TGM1/3-mediated transamidation of Exo70 promotes tumor metastasis upon LKB1 inactivation.

Exo70, a key exocyst complex component, is crucial for cell motility and extracellular matrix (ECM) remodeling in cancer metastasis. Despite its potential as a drug target, Exo70's post-translational modifications (PTMs) are poorly characterized. Here, we report that Exo70 is transamidated on Gln5 with Lys56 of cystatin A by transglutaminases TGM1 and TGM3, promoting tumor metastasis. This modification enhances Exo70's association with other exocyst subunits, essential for secreting matrix metalloproteinases, forming invadopodia, and delivering integrins to the leading edge. Tumor suppressor liver kinase B1 (LKB1), whose inactivation accelerates metastasis, phosphorylates TGM1 and TGM3 at Thr386 and Thr282, respectively, to inhibit their interaction with Exo70 and the following transamidation. Cantharidin, a US Food and Drug Administration (FDA)-approved drug, inhibits Exo70 transamidation to restrain tumor cell migration and invasion. Together, our findings highlight Exo70 transamidation as a key molecular mechanism and target and propose cantharidin as a therapeutic strategy with direct clinical translational value for metastatic cancers, especially those with LKB1 loss.

Ultrasensitive quantification of PD-L1+ extracellular vesicles in melanoma patient plasma using a parallelized high throughput droplet digital assay.

The expression of programmed death-ligand 1 (PD-L1) on extracellular vesicles (EVs) is an emerging biomarker for cancer, and has gained particular interest for its role mediating immunotherapy. However, precise quantification of PD-L1+ EVs in clinical samples remains challenging due to their sparse concentration and the enormity of the number of background EVs in human plasma, limiting applicability of conventional approaches. In this study, we develop a high-throughput droplet-based extracellular vesicle analysis (DEVA) assay for ultrasensitive quantification of EVs in plasma that are dual positive for both PD-L1 and tetraspanin (CD81) known to be expressed on EVs. We achieve a performance that significantly surpasses conventional approaches, demonstrating 360× enhancement in the limit of detection (LOD) and a 750× improvement in the limit of quantitation (LOQ) compared to conventional plate enzyme-linked immunoassay (ELISA). Underlying this performance is DEVA's high throughput analysis of individual EVs one at a time and the high specificity to targeted EVs versus background. We achieve a 0.006% false positive rate per droplet by leveraging avidity effects that arise from EVs having multiple copies of their target ligands on their surface. We use parallelized optofluidics to rapidly process 10 million droplets per minute, ∼100× greater than conventional approaches. A validation study on a cohort of 14 patients with melanoma confirms DEVA's ability to match conventional ELISA measurements with reduced plasma sample volume and without the need for prior EV purification. This proof-of-concept study demonstrates DEVA's potential for clinical utility to enhance prognosis as well as guide treatment for cancer.

Sex Influences the Extent of Physical Performance Adaptations in Response to Small-Sided Games and Running-Based High-Intensity Interval Training: A Parallel Study Design Involving Men and Women Soccer Players.

The aim of this study was two-fold: (i) to compare physical fitness adaptations following small-sided games (SSG) and running-based high-intensity interval training (HIIT), considering sex interactions; and (ii) to describe intra-individual variations of adaptations in both men and women developmental/trained soccer players over an 8-week randomized parallel study design involving 25 women and 27 men. Pre and post-intervention assessments included countermovement jump (CMJ), 10-meter linear sprint test, change-of-direction (COD) deficit, and final velocity at 30-15 Intermittent Fitness Test (VIFT). Significant interactions between time, groups and sex were found in 10-m sprint (F1,48 = 6.042; p = 0.018; ηp2 = 0.112). No significant interactions between time, groups and sex were found in CMJ (F F1,48 = 0.609; p = 0.439; ηp2 = 0.013), COD deficit (F F1,48 = 2.718; p = 0.106; ηp2 = 0.054) and VIFT (F F1,48 = 1.141; p = 0.291; ηp2 = 0.023). Significant interactions were found between time and sex in CMJ (F F1,48 = 29.342; p < 0.001; ηp2= 0.379), 10-m sprint (F F1,48 = 4.359; p = 0.042; ηp2 = 0.083), COD deficit (F F1,48 = 5.066; p = 0.029; ηp2= 0.095) and VIFT (F F1,48 = 11.248; p = 0.002; ηp2 = 0.190). In conclusion, this study suggests similar effects of HIIT in both sexes. However, for women, SSG may entail less efficacy and more inter-individual variability compared to men. Therefore, HIIT could potentially be a better solution for women, whereas both training approaches were equally effective in men.

A phosphoinositide switch mediates exocyst recruitment to multivesicular endosomes for exosome secretion.

Exosomes are secreted to the extracellular milieu when multivesicular endosomes (MVEs) dock and fuse with the plasma membrane. However, MVEs are also known to fuse with lysosomes for degradation. How MVEs are directed to the plasma membrane for exosome secretion rather than to lysosomes is unclear. Here we report that a conversion of phosphatidylinositol-3-phosphate (PI(3)P) to phosphatidylinositol-4-phosphate (PI(4)P) catalyzed sequentially by Myotubularin 1 (MTM1) and phosphatidylinositol 4-kinase type IIα (PI4KIIα) on the surface of MVEs mediates the recruitment of the exocyst complex. The exocyst then targets the MVEs to the plasma membrane for exosome secretion. We further demonstrate that disrupting PI(4)P generation or exocyst function blocked exosomal secretion of Programmed death-ligand 1 (PD-L1), a key immune checkpoint protein in tumor cells, and led to its accumulation in lysosomes. Together, our study suggests that the PI(3)P to PI(4)P conversion on MVEs and the recruitment of the exocyst direct the exocytic trafficking of MVEs for exosome secretion.

Stiff matrix induces exosome secretion to promote tumour growth.

Tissue fibrosis and extracellular matrix (ECM) stiffening promote tumour progression. The mechanisms by which ECM regulates its contacting cells have been extensively studied. However, how stiffness influences intercellular communications in the microenvironment for tumour progression remains unknown. Here we report that stiff ECM stimulates the release of exosomes from cancer cells. We delineate a molecular pathway that links stiff ECM to activation of Akt, which in turn promotes GTP loading to Rab8 that drives exosome secretion. We further show that exosomes generated from cells grown on stiff ECM effectively promote tumour growth. Proteomic analysis revealed that the Notch signalling pathway is activated in cells treated with exosomes derived from tumour cells grown on stiff ECM, consistent with our gene expression analysis of liver tissues from patients. Our study reveals a molecular mechanism that regulates exosome secretion and provides insight into how mechanical properties of the ECM control the tumour microenvironment for tumour growth.

Determine the Function of the Exocyst in Vesicle Tethering by Ectopic Targeting.

We describe an assay, in which ectopically targeting the exocyst subunit Sec3 to mitochondria is used to determine its role in tethering of post-Golgi vesicles to the plasma membrane. In the assay, we use a plasmid that encodes a fusion protein of the mitochondria protein Tom20 and Sec3 N-terminally tagged with the florescence protein mCherry, and coexpress the plasmid in yeast cells with CIT1-GFP, a marker protein of mitochondria. We then detect the colocalization between Sec3 and CIT1 and other exocyst subunits such as Sec5 on mitochondria using fluorescence microscopy. We further detect the colocalization between Sec3 and Sec4, a Rab protein and a marker of post-Golgi vesicles. Through this assay, we propose that the exocyst subunit Sec3 recruits the other exocyst subunits and secretory vesicles to a target membrane, suggesting that it plays a pivotal role in vesicle tethering. This approach is likely appropriate for studying other tethering complexes at their specific stages of trafficking and may also be used in other eukaryotic cells such as the cultured mammalian cells.

Auto-ubiquitination of NEDD4-1 Recruits USP13 to Facilitate Autophagy through Deubiquitinating VPS34.

The class III phosphoinositide 3-kinase vacuolar protein sorting 34 (VPS34) is a core protein of autophagy initiation, yet the regulatory mechanisms responsible for its stringent control remain poorly understood. Here, we report that the E3 ubiquitin ligase NEDD4-1 promotes the autophagy flux by targeting VPS34. NEDD4-1 undergoes lysine 29 (K29)-linked auto-ubiquitination at K1279 and serves as a scaffold for recruiting the ubiquitin-specific protease 13 (USP13) to form an NEDD4-1-USP13 deubiquitination complex, which subsequently stabilizes VPS34 to promote autophagy through removing the K48-linked poly-ubiquitin chains from VPS34 at K419. Knockout of either NEDD4-1 or USP13 increased K48-linked ubiquitination and degradation of VPS34, thus attenuating the formation of the autophagosome. Our results identify an essential role for NEDD4-1 in regulating autophagy, which provides molecular insights into the mechanisms by which ubiquitination regulates autophagy flux.

A Patient-Derived Glioblastoma Organoid Model and Biobank Recapitulates Inter- and Intra-tumoral Heterogeneity.

Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. Here, we report methods for generating and biobanking patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies. Additionally, our live biobank establishes a rich resource for basic and translational glioblastoma research.

Ultrasensitive Silicon Nanowire Sensor Developed by a Special Ag Modification Process for Rapid NH3 Detection.

Surface functionalization is very effective in enhancing sensing properties of a chemiresistive gas sensor. In this work, we develop a novel and cost-effective process to prepare Ag-modified silicon nanowire (SiNW) sensors and further suggest a resistance effect model to clarify the enhanced sensing mechanism of Ag-modified SiNWs. The SiNWs were formed via metal-assisted chemical etching (MACE), and the Ag nanoparticle (NP) modification was achieved in situ based on the MACE-produced Ag dendrites by involving a crucial anisotropic postetching of TMAH. The TMAH etching induces a loose array of needle-like, rough SiNWs (RNWs) with firm attachment of tiny Ag NPs. Comparative investigations for NH3-sensing properties indicate that the RNWs modified by discrete Ag NPs (Ag@RNWs) display an ∼3-fold enhancement in gas response at room temperature compared with pristine SiNWs. Meanwhile, transient response and ultrafast recovery are observed for the Ag@RNW sensor (tres ≤ 2 s and trec ≤ 9 s to 0.33-10 ppm of NH3). The study demonstrates the considerable effect and potential of the Ag modification process developed in this work. A resistance effect model was further suggested to clarify the underlying mechanism of the enhanced response and the response saturation characteristic of the Ag@RNWs. The promotion of TMAH etching-induced microstructure modulation to sensing properties was also demonstrated.