Cancer Cell Exosomes Research Articles

Abstract 809: nCounter for detection of clinically relevant alterations in exosomes of non-small cell lung cancer cells and patients

Abstract Background: ALK, ROS1 and RET fusions and MET exon 14 skipping variant (METex14) are present in 10-15% of advanced non-small-cell lung cancer (NSCLC) patients and their accurate identification is critical to guide targeted therapies. In a significant number of cancer patients, the tumor tissue available is insufficient for genetic analysis and repeated tissue biopsies for monitoring the course of the disease and the emergence of resistance are not feasible. Liquid biopsies constitute the only alternative available in these cases, but NGS techniques have shown insufficient sensitivity for fusion detection in blood samples. The nCounter technology has been adapted to detect fusions and skipping variants in FFPE tumor biopsies and we aimed to validate it for exosomes. Methods: Exosomes were purified using a miRCURY kit (Qiagen) and RNA was extracted using the TRI reagent (MRC Inc). A customized nCounter panel (Nanostring) for detection ALK, ROS1 and RET fusion transcripts and MET ex14 mRNA was used with a 10-cycles preamp step. First, proof-of-concept experiments were run by testing exosomes isolated from the culture medium of cell lines. Next, we tested exosomes isolated from the blood of NSCLC patients with know genotypes. Results: nCounter fusion probes successfully detected ALK, RET and ROS1 fusion transcripts in exosomes isolated from the culture medium of the cell lines H3122 (EML4-ALKv1), H2228 (EML4-ALKv3), HCC78 (SLC34A2-ROS1) and LC/2-Ad (CCDC6-RET). Exosomes from a cell line established from a patient progressing to alectinib were also positive for EML4-ALKv1and showed high MET expression levels, while exosomes from the fusion-negative cell lines A549 and H23 tested negative. Finally, fusion transcripts were detected in exosomes purified from the blood of fusion positive NSCLC patients but not in fusion negative cases. Conclusions: nCounter can detect ALK, RET and ROS1 fusion transcripts in exosomes purified from the blood of advanced NSCLC patients Citation Format: Ana Giménez Capitán, Jill Bracht, Chung-Ying Huang, Cristina Teixidó, Noemí Reguart, Rich Boykin, Sarah Warren, Joseph M Beechem, Santiago Viteri Ramirez, Juan José García, Andrés Aguilar, Rafael Rosell Costa, Jay Gerlach, Miguel Angel Molina-Vila. nCounter for detection of clinically relevant alterations in exosomes of non-small cell lung cancer cells and patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 809.

Abstract 6352: Potential anticancer role of secretion modification region (SMR) peptide reduces mortalin and HER2-overexpressing exosomes in human HER2-positive breast cancer cells

Abstract Background/Significance: Exosomes are 30-100 nm membrane vesicles secreted by many cell types. Tumors secrete large numbers of exosomes that transport oncoproteins and immune suppressive molecules, supporting tumor growth and metastasis. The role of exosomes in intercellular signaling is shown by human epidermal growth factor receptor type 2 (HER2)-expressing breast tumors, where exosomes with HER2 stimulate tumor growth and interfere with activity of the therapeutic antibody Herceptin, which binds to the extracellular domain of HER2 and prolongs the overall survival of HER2-positive breast cancer patients. Exosome release is modulated by growth factors EGF and heregulin, HER2 receptor-activating ligands naturally present in the surrounding tumor microenvironment. The SMRwt peptide reduces secretion of HER2 exosomes; thus, it can potentially be exploited as an anticancer agent. Methods: BT-474 and HCC1569 HER2-positive breast cancer cells overexpressing HER2 and mortalin were used in these experiments. Exosome size and concentration were measured by NanoSight. Purified tumor exosomes were prepared using paramagnetic beads coated with antibodies against tumor specific proteins and confirmed by flow cytometry and electron microscopy. To identify biological functions, we cultured tumor exosomes with breast cancer cells, which increased tumor cell proliferation. To determine SMRwt peptide inhibition effects, we assayed tumor exosome secretion and cell proliferation by NanoSight, Western blot and MTT. Results: We characterized exosomes secreted by mortalin and HER2-overexpressing breast cancer cell lines and analyzed SMRwt peptide's role in reducing tumor exosome release and cell proliferation. Exosome secretion increased in HER2-overexpressing tumor cell lines HCC1596 and BT474. Exosomes from HER2-positive tumor cell-conditioned supernatants stimulated tumor cell growth and tumor exosome release and inhibited Herceptin activity on HCC1569 and BT474 cell proliferation. Addition of SMRwt peptide reduced tumor cell proliferation and tumor exosome release. Conclusions: These data show the role of HER2-positive exosomes in modulating sensitivity to Herceptin and thus, to HER2-driven tumor aggressiveness; and indicate that the SMRwt peptide has potential anticancer functions in HER2-positive HCC1569 and BT474 human breast cancer cells. The data also indicate tumor exosomes affect the proliferation of parental cells. Tumor exosomes derived from BT-474 cells and HCC1569 cells stimulated the proliferation of BT-474 cells and HCC1569 cells, suggesting a biological function for tumor exosomes. Citation Format: Ming Bo Huang. Potential anticancer role of secretion modification region (SMR) peptide reduces mortalin and HER2-overexpressing exosomes in human HER2-positive breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6352.

Towards analyzing the potential of exosomes to deliver microRNA therapeutics.

Exosome selectivity mechanisms underlying exosome-target cell interactions and the specific traits affecting their capability to communicate still remain unclear. Moreover, the capacity of exosomes to efficiently deliver their molecular cargos intracellularly needs precise investigationtowards establishing functional exosome-based delivery platforms exploitable in the clinical practice. The current study focuses on: (a) exosome production from normal MRC-5 and Vero cells growing in culture, (b) physicochemical characterization by dynamic light scattering (DLS) and cryo-transmission electron microscopy; (c) cellular uptake studies of rhodamine-labeled exosomes in normal and cancer cells, providing to exosomes either "autologous" or "heterologous" cellular delivery environments; and (d) loading exogenous Alexa Fluor 488-labeled siRNA into exosomes for the assessment of their delivering capacity by immunofluorescence in a panel of recipient cells. The data obtained thus far indicate that MRC-5 and Vero exosomes, indeed exhibit an interesting delivering profile, as promising "bio-shuttles," being pharmacologically exploitable in the context of theranostic applications.

Glutamine deprivation alters the origin and function of cancer cell exosomes

Exosomes are secreted extracellular vesicles carrying diverse molecular cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11‐positive recycling endosomal MVBs. Release of Rab11‐positive exosomes from cancer cells is increased relative to late endosomal exosomes by reducing growth regulatory Akt/mechanistic Target of Rapamycin Complex 1 (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. Vesicles produced under these conditions promote tumour cell proliferation and turnover and modulate blood vessel networks in xenograft mouse models in vivo. Their growth‐promoting activity, which is also observed in vitro, is Rab11a‐dependent, involves ERK‐MAPK‐signalling and is inhibited by antibodies against amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release from Rab11a compartments of exosomes with pro‐tumorigenic functions, which we propose promote stress‐induced tumour adaptation.

Effect of Palmitic Acid on Exosome-Mediated Secretion and Invasive Motility in Prostate Cancer Cells.

High fat consumption can enhance metastasis and decrease survival in prostate cancer, but the picture remains incomplete on the epidemiological and cell-biological level, impeding progress toward individualized recommendations in the clinic. Recent work has highlighted the role of exosomes secreted by prostate cancer cells in the progression of the disease, particularly in metastatic invasion, and also the utility of targeting these extracellular vesicles for diagnostics, as carriers of disease progression markers. Here, we investigated the question of a potential impact of the chief nutritional saturated fatty acid on the exosome secretion. Palmitic acid decreased the secretion of exosomes in human prostate cancer cells in vitro in a concentration-dependent manner. At the same time, the content of some prospective metastatic markers in the secreted exosomal fraction was also reduced, as was the ability of the cells to invade across extracellular matrix barriers. While by themselves our in vitro results imply that on the cell level, palmitic acid may be beneficial vis-à-vis the course of the disease, they also suggest that, by virtue of the decreased biomarker secretion, palmitic acid has the potential to cause unjustified deprioritization of treatment in obese and lipidemic men.

Liquid biopsy of lung cancer by deep learning and spectroscopic analysis of circulating exosomes.

e15532 Background: Lung cancer has a high mortality rate because of belated diagnosis at advanced stages beyond the treatable condition. Early detection of lung cancer can improve the survival rate. A liquid biopsy that detects tumor-related biomarkers in body fluids has a great potential for the purpose. Particularly, tumor-derived exosomes in blood have been proposed as a promising biomarker. The tumor-derived exosomes carry molecules of their parental cells; thus, they provide information about the tumor in the body. Unfortunately, exosomal markers conducive to the early detection of lung cancer are still obscure. Therefore, using the molecular fingerprint of exosomes markers can be useful to detect the tumor exosomes. Raman spectroscopy is one of the representative methods for the purpose. However, because the exosomes have a heterogeneous composition in blood, interpreting their spectroscopic signals is hard. Thus, we utilized a deep learning approach to analyze the spectroscopic signal of the exosomes for liquid biopsy of lung cancer. Methods: The basic concept was to evaluate how much the exosomes in human plasma resemble cancer cell exosomes. As a proof of concept, exosomes of 43 non-small cell lung cancer (NSCLC) adenocarcinoma patients and 20 healthy controls were isolated from plasma of peripheral blood. Also, cell exosomes were isolated from culture media of adenocarcinoma cell lines and a human pulmonary alveolar epithelial cell line. Then, the spectroscopic signals were detected using surface-enhanced Raman spectroscopy (SERS). Further, the deep learning algorithm was employed to classify the signals. Then, we calculated the relative similarity to cancerous exosomes against human plasma exosomes. Results: Our method was able to classify cancer and normal cell exosomes with 95% accuracy. Also, Raman signals of cancer patients’ exosomes were more similar to the cancer cell exosomes than those of healthy controls. Notably, the similarity was proportional to cancer stages. Importantly, our method even detected stage I patients. The area under the curve (AUC) of receiver operating characteristic (ROC) curves was 0.912 for stage I and II, and 0.910 for stage I. Conclusions: We reported a novel diagnostic method using deep learning analysis against spectroscopic signals of circulating exosomes. Our method that evaluates the similarity to cancer exosomes accurately identified lung cancer patients, even stage I with high accuracy.

Exosomal miR-21 promotes proliferation, invasion and therapy resistance of colon adenocarcinoma cells through its target PDCD4

Exosomes contain cell-specific collections of bioactive materials including proteins, lipids, and RNAs that are transported to recipient cells to exert their impacts. MicroRNAs (miRNAs) can function as tumor suppressor or oncogenic genes and miR-21 is one of the most frequently up-regulated miRNAs in solid tumors including colon cancer. The aim of this study was to investigate the role of miR-21, secreted from exosomes, in proliferation and invasion of colon cancer, along with the mechanistic details. We used a variety of biochemical techniques including ultracentrifugation-based exosome purification, electron transmission microscopy, western blot and RT-qPCR to detect the expression levels of miR-21 in exosomes purified from culture media of human colonic adenocarcinoma cell lines. We then performed functional and mechanistic studies using three colon cancer cell lines HT29, T84 and LS174 as well as the normal colon epithelial cells CRL1831. miR-21 target PDCD4 was investigated for its role in mediating miR-21 effects. Expression of miR-21 was significantly up-regulated in exosomes of colon cancer cells, compared to the normal human colon epithelial cells. Treatment of colon cancer cells with isolated exosomes or miR-21 led to an increased expression of genes involved in cell proliferation, invasion and extracellular matrix formation. miR-21 targets PDCD4, TPM1 and PTEN were down-regulated by exosomes and silencing of PDCD4 mimicked miR-21 functional effects, even the induced resistance against 5-FU. Our study suggests that targeted inhibition of exosomes, particularly those carrying miR-21, may represent a novel approach for treatment of colorectal cancer.

Exosomes derived from endoplasmic reticulum-stressed liver cancer cells enhance the expression of cytokines in macrophages via the STAT3 signaling pathway.

Previous studies have shown that endoplasmic reticulum (ER) stress serves an important role in shaping the immunosuppressive microenvironment by modulating resident immune cells. However, the communication between ER-stressed tumor cells and immune cells is not fully understood. Exosomes have been reported to play a vital role in intercellular communication. Therefore, in order to investigate the role of ER stress-related exosomes in liver cancer cells mediated macrophage function remodeling, immunohistochemical analysis, western-blotting immunofluorescence and cytokine bead array analyses were performed. The results demonstrated that glucose-regulated protein 78 (GRP78) expression was upregulated in human liver cancer tissue. Moreover, 69.09% of GRP78-positive liver cancer tissues possessed macrophages expressing CD68+ (r=0.55; P<0.001). In addition to these CD68+ macrophages, interleukin (IL)-10 and IL-6 expression levels were increased in liver cancer tissues. It was also demonstrated that exosomes released by ER-stressed HepG2 cells significantly enhanced the expression levels of several cytokines, including IL-6, monocyte chemotactic protein-1, IL-10 and tumor necrosis factor-α in macrophages. Furthermore, incubation of cells with ER stress-associated exosomes resulted inactivation of the Janus kinase 2/STAT3 pathway, and inhibition of STAT3 using S3I-201 in RAW264.7 cells significantly reduced cytokine production. Collectively, the present study identified a novel function of ER stress-associated exosomes in mediating macrophage cytokine secretion in the liver cancer microenvironment, and also indicated the potential of treating liver cancer via an ER stress-exosomal-STAT3 pathway.

MiRViz: a novel webserver application to visualize and interpret microRNA datasets.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in the regulation of major pathways in eukaryotic cells through their binding to and repression of multiple mRNAs. With high-throughput methodologies, various outcomes can be measured that produce long lists of miRNAs that are often difficult to interpret. A common question is: after differential expression or phenotypic screening of miRNA mimics, which miRNA should be chosen for further investigation? Here, we present miRViz (http://mirviz.prabi.fr/), a webserver application designed to visualize and interpret large miRNA datasets, with no need for programming skills. MiRViz has two main goals: (i) to help biologists to raise data-driven hypotheses and (ii) to share miRNA datasets in a straightforward way through publishable quality data representation, with emphasis on relevant groups of miRNAs. MiRViz can currently handle datasets from 11 eukaryotic species. We present real-case applications of miRViz, and provide both datasets and procedures to reproduce the corresponding figures. MiRViz offers rapid identification of miRNA families, as demonstrated here for the miRNA-320 family, which is significantly exported in exosomes of colon cancer cells. We also visually highlight a group of miRNAs associated with pluripotency that is particularly active in control of a breast cancer stem-cell population in culture.

Early-Stage Lung Cancer Diagnosis by Deep Learning-Based Spectroscopic Analysis of Circulating Exosomes.

Lung cancer has a high mortality rate, but an early diagnosis can contribute to a favorable prognosis. A liquid biopsy that captures and detects tumor-related biomarkers in body fluids has great potential for early-stage diagnosis. Exosomes, nanosized extracellular vesicles found in blood, have been proposed as promising biomarkers for liquid biopsy. Here, we demonstrate an accurate diagnosis of early-stage lung cancer, using deep learning-based surface-enhanced Raman spectroscopy (SERS) of the exosomes. Our approach was to explore the features of cell exosomes through deep learning and figure out the similarity in human plasma exosomes, without learning insufficient human data. The deep learning model was trained with SERS signals of exosomes derived from normal and lung cancer cell lines and could classify them with an accuracy of 95%. In 43 patients, including stage I and II cancer patients, the deep learning model predicted that plasma exosomes of 90.7% patients had higher similarity to lung cancer cell exosomes than the average of the healthy controls. Such similarity was proportional to the progression of cancer. Notably, the model predicted lung cancer with an area under the curve (AUC) of 0.912 for the whole cohort and stage I patients with an AUC of 0.910. These results suggest the great potential of the combination of exosome analysis and deep learning as a method for early-stage liquid biopsy of lung cancer.

Molecular Mechanisms of Hypoxia‐Induced Extracellular Vesicle Release

BackgroundExtracellular vesicles (EVs) are released by many cells and provide a mechanism for intercellular communication in autocrine and paracrine fashion. EVs of various sizes, namely microvesicles (ranging from 100–1000 nm) or exosomes (with sizes between 30–150 nm) are released by most eukaryotic cells. EVs are known to contain bioactive molecules including proteins lipids and nucleic acids including miRNAs and LNC RNAs that regulate gene expression and cell functions in recipient cells. For example, tumour released exosomes remodel the microenvironment allowing cancer cells to survive and thrive. Low oxygen (hypoxia) is known to increase the release of exosomes in cancer cells. Hypoxia leads to the activation of hypoxia inducible factor (HIF) transcription factors, considered master regulators of the hypoxic response. The objective of this study was to investigate whether hypoxia induced EV (exosome) release is dependent on hypoxia inducible factor (HIF) proteins.MethodsWe isolated exosomes and microvesicles by differential centrifugation of conditioned media of cells exposed to normoxia or hypoxia (1% Oxygen) for different lengths of time ranging from 1–24hrs. We determined, size distribution and concentration of vesicles by nanoparticle tracking analysis (NTA). Expression of HIF1 and HIF2 was monitored by western blot of cellular lysates. We expressed oxygen‐stable mutated HIF1 and HIF2 isoforms either alone or in combination and determined EV release by NTA. We used chemical inhibitors of dioxygenase (PHD) and von Hippel–Lindau (VHL) proteins, the two proteins involved in HIF degradation under oxygen conditions, to stabilize HIF expression in normoxic cells and analysed EV release and HIF expression. We also analysed EV release in a cell line lacking VHL and after a VHL knock‐in. Western blotting of cellular lysates was used to monitor HIFs, VHL, and VEGF proteins. NTA was used to determine EV concentration and size. ANOVA statistical analysis was performed to analyse the data.ResultsHypoxia increased the amount extracellular vesicles in the conditioned media of all cells tested. The release of small vesicles (exosomes) was most increased following the exposure to hypoxia. HIF protein expression increased steadily during the time course of hypoxia, reaching a plateau after 2hrs following exposure. Expression of mutated (oxygen stable) HIF1 and HIF2 proteins in normoxic cells, either alone or in combination, did not increase exosome release. Chemical inhibition of PHD or VHL did not affect EV release. Exosome release was not different in VHL KD or KI cells.Conclusionthese data suggest that hypoxia driven exosome release is HIF independent.Support or Funding InformationSponsored by Fundacion Ramon Areces

Small‐Molecule Sigma1 Modulator Suppresses PD‐L1‐Mediated Cancer Cell Adaptive Immune Resistance in Response to Interferon‐Gamma

BackgroundSigma1 (also known as sigma‐1 receptor) is a unique ligand‐regulated integral membrane protein enriched in the endoplasmic reticulum (ER) of cancer cells. Sigma1 acts as a multi‐functional scaffolding or chaperone protein to maintain protein and lipid homeostasis in the secretory pathway of these cells. Programmed Death‐Ligand 1 (PD‐L1) is a type I integral membrane glycoprotein that is processed through the secretory pathway. Upon reaching the cell surface, PD‐L1 acts as an inhibitory immune checkpoint molecule by binding to its cognate receptor, programmed death‐1 (PD‐1), on activated T cells, resulting in T cell inactivation, thus suppressing the anti‐tumor immune response. PD‐L1 protein expression and transport to the cell surface can be upregulated by interferon‐gamma (IFN‐γ). This mechanism is known as adaptive immune resistance, whereby cancer cells change their phenotype in response to a proinflammatory immune response. PD‐L1 is also incorporated onto cancer cell‐derived extracellular vesicles called exosomes, which can inactivate T cells within the tumor microenvironment and at distant sites. We previously reported that Sigma1 associates with PD‐ L1 and that small molecule Sigma1 inhibitors (Sigma1i) can trigger lysosomal degradation of PD‐L1.ObjectiveThe purpose of our present study is to examine the role of Sigma1 in the PD‐L1 life cycle, including interferon gamma (IFN‐γ) signal transduction, upregulation of PD‐L1 mRNA, post‐translational modification of PD‐L1, transport to the cell surface, and incorporation onto exosomes.MethodsSix cancer cell lines from multiple cancer types were assessed for PD‐L1 protein expression by immunoblot and PD‐L1 mRNA by qRT‐PCR. PD‐L1 surface expression was assessed by flow cytometry and biotinylation assays. Exosomes were isolated by ultracentrifugation and concentrations and particle size profiles were determined using nanoparticle tracking. We used Sigma1i to examine the role of Sigma1 in the PD‐L1 life cycle.ResultsWe found that Sigma1i blocks IFN‐γ induced PD‐L1 at a post‐translational step. Cotreatment of Sigma1i with IFN‐γ results in accumulation of Endoglycosidase H (EndoH) sensitive PD‐L1 in the ER and thus prevents cell‐surface expression of PD‐L1. Furthermore, Sigma1i blocked IFN‐γ‐induced incorporation of PD‐L1 on cancer cell exosomes.ConclusionWe have found that Sigma1i causes nascent PD‐L1 to be retained in the ER, preventing it from progressing through the secretory pathway, reaching the cell surface, and from being packaged onto exosomes. By reducing cell surface PD‐L1 and exosomal PD‐L1, Sigma1 lation could provide a novel approach to enhancing anti‐tumor immunity.

ADAPT identifies an ESCRT complex composition that discriminates VCaP from LNCaP prostate cancer cell exosomes

Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC–MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.

Identification of exosomal miR-455-5p and miR-1255a as therapeutic targets for breast cancer.

Accumulated evidence has demonstrated exosomes of cancer cells carry microRNAs (miRNAs) to non-malignant cells to induce metastasis. The present study aimed to identify crucial exosomal miRNAs for breast cancer (BC) using microarray data (GSE83669 and GSE50429) from Gene Expression Omnibus database, including exosomal samples from human BC cells (MCF7, MDA-MB-231) and normal mammary epithelial cell line (MCF10, MCF-10A), as well as original cell samples. Differentially expressed miRNAs (DEMs) were identified using EdgeR package, and mRNA targets were predicted using miRWalk2 database. The target genes were overlapped with BC genes from Comparative Toxicogenomics Database (CTD) to construct BC-related interaction network. Potential functions were analyzed by DAVID. The expression of crucial miRNAs and target genes were confirmed in other microarray datasets or TCGA sequencing data. Their associations with survival and other clinical characteristics were validated by Kaplan–Meier plotter and LinkedOmics database. As a result, 9 and 8 DEMs were identified to be shared in two datasets for exosomal and original cells, respectively. Further comparison showed that miR-455-5p was specifically differentially expressed in exosomes, and miR-1255a was commonly expressed in exosomal and original cells samples. miR-455-5p could interact with CDKN1B to influence cell cycle process and miR-1255a could regulate SMAD4 to participate in TGF-β signaling pathway. High expressed miR-455-5p (basal-like) and miR-1255a (overall) were associated with poor overall survival, while the high expression of their target genes was associated with excellent overall, recurrence-free or distant metastasis-free survival. In conclusion, the present study preliminarily indicates that exosomal miR-455-5p and miR-1255a may be novel therapeutic targets for BC.

Functional Passenger-Strand miRNAs in Exosomes Derived from Human Colon Cancer Cells and Their Heterogeneous Paracrine Effects.

Exosome-mediated microRNAs (miRNAs) are closely related to the occurrence, development, invasion, metastasis, therapeutic resistance, diagnosis and treatment of malignant tumors. Guide-strand miRNA and passenger-strand miRNA (miRNA*) exist in miRNA processing, but the function of passenger-strand miRNA is often overlooked. In this study, we attempted to identify functional miRNA*s in exosomes derived from human colon cancer SW620 cells. miRNA expression profiles of human normal colonic epithelial cells NCM460 and colon cancer cells SW620 were compared by high-throughput sequencing. According to the sequencing results, we defined two sets of differentially expressed miRNAs: “high in exosome and high in cell” (HEHC) and “high in exosome but low in cell” (HELC). As passenger-strand miRNAs, miR-2277-3p and miR-26b-3p, which belong to different sets, have diametrically opposite functions. MiR-2277-3p promotes proliferation, migration, and invasion of SW620 cells by targeting NUPR1L, while miR-26b-3p exerts an inhibitory effect by targeting PFDN1. Using exosomes as transport vectors, the effect of exosomes rich in miR-2277-3p on cells is consistent with the effect of liposome-transfected overexpressed miR-2277-3p, resulting in a cancer-promoting effect. However, exosomes rich in miR-26b-3p did not have a tumor suppressor effect. Further analysis revealed that exosomes rich in miR-2277-3p also had a high abundance of integrin β4. Altering the abundance of integrin β4 in exosomes changes the ability of exosomes to be taken up by cells, thereby altering the paracrine effects of exosomes. In summary, we revealed the fact that a large number of passenger-strand miRNAs exist in exosomes of colon cancer cells, these miRNAs are preliminarily categorized into two sets, and miR-2277-3p and miR-26b-3p, as representatives of each set, showed opposite functions. In addition, we revealed that integrin β4 is a marker of exosome heterogeneity in colon cancer cells, which directly correlates with the ability of exosomes to be uptaken by cells of the same kind, thus regulating the paracrine effect of exosomes.

Isolation and Profiling of Circulating Tumor-Associated Exosomes Using Extracellular Vesicular Lipid-Protein Binding Affinity Based Microfluidic Device.

Extracellular vesicles (EVs) are emerging as a potential diagnostic test for cancer. Owing to the recent advances in microfluidics, on-chip EV isolation is showing promise with respect to improved recovery rates, smaller necessary sample volumes, and shorter processing times than ultracentrifugation. Immunoaffinity-based microfluidic EV isolation using anti-CD63 is widely used; however, anti-CD63 is not specific to cancer-EVs, and some cancers secrete EVs with low expression of CD63. Alternatively, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, is shown to be expressed on the outer surface of cancer-associated EVs. A new exosome isolation microfluidic device (new ExoChip), conjugated with a PS-specific protein, to isolate cancer-associated exosomes from plasma, is presented. The device achieves 90% capture efficiency for cancer cell exosomes compared to 38% for healthy exosomes and isolates 35% more A549-derived exosomes than an anti-CD63-conjugated device. Immobilized exosomes are then easily released using Ca2+ chelation. The recovered exosomes from clinical samples are characterized by electron microscopy and western-blot analysis, revealing exosomal shapes and exosomal protein expressions. The new ExoChip facilitates the isolation of a specific subset of exosomes, allowing the exploration of the undiscovered roles of exosomes in cancer progression and metastasis.

Screening miRNAs associated with resistance gemcitabine from exosomes in A549 lung cancer cells.

PurposeTo establish a gemcitabine-resistant lung adenocarcinoma cell line, A549/G+, and to screen the differences of miRNA expression in exosomes from A549 and A549/G+ cells.MethodsA549 cells were exposed in gemcitabine until they were resistant to gemcitabine, and extracted exosomes from A549 and A549/G+. The RNAs from exosomes were subjected to miRNA expression microarray experiments.ResultsAfter 39 weeks of continuous induction, we induced drug resistance in A549 cells. The resistance index was 6. Via GeneChip miRNA 4.0 analysis, there were 446 differential miRNAs between A549 and A549/G+. Target gene prediction and pathway analysis discovered the microRNAs in the intersections may participate in drug resistance.ConclusionThese differential miRNAs help to do in-depth research to elucidate the mechanism of resistance to gemcitabine in non-small cell lung cancer.

Abstract LB-232: A novel approach for liquid biopsy by using nExo

Abstract Exosomes are small extracellular vesicles secreted from all living cells; its contents are highly varied, including proteins, small RNAs and recently discovered, genomic DNA (gDNA). The presence of nuclear content in exosomes suggests that different cell compartments can contribute to exosome cargo; however, little is known about this sub population of nuclear derived exosomes (nExo). Here, we analyzed nExo isolated from ovarian cancer cells and characterized them to explore potential clinical relevance such as cancer detection and therapeutic response. Exosomes were isolated from ovarian cancer cell lines, normal human fallopian epithelial tube cells and bio-fluids from mouse and human samples. To determine the purity of exosomes, nanoparticle tracking system, immunoblotting assay and cryo-transmission electron microscopy were used. To analyze the content of exosomes, protein mass spectrometry and imaging flow cytometry analysis was performed. First, we determined that mass spectrometry analyses for ovarian cancer cell exosomes revealed that 12.5% of proteins are derived from the cell nucleus sub-population. Additionally, using a novel imaging flow cytometry technique which enables us to identify single exosome and to quantify them, we determined that 10% of exosomes carried gDNA. Conversely, normal cells secreted only 0.2% of nExo. Then we hypothesized that the prevalence of gDNA is affected by genotoxic drugs. Treating cells with either the PARP inhibitor olaparib or topoisomerase inhibitor topotecan increased the proportion of nExo secreted from ovarian cancer cells. In addition, PARP proteins exist in ovarian cancer cell exosomes and 80.4 % of the exosome co-localized with gDNA. Using in vivo preclinical models, serum from ovarian cancer bearing mice contained a higher number of nExo than non-tumor controls, and this population was increased in response to treatment of genotoxic drugs. Furthermore, plasma and ascites from ovarian cancer patients contain around 0.3 % of nExo, which include nuclear proteins and gDNA. We demonstrate that nExo are predominantly secreted from cancer cells and could serve as an important biomarker. Although the subpopulation is relatively rare, their presence in bio-fluids isolated from cancer patients holds the potential of serving as novel biomarkers for early detection and therapeutic response to genotoxic agents. Citation Format: Akira Yokoi, Alejandro Villar-Prados, Paul Allen Oliphint, Theresa J. O'Halloran, Jason Roszik, Anil K. Sood. A novel approach for liquid biopsy by using nExo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-232.

Human pancreatic cancer cell exosomes, but not human normal cell exosomes, act as an initiator in cell transformation.

This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that major issues remain unresolved (see decision letter).

Low Long Noncoding RNA Growth Arrest-Specific Transcript 5 Expression in the Exosomes of Lung Cancer Cells Promotes Tumor Angiogenesis.

Angiogenesis plays a key role in the development and progression of lung cancer. Recent studies have found that tumor cells can stimulate angiogenesis by secreting exosomes, which contain many long noncoding RNAs (lncRNAs), some of which are important for the development of lung cancer. However, the roles and mechanisms of exosomal lncRNAs in lung cancer angiogenesis have not yet been reported. In this study, lung cancer in mice was induced by urethane; we found that growth arrest specific 5 (GAS5) was lowly expressed in the serum exosomes and lung cancer tissues of mice with lung cancer. And there was a significant positive correlation between GAS5 expression in serum exosomes and lung cancer tissues. Furthermore, GAS5 was lowly expressed in human lung cancer tissues, lung cancer cells, and cells culture supernatant exosomes. The exosomes of lung cancer cells promoted human umbilical vein endothelial cells (HUVECs) proliferation and tube formation and inhibited their apoptosis. GAS5 overexpression in lung cancer cells increased GAS5 level in cell culture supernatant exosomes. And the exosomes of lung cancer cells containing high GAS5 level inhibited HUVECs proliferation and tube formation and increased their apoptosis. In addition, we found that GAS5 competitively bound miRNA-29-3p with phosphatase and tensin homolog (PTEN), upregulating PTEN mRNA and protein expression, and inhibited level of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3K) and serine/threonine kinase 1 (AKT) phosphorylation in HUVECs. Overall, our results suggest that exosomal GAS5 could be a new therapeutic target for lung cancer which inhibits angiogenesis.