Exosomal Cargo Research Articles

A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture.

Spermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Alternatively, testicular biopsies taken prior to treatment may be used to restore fertility in adulthood. Testicular SSC populations are limited, and in vitro culture systems are required to increase numbers of SSCs for treatment, demanding culture systems for SSC propagation. Using the pig as a non-rodent model, we developed culture systems to expand spermatogonia from immature testis tissue, comparing different feeders (Sertoli cells, peritubular myoid cells (PMCs) and pig fetal fibroblasts (PFFs)). Spermatogonia co-cultured with Sertoli cells, PMCs and PFFs had comparable rates of proliferation and apoptosis. To elucidate the mechanism behind the beneficial nature of feeder layers, we investigated the role of extracellular vesicles in crosstalk between spermatogonia and feeder cells. Sertoli cell-released exosomes are incorporated by spermatogonia, and inhibition of exosomal release reduces spermatogonial proliferation. Together, these results show that PMCs, PFFs and Sertoli cells promote spermatogonial proliferation in co-culture, with exosomal exchange representing one possible mechanism. Further characterization of exosomal cargo may ultimately allow the development of feeder-free culture systems for clinical use.

Exosomes Derived from Senescent Endothelial Cells Contain Distinct Pro-angiogenic miRNAs and Proteins.

Exosomes from senescence cells play pivotal roles in endothelium dysfunction. We investigated the exosomal angiogenic cargo of endothelial cells (ECs) in a model of senescence in vitro. After inducing aging by H2O2, the expression of P53, P21, and P16 was investigated by western blotting, while the expression of FMR1, miR-21, and miR-126 were measured by real-time PCR (q-PCR). Oil Red O dye was used to stain cells. Acetylcholinesterase (AChE) assay, transmission electron microscopy (TEM), and western blotting characterized Exosomes. Exosomal miR-21, miR-126, matrix metallopeptidase-9 (MMP-9), and tumor necrosis factor- ɑ (TNF-ɑ) proteins were measured by Q-PCR and western blotting. A wound-healing assay was used to explore the effect of exosomes on ECs migration rate. The results showed that the expression of P53, P21, P16, FMR1, and miR-21 was increased in treated cells as compared with control cells (P < 0.05). In addition, the expression of miR-126 was decreased in treated cells (P < 0.05). The number of Oil Red O-positive-treated cells increased (P < 0.05). The AChE activity of exosomes from treated cells was increased (P < 0.05). In comparison with control cells, an increase in the expression levels of exosomal miR-21 and TNF-ɑ of treated cells coincided with a decrease in the expression levels of miR-126 and MMP-9 levels (P < 0.05). We found that the migration rate of ECs co-cultured with exosomes from treated cells was decreased (P < 0.05). The data indicate ECs under H2O2 condition produce exosomes with distinct cargo that may be useful as a biomarker of age-related vascular disease.

Long non-coding RNA and transfer RNA-derived small fragments in exosomes are potential biomarkers for canine oral melanoma.

Novel small non-coding RNAs (sRNAs) represent an emerging line of research in both human and canine oncology, due to their diverse regulatory and functional roles. Novel sRNAs are regarded as distinct from microRNAs, although both are part of the exosomal cargo. Recently, we reported on exosomal miRNAs as biomarkers for canine melanoma; however, it is unknown if novel sRNAs hold similar potential. Accordingly, we aimed to identify and validate novel sRNAs as potential biomarkers of canine oral melanoma, as part of our larger project on sequencing small exosomal RNA for this disease. Next generation sequencing revealed several differentially expressed novel sRNAs in exosomes from two melanoma cell lines (KMeC and LMeC) when compared with reference exosomes (from tumour-free dogs). Among these novel sRNAs, long noncoding RNA fragments, tRNA-derived fragments, snoRNAs and snRNAs were abundantly expressed. We selected four novel sRNAs upregulated in each cell line, and validated their aberrant expression with qPCR. In analysis using plasma-derived exosomes from melanoma patients, six out of the eight selected novel sRNAs showed significantly elevated expression. Receiver operating curve (ROC) analysis showed that one long non-coding RNA-derived small fragment (ENSCAFT00000069599.1) and one transfer RNA-derived small fragment (tRNA-Ala-TGC-5-1) have more than 85% sensitivity and specificity for differentiating melanoma patients from tumour-free dogs. Therefore, we consider that novel sRNAs may serve as candidate biomarkers to facilitate more accurate diagnosis of canine oral melanoma in clinical settings.

Differential expression and sorting of exosomal microRNAs upon activation of the human monocyte-like cell line U937

Extracellular vesicles such as exosomes in eukaryotes have drawn scrutiny due to their various roles in intercellular communication. Small RNAs, including microRNAs (miRNAs), are more abundant among the cargo of exosomes than other RNA types. MiRNAs loaded in secreted exosomes (or extracellular microRNAs) can be transported to recipient cells and may play a regulatory role although the miRNA loading (or sorting) mechanism in exosomes has not been clearly elucidated. Therefore, this study analyzed exosomal miRNA sequencing data from human myeloid U937 cells treated with phorbol 12-myristate 13-acetate (PMA) and compared it with data from PMA-untreated U937 cells. MiR-24 was highly expressed in the cytoplasm and exosomes of PMA-treated U937 cells. Also, miRNA pull-down and mass spectrophotometry analysis of PMA-treated U937 cells revealed that miR-24 was specifically associated with α-tubulin and hnRNP-E1 proteins. Furthermore, exosomal miR-24 was dramatically reduced when those proteins were inactivated with siRNAs, whereas cellular miR-24 showed no significant effect. We conclude that miR-24 is transported into exosomes from activated macrophages with the support of α-tubulin and hnRNP-E1.

Message in a Bottle: Endothelial Cell Regulation by Extracellular Vesicles.

Simple SummaryElucidating the role of extracellular vesicles (EVs) in the communication mechanisms between cancer and endothelial cells (ECs) within the tumor microenvironment is an exciting challenge. At the same time, due to their ability to convey bioactive molecules, EVs may be potentially relevant from a therapeutic perspective for diverse vascular pathologies.Intercellular communication is a key biological mechanism that is fundamental to maintain tissue homeostasis. Extracellular vesicles (EVs) have emerged as critical regulators of cell–cell communication in both physiological and pathological conditions, due to their ability to shuttle a variety of cell constituents, such as DNA, RNA, lipids, active metabolites, cytosolic, and cell surface proteins. In particular, endothelial cells (ECs) are prominently regulated by EVs released by neighboring cell types. The discovery that cancer cell-derived EVs can control the functions of ECs has prompted the investigation of their roles in tumor angiogenesis and cancer progression. In particular, here, we discuss evidence that supports the roles of exosomes in EC regulation within the tumor microenvironment and in vascular dysfunction leading to atherosclerosis. Moreover, we survey the molecular mechanisms and exosomal cargoes that have been implicated in explanations of these regulatory effects.

Modified Bovine Milk Exosomes for Doxorubicin Delivery to Triple-Negative Breast Cancer Cells.

Biological nanoparticles, such as exosomes, offer an approach to drug delivery because of their innate ability to transport biomolecules. Exosomes are derived from cells and an integral component of cellular communication. However, the cellular cargo of human exosomes could negatively impact their use as a safe drug carrier. Additionally, exosomes have the intrinsic yet enigmatic, targeting characteristics of complex cellular communication. Hence, harnessing the natural transport abilities of exosomes for drug delivery requires predictably targeting these biological nanoparticles. This manuscript describes the use of two chemical modifications, incorporating a neuropilin receptor agonist peptide (iRGD) and a hypoxia-responsive lipid for targeting and release of an encapsulated drug from bovine milk exosomes to triple-negative breast cancer cells. Triple-negative breast cancer is a very aggressive and deadly form of malignancy with limited treatment options. Incorporation of both the iRGD peptide and hypoxia-responsive lipid into the lipid bilayer of bovine milk exosomes and encapsulation of the anticancer drug, doxorubicin, created the peptide targeted, hypoxia-responsive bovine milk exosomes, iDHRX. Initial studies confirmed the presence of iRGD peptide and the exosomes' ability to target the αvβ3 integrin, overexpressed on triple-negative breast cancer cells' surface. These modified exosomes were stable under normoxic conditions but fragmented in the reducing microenvironment created by 10 mM glutathione. In vitro cellular internalization studies in monolayer and three-dimensional (3D) spheroids of triple-negative breast cancer cells confirmed the cell-killing ability of iDHRX. Cell viability of 50% was reached at 10 μM iDHRX in the 3D spheroid models using four different triple-negative breast cancer cell lines. Overall, the tumor penetrating, hypoxia-responsive exosomes encapsulating doxorubicin would be effective in reducing triple-negative breast cancer cells' survival.

Message in the bottle: regulation of the tumor microenvironment via exosome-driven proteolysis.

Exosomes comprise a subtype of extracellular vesicles involved in cell-to-cell communication, specifically by transporting biological molecules, such as proteins and nucleic acids, to either local or more distant recipient cells, thus triggering distinct biological behaviors. Included in the exosome cargo is frequently a wide range of proteolytic enzymes, such as the matrix metalloproteinases (MMPs), the disintegrin and metalloproteinases (ADAMs), and the ADAM with thrombospondin-like motifs (ADAMTSs), whose functions contribute to the development and progression of cancer. In recent years, extensive research on the potential use of exosomes in diagnostic and therapeutic applications for personalized medicine has emerged, but the targeting of the proteolytic cargo of exosomes has not been fully exploited in this direction. In this review, we aim to explore both the mechanistic and the translational importance of proteolytic enzymes carried by the tumor cell-derived exosomes, as well as their role in the acquisition and support of certain hallmarks of cancer.

Hypoxia Induced Changes of Exosome Cargo and Subsequent Biological Effects.

Exosomes are small extracellular vesicles that are secreted by almost all types of cells and exist in almost all extracellular spaces. As an important mediator of intercellular communication, exosomes encapsulate the miRNA, lncRNA, cirRNA, mRNA, cytokine, enzyme, lipid, and other components from the cytoplasm into its closed single membrane structure and transfer them to recipient units in an autocrine, paracrine, or endocrine manner. Hypoxia is a state of low oxygen tension and is involved in many pathological processes. Hypoxia influences the size, quantity, and expression of exosome cargos. Exosomes derived from hypoxic tumor cells transfer genetics, proteins, and lipids to the recipient units to exert pleiotropic effects. Different donor cells produce different cargo contents, target different recipient units and lead to different biological effects. Hypoxic exosomes derived from tumor cells uptaken by normoxic tumor cells lead to promoted proliferation, migration, and invasion; uptaken by extracellular space or liver lead to promoted metastasis; uptaken by endothelial cells lead to promoted angiogenesis; uptaken by immune cells lead to promoted macrophage polarization and changed tumor immune microenvironment. In addition to various types of tumors, hypoxic exosomes also participate in the development of diseases in the cardiovascular system, neuron system, respiratory system, hematology system, endocrine system, urinary system, reproduction system, and skeletomuscular system. Understanding the special characteristics of hypoxic exosomes provide new insight into elaborating the pathogenesis of hypoxia related disease. This review summarizes hypoxia induced cargo changes and the biological effects of hypoxic exosomes in tumors and non-malignant diseases in different systems.

Energy Sources for Exosome Communication in a Cancer Microenvironment.

Simple SummaryExosomal communication in the tumor microenvironment plays a crucial role in cancer development, progression, and metastasis, and is achieved by either short-distance communication with neighboring cells or long-distance communication with distant organs. Nevertheless, how exosomes gain energy to establish such communication and the different sources of energy are unclear. Recently, a handful of studies have demonstrated the presence of mitochondria, adenosine triphosphate, and glycolytic enzymes, which may serve as potential energy sources for exosomes. This review clarifies how exosomes maintain their structural integrity and stability during their intracellular communication, and reviews evidence of their energy source. Exosomes are crucial extracellular vesicles (EVs) with a diameter of approximately 30–200 nm. They are released by most cell types in their extracellular milieu and carry various biomolecules, including proteins and nucleic acids. Exosomes are increasingly studied in various diseases, including cancer, due to their role in local and distant cell–cell communication in which they can promote tumor growth, cancer progression, and metastasis. Interestingly, a tremendous number of exosomes is released by malignant cancer cells, and these are then taken up by autologous and heterologous recipient stromal cells such as immune cells, cancer stem cells, and endothelial cells. All these events demand an enormous amount of energy and require that exosomes remain stable while having the capacity to reach distant sites and cross physical barriers. Nevertheless, there is a dearth of research pertaining to the energy sources of exosomes, and questions remain about how they maintain their motility in the tumor microenvironment (TME) and beyond. Moreover, exosomes can produce adenosine triphosphate (ATP), an important energy molecule required by all cells, and mitochondria have been identified as one of the exosomal cargoes. These findings strengthen the prospect of exosomal communication via transfer of mitochondria and the bioenergetics of target recipient cells. In the TME, the accumulation of ATP and lactate may facilitate the entry of exosomes into cancer cells to promote metastasis, as well as help to target cancer cells at the tumor site. This review highlights how exosomes obtain sufficient energy to thrive in the TME and communicate with distant physiological destinations.

Degron tagging of BleoR and other antibiotic-resistance genes selects for higher expression of linked transgenes and improved exosome engineering

Five antibiotic resistance (AR) genes have been used to select for transgenic eukaryotic cell lines, with the BleoR, PuroR, HygR, NeoR, and BsdR cassettes conferring resistance to zeocin, puromycin, hygromycin, geneticin/G418, and blasticidin, respectively. We recently demonstrated that each AR gene establishes a distinct threshold of transgene expression below which no cell can survive, with BleoR selecting for the highest level of transgene expression, nearly ∼10-fold higher than in cells selected using the NeoR or BsdR markers. Here, we tested the hypothesis that there may be an inverse proportionality between AR protein function and the expression of linked, transgene-encoded, recombinant proteins. Specifically, we fused each AR protein to proteasome-targeting degron tags, used these to select for antibiotic-resistant cell lines, and then measured the expression of the linked, recombinant protein, mCherry, as a proxy marker of transgene expression. In each case, degron-tagged AR proteins selected for higher mCherry expression than their cognate WT AR proteins. ER50BleoR selected for the highest level of mCherry expression, greater than twofold higher than BleoR or any other AR gene. Interestingly, use of ER50BleoR as the selectable marker translated to an even higher, 3.5-fold increase in the exosomal loading of the exosomal cargo protein, CD63/Y235A. Although a putative CD63-binding peptide, CP05, has been used to decorate exosome membranes in a technology known as “exosome painting,” we show here that CP05 binds equally well to CD63−/− cells, WT 293F cells, and CD63-overexpressing cells, indicating that CP05 may bind membranes nonspecifically. These results are of high significance for cell engineering and especially for exosome engineering.

Tumour generated exosomal miRNAs: A major player in tumour angiogenesis

One of the major reasons for the non-effectiveness of anticancer therapeutics designed to target tumour angiogenesis is the involvement of multiple layers of cellular communication that exists within the tumour microenvironment. Any attempt to block one signaling pathway by targeting one of its components led to the activation of alternate pathway that overcome this block rendering the therapy ineffective. During the previous decades most of the research was focused on manipulating signaling pathways initiated by soluble factors and their intermediates. More recently the work from our lab and many advanced labs all over the globe have brought into light, the significance of signaling events mediated by exosomes in regulation of tumour progression and angiogenesis. In this mini review we intend to collate the information available from several groups including ours related to the role of exosomal cargo especially exosomal miRNAs in modulating tumour angiogenesis.

Small but Mighty—Exosomes, Novel Intercellular Messengers in Neurodegeneration

Simple SummaryExosomes are biological nanoparticles recently recognized as intercellular messengers. They contain a cargo of lipids, proteins, and RNA. They can transfer their content to not only cells in the vicinity but also to cells at a distance. This unique ability empowers them to modulate the physiology of recipient cells. In brain, exosomes play a role in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease and amyotrophic lateral sclerosis.Exosomes of endosomal origin are one class of extracellular vesicles that are important in intercellular communication. Exosomes are released by all cells in our body and their cargo consisting of lipids, proteins and nucleic acids has a footprint reflective of their parental origin. The exosomal cargo has the power to modulate the physiology of recipient cells in the vicinity of the releasing cells or cells at a distance. Harnessing the potential of exosomes relies upon the purity of exosome preparation. Hence, many methods for isolation have been developed and we provide a succinct summary of several methods. In spite of the seclusion imposed by the blood–brain barrier, cells in the CNS are not immune from exosomal intrusive influences. Both neurons and glia release exosomes, often in an activity-dependent manner. A brief description of exosomes released by different cells in the brain and their role in maintaining CNS homeostasis is provided. The hallmark of several neurodegenerative diseases is the accumulation of protein aggregates. Recent studies implicate exosomes’ intercellular communicator role in the spread of misfolded proteins aiding the propagation of pathology. In this review, we discuss the potential contributions made by exosomes in progression of Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Understanding contributions made by exosomes in pathogenesis of neurodegeneration opens the field for employing exosomes as therapeutic agents for drug delivery to brain since exosomes do cross the blood–brain barrier.

Isolation of Platelet-Derived Exosomes from Human Platelet-Rich Plasma: Biochemical and Morphological Characterization.

Platelet-Rich Plasma (PRP) is enriched in molecular messengers with restorative effects on altered tissue environments. Upon activation, platelets release a plethora of growth factors and cytokines, either in free form or encapsulated in exosomes, which have been proven to promote tissue repair and regeneration. Translational research on the potential of exosomes as a safe nanosystem for therapeutic cargo delivery requires standardizing exosome isolation methods along with their molecular and morphological characterization. With this aim, we isolated and characterized the exosomes released by human PRP platelets. Western blot analysis revealed that CaCl2-activated platelets (PLT-Exos-Ca2+) released more exosomes than non-activated ones (PLT-Exos). Moreover, PLT-Exos-Ca2+ exhibited a molecular signature that meets the most up-to-date biochemical criteria for platelet-derived exosomes and possessed morphological features typical of exosomes as assessed by transmission electron microscopy. Array analysis of 105 analytes including growth factors and cytokines showed that PLT-Exos-Ca2+ exhibited lower levels of most analytes compared to PLT-Exos, but relatively higher levels of those consistently validated as components of the protein cargo of platelet exosomes. In summary, the present study provides new insights into the molecular composition of human platelet-derived exosomes and validates a method for isolating highly pure platelet exosomes as a basis for future preclinical studies in regenerative medicine and drug delivery.

β-catenin-controlled tubular cell-derived exosomes play a key role in fibroblast activation via the OPN-CD44 axis.

Tubular injury and peripheral fibroblast activation are the hallmarks of chronic kidney disease (CKD), suggesting intimate communication between the two types of cells. However, the underlying mechanisms remain to be determined. Exosomes play a role in shuttling proteins and other materials to recipient cells. In our study, we found that exosomes were aroused by β‐catenin in renal tubular cells. Osteopontin (OPN), especially its N‐terminal fragment (N‐OPN), was encapsulated in β‐catenin‐controlled tubular cell‐derived exosome cargo, and subsequently passed to fibroblasts. Through binding with CD44, exosomal OPN promoted fibroblast proliferation and activation. Gene deletion of β‐catenin in tubular cells (Ksp‐β‐catenin−/−) or gene ablation of CD44 (CD44−/−) greatly ameliorated renal fibrosis. Notably, N‐OPN was carried by exosome and secreted into the urine of patients with CKD, and negatively correlated with kidney function. The urinary exosomes from patients with CKD greatly accelerated renal fibrosis, which was blocked by CD44 deletion. These results suggest that exosome‐mediated activation of the OPN/CD44 axis plays a key role in renal fibrosis, which is controlled by β‐catenin.

Modified Exosomes: a Good Transporter for miRNAs within Stem Cells to Treat Ischemic Heart Disease.

Stem cell-based therapy for ischemic heart disease (IHD) has become a promising but controversial strategy during the past two decades. The fate and effects of stem cells engrafted into ischemia myocardium are still not fully understood. Stem cell-derived exosomes, a subcategory of extracellular vesicles with nano size, have been considered as an efficient and safe transporter for microRNAs (miRNAs) and a central mediator of the cardioprotective potentials of the parental cells. Hypoxia, pharmacological intervention, and gene manipulation could alter the exosomal miRNAs cargos from stem cells and promote therapeutic potential. Furthermore, several bioengineering methods were also successfully applied to modify miRNAs content and components of exosomal membrane proteins recently. In this review, we outline relevant results about exosomal miRNAs from stem cells and focus on the current strategies to promote their therapeutic efficiency in IHD.

Exosomal microRNAs synergistically trigger stromal fibroblasts in breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. TNBC progression is sustained by recruitment of a strong tumor microenvironment (TME) mainly composed of cancer-associated fibroblasts (CAFs) able to endorse tumor hallmarks. Increasing evidences demonstrate that exosomes mediate the crosstalk between cancer cells and the TME. We examined TNBC-derived exosomes and their microRNA (miRNA) cargo in activation of normal fibroblasts (NFs) toward CAFs. We demonstrated that TNBC cell-derived exosomes increased NF collagen contraction and migration alongside CAF molecular markers. Exosome-activated fibroblasts promoted the invasion potential of normal breast epithelial cells, as assessed by an organotypic co-culture assay that resembled the in vivo context. We also investigated TNBC cell-derived exosome cargo in activating NFs to CAFs by performing small RNA sequencing. We found that the synergistic action of miR-185-5p, miR-652-5p, and miR-1246 boosted fibroblast migration and contraction, promoting specific CAF subspecialization toward a pro-migratory functional state. These data highlight the role of breast cancer cells in re-education of the TME and their contribution to tumor evolution.

Abstract P2-12-24: Exosomal metabolic signatures are associated with differential response to neoadjuvant chemotherapy in patients with breast cancer

Abstract Background: Neoadjuvant chemotherapy (NAC) is becoming the standard of care for aggressive breast cancer (BC). In patients with BC, NAC can increase eligibility for breast-conserving surgery, lower the excision volume, and assist in evaluating the disease course. NAC may shrink (residual disease, RD) or even eliminate (pathologic complete response, pCR) the tumor. Patients with RD often have a poor long-term prognosis. In contrast, pCR is associated with better long-term survival outcomes. Approximately 30% of patients with BC show pCR after NAC, while the rest exhibit RD. A critical barrier to improving NAC outcomes in patients with BC is the lack of precise molecular targets and limited understanding of the molecular mechanisms underlying differential treatment outcomes in patients with BC receiving NAC. Thus, there is an urgent clinical need to elucidate the underlying molecular regulation associated with NAC resistance. Studies have shown that cellular stress induces the release of small extracellular vesicles known as exosomes, contributing to drug resistance. In this study, we evaluated the ability of exosomal metabolic profiles to predict NAC response in BC patients. Methods: We collected blood samples of 16 patients with BC who received NAC at Grady Memorial Hospitals, USA. Further, total exosomes were isolated from plasma by an ultracentrifugation method and characterized for their concentration (number/ml), size distribution, and exosomal content. Exosome sizes were validated using nanoparticle tracking analysis. RNA, proteins, and metabolites were isolated from exosomes to perform deep multiplatform analyses (viz. miRNA seq, proteomics, and metabolomics) of exosomal cargo to identify various potential biological determinants of NAC response and evaluate their contributions to the risk of RD in patients with BC after NAC. KEGG and HMDB IDs were used for metabolomics data analyses. Various analyses viz. enriched metabolomic pathway analysis, MS peak to pathway analysis (using GSEA and Mummichog), and network explorer analysis were performed using Metaboanalyst online portal to reveal the metabolic signatures in exosomes of BC patients who exhibited pCR and RD after NAC treatment. Results: Among the 16 patients with BC who received NAC, eight patients attained pCR, and eight had RD. Interestingly, we found that patients with pCR secreted 61% fewer exosomes but had 24% larger-sized exosomes than patients with RD following NAC. Furthermore, significant differences in the loading of exosomal cargo were observed between the two groups. Based on these results, we proposed that the differential RNA, protein, and metabolite profile of exosomes might contribute to the higher drug sensitivity in patients with pCR. Our proteomics and miRNA-seq analyses of exosomes from patients with pCR and RD after NAC did not show any significant differences between the groups. However, metabolite set enrichment analysis using MetaboAnalyst revealed that compared to exosomes from patients with pCR, patients with RD were significantly enriched in various metabolic pathways. These pathways included valine, leucine, and isoleucine biosynthesis; phenylalanine metabolism; one-carbon pool folate; and inositol phosphate metabolism. Additionally, MS peak to pathway analysis revealed differential enrichment of various other metabolic pathways, particularly in patients with RD.Conclusions: These data strongly suggest that exosomal metabolic signatures are associated with differential NAC outcomes in patients with BC. We believe that in-depth studies of these metabolite signatures will give essential clues about differential NAC response in patients with BC. Citation Format: Shriya Joshi, Chakravarthy Garalapati, Shristi Bhattarai, Darshan Shimoga Chandrashekar, Sooryanarayana Varambally, Gagan Deep, Ritu Aneja. Exosomal metabolic signatures are associated with differential response to neoadjuvant chemotherapy in patients with breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-12-24.

Transcriptome analysis of cervical cancer exosomes and detection of HPVE6*I transcripts in exosomal RNA

BackgroundExosomes play a key role in cell-to-cell communication and are integral component of the tumor microenvironment. Recent observations suggest transfer of RNA through tumor-derived exosomes that can potentially translate into regulatory proteins in the recipient cells. Role of cervical cancer-derived exosomes and their transcript cargo is poorly understood.Materials and methodsThe total RNA of exosomes from HPV-positive (SiHa and HeLa) and HPV-negative (C33a) cervical cancer cell lines were extracted and the transcripts were estimated using Illumina HiSeq X. Further, validation of HPV transcripts were performed using RT-PCR.Results3099 transcripts were found to be differentially-exported in HPV-positive vs. HPV-negative exosomes (p value <0.05). Analysis of top 10 GO terms and KEGG pathways showed enrichment of transcripts belonging to axon guidance and tumor innervation in HPV-positive exosomes. Among top 20 overexpressed transcripts, EVC2, LUZP1 and ANKS1B were the most notable due to their involvement in Hh signaling, cellular migration and invasion, respectively. Further, low levels of HPV-specific reads were detected. RT-PCR validation revealed presence of E6*I splice variant of HPV18 in exosomal RNA of HeLa cells. The E6*I transcripts were consistently retained in exosomes obtained from HeLa cells undergoing 5-FU and cisplatin-induced oxidative stress.ConclusionOur data suggests the enrichment of poly-A RNA transcripts in the exosomal cargo of cervical cancer cells, which includes pro-tumorigenic cellular RNA and viral transcripts such as HPV E6, which may have clinical utility as potential exosomal biomarkers of cervical cancer.

Developing Biomarkers of Mild Traumatic Brain Injury: Promise and Progress of CNS-Derived Exosomes.

Mild traumatic brain injuries (mTBI) are common injuries across civilian and military populations. Although most individuals recover after mTBI, some individuals continue to show long-term symptoms as well as increased risk for neurodegenerative and neuropsychiatric disorders. Currently, diagnosing TBI severity relies primarily on self-report and subjective symptoms, with limited tools for diagnosis or prognosis. Brain-derived exosomes, a form of extracellular vesicle, may offer a solution for interpreting injury states by aiding in diagnosis as well as outcome prediction with relatively low patient burden. Exosomes, which are released into circulation, contain both protein and RNA cargo that can be isolated and quantified, providing a molecular window into molecular status of the exosome source. Here we examined the current literature studying the utility of exosomes, in particular neuronal- and astrocyte-derived exosomes, to identify protein and miRNA biomarkers of injury severity, trajectory, and functional outcome. Current evidence supports the potential for these emerging new tools to capture an accessible molecular window into the brain as it responds to a traumatic injury, however a number of limitations must be addressed in future studies. Most current studies are relatively small and cross sectional; prospective, longitudinal studies across injury severity, and populations are needed to track exosome cargo changes after injury. Standardized exosome isolation as well as advancement in identifying/isolating exosomes from CNS-specific tissue sources will improve mechanistic understanding of cargo changes as well as reliability of findings. Exosomes are also just beginning to be used in model systems to understand functional effects of TBI-associated cargo such as toxicity. Finally linking exosome cargo changes to objective markers of neuronal pathology and cognitive changes will be critical in validating these tools to provide insights into injury and recovery states after TBI.

Wound Healing Properties of Exosomes - A Review and Modelling of Combinatorial Analysis Strategies.

Wound healing is an elaborated process, well-regulated via cell migration and proliferation. Although the physiological basics of wound healing have been thoroughly investigated and reported, much remains to be studied. Particularly, various studies have demonstrated the immunomodulatory roles of exosomes derived from plant cells, mammalian cells, and mesenchymal stem cells (MSCs) in the healing and repairing system. The paracrine and therapeutic effects of exosomes are mainly associated with the broad exosomal cargo content comprising growth factors, cytokines, enzymes, nucleic acids, proteins, and lipid signaling molecules. Nevertheless, the functional or mechanism pathway of exosomes with reference to overall exosomal cargo remains undetermined. To date, combinatorial analysis strategies employing Database for Annotation, Visualization, and Integrated Discovery (DAVID), STRING tools, Gene Ontology (GO), Kyoto Encyclopedia of Genes, Genomes (KEGG) pathway enrichment analysis, as well as Ingenuity Pathway Analysis (IPA) have been applied in elucidating network interaction and functional pathway of exosomes. In this review paper, the application of combinatorial analysis strategies is demonstrated to better understand the therapeutic potentials of exosomes in the wound healing process. In conclusion, functional modulation of exosomal cargo for specific biological treatment is achievable, and modelling of combinatorial analysis strategies will hopefully bridge the research gap and provide a paradigm shift to regenerative processes.