Exosome Isolation Research Articles

The Role of Exosomes in Epithelial–to-Mesenchymal Transition and Cell Functional Properties in Head and Neck Cancer

Exosomes are nanosized vesicles that are produced in normal and cancer cells, promoting intracellular communication. In head and neck cancer (HNC), exosomes are involved in many undesirable events of cancer development and progression, including angiogenesis, tumor microenvironment (TME) remodeling, invasion, epithelial-to-mesenchymal transition (EMT), metastasis, extracellular matrix (ECM) degradation, and drug resistance. Exosomes are involved in altering the signaling pathways in recipient cells by the cargoes they carry. Proteins, lipids, and nucleic acids such as DNA fragments and RNAs (i.e., mRNAs, miRNAs, and long non-coding RNAs) are carried in the exosomes to promote cell communication. EMT is a critical cellular process in which epithelial cells are forced to become mesenchymal cells by the actions of SNAIL/SLUG, TWIST, and ZEB family transcription factors carried in exosomes that facilitate metastasis. In this critical review, we focused on exosome biogenesis, their cargoes, and their involvement in EMT induction and metastasis during HNC. Insights into exosome isolation and characterization, as well as their key role in ECM remodeling and degradation, are also presented and critically discussed. More importantly, this article addresses the role of exosomes in HNC and drug resistance induced in drug-sensitive cancer cells. In addition, exosomes have a great potential to be used as diagnostic and therapeutic tools. A better understanding on exosome biogenesis, composition, and functions in HNC will aid in developing novel therapeutic strategies to treat HNC, overcome therapy resistance, and avoid metastasis, which is a significant cause of cancer death.

Abstract 833: Exosomes isolation via lyophilization for Philadelphia-positive leukemia therapy

Abstract Background: Exosomes are extracellular vesicles having less than 200 nm size, secreted extracellularly by almost all types of cells. They regulate intercellular communication by delivering RNAi, proteins, viruses, and soluble factors as inherited payloads. In recent years, the application of exosomes to deliver small molecules have been widely investigated in tissue regeneration, therapeutics, as well as diagnostics. However, the isolation of exosomes has remained challenging mainly due to limitations of specialized instruments and the least cost-effective reagents. In this study, we developed a novel method to isolate exosomes from cultured cells. Experimental: The supernatant from cultured cells was collected and subjected to lyophilization after low-speed centrifugation. The characterization and quantification of exosomes were performed using Bradford assay. Scanning Electron Microscopy (SEM) and Nanoparticles Tracking Analysis (NTA) were carried out for shape and size distribution. Western blot was performed for the evaluation of the exosome specific surface markers (CD63, CD9, CD81, calnexin, HSP70, and TSG101). The as-isolated exosomes were loaded with commercial dyes (Cy5, Eosin) for the evaluation of their drug delivery properties. Furthermore, their drug loading and release were confirmed by loading tyrosine kinase inhibitors (TKIs) as a payload. Finally, the anti-leukemic drugs (dasatinib and ponatinib) were loaded on the isolated exosomes for targeting leukemia cell line i.e., K562 and BCR-ABL expressing Ba/F3 cells in in vitro model, using MTT assay for cell proliferation and Annexin-V/PI for apoptosis detection. Results: Here we found that the isolated exosomes have round shape with an average size of ~130 nm. They expressed known exosome markers including CD63, CD9, CD81, calnexin, HSP70, and TSG101. These exosomes could efficiently target and deliver dyes (Eosin and Cy5) and TKIs (dasatinib and ponatinib) to K562 and Ba/F3-cells expressing BCR-ABL. The TKIs-loaded exosomes inhibited the growth of leukemic cells and induced apoptosis in a dose dependent manner. Conclusion: We claim the development of a new method for active exosome isolation from cell culture. These exosomes could qualify for all the essential characterization, drug loading, and release ability by harnessing proliferation and inducing apoptosis in Ph+ leukemia cells. Citation Format: Fawad Ur Rehman, Rida e Maria Qazi, Zahra Sajid, Afsar Ali Mian. Exosomes isolation via lyophilization for Philadelphia-positive leukemia therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 833.

Abstract 3347: Characterization of extracellular POTE-2 protein in liver cancer

Abstract Liver cancer is projected to be the 5th and 7th leading cause of mortality in males and females in 2022. The top two forms of liver cancer diagnosed are hepatocellular carcinoma and cholangiocarcinoma, with a percentage of 75% and 11%. In recent years, Texas has had the highest incidence, with a rate of 11.6 per 100,000, with the Hispanic population having one of the highest rates. In South Texas, the Rio Grande Valley contains a high Hispanic population and faces multiple health disparities leading to a higher prevalence of liver cancer. These factors and the insufficient diagnostic methods for liver cancer lead to a need to identify faster and early biomarkers. Recently, we have identified an extracellular cancer-testis antigen, POTE-2, which belongs to the POTE gene family. Our preliminary data show that POTE-2 has an increased expression in liver cancer and can be associated with cancer progression. In this study, we will discuss the characterization of the extracellular face of POTE-2 in liver cancer. Lentiviral plasmids created an overexpressing and a knockdown POTE-2 cell line. The exosomes and conditioned media were collected from the cell lines to analyze POTE-2 protein expression through immunoblotting and ELISA. Antibodies targeting varying protein regions were produced and characterized for use in extracellular characterization. Exosome isolation and collection of conditioned media showed the expression of extracellular POTE-2 protein. Using varying antibodies targeting different regions determined the protein domain cleaved through secretion. These studies will aid in discovering novel characteristics of POTE-2 protein function and further explore its potential in liver cancer progression. Citation Format: Samantha Lopez, Kyle D. Doxtater, Adithya Anilkumar, Sophia Leslie, Amayrani Sanchez, Kristopher Ezell, Subhash Chauhan, Manish K. Tripathi. Characterization of extracellular POTE-2 protein in liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3347.

Abstract 867: Identifying plasma exosomal small non-coding RNAs as potential biomarkers for lung cancer early detection

Abstract Introduction: Lung cancer is the second most common cancer and the leading cause of cancer death in the US and worldwide. The National Lung Screening Trial (NLST) through thoracic low-dose computed tomography (LDCT) scans has shown that screening decreases mortality by 20%, but nearly 40% of patients in the NLST had a positive screen at one point, with 96.4% of these being false positive. It is urgent to develop new non-invasive methods with high efficacy, such as blood molecular biomarkers as companion diagnostic tools to complement LDCT screening. Recent studies have shown that small non-coding RNAs (ncRNAs) play important roles in cancer development, and exosomes are actively secreted extracellular vesicles that contain proteins, lipids, and nucleic acids, including miRNAs and other classes of small ncRNAs. Our study aimed to screen and identify noval plasma exosomal small ncRNAs for lung cancer early detection. Methods: A supersensitive human plasma exosome isolation and purification as well as exosome small RNA sequencing were conducted on 233 human subjects including 117 lung cancer early-stage patients and 116 non-cancer subjects (54 benign and 62 normal samples). Sequencing reads were first processed by trimming off the adapters and low-quality bases using cutadapt. Following trimming, the sequences were aligned to the human genome (Genome Reference Consortium GRCh38) and mapped to different small ncRNA databases using STAR. Read counts for each RNA category (miRNA, piRNA, snRNA, snoRNA, tRNA, tsRNA) were calculated from the mapping results. Differential gene expression analysis was performed by the DESeq2 bioconductor package. The genes with Benjamini and Hochberg q-value < 0.05 were called differentially expressed. Based on the differential gene lists, diagnostic models were built by 10-fold cross validated Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression. Results: A total of 5470 small ncRNAs were initially identified, including 1094 miRNA, 2172 piRNA, 1185 snRNA, 376 snoRNA, 582 tRNA, 61 tsRNA. Using DESeq2 with an FDR q-value < 0.05 with a cut off of 1.5-fold change, we found 508 small ncRNAs significantly differentially expressed between cancer and non-cancer groups, consisting of 231 small ncRNAs up-regulated and 277 down-regulated in tumor. By diagnostic modeling, a set of 16 small ncRNAs were identified as promising biomarkers, which could differentiate patients with cancer from non-cancer with an area under the receiver operating (ROC) curve (AUC) of 96.71%. Using the same sixteen-smallRNA signature to distinguish cancer from benign, the AUC achieved 97.23%; while the same panel could distinguish cancer from normal with an AUC of 96.26%. Conclusion: This preliminary study demonstrated the potential diagnostic value of the exosomal small ncRNA biomarkers in lung cancer early detection. The results will be evaluated by more cases in the future study. Citation Format: Yuanyuan Fu, Masaki Nasu, Mayumi Jijiwa, Yu Chen, Zitong Gao, Isam Ibrahim, Youping Deng. Identifying plasma exosomal small non-coding RNAs as potential biomarkers for lung cancer early detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 867.

Abstract 3297: Deep reactive ion etched microneedle array for in-vivo melanoma cancer monitoring via cancer exosome isolation

Abstract This study reports a deep-reactive-ion-etched microneedle device (ExoNeedle chip) coated with a hybrid hydrogel that can directly capture cancer-associated extracellular vesicles (EVs) in interstitial fluid (ISF) in a minimally invasive manner, as opposed to conventional liquid biopsy methods involving sample extraction and EV isolation in-vitro or tissue biopsies for melanoma. Tumor derived EVs give insight into a tumor’s mutational burden and provide both prognostic and diagnostic value as a liquid biopsy tool. Melanoma is an aggressive cancer with a lack of promising markers for early detection, onset of metastasis, and monitoring of recurrence. The ExoNeedle chip is intended to pierce through the epidermis and parts of the dermis to isolate melanoma secreted EVs from the ISF to alleviate these unmet needs. The microneedle arrays were created through the Deep Reactive Ion Etching (DRIE) process and a subsequent wet etching step to sharpen the needles. A 4 inch silicon wafer spin coated with 3 microns of positive photoresist was lithographically patterned and etched by DRIE using the standard Bosch processes to expose arrays of cylindrical pillars. Next, the cylindrical features were sharpened using an isotropic wet etch consisting of a mixture of hydrofluoric and nitric acid. The microneedle arrays were made and cleaved into individual patches. Meanwhile, hybrid hydrogel solution was made from Polyvinyl alcohol (PVA) and alginate by dissolving each of them separately in water under heat, followed by mixing the solutions. After centrifuging and filtering the hydrogel, it was conjugated with Annexin V (Av) protein to give it affinity for cancerous exosomes. Lastly, this hydrogel complex was added onto the microneedle patches and gelated with the help of a humidifier that deposited Ca2+ ion vapor over the hydrogel. The microneedles were first optimized and validated using melanoma cell line secreted EVs. Melanoma EVs were successfully isolated and quantified to evaluate the efficacy of the microneedle patches yielding above an 80% capture efficiency from purified serum samples. The microneedle patches were DiO stained, fluorescently imaged, visualized in a scanning electron microscope, and protein concentration was determined respectfully. Additionally, the functional hydrogel coatings on the microneedles are dissolved in ethylenediaminetetraacetic acid (EDTA), subsequently unbinding EVs from Av due to EDTA-based Ca2+ chelation. The dissolved solution is then used for enumeration and quantification of EVs by nanoparticle tracking analysis. We are further validating these patches in skin mimicking models and patient derived xenograft models of melanoma. The technology developed in this study for the isolation of melanoma EVs can broadly pave the way for minimally invasive point of care cancer diagnostics and tracking for melanoma patients. Citation Format: Scott M. Smith, Abha Kumari, Thiago Reis, Yoon-Tae Kang, Sunitha Nagrath. Deep reactive ion etched microneedle array for in-vivo melanoma cancer monitoring via cancer exosome isolation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3297.

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes

AbstractPhospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes.

Phospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.

Exosomes and ultrasound: The future of theranostic applications.

Biomaterials and pertaining formulations have been very successful in various diagnostic and therapeutic applications because of its ability to overcome pharmacological limitations. Some of them have gained significant focus in the recent decade for their theranostic properties. Exosomes can be grouped as biomaterials, since they consist of various biological micro/macromolecules and possess all the properties of a stable biomaterial with size in nano range. Significant research has gone into isolation and exploitation of exosomes as potential theranostic agent. However, the limitations in terms of yield, efficacy, and target specificity are continuously being addressed. On the other hand, several nano/microformulations are responsive to physical or chemical alterations and were successfully stimulated by tweaking the physical characteristics of the surrounding environment they are in. Some of them are termed as photodynamic, sonodynamic or thermodynamic therapeutic systems. In this regard, ultrasound and acoustic systems were extensively studied for its ability towards altering the properties of the systems to which they were applied on. In this review, we have detailed about the diagnostic and therapeutic applications of exosomes and ultrasound separately, consisting of their conventional applications, drawbacks, and developments for addressing the challenges. The information were categorized into various sections that provide complete overview of the isolation strategies and theranostic applications of exosomes in various diseases. Then the ultrasound-based disease diagnosis and therapy were elaborated, with special interest towards the use of ultrasound in enhancing the efficacy of nanomedicines and nanodrug delivery systems, Finally, we discussed about the ability of ultrasound in enhancing the diagnostic and therapeutic properties of exosomes, which could be the future of theranostics.

RNA profile of immuno‐magnetically enriched lung cancer associated exosomes isolated from clinical samples

Exosomal cargo secreted from cancer cells has been associated with the development and progression of the tumour. Enriching clinically relevant tissue-specific exosomes may assist in the focused analysis of RNA molecules packaged during cancer. Therefore, this study utilized a rapid immunomagnetic enrichment approach for targeted isolation of lung cancer cell-derived exosomes from human plasma, followed by analysing their cargo RNA using high throughput sequencing. The total RNA purified from these immunomagnetically enriched exosomes provided adequate RNA quality for characterization through the Illumina platform. Differential expression analysis was performed between patients and healthy controls to study the altered exosomal RNA profile during lung cancer. Further, functional enrichment analysis was performed with the list of identified differentially expressed genes (DEGs). In total, 1383 mRNAs and 64 lncRNA were identified as differentially expressed between patient plasma exosomes than healthy controls (fold change > 2, P < 0.05). Kyoto encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that the DEGs were mainly associated with cancer-related pathways, purine metabolism, calcium, and cGMP-PKG signalling pathways. In conclusion, the presented approach successfully demonstrated a novel strategy for focused disease-specific transcriptome analysis, which provides a feasible option for identifying disease-specific exosome biomarkers for detecting non-small lung cancer.

SARS CoV-2 spike protein-guided exosome isolation facilitates detection of potential miRNA biomarkers in COVID-19 infections.

Nearly three years into the pandemic, SARS-CoV-2 infections are occurring in vaccinated and naturally infected populations. While humoral and cellular responses in COVID-19 are being characterized, novel immune biomarkers also being identified. Recently, an increase in angiotensin-converting enzyme 2 expressing (aka, ACE2 positive) circulating exosomes (ExoACE2) were identified in the plasma of COVID-19 patients (El-Shennawy etal.). In this pilot study, we describe a method to characterize the exosome-associated microRNA (exo-miRNA) signature in ACE2-positive and ACE2-negative exosomal populations (non-ExoACE2). We performed a sorting protocol using the recombinant biotin-conjugated SARS CoV-2 spike protein containing the receptor binding domain (RBD) on plasma samples from six patients. Following purification, exo-miRNA were characterized for ACE2-positive and ACE2-negative exosome subpopulations by RT-PCR. We identified differential expression of several miRNA. Specifically let-7g-5p and hsa-miR-4454+miR-7975 were upregulated, while hsa-miR-208a-3p and has-miR-323-3p were downregulated in ExoACE2 vs. non-ExoACE2. The SARS CoV-2 spike-protein guided exosome isolation permits isolation of ExoACE2 exosomes. Such purification facilitates detailed characterization of potential biomarkers (e.g. exo-miRNA) for COVID-19 patients. This method could be used for future studies to further the understanding mechanisms of host response against SARS CoV-2.

Improved exosome isolation methods from non-small lung cancer cells (NC1975) and their characterization using morphological and surface protein biomarker methods.

This study has demonstrated improved methods for isolating exosomes from non-small lung cancer cells, which address the problems characterized by exosome morphological and chemical methods. To improve the isolation methods, cells from the NCI 1975 cell line were used as the source for exosomes. The isolation processes were carried out using serial isolation techniques in addition to specific preservation tools. The isolated exosomes were characterized using transmission electron microscopy (TEM), and scanning electron microscopy (SEM) was added for further assurance of the investigation results. The statistical analysis results showed that the size distributions of apoptotic vesicles (APV) 450nm and necrotic bodies (NCB) 280nm (extracellular vesicles) were significantly different from exosomes (P < 0.001). In contrast, the exosome size distribution was not significantly different from the published exosome sizes, as demonstrated by statistical analysis tools. This study confirmed the improved methods for isolating exosomes that make exosomes accessible for use in the diagnosis and prognosis of non-small cell lung cancer (NSCLC).

Microfluidic device using metallic nanostructure arrays for the isolation, detection, and purification of exosomes

Microfluidic devices are widely used in disease detection and specimen separation due to their low cost, rapid processing, and the ability to use even minuscule samples. This study reports on the embedding of metallic nanostructure arrays (MeNTAs) with tube-like features in microfluidic devices for the immunoaffinity-based detection and efficient isolation of exosomes. We assessed MeNTA candidates in terms of their ability to withstand the mechanical stress of microfluidic operations (indentation analysis) as well as X-ray diffraction, zeta potential, and electrostatic interactions involving antibody coatings. Note that Zr60Cu25Al10Ni5 thin film metallic glass (Zr-TFMG) features outstanding mechanical properties and a negative zeta potential exceeding those of other common materials (e.g., Cu, Bronze, Ag, 7075Al, Ti64, 718Ni, SS316, Cu-TFMG, W-TFMG, and Al-TFMG). The resulting microfluidic device featured Zr-based MeNTAs with an interdigital electrode embedded in a microchannel. When applied to MCF-7 derived exosomes using a liquid biopsy of only 500 μL, the proposed device (∼6.25 million nanostructures/cm2) achieved an exosome recovery rate of 95.3% within 1 h, while resisting nonspecific binding to HeLa-derived exosomes (recovery rate of<0.1%). The device enabled the isolation of 1 × 108 exosome particles per mL–1 for detection via electrochemical impedance spectroscopy. It also enabled the efficient release of captured exosomes via cyclic voltammetry (CV) operations over a potential range of –0.8 to +0.8 V. The proposed Zr-MeNTA microfluidic device has considerable potential for the isolation, detection, and purification of exosomes in liquid biopsy samples for cancer diagnosis.

Exosome-Based Liquid Biopsy Approaches in Bone and Soft Tissue Sarcomas: Review of the Literature, Prospectives, and Hopes for Clinical Application.

The knowledge of exosome impact on sarcoma development and progression has been implemented in preclinical studies thanks to technological advances in exosome isolation. Moreover, the clinical relevance of liquid biopsy is well established in early diagnosis, prognosis prediction, tumor burden assessment, therapeutic responsiveness, and recurrence monitoring of tumors. In this review, we aimed to comprehensively summarize the existing literature pointing out the clinical relevance of detecting exosomes in liquid biopsy from sarcoma patients. Presently, the clinical utility of liquid biopsy based on exosomes in patients affected by sarcoma is under debate. The present manuscript collects evidence on the clinical impact of exosome detection in circulation of sarcoma patients. The majority of these data are not conclusive and the relevance of liquid biopsy-based approaches in some types of sarcoma is still insufficient. Nevertheless, the utility of circulating exosomes in precision medicine clearly emerged and further validation in larger and homogeneous cohorts of sarcoma patients is clearly needed, requiring collaborative projects between clinicians and translational researchers for these rare cancers.

Abstract P5-02-47: Quantitative proteomic analysis of plasma exosomes from patients with advanced hormone receptor-positive/HER2-negative breast cancer receiving palbociclib and tamoxifen

Abstract Background: A CDK4/6 inhibitor (CDK4/6i) in combination with endocrine therapy (ET) is standard first-line therapy for advanced, hormone receptor (HR)-positive, HER2-negative breast cancer (BC). However, not all patients respond and responders eventually develop drug resistance and disease progression. Exosomes are small extracellular vesicles that are secreted by both normal and tumor cells as a mechanism for intercellular communication. The protein cargo of exosomes reflects biological processes activated in cancer cells and may serve as predictive biomarkers to select patients most likely to benefit from treatment and to identify mechanisms of resistance. Methods: Whole blood was collected in Streck tubes at baseline and at time points during treatment from patients with advanced, HR+/HER2- BC enrolled in a single arm, phase 2 trial of first line therapy with palbociclib plus tamoxifen that was conducted by The Big Ten Cancer Research Consortium (NCT02668666). Plasma was separated and stored at -80C within 48 hours of collection. Different exosome protein isolation methods were evaluated and optimized to maximize protein recovery. Exosome and plasma proteins were extracted, purified, and digested with trypsin. Tryptic peptides were isotopically labeled with Tandem Mass Tag (TMT) 10plex for protein expression level quantitation. Triplicate samples from each patient were analyzed by LC-MS/MS with QExactive HF Orbitrap mass spectrometer. An unsupervised clustering method was used to classify patients based on exosomal proteomic profiles. Results: We developed a sensitive and efficient exosome extraction method to obtain exosome protein from minimal volumes of patient plasma. The optimized exosome isolation method quantitatively identified 800 proteins from a 100 µl plasma sample. Significant enrichment of exosome-specific markers was observed when comparing patient samples with healthy donor samples. A network model was developed to differentiate responders/stable disease patients from non-responders using exosome proteomics data generated from pretreatment plasma samples. Preliminary data from the first 22 patients analyzed (responders, n= 6; stable disease, n=12, and non-responder, n=4) identified a network of 45 proteins that predicted response/stable disease vs progressive disease with high specificity (95%) and sensitivity (89%). We also noted significant differences in the exosome proteomic profiles of patients with de novo vs. recurrent metastatic disease. A network of 22 proteins differentiated de novo vs recurrent metastatic disease with &amp;gt; 85% sensitivity and 78% specificity, providing molecular evidence differentiating the two disease states. This finding is relevant in light of the higher response rate and improved PFS in patients with de novo metastatic disease in this trial, and confirms that this approach may provide molecular insight into mechanisms of primary resistance to CDK4/6i. Results for the entire trial cohort of 46 patients will be presented, along with analysis of serial samples collected at various time points during treatment. Conclusion: This proof-of-concept study demonstrates that an ultrasensitive exosome proteomics platform combined with deep learning methods is ideally suited for developing predictive protein biomarkers and for exploring molecular mechanisms of drug resistance. If results are confirmed, this novel approach holds great promise for identifying protein biomarkers that could be used to select patients unlikely to respond to ET and CDK4/6i in order to spare them ineffective treatment and for selecting participants for clinical trials of novel agents. Additionally, exosome proteomics data generated from serially collected specimens can be used to identify mechanisms of resistance that emerge during therapy. This approach can be widely applied to other treatment regimens and disease sites. This study was funded by Pfizer. Citation Format: Ziwei Zhang, Xiuyuan Ma, Julia Ekiert, Gayatry Mohapatra, Louis Coleman, Cristina I. Truica, Anne Blaes, Jatin Rana, Tandra Pavankumar, Lauren Green, Menggang Yu, Deborah Toppmeyer, Ruth O’Regan, Kari B. Wisinski, Oana C. Danciu, Kent Hoskins, Yu Gao. Quantitative proteomic analysis of plasma exosomes from patients with advanced hormone receptor-positive/HER2-negative breast cancer receiving palbociclib and tamoxifen [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-47.

Comparative analysis of magnetically activated cell sorting and ultracentrifugation methods for exosome isolation.

Mesenchymal stem cell-derived exosomes regulate cell migration, proliferation, differentiation, and synthesis of the extracellular matrix, giving great potential for the treatment of different diseases. The ultracentrifugation method is the gold standard method for exosome isolation due to the simple protocol, and high yield, but presents low purity and requires specialized equipment. Amelioration of technical optimization is required for quick and reliable confinement of exosomes to translate them to the clinic as cell therapeutics In this study, we hypothesized that magnetically activated cell sorting may provide, an effective, reliable, and rapid tool for exosome isolation when compared to ultracentrifugation. We, therefore, aimed to compare the efficiency of magnetically activated cell sorting and ultracentrifugation for human mesenchymal stem cell-derived exosome isolation from culture media by protein quantification, surface biomarker, size, number, and morphological analysis. Magnetically activated cell sorting provided a higher purity and amount of exosomes that carry visible magnetic beads when compared to ultracentrifugation. The particle number of the magnetically activated cell sorting group was higher than the ultracentrifugation. In conclusion, magnetically activated cell sorting presents a quick, and reliable method to collect and present human mesenchymal stem cell exosomes to clinics at high purity for potential cellular therapeutic approaches. The novel isolation and purification method may be extended to different clinical protocols using different autogenic or allogeneic cell sources.

Tandem enrichment of serum exosomes and exosomal RNA with titanium dioxide

Exosomes have great potential as biomarker carriers for disease diagnosis and prognosis. In recent years, exosomal RNA (exoRNA) has become a promising candidate for the early diagnosis and prognosis of cancers, and its pathophysiological roles in various diseases have been revealed. For example, exosome-derived mRNAs, miRNAs, circRNAs, and lncRNAs function as signalling molecules to regulate tumour growth, angiogenesis, invasion, metastasis, and the response to chemotherapy. However, the isolation of exosomes and exoRNA with high quality and purity remains challenging due to the relatively small size of exosomes and the limited amount of RNA in exosomes. In this work, we developed a novel tandem enrichment method to isolate exoRNA from serum based on the specific interaction between titanium dioxide (TiO2) and the phosphate groups on the lipid bilayer of exosomes and of the exoRNA. TiO2-based RNA isolation was first demonstrated and optimized in HeLa cells. A total of 130.9 ± 8.34 µg of RNA was rapidly enriched from approximately 5 × 106 HeLa cells within 10 min. This was a 41.5% higher yield than that using a commercial Ultrapure RNA Kit. TiO2-based tandem enrichment of exoRNA was then performed using human serum, obtaining 64.53±3.41 ng of exoRNA from 500 µL of human serum within 30 min. A total of 2,137,902 reads, including seven types of exoRNAs, were identified from the exosomes. This method is compatible with various downstream RNA processing techniques and does not use toxic or irritating reagents, such as phenol or chloroform, providing a simple, economical, rapid, and safe approach for exoRNA extraction from biological samples.

EFFECTS OF TRAUMA PLASMA-DERIVED EXOSOMES ON HEMATOPOIETIC PROGENITOR CELLS.

Background: Severe trauma disrupts bone marrow function resulting in persistent anemia and immunosuppression. Exosomes are extracellular vesicles implicated in disease, cellular functions, and immunomodulation. The effects of trauma plasma-derived exosomes on bone marrow hematopoiesis are unstudied; we hypothesized that trauma plasma-derived exosomes suppress bone marrow hematopoietic progenitor cell (HPC) growth and contribute to increased inflammatory cytokines and HPC mobilization. Methods: Plasma was collected from a prospective, cohort study of trauma patients (n = 15) with hip and/or femur fractures and an ISS > 15 and elective total hip arthroplasty (THA) patients (n = 15). Exosomes were isolated from both groups using the Invitrogen Total Exosome Isolation Kit. Healthy bone marrow was cultured with 2% plasma, 50 μg, 100 μg, or 200 μg of exosomal protein and HPC (granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units [CFU-GEMM], erythroid burst-forming units [BFU-E], and macrophage colony-forming units [CFU-GM]) growth assessed. After culturing healthy bone marrow stroma with 100 μg of exosomal protein, expression of cytokines and factors influencing HPC mobilization were assessed by qPCR. Differences were compared using the ANOVA, with significance defined as P < 0.05. Results: The only demographic difference was age; trauma patients were significantly younger than THA (mean 44 vs. 63 years). In vitro exposure to trauma plasma significantly decreased growth of all HPCs. In vitro exposure to 100 μg or 200 μg of trauma exosomal protein significantly decreased growth of BFU-E and CFU-GM, whereas 50 μg had no effect. Culture of trauma exosomal protein with bone marrow stromal cells resulted in increased expression of IFN-γ, IL-1α, TNF-α, G-CSF, CXCR4, SDF-1, and VCAM-1 in bone marrow stroma. Conclusions: Both plasma and plasma-derived exosomes from trauma patients adversely affect bone marrow function. Plasma-derived exosomes may contribute to altered hematopoiesis after severe trauma; analysis of exosomal content may improve our understanding of altered bone marrow function.

Mesenchymal and Neural Stem Cell-Derived Exosomes in Treating Alzheimer's Disease.

As the most common form of dementia and a progressive neurodegenerative disorder, Alzheimer's disease (AD) affects over 10% world population with age 65 and older. The disease is neuropathologically associated with progressive loss of neurons and synapses in specific brain regions, deposition of amyloid plaques and neurofibrillary tangles, neuroinflammation, blood-brain barrier (BBB) breakdown, mitochondrial dysfunction, and oxidative stress. Despite the intensive effort, there is still no cure for the disorder. Stem cell-derived exosomes hold great promise in treating various diseases, including AD, as they contain a variety of anti-apoptotic, anti-inflammatory, and antioxidant components. Moreover, stem cell-derived exosomes also promote neurogenesis and angiogenesis and can repair damaged BBB. In this review, we will first outline the major neuropathological features associated with AD; subsequently, a discussion of stem cells, stem cell-secreted exosomes, and the major exosome isolation methods will follow. We will then summarize the recent data involving the use of mesenchymal stem cell- or neural stem cell-derived exosomes in treating AD. Finally, we will briefly discuss the challenges, perspectives, and clinical trials using stem cell-derived exosomes for AD therapy.

Potential Avenues for Exosomal Isolation and Detection Methods to Enhance Small-Cell Lung Cancer Analysis.

Around the world, lung cancer has long been the main factor in cancer-related deaths, with small-cell lung cancer (SCLC) being the deadliest form of lung cancer. Cancer cell-derived exosomes and exosomal miRNAs are considered promising biomarkers for diagnosing and prognosis of various diseases, including SCLC. Due to the rapidity of SCLC metastasis, early detection and diagnosis can offer better diagnosis and prognosis and therefore increase the patient's chances of survival. Over the past several years, many methodologies have been developed for analyzing non-SCLC-derived exosomes. However, minimal advances have been made in SCLC-derived exosome analysis methodologies. This Review discusses the epidemiology and prominent biomarkers of SCLC. Followed by a discussion about the effective strategies for isolating and detecting SCLC-derived exosomes and exosomal miRNA, highlighting the critical challenges and limitations of current methodologies. Finally, an overview is provided detailing future perspectives for exosome-based SCLC research.

Isolation and proteomic profiling of urinary exosomes from patients with colorectal cancer

Exosomes in the body fluid are effective cell-derived membranous structures transferring various molecules to mediate intercellular communication. The expression of protein in the urinary exosomes from the colorectal cancer (CRC) patients could reflect the characteristics of tumorigenesis. The urinary exosomes with globular membrane structure, the size of 30 ~ 100 nm and positive expression of CD9, CD63 and CD81 were successfully isolated from 9 CRC patients and 3 heathy adults using the density gradient ultracentrifugation. Proteome profiles revealed by label-free liquid chromatography-tandem mass spectrometry (LC–MS/MS) indicated that several proteins were differentially expressed among different stages of CRC. Compared with normal controls, 67 proteins in CRC urinary exosomes were upregulated and 74 proteins were downregulated. The bioinformatics analysis revealed the decreased proteins were related to ESCRT III complex disassembly. The CHMP family was further determined to be the hub of interaction network of proteins enriched in ESCRT signaling. The significant decrease of CHMP4A, CHMP4B, CHMP2A, CHMP2B and CHMP1B were respectively found in the total CRC group and distant metastasis group compared with NC group. Moreover, the CEACAM family also showed significant aberrant changes in the urinary exosomes of CRC patients. The CEACAM7 and CEACAM1 were increased in the CRC patients compared with healthy individuals (P < 0.05). Significant changes of proteomic profile could be found in the urinary exosomes in the CRC patients. The differential expressed urinary exosomes derived proteins showed potential usage in diagnosis and prognosis of CRC.