Single-cell analysis of white adipose tissue reveals the tumor-promoting adipocyte subtypes.

BACKGROUND: The tumor-adipose microenvironment (TAME) is characterized by the enrichment of adipocytes, and is considered a special ecosystem that supports cancer progression. However, the heterogeneity and diversity of adipocytes in TAME remains poorly understood. METHODS: We conducted a single-cell RNA sequencing analysis of adipocytes in mouse and human white adipose tissue (WAT). We analyzed several adipocyte subtypes to evaluate their relationship and potential as prognostic factors for overall survival (OS). The potential drugs are screened by using bioinformatics methods. The tumor-promoting effects of a typical adipocyte subtype in breast cancer are validated by performing in vitro functional assays and immunohistochemistry (IHC) in clinical samples. RESULTS: We profiled a comprehensive single-cell atlas of adipocyte in mouse and human WAT and described their characteristics, origins, development, functions and interactions with immune cells. Several cancer-associated adipocyte subtypes, namely DPP4+ adipocytes in visceral adipose and ADIPOQ+ adipocytes in subcutaneous adipose, are identified. We found that high levels of these subtypes are associated with unfavorable outcomes in four typical adipose-associated cancers. Some potential drugs including Trametinib, Selumetinib and Ulixertinib are discovered. Emphatically, knockdown of adiponectin receptor 1 (AdipoR1) and AdipoR2 impaired the proliferation and invasion of breast cancer cells. Patients with AdipoR2-high breast cancer display significantly shorter relapse-free survival (RFS) than those with AdipoR2-low breast cancer. CONCLUSION: Our results provide a novel understanding of TAME at the single-cell level. Based on our findings, several adipocyte subtypes have negative impact on prognosis. These cancer-associated adipocytes may serve as key prognostic predictor and potential targets for treatment in the future.

A Cryptic Plant Terpene Cyclase Producing Unconventional 18- and 14-Membered Macrocyclic C25 and C20 Terpenoids with Immunosuppressive Activity.

A versatile terpene synthase (LcTPS2) producing unconventional macrocyclic terpenoids was characterized from Leucosceptrum canum. Engineered Escherichia coli and Nicotiana benthamiana expressing LcTPS2 produced six 18-/14-membered sesterterpenoids including five new ones and two 14-membered diterpenoids. These products represent the first macrocyclic sesterterpenoids from plants and the largest sesterterpenoid ring system identified to date. Two variants F516A and F516G producing approximately 3.3- and 2.5-fold, respectively, more sesterterpenoids than the wild-type enzyme were engineered. Both 18- and 14-membered ring sesterterpenoids displayed significant inhibitory activity on the IL-2 and IFN-γ production of T cells probably via inhibition of the MAPK pathway. The findings will contribute to the development of efficient biocatalysts to create bioactive macrocyclic sesterterpenoids, and also herald a new potential in the well-trodden territory of plant terpenoid biosynthesis.

Embossed Mie resonator arrays composed of compacted TiO2 nanoparticles for broadband anti-reflection in solar cells.

Mie resonator arrays formed by embossing titanium dioxide (TiO2) nanoparticles (NPs) from solution are investigated as optical coatings for anti-reflection applications. Compacted nanoparticle assemblies offer unique possibilities to tailor the effective refractive index (RI). Here, we demonstrate a simple table-top, low pressure, and low temperature method to fabricate structured optical coatings. TiO2 nanostructures in the form of nanodisks support Mie resonances in the visible wavelength spectrum and exhibit strong forward scattering into the high index substrates, making them suitable as broadband anti-reflection coatings for solar cells. TiO2 NP-based nanodisk arrays are designed, fabricated, and characterized regarding their anti-reflection properties on Si, GaAs, and InP substrates and solar cells. Detailed finite-difference time-domain simulations are performed to optimize the TiO2 NP-based Mie resonator arrays for the broadband anti-reflection as well as to explain the measured reflectance spectra. The solar-weighted reflectance is used as a figure of merit (FoM). TiO2 nanodisk arrays on Si show a FoM of ~ 7% in the 400-1,100 nm wavelength spectrum; similar values are obtained for GaAs and InP substrates. TiO2 nanodisk arrays embossed directly on prefabricated planar single-junction Si, GaAs, and InP solar cells result in an appreciable increase (~ 1.3 times) in the short-circuit current densities.

Injectable cell constructs fabricated via culture on a thermoresponsive methylcellulose hydrogel system for the treatment of ischemic diseases.

Cell transplantation via direct intramuscular injection is a promising therapy for patients with ischemic diseases. However, following injections, retention of transplanted cells in engrafted areas remains problematic, and can be deleterious to cell-transplantation therapy. In this Progress Report, a thermoresponsive hydrogel system composed of aqueous methylcellulose (MC) blended with phosphate-buffered saline is constructed to grow cell sheet fragments and cell bodies for the treatment of ischemic diseases. The as-prepared MC hydrogel system undergoes a sol-gel reversible transition upon heating or cooling at ≈32 °C. Via this unique property, the grown cell sheet fragments (cell bodies) can be harvested without using proteolytic enzymes; consequently, their inherent extracellular matrices (ECMs) and integrative adhesive agents remain well preserved. In animal studies using rats and pigs with experimentally created myocardial infarction, the injected cell sheet fragments (cell bodies) become entrapped in the interstices of muscular tissues and adhere to engraftment sites, while a minimal number of cells exist in the group receiving dissociated cells. Moreover, transplantation of cell sheet fragments (cell bodies) significantly increases vascular density, thereby improving the function of an infarcted heart. These experimental results demonstrate that cell sheet fragments (cell bodies) function as a cell-delivery construct by providing a favorable ECM environment to retain transplanted cells locally and consequently, improving the efficacy of therapeutic cell transplantation.

The factors influence compatibility of pulse-pulse intervals with R-R intervals.

Cardiac autonomic dysfunction assessed by power spectral analysis of electrocardigographic (ECG) R-R intervals (RRI) is a useful method in clinical research. The compatibility of pulse-pulse intervals (PPI) acquired by photoplethysmography (PPG) with RRI is equivocal. In this study, we would like to investigate factors influence the compatibility. We recruited 25 young and health subjects divided into two groups: normal subjects (Group1, BMI < 24, n=15) and overweight subjects (Group2, BMI >/= 24, n=10). ECG and PPG were measured for 5 minutes. Used cross-approximate entropy (CAE) and Fast Fourier transform (FFT) to obtained compatibility between RRI and PPI. The CAE value in Group1 were significantly lower than in Group2 (1.71 ± 0.12 vs. 1.83 ± 0.11, P = 0.011). A positive linear relationship between CAE value and risk factors of metabolic syndrome. No significantly difference between LFP/HFP ratio of RRI (LHRRRI) and LFP/HFP ratio of PPI (LHRPPI) in Group1 (1.42 ± 0.19 vs. 1.38 ± 0.17, P = 0.064), LHRRRI significantly higher than LHRPPI in Group2 (2.18 ± 0.37 vs. 1.93 ± 0.30, P = 0.005). It should be careful that using PPI to assess autonomic function in the obese subjects or the patients with metabolic syndrome.

Hypoxia-induced therapeutic neovascularization in a mouse model of an ischemic limb using cell aggregates composed of HUVECs and cbMSCs.

Cell transplantation for therapeutic neovascularization holds great promise for treating ischemic diseases. This work prepared three-dimensional aggregates of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs) with different levels of internal hypoxia by a methylcellulose hydrogel system. We found that few apoptosis occurred in these cell aggregates, despite developing a hypoxic microenvironment in their inner cores. Via effectively switching on the hypoxia-inducible factor-1α-dependent angiogenic mechanisms, culturing the internally hypoxic HUVEC/cbMSC aggregates on Matrigel resulted in formation of extensive and persistent tubular networks and significant upregulation of pro-angiogenic genes. As the level of internal hypoxia created in cell aggregates increased, the robustness of the tubular structures developed on Matrigel increased, and expression levels of the pro-angiogenic genes also elevated. Transplantation of hypoxic HUVEC/cbMSC aggregates into a mouse model of an ischemic limb significantly promoted formation of functional vessels, improved regional blood perfusion, and attenuated muscle atrophy and bone losses, thereby rescuing tissue degeneration. Notably, their therapeutic efficacy was clearly dependent upon the level of internal hypoxia established in cell aggregates. These analytical results demonstrate that by establishing a hypoxic environment in HUVEC/cbMSC aggregates, their potential for therapeutic neovascularization can be markedly enhanced.

A translational approach in using cell sheet fragments of autologous bone marrow-derived mesenchymal stem cells for cellular cardiomyoplasty in a porcine model.

Based on a porcine model with surgically created myocardial infarction (MI) as a pre-clinical scheme, this study investigates the clinical translation of cell sheet fragments of autologous mesenchymal stem cells (MSCs) for cellular cardiomyoplasty. MSC sheet fragments retaining endogenous extracellular matrices are fabricated using a thermo-responsive methylcellulose hydrogel system. Echocardiographic observations indicate that transplantation of MSC sheet fragments in infarcted hearts can markedly attenuate the adverse ventricular dilation and preserve the cardiac function post MI, which is in contrast to the controlled groups receiving saline or dissociated MSCs. Additionally, histological analyses suggest that administering MSC sheet fragments significantly prevents the scar expansion and left ventricle remodeling after MI. Immunohistochemistry results demonstrate that the engrafted MSCs can differentiate into endothelial cells and smooth muscle cells, implying that angiogenesis and the subsequent regional perfusion improvement is a promising mechanism for ameliorating post-infarcted cardiac function. However, according to the data recorded by an implantable loop recorder, the transplanted MSCs may provoke arrhythmia. Nevertheless, the proposed approach may potentially lead to the eventual translation of MSC-based therapy into practical and effective clinical treatments.

Three-dimensional cell aggregates composed of HUVECs and cbMSCs for therapeutic neovascularization in a mouse model of hindlimb ischemia.

The proximity of cells in three-dimensional (3D) organization maximizes the cell-cell communication and signaling that are critical for cell function. In this study, 3D cell aggregates composed of human umbilical vein endothelial cells (HUVECs) and cord-blood mesenchymal stem cells (cbMSCs) were used for therapeutic neovascularization to rescue tissues from critical limb ischemia. Within the cell aggregates, homogeneously mixed HUVECs and cbMSCs had direct cell-cell contact with expressions of endogenous extracellular matrices and adhesion molecules. Although dissociated HUVECs/cbMSCs initially formed tubular structures on Matrigel, the grown tubular network substantially regressed over time. Conversely, 3D HUVEC/cbMSC aggregates seeded on Matrigel exhibited an extensive tubular network that continued to expand without regression. Immunostaining experiments show that, by differentiating into smooth muscle cell (SMC) lineages, the cbMSCs stabilize the HUVEC-derived tubular network. The real-time PCR analysis results suggest that, through myocardin, TGF-ß signaling regulates the differentiation of cbMSCs into SMCs. Transplantation of 3D HUVEC/cbMSC aggregates recovered blood perfusion in a mouse model of hindlimb ischemia more effectively compared to their dissociated counterparts. The experimental results confirm that the transplanted 3D HUVEC/cbMSC aggregates enhanced functional vessel formation within the ischemic limb and protected it from degeneration. The 3D HUVEC/cbMSC aggregates can therefore facilitate the cell-based therapeutic strategies for modulating postnatal neovascularization.

Intramuscular delivery of 3D aggregates of HUVECs and cbMSCs for cellular cardiomyoplasty in rats with myocardial infarction.

Cell-based therapeutic neovascularization is a promising method for treating ischemic disorders. In this work, human umbilical vein endothelial cells (HUVECs) were thoroughly premixed with cord-blood mesenchymal stem cells (cbMSCs) and cultivated to form three-dimensional (3D) cell aggregates for cellular cardiomyoplasty. In the in vitro study, tubular networks were formed at day 1 after the co-culturing of dissociated HUVECs and cbMSCs on Matrigel; however, as time progressed, the grown tubular networks regressed severely. Conversely, when 3D cell aggregates were grown on Matrigel, mature and stable tubular networks were observed over time, under the influence of their intensive cell-extracellular matrix (ECM) interactions and cell-cell contacts. 3D cell aggregates were transplanted into the peri-infarct zones of rats with myocardial infarction (MI) via direct intramyocardial injection. Based on our pinhole single photon emission computed tomography (SPECT) myocardial-perfusion observations, echocardiographic heart-function examinations and histological analyses, the engrafted 3D cell aggregates considerably enhanced the vascular densities and the blood flow recovery in the ischemic myocardium over those of their dissociated counterparts, thereby reducing the size of perfusion defects and restoring cardiac function. These results demonstrate that the intramuscular delivery of 3D cell aggregates of HUVECs/cbMSCs can be a valuable cell-based regenerative therapeutic strategy against MI.

Vascularization and restoration of heart function in rat myocardial infarction using transplantation of human cbMSC/HUVEC core-shell bodies.

Cell transplantation is a promising strategy for therapeutic treatment of ischemic heart diseases. In this study, cord blood mesenchymal stem cells (cbMSCs) and human umbilical vein endothelial cells (HUVECs) in the form of core-shell bodies (cbMSC/HUVEC bodies) were prepared to promote vascularization and restore heart functions in an experimentally-created myocardial infarction (MI) rat model. Saline, cbMSC bodies and HUVEC bodies were used as controls. In vitro results indicated that cbMSC/HUVEC bodies possessed the capability of heterotypic assembly of cbMSCs and HUVECs into robust and durable tubular networks on Matrigel. The up-regulated gene expressions of VEGF and IGF-1 reflected the robust expansion of tubular networks; in addition, the augmented levels of SMA and SM22 suggested smooth muscle differentiation of cbMSCs, possibly helping to improve the durability of networks. Moreover, according to the in vivo echocardiographic, magnetic resonance and computed-tomographic results, transplantation of cbMSC/HUVEC bodies benefited post-MI dysfunction. Furthermore, the vascularization analyses demonstrated the robust vasculogenic potential of cbMSC/HUVEC bodies in vivo, thus contributing to the greater viable myocardium and the less scar region, and ultimately restoring the cardiac function. The concept of core-shell bodies composed of perivascular cells and endothelial cells may serve as an attractive cell delivery vehicle for vasculogenesis, thus improving the cardiac function significantly.

Core-shell cell bodies composed of human cbMSCs and HUVECs for functional vasculogenesis.

Rapid induction and creation of functional vascular networks is essential for the success of treating ischemic tissues. The formation of mature and functional vascular networks requires the cooperation of endothelial cells (ECs) and perivascular cells. In the study, we used a thermo-responsive hydrogel system to fabricate core-shell cell bodies composed of cord-blood mesenchymal stem cells (cbMSCs) and human umbilical vascular ECs (HUVECs) for functional vasculogenesis. When seeded on Matrigel, the shelled HUVECs attempted to interact and communicate vigorously with the cored cbMSCs initially. Subsequently, HUVECs migrated out and formed tubular structures; cbMSCs were observed to coalesce around the HUVEC-derived tubes. With time progressing, the tubular networks continued to expand without regression, indicating that cbMSCs might function as perivascular cells to stabilize the nascent networks. In the in vivo study, cbMSC/HUVEC bodies were embedded in Matrigel and implanted subcutaneously in nude mice. At day 7, visible blood-filled vessels were clearly identified within the implant containing cbMSC/HUVEC bodies, indicating that the formed vessels anastomosed with the host vasculature. The cored cbMSCs were stained positive for smooth muscle actin, suggesting that they underwent smooth muscle differentiation and formed microvessels with the shelled HUVECs, as the role of perivascular cells. These data confirm that the formation of mature vessels requires heterotypic cooperation of HUVECs and MSCs. This study provides a new strategy for therapeutic vasculogenesis, by showing the feasibility of using cbMSC/HUVEC bodies to create functional vascular networks.