Automated disc device for multiplexed extracellular vesicle isolation and labelling from liquid biopsies in cancer diagnostics.

Renal cell carcinoma (RCC) is a fairly common and lethal cancer. The wide variety of RCC histological subtypes constitutes a challenge in treatment decision-making. Exosomes are extracellular membrane vesicles that are produced by all cell types in physiological conditions. Extracellular vesicles (EVs) are now accepted as a mode of intercellular communication and transport proteins, RNAs, DNA, and lipids to surrounding and distant cells. The lipid bilayer membrane of the EVs helps to protect these cargos. EVs are involved in many pathological processes, such as cancer, and can be easily obtained through liquid biopsy. Currently, EVs are rarely considered as candidate biomarkers for kidney cancer. However, improvements in the characterization of tumor-derived EVs could lead to the implementation of blood- and urine-derived EVs as biomarkers in the management of oncologic patients. Since all organs, not just the tumor, contribute to EV population, the thoughtful discrimination of tumor-derived EVs remains an unmet need for the clinical application of this kind of liquid biopsy technology. To determine the contribution of the tumor to blood- and urine-derived EVs, we utilize a novel approach to isolate tissue-derived EVs in parallel with liquid biopsy-derived EVs. To our knowledge, only a handful of studies (only one in RCC) have studied EVs directly derived from tissue. We propose the use tissue-derived EVs to screen for candidate EV biomarkers in plasma and/or urine. We hypothesize that using tissue-derived EVs would increase the tumor specificity for the characterization of EVs as liquid biopsy biomarkers. We optimized a protocol in which we used tissue of RCC patients (normal kidney or tumor) to condition media and isolate tumor-derived EVs alone by ultracentrifugation. Additionally, we isolated plasma- and urine EVs by ultracentrifugation using standard protocols. Nanoparticle Tracking Analysis (NTA) showed normalized concentrations of >2 x 109 particles/mL with a size distribution in the small EV-range. Transmission Electron Microscopy (TEM) images showed typical exosome morphology, with the characteristic cup-shaped membrane vesicles. Western Blot (WB) confirmed the presence of exosome markers. We successfully isolated EVs from human RCC and healthy kidney tissue. We will proceed with screening the EV-samples for candidate miRNA-biomarkers by multiplexed gene expression analysis, followed by confirmation of the candidate markers by RT-qPCR. Citation Format: Richard C. Zieren, Liang Dong, Sarah R. Amend, Philip M. Pierorazio, Theo M. de Reijke, Kenneth J. Pienta. Tumor-derived extracellular vesicles as kidney cancer biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1358.

Abstract: Meningioma (MGM) comprises 39% of all primary intracranial tumors and is classified by the WHO into three grades depending on potential for recurrence. Risk stratification plays a crucial role in determining post-operative therapy and treatment response. Current classifications depend on tumor histology and a small number of genomic markers, though molecular profiling (DNA methylation, copy number, gene expression) may carry higher prognostic value. Minimally invasive liquid biopsy approaches could improve clinical management of MGM by identifying high-risk patients prior to surgery, as well as tumors that have transformed post-operatively. Extracellular vesicles (EVs) are nanometer sized membrane-bound capsules that carry cellular cargo and are abundant in plasma. In this work, we demonstrate the use of a microfluidic based technology (MGMExoChip) to isolate EVs specifically from MGM tissues using a specific protein (MGM-p). Using various characterization methods, we confirmed high capture efficiency and abundant proteins and RNA from 100 µL of plasma. These EVs were isolated from patients with high- and low-grade meningiomas, and then studied using proteomics and mRNA sequencing to identify features that associate with tumor aggression. Methods: MGMExoChip has a polydimethylsiloxane top and a glass bottom. Experiments were performed using MGM cell-line CH157, as well as primary cell cultures and plasma samples collected from MGM patients. Isolated EVs were characterized using nanoparticle tracking analysis, scanning electron microscopy, western blot, and proteomics. RNA extraction was performed using Norgen Biotek kit, sequenced in Illumina platform at 40M raw reads. Data analyses were done using R. Results: MGMExoChip was optimized for flow and capture conditions using EVs harvested from CH157 and two patient-derived primary cell cultures. Using MGM-p-based capture, the capture efficiency of EVs was significantly higher in CH157 relative to lung cancer (A549) (80.5±0.9% vs 6.2±4.2%). Among three antibodies tested for capture (anti-CD63, anti-MGM-p, and annexin-V), western blot confirmed that MGM-p had the highest specificity to isolate MGM EVs. We determined that only 100 µL of plasma was needed to extract an average of 7 µg of proteins and 5 ng of RNA without saturating the device. We further utilized proteomics, and mRNA-seq to explore molecular features that distinguish EVs in low and high grade meningiomas from normal controls among a cohort of 19 plasma samples. Conclusions: The use of microfluidics for liquid biopsy is a novel approach to study meningioma. MGMExoChip isolates MGM-specific EVs from patient plasma that allows extraction of molecular cargo for downstream characterization. Investigation of MGM EVs in a larger cohort could identify promising biomarkers for risk stratification of meningioma and determination of post-operative therapy. Citation Format: Abha Kumari, Harika Lingamarla, Mateo Gomez, Scott Smith, Kathleen McCortney, James Chandler, Craig Horbinski, David Raleigh, Adam Sonabend, Stephen Magill, Mark Youngblood, Sunitha Nagrath. Selective microfluidic capture and molecular analysis of plasma extracellular vesicles from meningiomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1922.

3046 Background: Extracellular Vesicles (EV) are of broad interest as carriers of molecular signatures of tumor progression and cancer treatment response. EVs, which contain nucleic acids, lipids, and proteins, are released from cells for waste excretion and communication. Numerous proteins and markers are expressed within and on the surface of EVs, but classification markers for murine EV subsets are lacking. To identify tumor and dendritic cell- derived EV markers for preclinical models of breast cancer, we investigated surface marker repertoires of EVs produced by the murine breast cancer and dendritic cell lines, 4T1 and DC2.4. Methods: Cells were cultured in serum free media for 2 days. EVs were harvested and isolated by ultrafiltration followed by size exclusion chromatography. EV particle size and concentration were estimated by nanoparticle tracking analysis and microBCA. To identify highly expressed EV markers, a mouse EV multiplex flow cytometry assay was performed using detection antibodies, CD9, CD63, and CD81, with sets of >35 barcoded capture beads, representing more than 100 specific capture: detection combinations. EV marker expression was analyzed using the FCMPASS/MPAPASS software (nano.ccr.cancer.gov). > 250 beads were assessed for each capture- and detection- antibody combination for each EV type and dilution tested; mean fluorescent intensity was determined; and pairwise comparisons between test and control sample sets were evaluated by t-tests. Results: Breast cancer (4T1)-derived EVs but not dendritic cell (DC2.4)-derived EVs were strongly detected with CD326 (EpCAM) and CD49b (integrin alpha5, VLA-2) capture beads, using each of the three tetraspanin antibodies. Both types of EVs were detected with anti-CD9 and anti-CD81 when captured by anti-CD44 and anti-CD49e (integrin beta1, VLA-5) beads. DC2.4 EVs were distinctively identified by CD11b capture. CD63 capture and detection antibodies robustly recognized EVs from 4T1 but provided minimal recognition of DC2.4 EVs. Mouse serum EVs from non-tumor bearing mice, showed minimal or no detectable CD326 or CD11b. Conclusions: Multiparametric MPAPASS-processed EV repertoire analysis of EVs from murine breast cancer and dendritic cell lines identified CD9, CD81, CD44, and CD49e as common epitopes among both types of evaluated EVs. CD326, CD49b, and CD63 distinguished 4T1 from DC2.4 EVs, and CD11b distinctively identified the DC2.4 EVs. The absence of detected CD326+ and CD11b+ in the serum of non-tumor bearing mice indicates the potential of these two markers for detection of specific tumor and antigen presenting cell EV subsets in serum from mice bearing CD326+ tumors such as 4T1. These results establish a foundation for further tests of detection and tracking of tumor-specific CD326+ EVs as "liquid biopsies" in blood samples as correlates to tumor progression and/or response to treatment.

A fatal affair: Circulating tumor cell relationships that shape metastasis

MP14-05 EXTRACELLULAR VESICLE BIOMARKER SIGNATURE DETECTION PLATFORM FOR GUIDING CANCER DETECTION AND THERAPY

Introduction: Tissue rebiopsy has limited value for detecting T790M mutation to prescribe the third-generation EGFR-TKIs. Liquid biopsy using cell-free DNA is being adopted but it plays a complementary role because of low sensitivity. Recently, extracellular vesicles (EV) are proven to contain double-stranded DNA and liquid biopsy using EV DNA is highly promising owing to its intact stability. We aimed to investigate the feasibility of liquid biopsy using EV DNA isolated from bronchoalveolar lavage fluid (BALF) and plasma in addition to tissue rebiopsy to detect T790M mutation in NSCLC patients who developed acquired resistance to EGFR-TKIs Methods: From October 2014 to December 2016, we enrolled 30 patients who had disease progression after previous treatment with EGFR-TKIs. Rebiopsy was performed in 23 patients and liquid biopsy was done in 16 BALF and 6 plasma samples. Sixteen BALF samples were obtained in 9 T790M-negative cases by rebiopsy and 7 patients ineligible for rebiopsy. Plasma testing was done in 6 BALF T790M-negative cases. EV were isolated from BALF and plasma by ultracentrifuge method. After DNA extraction from the isolated EV, EGFR genotyping to detect T790M mutation was done by peptide nucleic acid (PNA)-clamping method. We retrospectively reviewed patient characteristics, types of tumor rebiopsy procedure, and responses to third generation EGFR-TKIs. Results: Thirty patients who experienced progression to EGFR-TKIs were evaluated. Tumor rebiopsy was not possible in 7 patients. Out of 23 patients who performed tumor rebiopsy, 22 patients achieved adequate tissue specimen for evaluation, among whom 5 patients conducted rebiopsy twice (4 patients) or three times (1 patient), but 1 patient did not get evaluable tumor cells. Twenty two evaluable tumor rebiopsy revealed 54.5% (12/22) of T790M positivity. Among 17 patients who showed T790M negativity for tumor rebiopsy (n=10) or were ineligible for tumor rebiopsy (n=7), BAL was done in 16 patients and EGFR genotyping using BALF EV DNA showed 31.3% (5/16) of T790M positivity. In 11 patients with T790M negativity from EGFR genotyping using BALF EV DNA, 6 available plasma samples were tested for EGFR genotyping using plasma EV DNA and 3 patients revealed T790M positivity (3/6 = 50%). Finally, we found 8 more T790M positive patients by liquid biopsy (BALF EV DNA = 5, plasma EV DNA = 3) in addition to standard tissue rebiopsy (n=12). Conclusion: Liquid biopsy using EV DNA isolated from BALF and plasma could be an alternative and promising method to detect T790M mutation in NSCLC patients with acquired resistance to EGFR-TKIs. Citation Format: Jong Sik Lee, Hee Joung Kim, In Ae Kim, Jae Young Hur, Kye Young Lee. Feasibility of liquid biopsy using extracellular vesicle-derived DNA isolated from bronchoalveolar lavage fluid and plasma in addition to tissue rebiopsy to detect T790M mutation in NSCLC patients with acquired resistance to EGFR-TKIs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1596.

Extracellular Vesicle-Based Liquid Biopsy Shows Clinical Relevance and Reveals Links with Single-Cell Metabolic Signature in Acute Myeloid Leukemia

The field of liquid biopsies is of enormous interest in cancer using a noninvasive process. There are currently three major approaches: Circulating Cell Tumors (CTCs), circulating cell free DNA (cfDNA) and Extracellular Vesicles (EVs) such as exosomes, microvesicles and apoptotic bodies. The EVs are cell-derived small membrane vesicles secreted by cells that convey biological messages, either by surface-to-surface interaction or by shuttling bioactive molecules to a recipient cell's cytoplasm. The EVs released by cells harbor the cargos of their original cells and could be a good tool to follow drug activity. We are developing anti-TGFβ antibody SAR439459 for the treatment of cancer (NCT03192345), and in this vitro study we explore the effect of anti-TGFβ antibody in EVs carrying TGFβ. The cargo of EVs were analyzed by an Elisa test for both latent and active free TGFβ and by miRNA expression. Cell lines were selected based on their expression of TGFβ (A549, DU45, PC3, MCF7 and Caco2), treated for 72h and compared with respective untreated controls. The EVs were isolated from the supernatant and purified. Nanoparticle Tracking Analysis was used to quantify and determine size distribution of EVs. The expression of active free and latent TGFβ was done using Elisa tests. TGFβ antibody-coated magnetic beads were used to capture EVs, and miRNA expression sequencing was done using the Illumina NextSeq. Anti-TGFβ treatment reduced notably the expression of latent TGFβ in the supernatant of cultured tumor cells; the active free TGFβ was very low expressed or not detected in the supernatants. EVs express both latent and active free TGFβ; the anti-TGFβ antibody treatment significantly reduced the active free TGFβ and altered miRNA expression in EVs. Finally, using TGFβ Antibody-coated magnetic beads, we demonstrate that intact EVs express TGFβ at their surface. In conclusion, these results suggest that EVs express TGFβ at their surface and carry the active free TGFβ. Finally, the expression of active free TGFβ in EVs is decreased with anti-TGFβ antibody, suggesting that it might be a biomarker for monitoring anti-TGFβ antibody activity in patients by liquid biopsies. Citation Format: Manoel Nunes, Camille Bernard, Gaelle Muzard, Wilson Dos-Santos-Bele, Jack Pollard, Alexei Protopopov, Colette Dib. Tumor-derived Extracellular Vesicles (EVs) expressing TGFb as potential biomarkers for anti-TGFb antibody activity and drug activity in liquid biopsy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 773.

Editorial: Novel Clinical Applications of Extracellular Vesicles

Extracellular vesicles (EVs) are nano-sized, membrane-bound particles secreted by cells under physiological conditions and are involved in multiple functions, especially cell signaling. They are found in body fluids including blood, saliva, semen, bile, cerebrospinal fluid, amniotic fluid, breast milk, and urine. The molecular cargoes encapsulated by the EVs provide information about the cells of their origin. More recent developments in liquid biopsies have enabled uncovering the potential diagnostic uses of these vesicles offering an alternative to traditional biopsy enquiries in pathology. In the field of cancer, cancer-associated exosomes have been explored for early diagnosis with biomarkers to determine prognosis and therapy responses. This review filters recent advancements in EV biology and focuses on EV biogenesis and how EVs play a pivotal role in mediating intercellular communication in cancer. We draw attention to EV molecular profile with cargo heterogeneity which warrants scientists to develop precision therapeutic strategies to enhance treatment efficacy. Integrating EV analysis into clinical practice holds significant potential to revolutionize cancer diagnostics and therapeutic approaches, paving the way for more effective patient-centered healthcare solutions.

MP18-13 IDENTIFICATION OF TUMOR-SPECIFIC MARKERS ON EXTRACELLULAR VESICLES IN PATIENTS WITH RENAL CELL CARCINOMA

Fusarium head blight, caused by Fusarium graminearum, is one of the most threatening fungal diseases of cereals worldwide. Current practices for control of F. graminearum are not always efficient, as epidemics still occur and there is low resistance in wheat varieties. Therefore, novel antifungal targets must be discovered by analyzing the molecular interaction between F. graminearum and its host. Fungal extracellular vesicles (EVs) are small membrane-bound compartments (30–1000 nm) that carry macromolecules and support fungal virulence, hence the disruption of EV production could lead to reduced fungal pathogenicity. However, EV study is limited by the lack of surface protein markers to aid in their characterization. Therefore, the aim of this report was to target a surface protein marker with an antibody, to unlock advanced EV characterization techniques. Using the list of potential EV markers for Candida albicans, we selected the tetraspanin-like Sur7 to perform immunogold microscopy, revealing that this protein is a surface marker of F. graminearum EVs. SUR7 is present on the surface of some but not all vesicles. EVs carrying SUR7 were larger than those without the marker, suggesting that there are subtypes of fungal EVs. The epitope recognized by the anti-Sur7 antibody is conserved in other Fusarium pathogens, making Sur7 a potential pan-Fusarium EV marker. Our results unlock techniques, such as immunoaffinity chromatography and antibody labeling, to track fungal EVs and understand their biogenesis, which may lead to the development of novel antifungals.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s40694-025-00206-8.

Ovarian cancer is the most lethal gynecologic cancer in the world. Most patients are diagnosed at advanced stages with the formation of ascites. Extracellular vesicles (EVs) have been elucidated to play a pivotal role in cancer development and progression, also emerging as promising resources for clinical biomarkers in liquid biopsy. In this study, we aim to identify cancer-specific protein biomarkers in ascites-derived EVs for ovarian cancer diagnosis. Malignant ascites from high grade ovarian cancer patients (HGSOC) and benign peritoneal fluids from female patients with benign gynecologic diseases were collected with informed consent. EVs were isolated from these biofluids using the Exoquick kit. Meanwhile, HGSOC cell lines and normal cell lines were cultured in conditioned media supplemented with EVs-depleted FBS. EVs were isolated from the culture media. Proteins were extracted from EVs and fragmented into peptides. Tandem mass tag (TMT) hyper multiplex quantitation assay was carried out to detect the EV proteomic profiling. EVs isolated from biofluids and cell culture media were identified as double-membrane nanoparticles sizing ranging from 30nm to 200nm. Proteomic analysis of biofluids-derived EVs showed that malignant ascites-derived EVs displayed different proteomic profiling compared to benign peritoneal fluids-derived EVs. The differently expressed proteins between malignant ascites-derived EVs and benign peritoneal fluids-derived EVs were defined as candidate EV protein biomarkers. In addition, proteins that were expressed in at least one cancer cell line-derived EVs but not expressed in normal cell line-derived EVs were also identified as ovarian cancer-specific proteins. A diagnostic model constructed based on the integrative analysis of candidate EV protein biomarkers and ovarian cancer-specific proteins yielded a good discrimination ability between benign and cancer patients.In conclusion, this study demonstrates the distinct proteomic profiling in ascites- and cancer cell-derived EVs in ovarian cancer, which sheds new light on implementing EV proteins as a potential diagnostic strategy in clinical settings. Citation Format: Wenyu Wang, Dohyun Han, Yong Sang Song. Proteomic analysis of ascites- and cancer cell-derived EVs for identifying ovarian cancer diagnostic biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5081.

Introduction: Extracellular vesicles (EV), recovered from a liquid biopsy, are emerging as a putative diagnostic test for cancer. To date, ultracentrifugation has been accepted as the gold standard for EV isolation despite its lengthy processing time and low recovery rate. Thanks to the recent technological advances in microfluidics, several microfluidic devices for EV isolation have been developed and are showing promise with respect to improved recovery rates, the ability to extract EVs from small sample volumes, and shorter processing time compared to ultracentrifugation. Among the techniques, immunoaffinity based microfluidic isolation using antibodies against tetraspanins, such as anti-CD63, has been widely applied for EV isolation in general, however, anti-CD63 is not specific to cancer-associated EVs and some cancer cells secrete exosomes with low expression of CD63, thus limiting exosome isolation for cancer study. Alternatively, one of the phospholipids, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, has also been shown to be expressed on the outer surface of cancer-associated EVs during its vesiculation procedure. Here, we present a new exosome isolation device (newExoChip), conjugated with PS-specific protein, to isolate cancer-associated exosomes from plasma Methods: The newExoChip, having 30x60 circular channels conjugated with PS-specific protein, isolates cancer-associated exosomes in a Ca2+ dependent manner. The isolated exosomes using the device were easily released using Ca2+ chelation and the released exosomes has been analyzed quantitatively and qualitatively using nanoparticle tracking analysis (NTA) and western blot analysis, respectively. For clinical studies, we obtained non-small cell lung cancer (NSCLC) blood samples and each blood sample was centrifuged and the 30-100µl of plasma supernatant was flowed through the device using a syringe pump. Results: The newExoChip achieves 84.58% capture efficiency for NSCLC-derived exosomes compared to 38.43% for healthy exosomes and isolates averagely 47.4% more A549-derived exosomes than anti-tetraspanin devices (anti-CD63, 9, and 81). From a clinical study using 4 NSCLC blood samples, we recovered 1.47x109 EVs per 1 milliliter and 75.03% of vesicles was within the exosomal size range (30-150nm). The isolated exosomes from clinical samples were characterized by western blot using CD9 and flotillin-1, revealing exosomal protein expressions. Discussion and conclusion: Our results demonstrated that the isolated and released vesicles using the newExoChip show conventional features of exosomes, such as exosomal marker expressions and the exosomal size. We believe that the newExoChip facilitates the isolation of a specific subset of exosomes, allowing us to explore the undiscovered roles of exosomes in cancer. Citation Format: Yoon-Tae Kang, Emma Purcell, Colin Palacios-Rolston, Ting-Wen Lo, Nithya Ramnath, Shruti Jolly, Sunitha Nagrath. Microfluidic isolation (newExoChip) and profiling of cancer-associated exosomes using extracellular vesicular lipid-protein binding affinity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 751.

Analysis of Extracellular Vesicles: New Avenues for Signaling Biology and Biomarker Discovery