Ceramide as an endothelial cell surface receptor and a lung-specific lipid vascular target for circulating ligands.

The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC.

Assembly of Hepatocyte Spheroids Using Magnetic 3D Cell Culture for CYP450 Inhibition/Induction.

There is a significant need for in vitro methods to study drug-induced liver injury that are rapid, reproducible, and scalable for existing high-throughput systems. However, traditional monolayer and suspension cultures of hepatocytes are difficult to handle and risk the loss of phenotype. Generally, three-dimensional (3D) cell culture platforms help recapitulate native liver tissue phenotype, but suffer from technical limitations for high-throughput screening, including scalability, speed, and handling. Here, we developed a novel assay for cytochrome P450 (CYP450) induction/inhibition using magnetic 3D cell culture that overcomes the limitations of other platforms by aggregating magnetized cells with magnetic forces. With this platform, spheroids can be rapidly assembled and easily handled, while replicating native liver function. We assembled spheroids of primary human hepatocytes in a 384-well format and maintained this culture over five days, including a 72 h induction period with known CYP450 inducers/inhibitors. CYP450 activity and viability in the spheroids were assessed and compared in parallel with monolayers. CYP450 activity was induced/inhibited in spheroids as expected, separate from any toxic response. Spheroids showed a significantly higher baseline level of CYP450 activity and induction over monolayers. Positive staining in spheroids for albumin and multidrug resistance-associated protein (MRP2) indicates the preservation of hepatocyte function within spheroids. The study presents a proof-of-concept for the use of magnetic 3D cell culture for the assembly and handling of novel hepatic tissue models.

Magnetically Bioprinted Human Myometrial 3D Cell Rings as A Model for Uterine Contractility.

Deregulation in uterine contractility can cause common pathological disorders of the female reproductive system, including preterm labor, infertility, inappropriate implantation, and irregular menstrual cycle. A better understanding of human myometrium contractility is essential to designing and testing interventions for these important clinical problems. Robust studies on the physiology of human uterine contractions require in vitro models, utilizing a human source. Importantly, uterine contractility is a three-dimensionally (3D)-coordinated phenomenon and should be studied in a 3D environment. Here, we propose and assess for the first time a 3D in vitro model for the evaluation of human uterine contractility. Magnetic 3D bioprinting is applied to pattern human myometrium cells into rings, which are then monitored for contractility over time and as a function of various clinically relevant agents. Commercially available and patient-derived myometrium cells were magnetically bioprinted into rings in 384-well formats for throughput uterine contractility analysis. The bioprinted uterine rings from various cell origins and patients show different patterns of contractility and respond differently to clinically relevant uterine contractility inhibitors, indomethacin and nifedipine. We believe that the novel system will serve as a useful tool to evaluate the physiology of human parturition while enabling high-throughput testing of multiple agents and conditions.

Nanoparticle Improved Stem Cell Therapy for Erectile Dysfunction in a Rat Model of Cavernous Nerve Injury.

PURPOSE: Recently intracavernous injection of stem cells has garnered great interest as a potential treatment of erectile dysfunction. However, most stem cells are washed out immediately after intracavernous injection. The goal of this study was to investigate using NanoShuttle™ magnetic nanoparticles to maintain stem cells in the corpus cavernosum after intracavernous injection, thereby improving stem cell therapy of erectile dysfunction in an animal model. MATERIALS AND METHODS: Adipose derived stem cells were magnetized with NanoShuttle magnetic nanoparticles to create Nano-adipose derived stem cells. A total of 24 rats underwent bilateral cavernous nerve crush and were randomly assigned to 3 groups, including adipose derived stem cells, Nano-adipose derived stem cells and Nano-adipose derived stem cells plus magnet. Cells were tracked at days 1, 3, 5 and 9 after intracavernous injection. Another 40 rats with bilateral cavernous nerve crush were randomly assigned to 4 groups, including bilateral cavernous nerve crush, bilateral cavernous nerve crush plus adipose derived stem cell intracavernous injection, bilateral cavernous nerve crush plus Nano-adipose derived stem cell intracavernous injection and bilateral cavernous nerve crush plus Nano-adipose derived stem cell intracavernous injection plus magnet. Functional testing and histological analysis were performed 4 weeks after intracavernous injection. RESULTS: In the in vitro study 1) NanoShuttle magnetic nanoparticles were successfully bound to adipose derived stem cells and 2) Nano-adipose derived stem cells migrated toward the magnet. In the in vivo study 1) cell tracking showed that Nano-adipose derived stem cells were successfully retained in the corpus cavernosum using the magnet for up to 3 days while most adipose derived stem cells were washed out in other groups by day 1 after intracavernous injection, and 2) intracavernous pressure/mean arterial pressure, and αSMA (α-smooth muscle actin) and PECAM-1 (platelet endothelial cell adhesion molecule 1) expression in the Nano-adipose derived stem cell group was significantly higher than in the other groups. CONCLUSIONS: Magnetization of adipose derived stem cells with NanoShuttle magnetic nanoparticles kept adipose derived stem cells in the corpus cavernosum and improved adipose derived stem cell therapy of erectile dysfunction in an animal model.

Bioprinting salivary gland models and their regenerative applications.

OBJECTIVE: Salivary gland (SG) hypofunction is a common clinical condition arising from radiotherapy to suppress head and neck cancers. The radiation often destroys the SG secretory acini, and glands are left with limited regenerative potential. Due to the complex architecture of SG acini and ducts, three-dimensional (3D) bioprinting platforms have emerged to spatially define these in vitro epithelial units and develop mini-organs or organoids for regeneration. Due to the limited body of evidence, this comprehensive review highlights the advantages and challenges of bioprinting platforms for SG regeneration. METHODS: SG microtissue engineering strategies such as magnetic 3D bioassembly of cells and microfluidic coaxial 3D bioprinting of cell-laden microfibers and microtubes have been proposed to replace the damaged acinar units, avoid the use of xenogeneic matrices (like Matrigel), and restore salivary flow. RESULTS: Replacing the SG damaged organ is challenging due to its complex architecture, which combines a ductal network with acinar epithelial units to facilitate a unidirectional flow of saliva. Our research group was the first to develop 3D bioassembly SG epithelial functional organoids with innervation to respond to both cholinergic and adrenergic stimulation. More recently, microtissue engineering using coaxial 3D bioprinting of hydrogel microfibers and microtubes could also supported the formation of viable epithelial units. Both bioprinting approaches could overcome the need for Matrigel by facilitating the assembly of adult stem cells, such as human dental pulp stem cells, and primary SG cells into micro-sized 3D constructs able to produce their own matrix and self-organize into micro-modular tissue clusters with lumenized areas. Furthermore, extracellular vesicle (EV) therapies from organoid-derived secretome were also designed and validated ex vivo for SG regeneration after radiation damage. CONCLUSION: Magnetic 3D bioassembly and microfluidic coaxial bioprinting platforms have the potential to create SG mini-organs for regenerative applications via organoid transplantation or organoid-derived EV therapies.

Advances in the application of iron oxide nanoparticles (IONs and SPIONs) in three-dimensional cell culture systems.

BACKGROUND: The field of tissue engineering has remarkably progressed through the integration of nanotechnology and the widespread use of magnetic nanoparticles. These nanoparticles have resulted in innovative methods for three-dimensional (3D) cell culture platforms, including the generation of spheroids, organoids, and tissue-mimetic cultures, where they play a pivotal role. Notably, iron oxide nanoparticles and superparamagnetic iron oxide nanoparticles have emerged as indispensable tools for non-contact manipulation of cells within these 3D environments. The variety and modification of the physical and chemical properties of magnetic nanoparticles have profound impacts on cellular mechanisms, metabolic processes, and overall biological function. This review article focuses on the applications of magnetic nanoparticles, elucidating their advantages and potential pitfalls when integrated into 3D cell culture systems. This review aims to shed light on the transformative potential of magnetic nanoparticles in terms of tissue engineering and their capacity to improve the cultivation and manipulation of cells in 3D environments.

In Vitro and In Vivo Drug-Response Profiling Using Patient-Derived High-Grade Glioma.

BACKGROUND: Genomic profiling cannot solely predict the complexity of how tumor cells behave in their in vivo microenvironment and their susceptibility to therapies. The aim of the study was to establish a functional drug prediction model utilizing patient-derived GBM tumor samples for in vitro testing of drug efficacy followed by in vivo validation to overcome the disadvantages of a strict pharmacogenomics approach. METHODS: High-throughput in vitro pharmacologic testing of patient-derived GBM tumors cultured as 3D organoids offered a cost-effective, clinically and phenotypically relevant model, inclusive of tumor plasticity and stroma. RNAseq analysis supplemented this 128-compound screening to predict more efficacious and patient-specific drug combinations with additional tumor stemness evaluated using flow cytometry. In vivo PDX mouse models rapidly validated (50 days) and determined mutational influence alongside of drug efficacy. We present a representative GBM case of three tumors resected at initial presentation, at first recurrence without any treatment, and at a second recurrence following radiation and chemotherapy, all from the same patient. RESULTS: Molecular and in vitro screening helped identify effective drug targets against several pathways as well as synergistic drug combinations of cobimetinib and vemurafenib for this patient, supported in part by in vivo tumor growth assessment. Each tumor iteration showed significantly varying stemness and drug resistance. CONCLUSIONS: Our integrative model utilizing molecular, in vitro, and in vivo approaches provides direct evidence of a patient's tumor response drifting with treatment and time, as demonstrated by dynamic changes in their tumor profile, which may affect how one would address that drift pharmacologically.

An unrecognized extracellular function for an intracellular adapter protein released from the cytoplasm into the tumor microenvironment.

Mammalian cell membranes provide an interface between the intracellular and extracellular compartments. It is currently thought that cytoplasmic signaling adapter proteins play no functional role within the extracellular tumor environment. Here, by selecting combinatorial random peptide libraries in tumor-bearing mice, we uncovered a direct, specific, and functional interaction between CRKL, an adapter protein [with Src homology 2 (SH2)- and SH3-containing domains], and the plexin-semaphorin-integrin domain of beta(1) integrin in the extracellular milieu. Through assays in vitro, in cellulo, and in vivo, we show that this unconventional and as yet unrecognized protein-protein interaction between a regulatory integrin domain (rather than a ligand-binding one) and an intracellular adapter (acting outside of the cells) triggers an alternative integrin-mediated cascade for cell growth and survival. Based on these data, here we propose that a secreted form of the SH3/SH2 adaptor protein CRKL may act as a growth-promoting factor driving tumorigenesis and may lead to the development of cancer therapeutics targeting secreted CRKL.

Magnetic bioassembly platforms for establishing craniofacial exocrine gland organoids as aging in vitro models.

A multitude of aging-related factors and systemic conditions can cause lacrimal gland (LG) or salivary gland (SG) hypofunction leading to degenerative dry eye disease (DED) or dry mouth syndrome, respectively. Currently, there are no effective regenerative therapies that can fully reverse such gland hypofunction due to the lack of reproducible in vitro aging models or organoids required to develop novel treatments for multi-omic profiling. Previously, our research group successful developed three-dimensional (3D) bioassembly nanotechnologies towards the generation of functional exocrine gland organoids via magnetic 3D bioprinting platforms (M3DB). To meet the needs of our aging Asian societies, a next step was taken to design consistent M3DB protocols to engineer LG and SG organoid models with aging molecular and pathological features. Herein, a feasible step-by-step protocol was provided for producing both LG and SG organoids using M3DB platforms. Such protocol provided reproducible outcomes with final organoid products resembling LG or SG native parenchymal epithelial tissues. Both acinar and ductal epithelial compartments were prominent (21 ± 4.32% versus 42 ± 6.72%, respectively), and could be clearly identified in these organoids. Meanwhile, these can be further developed into aging signature models by inducing cellular senescence via chemical mutagenesis. The generation of senescence-like organoids will be our ultimate milestone aiming towards high throughput applications for drug screening and discovery, and for gene therapy investigations to reverse aging.

Magnetic bioassembly platforms towards the generation of extracellular vesicles from human salivary gland functional organoids for epithelial repair.

Salivary glands (SG) are exocrine organs with secretory units commonly injured by radiotherapy. Bio-engineered organoids and extracellular vesicles (EV) are currently under investigation as potential strategies for SG repair. Herein, three-dimensional (3D) cultures of SG functional organoids (SGo) and human dental pulp stem cells (hDPSC) were generated by magnetic 3D bioassembly (M3DB) platforms. Fibroblast growth factor 10 (FGF10) was used to enrich the SGo in secretory epithelial units. After 11 culture days via M3DB, SGo displayed SG-specific acinar epithelial units with functional properties upon neurostimulation. To consistently develop 3D hDPSC in vitro, 3 culture days were sufficient to maintain hDPSC undifferentiated genotype and phenotype for EV generation. EV isolation was performed via sequential centrifugation of the conditioned media of hDPSC and SGo cultures. EV were characterized by nanoparticle tracking analysis, electron microscopy and immunoblotting. EV were in the exosome range for hDPSC (diameter: 88.03 ± 15.60 nm) and for SGo (123.15 ± 63.06 nm). Upon ex vivo administration, exosomes derived from SGo significantly stimulated epithelial growth (up to 60%), mitosis, epithelial progenitors and neuronal growth in injured SG; however, such biological effects were less distinctive with the ones derived from hDPSC. Next, these exosome biological effects were investigated by proteomic arrays. Mass spectrometry profiling of SGo exosomes predicted that cellular growth, development and signaling was due to known and undocumented molecular targets downstream of FGF10. Semaphorins were identified as one of the novel targets requiring further investigations. Thus, M3DB platforms can generate exosomes with potential to ameliorate SG epithelial damage.

Lead identification using 3D models of pancreatic cancer.

Recent technological advances have enabled 3D tissue culture models for fast and affordable HTS. We are no longer bound to 2D models for anti-cancer agent discovery, and it is clear that 3D tumor models provide more predictive data for translation of preclinical studies. In a previous study, we validated a microplate 3D spheroid-based technology for its compatibility with HTS automation. Small-scale screens using approved drugs have demonstrated that drug responses tend to differ between 2D and 3D cancer cell proliferation models. Here, we applied this 3D technology to the first ever large-scale screening effort completing HTS on over 150K molecules against primary pancreatic cancer cells. It is the first demonstration that a screening campaign of this magnitude using clinically relevant, ex-vivo 3D pancreatic tumor models established directly from biopsy, can be readily achieved in a fashion like traditional drug screen using 2D cell models. We identified four unique series of compounds with sub micromolar and even low nanomolar potency against a panel of patient derived pancreatic organoids. We also applied the 3D technology to test lead efficacy in autologous cancer associated fibroblasts and found a favorable profile for better efficacy in the cancer over wild type primary cells, an important milestone towards better leads. Importantly, the initial leads have been further validated in across multiple institutes with concordant outcomes. The work presented here represents the genesis of new small molecule leads found using 3D models of primary pancreas tumor cells.

Scaffold-free 3D cell culture of primary skin fibroblasts induces profound changes of the matrisome.

The human skin has a highly developed extracellular matrix (ECM) that is vital for proper skin functioning, its 3D architecture playing a pivotal role in support and guidance of resident and invading cells. To establish relevant in vitro models mimicking the complex design observed in vivo, scaffold-based and scaffold-free 3D cell culture systems have been developed. Here we show that scaffold-free systems are well suited for the analysis of ECM protein regulation. Using quantitative mass spectrometry-based proteomics in combination with magnetic 3D bioprinting we characterize changes in the proteome of skin fibroblasts and squamous cell carcinoma cells. Transferring cells from 2D to 3D without any additional scaffold induces a profound upregulation of matrisome proteins indicating the generation of a complex, tissue-like ECM.

Magnetic 3D cell culture: State of the art and current advances.

Cell culture is an important tool for the understanding of cell biology and behavior. In vitro cultivation has been increasingly indispensable for biomedical, pharmaceutical, and biotechnology research. Nevertheless, with the demand for in vitro experimentation strategies more representative of in vivo conditions, tridimensional (3D) cell culture models have been successfully developed. Although these 3D models are efficient and address critical questions from different research areas, there are considerable differences between the existing techniques regarding both elaboration and cost. In light of this, this review describes the construction of 3D spheroids using magnetization while bringing the most recent updates in this field. Magnetic 3D cell culture consists of magnetizing cells using an assembly of gold and iron oxide nanoparticles cross-linked with poly-l-lysine nanoparticles. Then, 3D culture formation in special plates with the assistance of magnets for levitation or bioprinting. Here, we discuss magnetic 3D cell culture advancements, including tumor microenvironment, tissue reconstruction, blood vessel engineering, toxicology, cytotoxicity, and 3D culture of cardiomyocytes, bronchial and pancreatic cells.

Combinatorial targeting and nanotechnology applications.

The development of improved methods for targeted cell detection is of general interest in many fields of research and drug development. There are a number of well-established techniques for the study and detection of biomarkers expressed in living cells and tissues. Many of them rely on multi-step procedures that might not meet ideal assay requirements for speed, cost, sensitivity, and specificity. Here we report and further validate an approach that enables spontaneous molecular assembly to generate biologically active networks of bacteriophage (phage) assembled with gold (Au) nanoparticles (termed Au-phage nanoshuttles). Here, the nanoshuttles preserve the cell binding and internalization attributes mediated by a displayed peptide targeted to a cell surface receptor. The organization of such targeted assemblies can be further manipulated to be used as a multimodal detection assembly, and they can be characterized as fractal nanostructures by angle-dependent light scattering fractal dimension analysis. Targeted Au-phage nanoshuttles offer multiple functionalities for nanotechnology-based sensing and reporting, including enhanced fluorescence and improved contrast for darkfield microscopy.

A magnetic three-dimensional levitated primary cell culture system for the development of secretory salivary gland-like organoids.

Salivary gland (SG) hypofunction and oral dryness can be induced by radiotherapy for head and neck cancers or autoimmune disorders. These are common clinical conditions that involve loss of saliva-secreting epithelial cells. Several oral complications arise with SG hypofunction that interfere with routine daily activities such as chewing, swallowing, and speaking. Hence, there is a need for replacing these saliva-secreting cells. Recently, researchers have proposed to repair SG hypofunction via various cell-based approaches in three-dimensional (3D) scaffold-based systems. However, majority of the scaffolds used cannot be translated clinically due to the presence of non-human-based substrates. Herein, saliva-secreting organoids/mini-glands were developed using a new scaffold/substrate-free culture system named magnetic 3D levitation (M3DL), which assembles and levitates magnetized primary SG-derived cells (SGDCs), allowing them to produce their own extracellular matrices. Primary SGDCs were assembled in M3DL to generate SG-like organoids in well-established SG epithelial differentiation conditions for 7 days. After such culture time, these organoids consistently presented uniform spheres with greater cell viability and pro-mitotic cells, when compared with conventional salisphere cultures. Additionally, organoids formed by M3DL expressed SG-specific markers from different cellular compartments: acinar epithelial including adherens junctions (NKCC1, cholinergic muscarinic receptor type 3, E-cadherin, and EpCAM); ductal epithelial and myoepithelial (cytokeratin 14 and α-smooth muscle actin); and neuronal (ß3-tubulin and vesicular acetylcholine transferase). Lastly, intracellular calcium and α-amylase activity assays showed functional organoids with SG-specific secretory activity upon cholinergic stimulation. Thus, the functional organoid produced herein indicate that this M3DL system can be a promising tool to generate SG-like mini-glands for SG secretory repair.

Automating a Magnetic 3D Spheroid Model Technology for High-Throughput Screening.

Affordable and physiologically relevant three-dimensional (3D) cell-based assays used in high-throughput screening (HTS) are on the rise in early drug discovery. These technologies have been aided by the recent adaptation of novel microplate treatments and spheroid culturing techniques. One such technology involves the use of nanoparticle (NanoShuttle-PL) labeled cells and custom magnetic drives to assist in cell aggregation to ensure rapid 3D structure formation after the cells have been dispensed into microtiter plates. Transitioning this technology from a low-throughput manual benchtop application, as previously published by our lab, into a robotically enabled format achieves orders of magnitude greater throughput but required the development of specialized support hardware. This effort included in-house development, fabrication, and testing of ancillary devices that assist robotic handing and high-precision placement of microtiter plates into an incubator embedded with magnetic drives. Utilizing a "rapid prototyping" approach facilitated by cloud-based computer-aided design software, we built the necessary components using hobby-grade 3D printers with turnaround times that rival those of traditional manufacturing/development practices at a substantially reduced cost. This approach culminated in a first-in-class HTS-compatible 3D system in which we have coupled 3D bioprinting to a fully automated HTS robotic platform utilizing our novel magnetic incubator shelf assemblies.

Ligand-directed surface profiling of human cancer cells with combinatorial peptide libraries.

A collection of 60 cell lines derived from human tumors (NCI-60) has been widely explored as a tool for anticancer drug discovery. Here, we profiled the cell surface of the NCI-60 by high-throughput screening of a phage-displayed random peptide library and classified the cell lines according to the binding selectivity of 26,031 recovered tripeptide motifs. By analyzing selected cell-homing peptide motifs and their NCI-60 recognition patterns, we established that some of these motifs (a) are similar to domains of human proteins known as ligands for tumor cell receptors and (b) segregate among the NCI-60 in a pattern correlating with expression profiles of the corresponding receptors. We biochemically validated some of the motifs as mimic peptides of native ligands for the epidermal growth factor receptor. Our results indicate that ligand-directed profiling of tumor cell lines can select functional peptides from combinatorial libraries based on the expression of tumor cell surface molecules, which in turn could be exploited as "druggable" receptors in specific types of cancer.

Engineering innervated secretory epithelial organoids by magnetic three-dimensional bioprinting for stimulating epithelial growth in salivary glands.

Current saliva-based stimulation therapies for radiotherapy-induced xerostomia are not fully effective due to the presence of damaged secretory epithelia and nerves in the salivary gland (SG). Hence, three-dimensional bio-engineered organoids are essential to regenerate the damaged SG. Herein, a recently validated three-dimensional (3D) biofabrication system, the magnetic 3D bioprinting (M3DB), is tested to generate innervated secretory epithelial organoids from a neural crest-derived mesenchymal stem cell, the human dental pulp stem cell (hDPSC). Cells are tagged with magnetic nanoparticles (MNP) and spatially arranged with magnet dots to generate 3D spheroids. Next, a SG epithelial differentiation stage was completed with fibroblast growth factor 10 (4-400 ng/ml) to recapitulate SG epithelial morphogenesis and neurogenesis. The SG organoids were then transplanted into ex vivo model to evaluate their epithelial growth and innervation. M3DB-formed spheroids exhibited both high cell viability rate (>90%) and stable ATP intracellular activity compared to MNP-free spheroids. After differentiation, spheroids expressed SG epithelial compartments including secretory epithelial, ductal, myoepithelial, and neuronal. Fabricated organoids also produced salivary α-amylase upon FGF10 stimulation, and intracellular calcium mobilization and trans-epithelial resistance was elicited upon neurostimulation with different neurotransmitters. After transplantation, the SG-like organoids significantly stimulated epithelial and neuronal growth in damaged SG. It is the first time bio-functional innervated SG-like organoids are bioprinted. Thus, this is an important step towards SG regeneration and the treatment of radiotherapy-induced xerostomia.

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.

Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways.

Klotho is a single-pass transmembrane protein with documented anti-cancer properties. Recent reports have implicated Klotho as an inhibitor of transforming growth factor ß1 induced cell migration in renal fibrosis. Overexpression of epidermal growth factor receptor (EGFR) is known to promote tumor initiation and progression in clear-cell renal cell carcinoma (cRCC). We tested our hypothesis that Klotho inhibits EGF-mediated cell migration in cRCC by interfering with the EGFR signaling complex and mitogen-activated protein kinase (MAPK) pathways. We performed cell adhesion, migration, and biochemical studies in vitro using Caki-1 cell line. In addition, we validated the cell culture studies with expression analysis of six de-identified FFPE tissues from primary and metastatic cRCC patients. Our studies show that Klotho inhibited EGF-induced Caki-1 de-adhesion and decreased spreading on collagen type 1. Klotho also inhibited EGF-induced α2ß1 integrin-dependent cell migration on collagen type 1. To test the involvement of MAPK pathways in EGF-induced Caki-1 cell motility, the cells were pretreated with either SB203580, a specific p38 MAPK inhibitor, or Klotho. SB203580 blocked the EGF-induced Caki-1 cell migration. Klotho had a comparable inhibitory effect. Our FFPE clinical specimens revealed decreased Klotho mRNA expression compared to a control, non-cancer kidney tissue. The decrease in Klotho mRNA levels correlated with increased c-Src expression, while E-Cadherin was relatively reduced in metastatic FFPE specimens where Klotho was least expressed. Taken together, these results suggest that secreted Klotho inhibits EGF-induced pro-migratory cell morphological changes and migration in Caki-1 cells. Our data additionally suggest that decreased Klotho expression may be involved in cRCC metastasis.