Mimicking angiogenic microenvironment of alveolar soft-part sarcoma in a microfluidic coculture vasculature chip.

Alveolar soft-part sarcoma (ASPS) is a slow-growing soft tissue sarcoma with high mortality rates that affects adolescents and young adults. ASPS resists conventional chemotherapy; thus, decades of research have elucidated pathogenic mechanisms driving the disease, particularly its angiogenic capacities. Integrated blood vessels that are rich in pericytes (PCs) and metastatic potential are distinctive of ASPS. To mimic ASPS angiogenic microenvironment, a microfluidic coculture vasculature chip has been developed as a three-dimensional (3D) spheroid composed of mouse ASPS, a layer of PCs, and endothelial cells (ECs). This ASPS-on-a-chip provided functional and morphological similarity as the in vivo mouse model to elucidate the cellular crosstalk within the tumor vasculature before metastasis. We successfully reproduce ASPS spheroid and leaky vessels representing the unique tumor vasculature to assess effective drug delivery into the core of a solid tumor. Furthermore, this ASPS angiogenesis model enabled us to investigate the role of proteins in the intracellular trafficking of bioactive signals from ASPS to PCs and ECs during angiogenesis, including Rab27a and Sytl2. The results can help to develop drugs targeting the crosstalk between ASPS and the adjacent cells in the tumoral microenvironment.

ASPSCR1::TFE3 orchestrates the angiogenic program of alveolar soft part sarcoma.

Alveolar soft part sarcoma (ASPS) is a soft part malignancy affecting adolescents and young adults. ASPS is characterized by a highly integrated vascular network, and its high metastatic potential indicates the importance of ASPS's prominent angiogenic activity. Here, we find that the expression of ASPSCR1::TFE3, the fusion transcription factor causatively associated with ASPS, is dispensable for in vitro tumor maintenance; however, it is required for in vivo tumor development via angiogenesis. ASPSCR1::TFE3 is frequently associated with super-enhancers (SEs) upon its DNA binding, and the loss of its expression induces SE-distribution dynamic modification related to genes belonging to the angiogenesis pathway. Using epigenomic CRISPR/dCas9 screening, we identify Pdgfb, Rab27a, Sytl2, and Vwf as critical targets associated with reduced enhancer activities due to the ASPSCR1::TFE3 loss. Upregulation of Rab27a and Sytl2 promotes angiogenic factor-trafficking to facilitate ASPS vascular network construction. ASPSCR1::TFE3 thus orchestrates higher ordered angiogenesis via modulating the SE activity.

G-Quadruplex-Functionalized Gold Nanoparticles for a Real-Time Biomolecule Sensor with On-Demand Tunable Properties.

G-quadruplex (G4) DNA-functionalized gold nanoparticles (AuNPs) were fabricated for a new sensing platform for a biomolecule, thrombin. Thrombin-binding aptamer (TBA), which forms a highly ordered G4 structure, was immobilized on AuNPs. The particles were induced to aggregate by binding of thrombin to G4 DNA. Thrombin was thus detected by the color change of the colloidal system from red to purple-blue. The aggregation was not due to the bridging between the particles through thrombin but to the reduction in steric repulsion attributable to the mobility and flexibility of G4 DNA. The change in the colloidal stability was quick and the bathochromic peak shift varied with the concentration of thrombin. The sensor showed a high specificity to the thrombin target over major proteins in human serum. The detection sensitivity and analytical performance were successfully tuned for an on-demand sensor with a linearity of 10.0-40.0 nM. The limits of detection and of quantification were 3.6 and 10.7 nM, respectively.

Three-dimensional tissue model in direct contact with an on-chip vascular bed enabled by removable membranes.

Three-dimensional (3D) tissue culture is a powerful tool for understanding physiological events. However, 3D tissues still have limitations in their size, culture period, and maturity, which are caused by the lack of nutrients and oxygen supply through the vasculature. Here, we propose a new method for culturing a 3D tissue-a spheroid-directly on an 'on-chip vascular bed'. The method can be applied to any 3D tissue because the vascular bed is preformed, so that angiogenic factors from the tissue are not necessary to induce vasculature. The essential component of the assay system is the removable membrane that initially separates the 3D tissue culture well and the microchannel in which a uniform vascular bed is formed, and then allows the tissue to be settled directly onto the vascular bed following its removal. This in vitro system offers a new technique for evaluating the effects of vasculature on 3D tissues.

Electrochemical Impedimetric Study of Non-Watson-Crick Base Pairs of DNA.

Electrochemical impedance spectroscopy (EIS) was used to detect non-Watson-Crick base pairs of DNA. Thiol-modified DNA as a probe and mercaptohexanol (MCH) were co-immobilized to form a DNA/MCH mixed self-assembled monolayer on a gold electrode surface and then hybridized with complementary DNAs. The DNA layers were measured by the EIS method and interpreted by equivalent circuits. Every terminal base mismatch of the DNA duplex brought about an increase in the charge-transfer resistance (Rct), unlike the case with a fully matched DNA duplex. The value of Rct was highly sensitive to the number of base mismatches for both unpaired and overhang DNA at the terminal. For internal base mismatches, however, no significant increase in Rct was observed. These experimental results proved that the charge transfer of redox molecules to the electrode surface is largely hindered by an end fraying motion due to base unpairing and dangling overhang. EIS was able to detect these steric properties of DNA strands. Furthermore, an electrode modified with G-quadruplex (G4) DNA demonstrated the influences of bulkiness and loop structure on the accessibility of the redox probe to the electrode.