGC radiolabeled with 67Ga for biodistribution analysis demonstrated selective probe homing to lung metastases. Moreover, systemic administration of 68Ga-labeled CRAGVGRGC enabled noninvasive imaging of lung metastases in mice by PET. A CRAGVGRGC-derived peptide induced apoptosis upon cell internalization in vitro and suppressed metastatic burden in vivo. Colocalization of CLRHSSKIC and CRAGVGRGC with N-cadherin+/E-cadherin- cells indicated that both peptides are selective for cancer cells that have undergone the epithelial-to-mesenchymal transition. We conclude that CRAGVGRGC is useful as a probe to facilitate the development of imaging modalities and therapies targeting metastases. SIGNIFICANCE: This study identifies new molecules that bind metastatic cells and demonstrates their application as noninvasive imaging probes and vehicles for cytotoxic therapy delivery in preclinical cancer models.

Partial Ablation of Non-Myogenic Progenitor Cells as a Therapeutic Approach to Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), caused by the loss of dystrophin, remains incurable. Reduction in muscle regeneration with DMD is associated with the accumulation of fibroadipogenic progenitors (FAPs) differentiating into myofibroblasts and leading to a buildup of the collagenous tissue aggravating DMD pathogenesis. Mesenchymal stromal cells (MSCs) expressing platelet-derived growth factor receptors (PDGFRs) are activated in muscle during DMD progression and give rise to FAPs promoting DMD progression. Here, we hypothesized that muscle dysfunction in DMD could be delayed via genetic or pharmacologic depletion of MSC-derived FAPs. In this paper, we test this hypothesis in dystrophin-deficient mdx mice. To reduce fibro/adipose infiltration and potentiate muscle progenitor cells (MPCs), we used a model for inducible genetic ablation of proliferating MSCs via a suicide transgene, viral thymidine kinase (TK), expressed under the Pdgfrb promoter. We also tested if MSCs from fat tissue, the adipose stromal cells (ASCs), contribute to FAPs and could be targeted in DMD. Pharmacological ablation was performed with a hunter-killer peptide D-CAN targeting ASCs. MSC depletion with these approaches resulted in increased endurance, measured based on treadmill running, as well as grip strength, without significantly affecting fibrosis. Although more research is needed, our results suggest that depletion of pathogenic MSCs mitigates muscle damage and delays the loss of muscle function in mouse models of DMD.

Prohibitin Inactivation in Adipocytes Results in Reduced Lipid Metabolism and Adaptive Thermogenesis Impairment.

Prohibitin-1 (PHB) is a multifunctional protein previously reported to be important for adipocyte function. PHB is expressed on the surface of adipose cells, where it interacts with a long-chain fatty acid (LCFA) transporter. Here, we show that mice lacking PHB in adipocytes (PHB adipocyte [Ad]-knockout [KO]) have a defect in fat tissue accumulation despite having larger lipid droplets in adipocytes due to reduced lipolysis. Although PHB Ad-KO mice do not display glucose intolerance, they are insulin resistant. We show that PHB Ad-KO mice are lipid intolerant due to a decreased capacity of adipocytes for LCFA uptake. Instead, PHB Ad-KO mice have increased expression of GLUT1 in various tissues and use glucose as a preferred energy source. We demonstrate that PHB Ad-KO mice have defective brown adipose tissue, are intolerant to cold, and display reduced basal energy expenditure. Systemic repercussions of PHB inactivation in adipocytes were observed in both males and females. Consistent with lower cellular mitochondrial content and reduced uncoupling protein 1 protein expression, brown adipocytes lacking PHB display decreased proton leak and switch from aerobic metabolism to glycolysis. Treatment of differentiating brown adipocytes with small molecules targeting PHB suppressed mitochondrial respiration and uncoupling. Our results demonstrate that PHB in adipocytes is essential for normal fatty acid uptake, oxidative metabolism, and adaptive thermogenesis. We conclude that PHB inhibition could be investigated as an approach to altering energy substrate utilization.

Fatty acid mobilization from adipose tissue is mediated by CD36 posttranslational modifications and intracellular trafficking.

The mechanism controlling long-chain fatty acid (LCFA) mobilization from adipose tissue is not well understood. Here, we investigated how the LCFA transporter CD36 regulates this process. By using tissue-specific KO mouse models, we showed that CD36 in adipocytes and endothelial cells mediated both LCFA deposition into and release from adipose tissue. We demonstrated the role of adipocytic and endothelial CD36 in promoting tumor growth and chemoresistance conferred by adipose tissue-derived LCFAs. We showed that dynamic cysteine S-acylation of CD36 in adipocytes, endothelial cells, and cancer cells mediated intercellular LCFA transport. We demonstrated that lipolysis induction in adipocytes triggered CD36 deacylation and deglycosylation, as well as its dissociation from interacting proteins, prohibitin-1 (PHB) and annexin 2 (ANX2). Our data indicate that lipolysis triggers caveolar endocytosis and translocation of CD36 from the cell membrane to lipid droplets. This study suggests a mechanism for both outside-in and inside-out cellular LCFA transport regulated by CD36 S-acylation and its interactions with PHB and ANX2.

Browning white adipose tissue using adipose stromal cell-targeted resveratrol-loaded nanoparticles for combating obesity.

Enhancing thermogenic energy expenditure via promoting the browning of white adipose tissue (WAT) is a potential therapeutic strategy to manage energy imbalance and the consequent comorbidities associated with excess body weight. Adverse effects and toxicities of currently available methods to induce browning of WAT have retarded exploration of this promising therapeutic approach. Targeted delivery of browning agents to adipose stromal cells (ASCs) in subcutaneous WAT to induce differentiation into beige adipocytes may overcome these barriers. Herein, we report for the first time, ASC-targeted delivery of trans-resveratrol (R), a representative agent, using ligand-coated R-encapsulated nanoparticles (L-Rnano) that selectively bind to glycanation site-deficient decorin receptors on ASCs. After biweekly intravenous administration of L-Rnano to obese C57BL/6 J mice for 5 weeks targeted R delivery significantly induced ASCs differentiation into beige adipocytes, which subsequently resulted in 40% decrease in fat mass, accompanied by improved glucose homeostasis and decreased inflammation. Our results suggest that the ASC-targeted nanoparticle delivery of browning agents could be a transformative technology in combating obesity and its comorbidities with high efficacy and low toxicity.

Progression of prostate carcinoma is promoted by adipose stromal cell-secreted CXCL12 signaling in prostate epithelium.

Aggressiveness of carcinomas is linked with tumor recruitment of adipose stromal cells (ASC), which is increased in obesity. ASC promote cancer through molecular pathways not fully understood. Here, we demonstrate that epithelial-mesenchymal transition (EMT) in prostate tumors is promoted by obesity and suppressed upon pharmacological ASC depletion in HiMyc mice, a spontaneous genetic model of prostate cancer. CXCL12 expression in tumors was associated with ASC recruitment and localized to stromal cells expressing platelet-derived growth factor receptors Pdgfra and Pdgfrb. The role of this chemokine secreted by stromal cells in cancer progression was further investigated by using tissue-specific knockout models. ASC deletion of CXCL12 gene in the Pdgfr + lineages suppressed tumor growth and EMT, indicating stroma as the key source of CXCL12. Clinical sample analysis revealed that CXCL12 expression by peritumoral adipose stroma is increased in obesity, and that the correlating increase in Pdgfr/CXCL12 expression in the tumor is linked with decreased survival of patients with prostate carcinoma. Our study establishes ASC as the source of CXCL12 driving tumor aggressiveness and outlines an approach to treatment of carcinoma progression.

Age-associated telomere attrition in adipocyte progenitors predisposes to metabolic disease.

White and beige adipocytes in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) are maintained by proliferation and differentiation of adipose progenitor cells (APCs). Here we use mice with tissue-specific telomerase reverse transcriptase (TERT) gene knockout (KO), which undergo premature telomere shortening and proliferative senescence in APCs, to investigate the effect of over-nutrition on APC exhaustion and metabolic dysfunction. We find that TERT KO in the Pdgfra+ cell lineage results in adipocyte hypertrophy, inflammation and fibrosis in SAT, while TERT KO in the Pdgfrb+ lineage leads to adipocyte hypertrophy in both SAT and VAT. Systemic insulin resistance is observed in both KO models and is aggravated by a high-fat diet. Analysis of human biopsies demonstrates that telomere shortening in SAT is associated with metabolic disease progression after bariatric surgery. Our data indicate that over-nutrition can promote APC senescence and provide a mechanistic link between ageing, obesity and diabetes.

Glycosaminoglycan Modification of Decorin Depends on MMP14 Activity and Regulates Collagen Assembly.

Proper processing of collagens COL1 and COL6 is required for normal function of adipose tissue and skeletal muscle. Proteoglycan decorin (DCN) regulates collagen fiber formation. The amino-terminus of DCN is modified with an O-linked glycosaminoglycan (GAG), the function of which has remained unclear. Previously, non-glycanated DCN (ngDCN) was identified as a marker of adipose stromal cells. Here, we identify MMP14 as the metalloprotease that cleaves DCN to generate ngDCN. We demonstrate that mice ubiquitously lacking DCN GAG (ngDCN mice) have reduced matrix rigidity, enlarged adipocytes, fragile skin, as well as skeletal muscle hypotrophy, fibrosis, and dysfunction. Our results indicate that DCN deglycanation results in reduced intracellular DCN-collagen binding and increased production of truncated COL6 chains, leading to aberrant procollagen processing and extracellular localization. This study reveals that the GAG of DCN functions to regulate collagen assembly in adipose tissue and skeletal muscle and uncovers a new mechanism of matrix dysfunction in obesity and aging.

PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer.

While plasminogen activator inhibitor-1 (PAI-1) is known to potentiate cellular migration via proteolytic regulation, this adipokine is implicated as an oncogenic ligand in the tumor microenvironment. To understand the underlying paracrine mechanism, here, we conduct transcriptomic analysis of 1,898 endometrial epithelial cells (EECs) exposed and unexposed to PAI-1-secreting adipose stromal cells. The PAI-1-dependent action deregulates crosstalk among tumor-promoting and tumor-repressing pathways, including transforming growth factor ß (TGF-ß). When PAI-1 is tethered to lipoprotein receptor-related protein 1 (LRP1), the internalized signaling causes downregulation of SMAD4 at the transcriptional and post-translational levels that attenuates TGF-ß-related transcription programs. Repression of genes encoding the junction and adhesion complex preferentially occurs in SMAD4-underexpressed EECs of persons with obesity. The findings highlight a role of PAI-1 signaling that renders ineffective intercellular communication for the development of adiposity-associated endometrial cancer.

Three-Dimensional Magnetic Levitation Culture System Simulating White Adipose Tissue.

White adipose tissue (WAT) has attracted interest for tissue engineering and cell-based therapies as an abundant source of adipose stem/stromal cells (ASC). However, technical challenges in WAT cell culture have limited its applications in regenerative medicine. Traditional two-dimensional (2D) cell culture models, which are essentially monolayers of cells on glass or plastic substrates, inadequately represent tissue architecture, biochemical concentration gradients, substrate stiffness, and most importantly for WAT research, cell phenotypic heterogeneity. Physiological cell culture platforms for WAT modeling must recapitulate the native diversity of cell types and their coordination within the organ. For this purpose, we developed a three-dimensional (3D) model using magnetic levitation. Here, we describe our protocol that we successfully employed to build adipose tissue organoids (adipospheres) that preserve the heterogeneity of the constituent cell types in vitro. We demonstrate the capacity of assembling adipospheres from multiple cell types, including ASCs, endohtelial cells, and leukocytes that recreate tissue organization. These adipospheres mimicked WAT organogenesis in that they enabled the formation of vessel-like endothelial structures with lumens and differentiation of unilocular adipocytes. Altogether, magnetic levitation is a cell culture platform that recreates tissue structure, function, and heterogeneity in vitro, and serves as a foundation for high-throughput WAT tissue culture and analysis.

PDGFRα/PDGFRß signaling balance modulates progenitor cell differentiation into white and beige adipocytes.

The relative abundance of thermogenic beige adipocytes and lipid-storing white adipocytes in adipose tissue underlie its metabolic activity. The roles of adipocyte progenitor cells, which express PDGFRα or PDGFRß, in adipose tissue function have remained unclear. Here, by defining the developmental timing of PDGFRα and PDGFRß expression in mouse subcutaneous and visceral adipose depots, we uncover depot specificity of pre-adipocyte delineation. We demonstrate that PDGFRα expression precedes PDGFRß expression in all subcutaneous but in only a fraction of visceral adipose stromal cells. We show that high-fat diet feeding or thermoneutrality in early postnatal development can induce PDGFRß+ lineage recruitment to generate white adipocytes. In contrast, the contribution of PDGFRß+ lineage to beige adipocytes is minimal. We provide evidence that human adipose tissue also contains distinct progenitor populations differentiating into beige or white adipocytes, depending on PDGFRß expression. Based on PDGFRα or PDGFRß deletion and ectopic expression experiments, we conclude that the PDGFRα/PDGFRß signaling balance determines progenitor commitment to beige (PDGFRα) or white (PDGFRß) adipogenesis. Our study suggests that adipocyte lineage specification and metabolism can be modulated through PDGFR signaling.

Non-glycanated Decorin Is a Drug Target on Human Adipose Stromal Cells.

Adipose stromal cells (ASCs) have been identified as a mesenchymal cell population recruited from white adipose tissue (WAT) by tumors and supporting cancer progression. We have previously reported the existence of a non-glycanated decorin isoform (ngDCN) marking mouse ASCs. We identified a peptide CSWKYWFGEC that binds to ngDCN and hence can serve as a vehicle for ASC-directed therapy delivery. We used hunter-killer peptides composed of CSWKYWFGEC and a pro-apoptotic moiety to deplete ASCs and suppress growth of mouse tumors. Here, we report the discovery of the human non-glycanated decorin isoform. We show that CSWKYWFGEC can be used as a probe to identify ASCs in human WAT and tumors. We demonstrate that human ngDCN is expressed on ASC surface. Finally, we validate ngDCN as a molecular target for pharmacological depletion of human ASCs with hunter-killer peptides. We propose that ngDCN-targeting agents could be developed for obesity and cancer treatment.

Human and Mouse Brown Adipose Tissue Mitochondria Have Comparable UCP1 Function.

Brown adipose tissue (BAT) plays an important role in mammalian thermoregulation. The component of BAT mitochondria that permits this function is the inner membrane carrier protein uncoupling protein 1 (UCP1). To the best of our knowledge, no studies have directly quantified UCP1 function in human BAT. Further, whether human and rodent BAT have comparable thermogenic function remains unknown. We employed high-resolution respirometry to determine the respiratory capacity, coupling control, and, most importantly, UCP1 function of human supraclavicular BAT and rodent interscapular BAT. Human BAT was sensitive to the purine nucleotide GDP, providing the first direct evidence that human BAT mitochondria have thermogenically functional UCP1. Further, our data demonstrate that human and rodent BAT have similar UCP1 function per mitochondrion. These data indicate that human and rodent BAT are qualitatively similar in terms of UCP1 function.

Prohibitin/annexin 2 interaction regulates fatty acid transport in adipose tissue.

We have previously identified prohibitin (PHB) and annexin A2 (ANX2) as proteins interacting on the surface of vascular endothelial cells in white adipose tissue (WAT) of humans and mice. Here, we demonstrate that ANX2 and PHB also interact in adipocytes. Mice lacking ANX2 have normal WAT vascularization, adipogenesis, and glucose metabolism but display WAT hypotrophy due to reduced fatty acid uptake by WAT endothelium and adipocytes. By using cell culture systems in which ANX2/PHB binding is disrupted either genetically or through treatment with a blocking peptide, we show that fatty acid transport efficiency relies on this protein complex. We also provide evidence that the interaction between ANX2 and PHB mediates fatty acid transport from the endothelium into adipocytes. Moreover, we demonstrate that ANX2 and PHB form a complex with the fatty acid transporter CD36. Finally, we show that the colocalization of PHB and CD36 on adipocyte surface is induced by extracellular fatty acids. Together, our results suggest that an unrecognized biochemical interaction between ANX2 and PHB regulates CD36-mediated fatty acid transport in WAT, thus revealing a new potential pathway for intervention in metabolic diseases.

Targeted Proapoptotic Peptides Depleting Adipose Stromal Cells Inhibit Tumor Growth.

Progression of many cancers is associated with tumor infiltration by mesenchymal stromal cells (MSC). Adipose stromal cells (ASC) are MSC that serve as adipocyte progenitors and endothelium-supporting cells in white adipose tissue (WAT). Clinical and animal model studies indicate that ASC mobilized from WAT are recruited by tumors. Direct evidence for ASC function in tumor microenvironment has been lacking due to unavailability of approaches to specifically inactivate these cells. Here, we investigate the effects of a proteolysis-resistant targeted hunter-killer peptide D-WAT composed of a cyclic domain CSWKYWFGEC homing to ASC and of a proapoptotic domain KLAKLAK2. Using mouse bone marrow transplantation models, we show that D-WAT treatment specifically depletes tumor stromal and perivascular cells without directly killing malignant cells or tumor-infiltrating leukocytes. In several mouse carcinoma models, targeted ASC cytoablation reduced tumor vascularity and cell proliferation resulting in hemorrhaging, necrosis, and suppressed tumor growth. We also validated a D-WAT derivative with a proapoptotic domain KFAKFAK2 that was found to have an improved cytoablative activity. Our results for the first time demonstrate that ASC, recruited as a component of tumor microenvironment, support cancer progression. We propose that drugs targeting ASC can be developed as a combination therapy complementing conventional cancer treatments.

A peptide probe for targeted brown adipose tissue imaging.

The presence of brown adipose tissue responsible for thermogenic energy dissipation has been revealed in adult humans and has high clinical importance. Owing to limitations of current methods for brown adipose tissue detection, analysing the abundance and localization of brown adipose tissue in the body has remained challenging. Here we screen a combinatorial peptide library in mice and characterize a peptide (with the sequence CPATAERPC) that selectively binds to the vascular endothelium of brown adipose tissue, but not of intraperitoneal white adipose tissue. We show that in addition to brown adipose tissue, this peptide probe also recognizes the vasculature of brown adipose tissue-like depots of subcutaneous white adipose tissue. Our results indicate that the CPATAERPC peptide localizes to brown adipose tissue even in the absence of sympathetic nervous system stimulation. Finally, we demonstrate that this probe can be used to identify brown adipose tissue depots in mice by whole-body near-infrared fluorescence imaging.

Adipose tissue engineering in three-dimensional levitation tissue culture system based on magnetic nanoparticles.

White adipose tissue (WAT) is becoming widely used in regenerative medicine/cell therapy applications, and its physiological and pathological importance is increasingly appreciated. WAT is a complex organ composed of differentiated adipocytes, stromal mesenchymal progenitors known as adipose stromal cells (ASC), as well as endothelial vascular cells and infiltrating leukocytes. Two-dimensional (2D) culture that has been typically used for studying adipose cells does not adequately recapitulate WAT complexity. Improved methods for reconstruction of functional WAT ex vivo are instrumental for understanding of physiological interactions between the composing cell populations. Here, we used a three-dimensional (3D) levitation tissue culture system based on magnetic nanoparticle assembly to model WAT development and growth in organoids termed adipospheres. We show that 3T3-L1 preadipocytes remain viable in spheroids for a long period of time, while in 2D culture, they lose adherence and die after reaching confluence. Upon adipogenesis induction in 3T3-L1 adipospheres, cells efficiently formed large lipid droplets typical of white adipocytes in vivo, while only smaller lipid droplet formation is achievable in 2D. Adiposphere-based coculture of 3T3-L1 preadipocytes with murine endothelial bEND.3 cells led to a vascular-like network assembly concomitantly with lipogenesis in perivascular cells. Adipocyte-depleted stromal vascular fraction (SVF) of mouse WAT cultured in 3D underwent assembly into organoids with vascular-like structures containing luminal endothelial and perivascular stromal cell layers. Adipospheres made from primary WAT cells displayed robust proliferation and complex hierarchical organization reflected by a matricellular gradient incorporating ASC, endothelial cells, and leukocytes, while ASC quickly outgrew other cell types in adherent culture. Upon adipogenesis induction, adipospheres derived from the SVF displayed more efficient lipid droplet accumulation than 2D cultures. This indicates that 3D intercellular signaling better recapitulates WAT organogenesis. Combined, our studies show that adipospheres are appropriate for WAT modeling ex vivo and provide a new platform for functional screens to identify molecules bioactive toward individual adipose cell populations. This 3D methodology could be adopted for WAT transplantation applications and aid approaches to WAT-based cell therapy.