Exosomal Cargo Research Articles

The Analysis of Inflammation-Related Proteins in a Cargo of Exosomes Derived from the Serum of Uveal Melanoma Patients Reveals Potential Biomarkers of Disease Progression.

Simple SummaryUveal melanoma (UM) is the most common intraocular tumour in adults with a poor prognosis and extremely high mortality rate due to the development of metastatic disease. Despite good knowledge of the histological and genetic background of metastases formation, there is still a lack of specific biomarkers which would allow early detection of UM progression. Due to their unique molecular cargo consisting of proteins and nucleic acids, exosomes have been widely studied as carriers of biomarkers for cancer development and progression. In this study, we analyzed the inflammation-related protein cargo of exosomes derived from the serum of primary and metastatic UM patients and healthy donors using multiplex immunoassay technology. We showed a significant correlation between the disease stage and the concentration of inflammation-related proteins from exosomal cargo. Based on the obtained results, we propose the panel of exosomal proteins for early detection of uveal melanoma progression into metastatic disease.Background: Uveal melanoma (UM) is the most common intraocular tumour in adults with a poor prognosis and extremely high mortality rate due to the development of metastatic disease. However, despite relatively good knowledge about the histological and genetic risk factors for metastasis development, there is no specific biomarker that would allow early detection of UM progression. Recently, exosomes and their molecular cargo have been widely studied in the search for potential biomarkers in several cancers. The purpose of this study was to analyze the inflammation-related protein cargo of exosomes derived from the serum of primary and metastatic UM patients and healthy donors. Methods: The exosomes were isolated from the serum of primary and metastatic UM patients and healthy donors. Using multiplex immunoassay technology, we analyzed the concentration of 37 inflammation-related proteins in obtained exosomes. Results: The analysis of protein cargo showed several molecules related to inflammation, such as interferon-gamma, interleukin 2, 22 and 12(p40), Pentraxin-3, TNFSF13B and TNFSF8 which were significantly enriched in metastatic UM exosomes. We showed a significant correlation between the disease stage and the concentration of these inflammation-related proteins from exosomal cargo. Conclusions: Based on the obtained results, we propose the panel of exosomal proteins for early detection of uveal melanoma progression into metastatic disease.

Abstract 317: Berberine-mediated reprogramming of the inflammatory environment in anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer's (ATC) undifferentiated, inflammatory nature makes it one of the most aggressive cancers, with a five-year survival rate of only 4% when metastatic. ATC is a rare cancer that is refractory to conventional therapeutic modalities. Without high expression of targetable genetic lesions, small molecule inhibitors alone have been insufficient in trials. Treatment of ATC could benefit from a holistic approach that reprograms the inflammatory tumor microenvironment (TME). Berberine (BBR), a natural plant-derived alkaloid used extensively in Traditional Chinese Medicine, is a compound shown to exhibit anti-microbial, anti-inflammatory, and anti-cancer properties. Our work aims to exploit the anti-inflammatory activity of BBR in ATC. As the inflammatory status of ATC defines its intractable nature, remodeling its secretome, including its cytokine and chemokine profile and its exosomal cargo, in the TME fundamentally targets ATC's progressive determinant - inflammation. Exosomes, membrane-bound extracellular vesicles, are secreted by cells in the TME, including activated tumor-associated macrophages and ATC cells. Exosomal cargo primarily consists of miRNAs. We observed distinct miRNA expression from ATC-secreted exosomes when compared to papillary thyroid cancer (PTC)-secreted exosomes. Comparative analysis revealed ten miRNAs specifically downregulated in exosomes secreted from anaplastic-like 8505C compared to papillary BCPAP, including: hsa-miR-26b-5p, hsa-miR-125b-5p, hsamiR-138-5p, hsa-miR-148a-5p, hsamiR-152-5p, hsa-miR-191-5p, hsa-miR-9-5p, hsa-miR-21-5p, hsa-miR-134-5p, and hsa-miR-379-5p. The first six miRNAs listed are tumor suppressors, and as such, their downregulation may be contributory to the metastatic propensity and aggressiveness of ATC and its refractory nature towards conventional treatments. In activated macrophage-derived exosomes, miR-21-5p and miR-138-5p were also upregulated. ATC-secreted exosomes activate macrophages, subsequently priming the TME to be pro-tumorigenic and pro-inflammatory. This reciprocal interaction between inflammatory macrophages and ATC cells mediated by exosomal miRNAs defines its metastatic and inflammatory phenotype. We also observed that BBR significantly downregulates phosphorylation of MEK, ERK, and ribosomal protein S6 in proliferating ATC cells with as low as 10 μM BBR treatment. These are important downstream regulators of the pro-proliferative, pro-survival, and metabolic MAPK and PI3K-PTEN-AKT signaling pathways. Overall, the ability for BBR to alleviate the pro-inflammatory phenotype of ATC and remodel its immune environment, while simultaneously depressing overactive signaling in these cell survival pathways, may mark it as an important agent to make ATC amenable to combination therapy with small molecule inhibitors (MEKi) or other immunotherapeutics. Citation Format: Tara Jarboe, Nicole DeSouza, Sarnath Singh, Augustine Moscatello, Jan Geliebter, Raj K. Tiwari, Xiu-Min Li. Berberine-mediated reprogramming of the inflammatory environment in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 317.

Abstract 671: Development of an exosomal biomarker signature to detect minimal residual disease in dogs with osteosarcoma using a novel xenograft platform and machine learning

Abstract Osteosarcoma, the most common primary bone tumor in humans and dogs, has a guarded prognosis. A major hurdle in developing more effective osteosarcoma therapies is the lack of disease-specific biomarkers to predict risk, prognosis, or therapeutic response. Exosomes are secreted extracellular microvesicles emerging as powerful diagnostic tools. However, the wide clinical use of exosomes is precluded by the challenges in identifying disease-associated cargo from the vastly larger background of normal exosome cargo. We developed a method using canine osteosarcoma xenografts to distinguish tumor-derived exosome mRNAs and host-response mRNAs, allowing for identification of osteosarcoma-specific gene signatures, which were then validated in samples from dogs with osteosarcoma. A canine osteosarcoma-associated gene signature was developed using exosomes from mouse xenograft experiments and a species-aware bioinformatics pipeline. Validation of the gene signature in canine serum exosomes was done by qRT-PCR analysis. Machine learning algorithms assigned dogs into healthy or disease groups based on the qRT-PCR data. Dogs in a validation set of clinical osteosarcoma cases with post-treatment samples were classified as “osteosarcoma -detected” or “osteosarcoma - NOT detected”, and clinical outcome measures were compared. An osteosarcoma-associated signature consisting of five mRNAs (SKA2, NEU1, PAF1, PSMG2, and NOB1) was identified using our canine osteosarcoma xenograft model. Serum exosomes were isolated from 53 dogs in distinct clinical groups, including “healthy”, “osteosarcoma”, “other bone tumor”, or “non-neoplastic disease”. Dogs in a validation set whose post-treatment samples were classified as “osteosarcoma - NOT detected” had longer remissions than dogs classified as “osteosarcoma - detected” for up to 15 months after treatment. In conclusion, we identified a gene signature associated with canine osteosarcoma for the detection of minimal residual disease. This gene signature was validated by qRT-PCR with serum exosomes from canine patients with osteosarcoma, and used to train artificial intelligence. The test results were predictive of molecular remissions in dogs up to 15 months after initiating therapy, suggesting it will have applications in the early detection and minimal residual disease settings. This study combines a bioinformatics approach to biomarker discovery with machine learning to correctly identify osteosarcoma in canine patients. These results set the stage for future discoveries to inform cancer risk, diagnosis, prognosis, and response to therapy. Citation Format: Kelly M. Makielski, Alicia J. Donnelly, Ali Khammanivong, Milcah C. Scott, Hirotaka Tomiyasu, John Garbe, Lauren J. Mills, Gary R. Cutter, Andrea Ortiz, Dana C. Galvan, Kristi Ward, Alexa N. Montoya, Brad A. Bryan, Joelle M. Fenger, William C. Kisseberth, Subbaya Subramanian, Jaime F. Modiano. Development of an exosomal biomarker signature to detect minimal residual disease in dogs with osteosarcoma using a novel xenograft platform and machine learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 671.

Plasma-Derived Exosomes Carrying Leukemia-Associated Antigens Associate with Leukemia Relapse in AML Patients after Chemotherapy

Introduction: Exosomes are the smallest (30-150nm) type of extracellular vesicles that are produced by normal and cancerous cells, including leukemia blasts. Exosomes accumulating in the plasma of patients with cancer carry molecular cargo resembling that of parental tumor cells. Patients newly diagnosed with AML prior to any therapy have significantly higher plasma levels of exosomes (in µg protein/mL plasma) compared to levels in the plasma of normal controls. We previously showed that exosomes in AML patients carry leukemia-associated antigens (LAAs) and hypothesized that exosomes could define disease progression and outcome. Here, we evaluated the molecular cargo of exosomes at AML diagnosis, monitored changes in the exosome cargo during and post therapy, and correlated these changes with the blast content of LAAs detected in leukemia blasts using multi-parameter flow cytometry.Methods: Venous blood (20-50 mL) was obtained from patients newly diagnosed with AML and following therapy. Exosomes were isolated from plasma by exclusion chromatography and characterized for protein levels, number and size by qNano, and for morphology by transmission electron microscopy. Exosome cargos, including the presence and level of LAA, were evaluated by Western blots using exosomes isolated from serial plasma specimens obtained during and post chemotherapy. The data were correlated for expression of LAAs.Results: Exosomes isolated from the plasma of 15 patients newly diagnosed with AML carried CD34, CD117, C-type lectin-like molecule-1 (CLL-1), and interleukin-3 receptor subunit alpha (CD123). These antigens were carried by exosomes of all patients, but the antigen levels or profiles were distinct for each patient. The exosome antigen profile detected at AML diagnosis was comparable to that detected in each patient's leukemic blasts by multi-parameter flow cytometry. In AML patients with persistent leukemia following therapy, at the time when blasts were detectable in the bone marrow but not in the peripheral blood, circulating exosomes carried the LAAs. In all patients who achieved complete remission (n=7), at the time when no blasts were detected by morphologic and flow cytometric evaluation of the bone marrow, circulating exosomes carried LAAs with the same profile as that detected at AML diagnosis. All complete remission patients with LAA+ exosomes in the plasma eventually relapsed.Conclusions: We demonstrated, for the first time, that exosomes in patients newly diagnosed with AML have the LAA profiles that are identical to the profiles detected on leukemic blasts by conventional multi-parameter flow cytometry. The persistence of LAA+ exosomes in patients' plasma post-chemotherapy at the time of complete remission suggests the presence of residual leukemia and may be predictive of leukemia relapse. DisclosuresNo relevant conflicts of interest to declare.

Exosomes from cervical cancer cells facilitate pro-angiogenic endothelial reconditioning through transfer of Hedgehog\u2013GLI signaling components

BackgroundAngiogenic switch is a hallmark feature of transition from low-grade to high-grade cervical intraepithelial neoplasia (CIN) in cervical cancer progression. Therefore, early events leading to locally-advanced cervical metastatic lesions demand a greater understanding of the underlying mechanisms. Recent leads indicate the role of tumor-derived exosomes in altering the functions of endothelial cells in cervical cancer, which needs further investigation.MethodsExosomes isolated from cervical cancer cell lines were assessed for their angiogenic effect on the human umbilical vein endothelial cells (HUVEC) using tube formation and wound healing assay. The exosomal uptake by HUVEC cells was monitored using PKH-67 labelling followed by fluorescence microscopy. Alterations in Hh-GLI signaling components, PTCH1 and GLI1, in HUVEC were measured by immunoblotting. Changes in angiogenesis-related transcripts of vascular endothelial growth factor VEGF-A, VEGF-B, VEGFR2 and angiopoietin-1, angiopoietin-2, osteopontin were measured in exosome-treated HUVEC and in the exosomal RNA by RT-PCR.ResultsEnhanced tube formation, with an increased number of nodes and branching was observed in HUVEC’s treated with exosomes derived from different cervical cancer cell lines. HPV-positive (SiHa and HeLa) cells’ exosomes were more angiogenic. Exosome-treated HUVEC showed increased migration rate. PKH-67 labelled exosomes were found internalized in HUVEC. A high level of PTCH1 protein was detected in the exosome—treated endothelial cells. Subsequent RT-PCR analysis showed increased transcripts of Hh-GLI downstream target genes VEGF-A, VEGFR2, angiopoietin-2, and decreased expression of VEGF-B, and angiopoietin-1, suggestive of active Hh-GLI signaling. These effects were more pronounced in HUVEC’s treated with exosomes of HPV-positive cells. However, these effects were independent of tumor-derived VEGF-A as exosomal cargo lacked VEGF-A transcripts or proteins.ConclusionOverall, the data showed cervical cancer exosomes promote pro-angiogenic response in endothelial cells via upregulation of Hh-GLI signaling and modulate downstream angiogenesis-related target genes. The study provides a novel exosome-mediated mechanism potentially favoring cervical angiogenesis and thus identifies the exosomes as potential pharmacological targets against locally-advanced metastatic cervical lesions.Graphic abstract

PHENSIM: Phenotype Simulator.

Despite the unprecedented growth in our understanding of cell biology, it still remains challenging to connect it to experimental data obtained with cells and tissues' physiopathological status under precise circumstances. This knowledge gap often results in difficulties in designing validation experiments, which are usually labor-intensive, expensive to perform, and hard to interpret. Here we propose PHENSIM, a computational tool using a systems biology approach to simulate how cell phenotypes are affected by the activation/inhibition of one or multiple biomolecules, and it does so by exploiting signaling pathways. Our tool's applications include predicting the outcome of drug administration, knockdown experiments, gene transduction, and exposure to exosomal cargo. Importantly, PHENSIM enables the user to make inferences on well-defined cell lines and includes pathway maps from three different model organisms. To assess our approach's reliability, we built a benchmark from transcriptomics data gathered from NCBI GEO and performed four case studies on known biological experiments. Our results show high prediction accuracy, thus highlighting the capabilities of this methodology. PHENSIM standalone Java application is available at https://github.com/alaimos/phensim, along with all data and source codes for benchmarking. A web-based user interface is accessible at https://phensim.tech/.

Advances in exosome therapies in ophthalmology-From bench to clinical trial.

During the last decade, the fields of advanced and personalized therapeutics have been constantly evolving, utilizing novel techniques such as gene editing and RNA therapeutic approaches. However, the method of delivery and tissue specificity remain the main hurdles of these approaches. Exosomes are natural carriers of functional small RNAs and proteins, representing an area of increasing interest in the field of drug delivery. It has been demonstrated that the exosome cargo, especially miRNAs, is at least partially responsible for the therapeutic effects of exosomes. Exosomes deliver their luminal content to the recipient cells and can be used as vesicles for the therapeutic delivery of RNAs and proteins. Synthetic therapeutic drugs can also be encapsulated into exosomes as they have a hydrophilic core, which makes them suitable to carry water-soluble drugs. In addition, engineered exosomes can display a variety of surface molecules, such as peptides, to target specific cells in tissues. The exosome properties present an added advantage to the targeted delivery of therapeutics, leading to increased efficacy and minimizing the adverse side effects. Furthermore, exosomes are natural nanoparticles found in all cell types and as a result, they do not elicit an immune response when administered. Exosomes have also demonstrated decreased long-term accumulation in tissues and organs and thus carry a low risk of systemic toxicity. This review aims to discuss all the advances in exosome therapies in ophthalmology and to give insight into the challenges that would need to be overcome before exosome therapies can be translated into clinical practice.

Pro-Inflammatory Microenvironment Modulates the Transfer of Mutated TP53 Mediated by Tumor Exosomes.

Exosomes released from tumor cells are instrumental in shaping the local tumor microenvironment to allow cancer progression. Recently, it has been shown that tumor exosomes carry large fragments of dsDNA, which may reflect the mutational status of parental cells. Although it has been described that a stressful microenvironment can influence exosomal cargo, the effects on DNA packing and its transfer into recipient cells have yet to be investigated. Here, we report that exosomes derived from SW480 (human colorectal adenocarcinoma cell line) cells can carry dsDNA fragments containing the entire coding sequence of both TP53 and KRAS genes, harboring the SW480-related TP53 c.818G > A and KRAS c.35G > T typical mutations. We also report the following: that cell stimulation with lipopolysaccharides (LPS) promotes the selective packaging of the TP53 gene, but not the KRAS gene; that exosomes secreted by SW480 cells efficiently transfer the mutated sequences into normal CCD841-CoN colon epithelial and THLE-2 hepatic cells; that this mechanism is more efficient when the cells had been previously incubated with pro-inflammatory cytokines; that the TP53 gene appears actively transcribed in both recipient cells; and that mutated mRNA levels are not influenced by cytokine treatment. Our data strongly suggest that pro-inflammatory stimulation promotes the horizontal transfer of an oncogene by exosomes, although this remains a rare event. Further studies are needed to assess the impact of the oncogenic transfer by exosomes in malignant transformation and its role in tumor progression.

An Examination of the Putative Role of Melatonin in Exosome Biogenesis.

During the last two decades, melatonin has been found to have pleiotropic effects via different mechanisms on its target cells. Data are abundant for some aspects of the signaling pathways within cells while other casual mechanisms have not been adequately addressed. From an evolutionary perspective, eukaryotic cells are equipped with a set of interrelated endomembrane systems consisting of intracellular organelles and secretory vesicles. Of these, exosomes are touted as cargo-laden secretory vesicles that originate from the endosomal multivesicular machinery which participate in a mutual cross-talk at different cellular interfaces. It has been documented that cells transfer various biomolecules and genetic elements through exosomes to sites remote from the original cell in a paracrine manner. Findings related to the molecular mechanisms between melatonin and exosomal biogenesis and cargo sorting are the subject of the current review. The clarification of the interplay between melatonin and exosome biogenesis and cargo sorting at the molecular level will help to define a cell’s secretion capacity. This review precisely addresses the role and potential significance of melatonin in determining the efflux capacity of cells via the exosomal pathway. Certain cells, for example, stem cells actively increase exosome efflux in response to melatonin treatment which accelerates tissue regeneration after transplantation into the injured sites.

Eribulin and Paclitaxel Differentially Alter Extracellular Vesicles and Their Cargo from Triple-Negative Breast Cancer Cells

Simple SummaryCancer cells release small vesicles called extracellular vesicles (EVs) and these vesicles and their cargo promote cancer progression. The release of these vesicles is coordinated by cellular structures called microtubules. Drugs used in the treatment of breast cancer include eribulin and paclitaxel and they function by inhibiting microtubules. We investigated if these drugs would alter the release of vesicles from breast cancer cells and change their cargo. While our results show that these drugs do not alter the concentration of vesicles released, eribulin but not paclitaxel reduced levels of an important cargo called ILK. This is significant because ILK promotes cancer progression in breast cancer models. These results show that drugs can differently affect the release and cargo of extracellular vesicles and this could be significant for their clinical actions.Extracellular vesicles play a central role in intercellular communication and contribute to cancer progression, including the epithelial-to-mesenchymal transition (EMT). Microtubule targeting agents (MTAs) including eribulin and paclitaxel continue to provide significant value in cancer therapy and their abilities to inhibit oncogenic signaling pathways, including eribulin’s capacity to reverse EMT are being revealed. Because microtubules are involved in the intracellular trafficking required for the formation and cargo loading of small extracellular vesicles (sEVs), we investigated whether MTA-mediated disruption of microtubule-dependent transport would impact sEV release and their cargo. Eribulin and paclitaxel caused an intracellular accumulation of CD63, a tetraspanin component of sEVs, in late/multivesicular endosomes of triple-negative breast cancer cells, consistent with the disruption of endosomal sorting and exosome cargo loading in these cells. While the concentrations of sEVs released from MTA-treated cells were not significantly altered, levels of CD63 and the CD63-associated cargos, ILK and β-integrin, were reduced in sEVs isolated from eribulin-treated HCC1937 cells as compared to vehicle or paclitaxel-treated cells. These results show that eribulin can reduce specific sEV cargos, including ILK, a major transducer of EMT in the tumor microenvironment, which may contribute to eribulin’s ability to reverse EMT to promote anticancer efficacy.

Proteomic Alterations in Salivary Exosomes Derived from Human Papillomavirus-Driven Oropharyngeal Cancer.

Increasing evidence supports the notion that human papillomavirus (HPV) DNA integration onto the human genome can influence and alter the molecular cargo in the exosomes derived from head and neck cancer cells. However, the molecular cargo of salivary exosomes derived from HPV-driven oropharyngeal cancer (HPV-driven OPC) remains unelucidated. Salivary exosomes morphology and molecular characterizations were examined using the nanoparticle tracking (NTA), western blot analysis, transmission electron microscopy (TEM) and mass spectrometry analysis. We report that HPV16 DNA was detected (80%) in isolated salivary exosomes of HPV-driven OPC patients. Importantly, we demonstrate elevated protein levels of six main glycolytic enzymes [i.e., aldolase (ALDOA), glyceraldehye-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A/B (LDHA and LDHB), phosphoglycerate kinase 1 (PGK1) and pyruvate kinase M1/2 (PKM)] in isolated salivary exosomes of HPV-driven OPC patients, suggesting a novel mechanism underlying the potential role of salivary exosomes in mediating the reciprocal interplay between glucose metabolism and HPV-driven OPC. Our data demonstrate the potential diagnostic value of HPV16 DNA and glycolytic enzymes in salivary exosomes in discriminating healthy controls from HPV-driven OPC patients, thereby opening new avenues in the future for clinical translation studies.

Intermittent Fasting Alters Serum Exosomes in Middle-Aged Male Mice on Long-Term High-Fat Diet

ObjectivesExtracellular vesicles (EVs), especially exosomes, have emerged as a critical regulator of intercellular and inter-organ communications. Dysregulation of exosome secretion and exosomal cargo composition has been associated with metabolic diseases. The objective of this study was to determine the effect of intermittent fasting (IF) on the size profile and cargoes of exosomes in diet-induced obesity. MethodsFemale and male mice (8–10 weeks old) were fed a high-fat diet (HFD) for 18 weeks prior to being placed either in an ad libitum feeding group (HFD-AL) or an IF feeding group (HFD-IF) for an additional 10 weeks. Mice on the normal chow ad libitum (NC-AL) for 28 weeks served as control. The IF group had food available for 10 hours and fasted for 14 hours daily. Serum EVs measured using Nanoparticle Tracking Analysis. ResultsHFD-AL mice had an increasing trend in mean diameter of serum EVs by 24 nm (P = 0.30) compared to the NC-AL mice. The IF caused a significant decrease in mean diameter of serum EVs by 47.9 nm (P < 0.03) when compared to the HFD-AL mice. However, neither HFD nor IF altered the overall particle amount per mL of serum when compared to NC-AL. The HFD-AL mice had a decreasing trend in the population of small-size EVs but an increase in large-size EVs compared to the NC-AL mice. Mice on long-term HFD with IF had a significant increase in the total amount of small EVs/exosomes (50–100 nm in size) per unit of serum, but a decrease in the amount of large EVs (250–500 nm in size). Moreover, the mean population (particles/ml) of small EVs/exosomes were increased in the HFD-IF mice by an average of 38% (P < 0.03), whereas the population of large-size micro-vesicles (250–300 nm in size) were decreased by an average of 4% (P < 0.01) when compared to the HFD-AL mice. In addition to exosome alterations, the weekly weigh-in differences were significantly higher in the HFD-IF mice compared to the HFD-AL mice (P < 0.02). Lastly, we found that IF could prevent the whitening of brown adipose tissue (BAT) and liver micro-vesicular steatosis induced by a long-term HFD. ConclusionsIF significantly alters the profile of serum EVs, which may serve as an important mechanism for the metabolic benefits of IF such as preventing the whitening of BAT and liver micro-vesicular steatosis in obesity. Funding SourcesThis work was supported by the General Mills Foundation Chair in Genomics for Healthful Foods to X.C.

Neuronal exosome proteins: novel biomarkers for predicting neonatal response to therapeutic hypothermia

ObjectiveCentral nervous system (CNS) derived exosomes can be purified from peripheral blood and have been used widely in adult neurological disease. Application to neonatal neurological disease deserves investigation in the...

Roles of exosomes in cancer chemotherapy resistance, progression, metastasis and immunity, and their clinical applications (Review)

Exosomes are a type of vesicle that are secreted by cells, with a diameter of 40-100 nm, and that appear as a cystic shape under an electron microscope. Exosome cargo includes a variety of biologically active substances such as non-coding RNA, lipids and small molecule proteins. Exosomes can be taken up by neighboring cells upon secretion or by distant cells within the circulatory system, affecting gene expression of the recipient cells. The present review discusses the formation and secretion of exosomes, and how they can remodel the tumor microenvironment, enhancing cancer cell chemotherapy resistance and tumor progression. Exosome-mediated induction of tumor metastasis is also highlighted. More importantly, the review discusses the manner in which exosomes can change the metabolism of cancer cells and the immune system, which may help to devise novel therapeutic approaches for cancer treatment. With the development of nanotechnology, exosomes can also be used as biomarkers and for the delivery of chemical drugs, serving as a tool to diagnose and treat cancer.

Exosome Traceability and Cell Source Dependence on Composition and Cell-Cell Cross Talk.

Exosomes are small vesicles with an average diameter of 100 nm that are produced by many, if not all, cell types. Exosome cargo includes lipids, proteins, and nucleic acids arranged specifically in the endosomes of donor cells. Exosomes can transfer the donor cell components to target cells and can affect cell signaling, proliferation, and differentiation. Important new information about exosomes’ remote communication with other cells is rapidly being accumulated. Recent data indicates that the results of this communication depend on the donor cell type and the environment of the host cell. In the field of cancer research, major questions remain, such as whether tumor cell exosomes are equally taken up by cancer cells and normal cells and whether exosomes secreted by normal cells are specifically taken up by other normal cells or also tumor cells. Furthermore, we do not know how exosome uptake is made selective, how we can trace exosome uptake selectivity, or what the most appropriate methods are to study exosome uptake and selectivity. This review will explain the effect of exosome source and the impact of the donor cell growth environment on tumor and normal cell interaction and communication. The review will also summarize the methods that have been used to label and trace exosomes to date.

Mutant Presenilin 1 Dysregulates Exosomal Proteome Cargo Produced by Human-Induced Pluripotent Stem Cell Neurons.

The accumulation and propagation of hyperphosphorylated tau (p-Tau) is a neuropathological hallmark occurring with neurodegeneration of Alzheimer’s disease (AD). Extracellular vesicles, exosomes, have been shown to initiate tau propagation in the brain. Notably, exosomes from human-induced pluripotent stem cell (iPSC) neurons expressing the AD familial A246E mutant form of presenilin 1 (mPS1) are capable of inducing tau deposits in the mouse brain after in vivo injection. To gain insights into the exosome proteome cargo that participates in propagating tau pathology, this study conducted proteomic analysis of exosomes produced by human iPSC neurons expressing A246E mPS1. Significantly, mPS1 altered the profile of exosome cargo proteins to result in (1) proteins present only in mPS1 exosomes and not in controls, (2) the absence of proteins in the mPS1 exosomes which were present only in controls, and (3) shared proteins which were upregulated or downregulated in the mPS1 exosomes compared to controls. These results show that mPS1 dysregulates the proteome cargo of exosomes to result in the acquisition of proteins involved in the extracellular matrix and protease functions, deletion of proteins involved in RNA and protein translation systems along with proteasome and related functions, combined with the upregulation and downregulation of shared proteins, including the upregulation of amyloid precursor protein. Notably, mPS1 neuron-derived exosomes displayed altered profiles of protein phosphatases and kinases involved in regulating the status of p-tau. The dysregulation of exosome cargo proteins by mPS1 may be associated with the ability of mPS1 neuron-derived exosomes to propagate tau pathology.

Exosomes: Innocent Bystanders or Critical Culprits in Neurodegenerative Diseases.

Extracellular vesicles (EVs) are nano-sized membrane-enclosed particles released by cells that participate in intercellular communication through the transfer of biologic material. EVs include exosomes that are small vesicles that were initially associated with the disposal of cellular garbage; however, recent findings point toward a function as natural carriers of a wide variety of genetic material and proteins. Indeed, exosomes are vesicle mediators of intercellular communication and maintenance of cellular homeostasis. The role of exosomes in health and age-associated diseases is far from being understood, but recent evidence implicates exosomes as causative players in the spread of neurodegenerative diseases. Cells from the central nervous system (CNS) use exosomes as a strategy not only to eliminate membranes, toxic proteins, and RNA species but also to mediate short and long cell-to-cell communication as carriers of important messengers and signals. The accumulation of protein aggregates is a common pathological hallmark in many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and prion diseases. Protein aggregates can be removed and delivered to degradation by the endo-lysosomal pathway or can be incorporated in multivesicular bodies (MVBs) that are further released to the extracellular space as exosomes. Because exosome transport damaged cellular material, this eventually contributes to the spread of pathological misfolded proteins within the brain, thus promoting the neurodegeneration process. In this review, we focus on the role of exosomes in CNS homeostasis, their possible contribution to the development of neurodegenerative diseases, the usefulness of exosome cargo as biomarkers of disease, and the potential benefits of plasma circulating CNS-derived exosomes.

Effect of caveolin-1 knockdown on the protein composition of extracellular vesicles secreted by non-small cell lung cancer cells

Background. Recent data show evidence that lipid rafts (LR) proteins could be involved in the formation of exosomes and the sorting of proteins that make up the exosomal cargo. Such data are available for flotillins, structural and functional components of flatted rafts. The presence of the main component of caveolar rafts, caveolin-1 (Cav-1), has been shown in exosomes produced by some cancer cells; however, its possible participation in the regulation of the protein composition of exosomes has not been studied previously.Materials and methods. Knockdown of Cav-1 by transduction of a lentiviral vector expressing precursors of short hairpin ribonucleic acid to Cav-1; isolation (by ultracentrifugation) and analysis (transmission electron microscopy, nanoparticle tracking analysis) of extracellular vesicles (EVs) from non-small cell lung cancer cells (NSCLC) H1299; analysis of proteins in cells and in EVs by immunoblotting.Results. Analysis of the effect of Cav-1 expression on the composition of EV proteins associated with exosome biogenesis revealed a decrease in the level of Alix and TSG101, an increase in the level of LR proteins and the absence of changes in the level of tetraspanin CD9. Conclusion. The obtained data demonstrate a Cav-1-dependent changes in the composition of EVs, indicating a change in the ratio of vesicles formed by the various molecular mechanisms.

Traditional and Emerging Biomarkers in Asymptomatic Left Ventricular Dysfunction-Promising Non-Coding RNAs and Exosomes as Biomarkers in Early Phases of Cardiac Damage.

Heart failure (HF) is one of the major causes of morbidity and mortality worldwide and represents an escalating problem for healthcare systems. The identification of asymptomatic patients with underlying cardiac subclinical disease would create an opportunity for early intervention and prevention of symptomatic HF. Traditional biomarkers are very useful as diagnostic and prognostic tools in the cardiovascular field; however, their application is usually limited to overt cardiac disease. On the other hand, a growing number of studies is investigating the diagnostic and prognostic potential of new biomarkers, such as micro-RNAs (miRNA), long non-coding RNAs, and exosome cargo, because of their involvement in the early phases of cardiac dysfunction. Unfortunately, their use in asymptomatic phases remains a distant goal. The aim of this review is to gather the current knowledge of old and novel biomarkers in the early diagnosis of cardiac dysfunction in asymptomatic individuals.

Effects of Growth Hormone on Pancreatic Cancer Derived Exosomes

In 2020, the National Cancer Institute (NCI) estimates 57,600 new cases and 47,050 deaths in the US due to pancreatic ductal adenocarcinoma (PDAC). A dismal 10% five-year overall survival rate in PDAC is attributed to late diagnosis, limited treatment options, a remarkably high metastasis rate, and resistance of this cancer to available therapies. Therefore, a better understanding of the mechanisms of how PDAC tumors acquire drug resistance and spread to distal parts of the body are necessary for developing novel therapeutic approaches. Exosomes, microscopic vesicles released from most cells (both tumor and non-tumor) have been recently established to play a significant role in cell to cell communication. Exosomes modulate their target cell responses systematically depending on the nature of exosomal cargoes (nucleic acids, proteins, and lipids). PDAC derived exosomes have been implicated to promote metastasis via forming a pre-metastatic niche of cells as well as enhancing drug resistance. Growth hormone (GH) secreted primarily by the pituitary gland promotes metastasis and drug resistance as shown by plethora of studies. No study has directly assessed the effect of GH on exosomal cargoes in terms of promoting metastases and drug resistance. In this report, we show that GH modulates various pancreatic cancer cell exosomal cargoes which in turn potentially amplifies tumor invasion and metastases. Our data shows that GH treatment on human and mouse PDAC cells increases the exosomal protein levels of TGFβ - a critical inducer of epithelial-to-mesenchymal transition (EMT, a process leading to metastasis). In addition, GH treatment also increases extracellular matrix-degrading enzymes, MMP2 and 9, as well as multi-drug efflux pump ABCC1, ABCB1, and ABCG2 in PDAC cells. These results strongly implicate GH action in driving EMT and chemoresistance via exosomes in pancreatic cancer. Exosomes have a crucial impact especially in the areas of diagnostics and therapeutics. This report is the first to show that GH modulates the effects of exosomes secreted by pancreatic cancer cells. Acknowledgement: This work was supported in part by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by the AMVETS, and Ohio University’s Student Enhancement Award and Edison Biotechnology Institute.