Kim, Yohan

Alcohol-associated liver disease (ALD) is a growing global health concern, with alcohol-associated hepatitis (AH) leading to the highest morbidity and mortality. Available therapies are limited and often inadequate. Platelets contribute in a variety of ways to liver disease pathogenesis, but their role in AH remains largely unexplored. In this study, we addressed the hypothesis that platelets contribute to pathological inflammation in AH. Using patient samples and a multiomics approach, we found that platelets undergo proinflammatory transcriptomic and proteomic changes in AH, with 2 alarmins, S100A8 and S100A9, being among the top upregulated genes/proteins. Additionally, the abundance of platelet-derived microparticles containing S100A8 and S100A9 in AH patient plasma was increased and correlated with disease severity (assessed by model for end-stage liver disease sodium [MELD-Na]) and endotheliopathy (assessed by ICAM1, CXCL8, and vWF). We mechanistically linked S100A9 with endotheliopathy via crosstalk between primary human liver sinusoidal endothelial cells and primary human monocytes. We also demonstrated that IL-6 upregulates S100A9 in megakaryocytic cells in a JAK/STAT-dependent manner, modeling changes occurring in the bone marrow in patients with AH. Our studies establish proinflammatory platelets as important contributors to AH pathology. Moreover, antiplatelet agents — or, more specifically, S100A9 targeted drugs — are potential therapeutic strategies in AH.

Abstract Extracellular vesicles (EV) are phospholipid‐encapsulated nanoparticles secreted by cells into their surrounding environment. EVs can transfer a variety of biomolecules that mediate intercellular communication and play a key role in physiological and pathological processes. Therefore, EVs are emerging as new biomarkers for diseases, therapeutic targets, and drug delivery vehicles. The isolation and detection of EVs requires time‐consuming and labor‐intensive processes, first to extract EVs from biological and physiological fluids and then to detect EV‐associated molecules with high sensitivity. The methodologies and instruments commonly used for EV analysis are not widely accessible outside of dedicated research laboratories, creating practical barriers for the study of EV‐associated molecules in biology research and clinical applications. To bridge this gap, we developed a proof‐of‐concept digital microfluidic device that can automatedly extract EVs from 20 µL of culture and human plasma samples within 25 min and detect EV‐bound proteins (e.g PD‐L1) on‐chip using an electrochemical sensor. This work serves as a framework for the development of streamlined EV analysis in both research and diagnostics.

Abstract Purpose: Two randomized clinical trials (STOMP and ORIOLE) demonstrated that stereotactic ablative radiotherapy (SABR) can prolong androgen-deprivation therapy–free survival or progression-free survival (PFS) in patients with metachronous oligometastatic castration-sensitive prostate cancer (omCSPC). Although most patients with omCSPC have a more modest delay in progression, a small subset achieves a durable response following SABR. We investigated the prognostic and predictive value of circulating prostate-specific membrane antigen-positive (PSMA+) extracellular vesicles (EV) and PSA in a biomarker correlative study using blood samples from three independent patient cohorts. Experimental Design: Plasma samples from 46 patients with omCSPC on the ORIOLE trial and 127 patients with omCSPC on the STOMP trial protocol treated with SABR were included in the study. Pre-SABR PSMA+EV levels (EV/mL) were measured by nanoscale flow cytometry. Kaplan–Meier curves and logistic regression models were used to determine the association of PSMA+EV and PSA levels with clinical outcomes. Results: In the pooled cohorts, the median biochemical PFS were 26.1 and 15.0 months (P = 0.005), and the median radiographic PFS were 36.0 and 25.0 months (P = 0.003) for PSMA+EV-low and -high groups, respectively. The combination of pre-SABR low levels of both PSMA+EV and PSA was associated with a lower risk of radiographic progression (HR, 0.34, 95% confidence interval, 0.18–0.58; P = 0.0002). In the ORIOLE cohort, which included both an SABR arm and an observation arm, low PSMA+EV was predictive of benefit from SABR (P = 0.012). Conclusions: PSMA+EV is a novel prognostic and predictive biomarker of radiographically occult tumor burden in omCSPC. PSMA+EV may inform clinical decisions about identifying patients who will achieve a durable benefit from consolidative SABR alone.

Abstract Background. Extracellular vesicles (EV) are emerging as new biomarkers for cancer diagnostics and therapeutic monitoring1. For example, in patients that receive PD1/PD-L1 immune checkpoint inhibitors (a key pillar of cancer immunotherapy), circulating EV-derived PD-L1 is gaining attention as a non-invasive biomarker for therapeutic efficacy. Standard methodologies to measure EV-derived PD-L1 requires ultracentrifugation to first separate EVs and then quantify those that express PD-L1 using nano flow cytometry. These are tedious processes that require trained technologists in centralized facilities, which is a key barrier to frequently monitoring EV-based biomarkers. Yet, frequent monitoring is crucial to quickly identify and stop an ineffective therapy while more fruitful options are still available. Method. To bridge the gap, we develop a proof-of-concept digital microfluidic (DMF) device that can separate EVs directly from biofluids and quantify PD-L1+ EVs automatedly. Briefly, DMF device supports automatedly droplet handling, including droplet transport, splitting and mixing, based on electrowetting principles2. Immunomagnetic beads were used to capture EVs from biofluids, and EVs were then detected by electrochemical sensors inserted into the device. These tips of the electrochemical sensors were modified with gold nanoparticle self-assembled layers to enhance target binding, and CD9 antibodies were immobilized on the tips to bind to EVs. We then used anti-PD-L1 as secondary antibodies for target detection. Differential Pulse Voltammetry (DPV)3 were performed to obtain the results. Results. We used the DMF device to extract 109- 1011 #/mL EVs secreted by a human breast cancer cell line (MDA-MB-231). Briefly, 10 mL of cell culture media was loaded into the DMF device, and the sample was mixed and incubated with a 10 mL droplet of immunomagnetic beads to capture EVs. After 5 min, EVs were eluted in 20 mL of elution buffer, and the beads were discarded. We estimated the number of EVs using Nanoparticle Tracking Analysis; the number of EVs extracted on-chip was comparable with those that were extracted using standard in-tube method. We also verified the purity of the EVs extracted on-chip using Western Blot. Our results showed that the electrochemical sensors can detect as low as 104 #/mL PD-L1+ EVs, with a linear range of 104 – 107 #/mL (R2=0.9701). The entire process can be completed within 1 hour. Conclusion. We integrated automated EV extraction and their surface marker detection in a single DMF device. Our next step is to extract and detect EV-based biomarkers from plasma samples. This platform holds promise for the regular monitoring of EV-based biomarkers that are indicators of therapeutic responses in patients that receive cancer immunotherapies. References1. F. Lucien, D. Gustafson, M. Lenassi et al, Journal of extracellular vesicles 12 (12), 12385 (2023). 2. Y. Zhang and Y. Liu, Sensors & Diagnostics (2022). 3. Y. Zhang, J. Liu, T.-W. Lo et al, Biosensors and Bioelectronics: X, 100490 (2024). Citation Format: Ting-Wen Lo, Yuqian Zhang, Jing Liu, Yohan Kim, Haidong Dong, Fabrice Lucien, Yuguang Liu. Rapid extraction and detection of extracellular vesicle-derived PD-L1 in a microfluidic platform [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A030.

Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 ( RAB31 ) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.