Cancer Cell Exosomes Research Articles

Abstract 2792: On-chip evaluation of cancer cell-extracellular vesicle interactions using a novel microfluidic microsystem (CellExoChip)

Abstract Introduction: Analytical methods of extracellular vesicles (EVs) is becoming an increasingly promising field of study due to them being an effective biomarker for cancers. EVs are present in various types of body fluids, which can be easily used for diagnostic purpose. Prior research has explored numerous techniques for isolating and analyzing these EVs based on their physical and biochemical properties, however, the complexity of biological samples makes conventional EV isolation difficult to exclusively extract EVs of a certain type of cell. Current downstream analysis methods lack the ability to differentiate exosomes of different origins in a sample. Recent studies suggested the presence of certain proteins in cancer exosomes that facilitates preferential uptake of the exosomes by organ-specific cancer cells, called organotropism. Using this unique property, we devised a microfluidic platform to examine the uptake of specific exosomes onto their respective progenitor cell lines, thus aiming to use the interaction for cancer diagnosis purposes. Methods: The CellExoChip was prepared following the standard PDMS-based soft lithography method. The device consists of an inlet and outlet, with dimensions of 22x22 mm with a cell capture area of about 50 mm2. The microfluidic device was functionalized by Streptavidin and the cancer cells were biotinylated with EZ-Link-NHS-Biotin to create an intense affinity between the cells and the device. We further injected dyed exosomes with different origins through the device and evaluated their specific uptake using fluorescence microscope. Results: The prepared CellExoChip successfully immobilized over 1,500 cells onto the surface and viability evaluation demonstrated that only 6.79% of the initial cells were sacrificed during the biotinylation and on-chip binding process. The average on-chip cell viability showed 75.47±7.68%. The uptake of lung cancer cell exosomes into three different cancer cell lines (lung, bladder and breast) was measured on chip. The relative uptake of lung cell exosomes by the respective lung cells was 100% compared to the bladder cells and breast cell which were 15.87% and 40.31%, respectively. We extended this specific uptake evaluation to other LungCell-LungExo combinations using H1650, A549, and in-house CTC cell line, CTCR5, and the results demonstrated the organotropism nature of the exosomes in lung cancer. Discussion and conclusion: We present a novel screening method to accurately characterize specific cancer-derived EVs using a microfluidic platform. This process bypasses the requirement of analyzing and profiling these embedded proteins prior to EV isolation. Our microfluidic device facilitates this interaction between cells and exosomes through the diagnosis process of liquid biopsy. Citation Format: Kruthi Srinivasa Raju, Zeqi Niu, Joseph Marvar, Shawn Fortna, Nna-Emeka Onukwugha, Yoon-Tae Kang, Sunitha Nagrath. On-chip evaluation of cancer cell-extracellular vesicle interactions using a novel microfluidic microsystem (CellExoChip) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2792.

Hydroxylated Graphene Porous Membrane-Based Biosensor for Exosome Isolation and Detection

Exosomes are nanoscale (about 100–200 nm) extracellular vesicles (EVs) that are secreted by cells into the extracellular space and in body liquid circulation. Recent studies on exosomes have shown that they play an important role in various biological processes and can be detected for an early diagnosis of cancer. In this work, we developed a graphene-based biosensor that could isolate exosomes of breast cancer cells from EVs and simultaneously achieve quantitative detection of exosomes. G-OH (hydroxylated graphene), a novel nanomaterial with high conductivity, good biocompatibility, and huge surface area, is quite suitable for the preparation of biosensors. First, the G-OH nanosheets were deposited to form the membrane with the micropores to screen exosomes and microvesicles in EVs. Then, rabbit α-human CD44 antibody was incubated in the membrane to capture MDA-MB-231 (MM-231) exosomes. Finally, modified mouse α-human CD9 antibodies and the third anti-horseradish peroxidase complex were incubated in the membrane, respectively. Under the optimal experimental conditions, this biosensor showed a wide linear range of 25 to 1 × 106 particles·μL–1 and a low detection limit of 9 particles·μL–1 (S/N = 3), spanning 5 orders of magnitude. The quantitative detection of the cancer cell exosomes could be achieved in a mixture of cancer cells and healthy cell exosomes.

Prognostic Biomarker NUMB Is Inhibited by Breast Cancer Cell Exosomes to Promote Breast Cancer Progression.

Objective To clarify the regulation of breast cancer cell-derived exosomes on breast cancer and the expression of the NUMB endocytic adaptor protein (NUMB) protein. Methods The exosomes of breast cancer cell line MDA-MB-231 were isolated. The exosomes were subsequently labeled with PKH67 and added to breast cancer MDA-MB-231 cells cultured in vitro. Transwell and clone formation assays were performed to detect cell migration, invasion, and clone formation. Meanwhile, Western blot and qPCR were conducted to determine the regulation of NUMB expression by exosomes in breast cancer cells. Furthermore, NUMB overexpressed lentivirus was supplemented to validate the recovery. Results The number of migrating and invasive breast cancer cells in the exosome-treated group was significantly increased compared with the control group. Moreover, the number of breast cancer cell clones in the exosome-treated group was increased than in the control group. However, the NUMB expression in breast cancer cells treated with exosomes revealed a substantial decrease, indicating that the exosomes of breast cancer cells could inhibit NUMB expression. NUMB overexpressed lentivirus supplementation markedly suppressed cell migration, invasion, and proliferation of breast cancer cells compared with exosome group. Conclusion Taken together, the exosomes of breast cancer cells could inhibit the expression of NUMB and promote the migration, invasion, and cell clone formation of breast cancer cells.

The Overexpression of TOB1 Induces Autophagy in Gastric Cancer Cells by Secreting Exosomes

We previously confirmed that transducer of ERBB2, 1 (TOB1) gene, can induce autophagy in gastric cancer cells. Studies have shown the biogenesis of exosomes overlaps with different autophagy processes, which helps to maintain the self-renewal and homeostasis of body cells. This study is aimed at verifying whether overexpressing TOB1 induces autophagy by secreting exosomes in gastric cancer cells and its underlying mechanisms. Differential ultracentrifugation was used to extracted the exosomes from the culture medium of gastric cancer cell line AGS-TOB1 ectopically overexpressing TOB1 (exo-AGS-TOB1, experimental group) and AGS-empty-vector cell line with low expression of endogenous TOB1 (exo-AGS-Vector, control group). Exosomal markers CD9 and TSG101 were determined in both the cell supernatants of exo-AGS-TOB1 and exo-AGS-Vector by Western blot. Under the transmission electron microscope (TEM), the exosomes were round and saucer-like vesicles with double-layer membrane structure, and the vesicles showed different translucency due to different contents. The peak size of exosomes detected by nanoparticle tracking analysis (NTA) was about 100 nm. When the exosomes of exo-AGS-TOB1 and exo-AGS-Vector were cocultured with TOB1 knockdown gastric cancer cell line HGC-27-TOB1-6E12 for 48 hours, the conversion of autophagy-related protein LC3-I to LC3-II in HGC-27-TOB1-6E12 gastric cancer cells cocultured with exo-AGS-TOB1 was significantly higher than that in the control group, and the ratio of LC3-II/LC3-I was statistically different (P < 0.05). More autophagosomes in HGC-27-TOB1-6E12 cells cocultured with exo-AGS-TOB1 for 48 hours were observed under TEM, while fewer autophagosomes were found in the control group. Lastly, miRNAs were differentially expressed by cell supernatant-exosomal whole transcriptome sequencing. Thus, our results provide new insights into TOB1-induced autophagy in gastric cancer.

Cancer-Associated Fibroblasts Influence the Biological Properties of Malignant Tumours via Paracrine Secretion and Exosome Production.

Cancer-associated fibroblasts (CAFs) are an essential component of the tumour microenvironment. They represent a heterogeneous group of cells that are under the control of cancer cells and can reversely influence the cancer cell population. They affect the cancer cell differentiation status, and the migration and formation of metastases. This is achieved through the production of the extracellular matrix and numerous bioactive factors. IL-6 seems to play the central role in the communication of noncancerous and cancer cells in the tumour. This review outlines the role of exosomes in cancer cells and cancer-associated fibroblasts. Available data on the exosomal cargo, which can significantly intensify interactions in the tumour, are summarised. The role of exosomes as mediators of the dialogue between cancer cells and cancer-associated fibroblasts is discussed together with their therapeutic relevance. The functional unity of the paracrine- and exosome-mediated communication of cancer cells with the tumour microenvironment represented by CAFs is worthy of attention.

Lysophosphatidylcholine acyltransferase 1 is involved in the regulation of exosome secretion and uptake in colorectal cancer cells

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a phospholipid acyltransferase that promotes phospholipid synthesis and plasma membrane reconstruction. Exosomes play an important role in tumor metastasis. The release and uptake of exosomes are key steps of their functions and depend on plasma membrane fusion and plasma membrane receptors, respectively. The purpose of this study was to explore whether LPCAT1-induced plasma membrane remodeling would change the secretion and uptake behavior of exosomes in tumor cells. We first confirmed the abnormally high expression of LPCAT1 in colorectal cancer cells by quantitative real-time PCR (qPCR) and Western blot analysis. Then, SW620 cells were used as exosome source cells, and SW480 cells were used as exosome receiver cells. Exosomes from SW620 cells could effectively promote the migration of SW480 cells. When LPCAT1 expression was reduced via siRNA knockdown in the source cells, the secretion of exosomes was downregulated, thus weakening the pro-migratory effects of exosomes on target cells. Conversely, when LPCAT1 was knocked down in target cells, the uptake of exosomes in target cells also decreased sharply. These results undoubtedly revealed that LPCAT1 is functionally associated with the release and internalization of exosomes in colorectal cancer cells and could affect the paracrine effects of exosomes, preliminarily extending the classical metabolic function of LPCAT1 to exosome-related pathways.

Lysophosphatidylcholine acyltransferase 1 is involved in the regulation of exosome secretion and uptake in colorectal cancer cells

Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is a phospholipid acyltransferase that promotes phospholipid synthesis and plasma membrane reconstruction. Exosomes play an important role in tumor metastasis. The release and uptake of exosomes are key steps of their functions and depend on plasma membrane fusion and plasma membrane receptors, respectively. The purpose of this study was to explore whether LPCAT1-induced plasma membrane remodeling would change the secretion and uptake behavior of exosomes in tumor cells. We first confirmed the abnormally high expression of LPCAT1 in colorectal cancer cells by quantitative real-time PCR (qPCR) and Western blot analysis. Then, SW620 cells were used as exosome source cells, and SW480 cells were used as exosome receiver cells. Exosomes from SW620 cells could effectively promote the migration of SW480 cells. When LPCAT1 expression was reduced via siRNA knockdown in the source cells, the secretion of exosomes was downregulated, thus weakening the pro-migratory effects of exosomes on target cells. Conversely, when LPCAT1 was knocked down in target cells, the uptake of exosomes in target cells also decreased sharply. These results undoubtedly revealed that LPCAT1 is functionally associated with the release and internalization of exosomes in colorectal cancer cells and could affect the paracrine effects of exosomes, preliminarily extending the classical metabolic function of LPCAT1 to exosome-related pathways.

Cervical cancer-derived exosomal miR-663b promotes angiogenesis by inhibiting vinculin expression in vascular endothelial cells

BackgroundAngiogenesis provides essential nutrients and oxygen for tumor growth and has become the main mechanism of tumor invasion and metastasis. Exosomes are nanoscale membrane vesicles containing proteins, lipids, mRNA and microRNA (miRNA), which mediate intercellular communication and play an important role in tumor progression. Accumulated evidence indicates that tumor-derived exosomal miRNAs participate in the tumor microenvironment and promote angiogenesis.MethodsBioinformatic target prediction and dual luciferase reporter assays were performed to identify the binding site between miR-663b and the 3′-UTR of vinculin (VCL). VCL overexpression lentivirus and miR-663b overexpression/inhibition lentivirus were used to create a VCL overexpression model and miR-663b overexpression/inhibition model in-vitro. Immunohistochemistry (IHC) assays and western blot assays were used to detect protein expression. Exosome-cell cocultures, wound healing assays, tube formation assays and transwell assays were used to measure the migration and tube formation ability of vascular endothelial cells [human umbilical vein endothelial cells (HUVECs)]. siRNA targeted VCL was used to knockdown VCL.ResultsIn the present study, we found that miR-663b was elevated in cervical cancer tissue and exosomes. miR-663b could bind the 3′-UTR of VCL and inhibit its expression. VCL is downregulated in cervical cancer, and decreased VCL has a negative correlation with a high level of miR-663b. Further studies demonstrated that exosomes secreted by cervical cancer cells can deliver miR-663b to HUVECs and inhibit the expression of VCL, thereby promoting angiogenesis and tumor growth.ConclusionsmiR-663b derived from cancer cell exosomes acts as a driving factor for angiogenesis and a potential target of antiangiogenic therapy in cervical cancer. Our findings illustrated a new signaling pathway, including exosomes, miRNAs and target genes, which provides potential targets for antiangiogenic therapy.

DAL-1/4.1B promotes the uptake of exosomes in lung cancer cells via Heparan Sulfate Proteoglycan 2 (HSPG2).

DAL-1/4.1B is frequently absent in lung cancer tissues, which is significantly related to the occurrence and development of lung cancer. In this research, we found that DAL-1/4.1B affected the uptake of exosomes by lung cancer cells. When the expression of DAL-1/4.1B increased and decreased, the ability of exosome uptake enhanced and attenuated correspondingly. And we found that when cells were treated with different vesicles uptake inhibitors (chlorpromazine, methyl-β-cyclodextrin (MβCD), cytochalasin D, chloroquine and heparin) and heparinase (HSPE), only heparin and HSPE counteracted the uptake enhancement effect caused by DAL-1/4.1B. Therefore, we speculated that DAL-1/4.1B might promote the uptake of exosomes through the heparan sulfate proteoglycans (HSPGs) pathway. After screening the expression of HSPGs and HSPE in H292 cells, the expression of heparan sulfate proteoglycan 2 (HSPG2) increased with overexpression of DAL-1/4.1B and decreased with knockdown of DAL-1/4.1B. Meanwhile, exosome uptake decreased with HSPG2 knockdown in H292 and DAL-1/4.1B-overexpressing H292 cells. Moreover, knockdown of DAL-1/4.1B and HSPG2 in lung cancer A549 cells resulted in a similar decrease in exosome uptake, and the expression of HSPG2 was also decreased with DAL-1/4.1B knockdown. These results indicated that HSPG2 directly affected the uptake of exosomes, while DAL-1/4.1B positively affected the expression of HSPG2. Therefore, DAL-1/4.1B may promote cellular adhesion and inhibit migration in cancer cells.

Multicolor Covalent Organic Framework-DNA Nanoprobe for Fluorescence Imaging of Biomarkers with Different Locations in Living Cells.

Precisely detecting biomarkers in living systems holds tremendous promise for disease diagnosis and monitoring. Herein, we developed a covalent organic framework (COF)-based tricolor fluorescent nanoprobe for simultaneously imaging biomarkers with different spatial locations in living cells. Briefly, a TAMRA-labeled survivin mRNA antisense nucleotide and a Cy5-labeled transmembrane glycoprotein mucin 1 (MUC1) aptamer were adsorbed on a nanoscale fluorescent COF. To enhance the interactions between COF nanoparticles (NPs) and nucleic acid molecules, a freezing method was employed for improving the nucleic acid loading density and ensuring detection performance. The fluorescence signals of dyes on DNAs were first quenched by the COF NPs. Internalization and distribution of the nanoprobes can be real-time visualized by the autofluorescence of COF NPs. In living cells, recognition between MUC1 with MUC1 aptamers causes fluorescence signal recovery of Cy5, while hybridization between survivin mRNA and its antisense DNA induces the signal recovery of TAMRA. Therefore, this COF-based multicolor nanoprobe could be employed for visualizing MUC1 on the cell membrane and survivin mRNA in the cytoplasm. Cancer cell-specific diagnostic imaging and monitoring of the process of cancer cell exosomes infecting normal cells using the nanoprobe were achieved. This work not only offers a versatile nanoprobe for bioanalysis but also provides new insights for developing novel COF-based nanoprobes.

Exosomal lncRNA SCIRT/miR-665 Transferring Promotes Lung Cancer Cell Metastasis through the Inhibition of HEYL.

Lung cancer remains the leading cause of cancer-related death worldwide. Recently, extracellular vesicles such as exosomes have attracted considerable interest both as a source for theranostic biomarkers and an essential participant in lung cancer progression. However, how specific exosomal cargos, such as noncoding RNAs, are selectively packaged into exosomes and promote lung cancer progression remains unclear. In this study, we identified miR-665 as the most elevated exosomal miRNA from both non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) patients. We further demonstrated that lncRNA SCIRT was also increased in cancer cell exosomes and may facilitate the exosomal loading of miR-665 with the help of hnRNPA1. As a consequence, exosomal miR-665 promoted lung cancer cell invasion and migration by targeting Notch downstream transcription factor HEYL. In addition, we found that miR-665 and SCIRT were significantly upregulated in tumor tissue and plasma of patients with lung cancer, and both of them showed increased expression in metastatic disease samples. Our findings suggest that the exosomal transferring of miR-665 and SCIRT is a functional and mechanism-driven pathway that contributes to cancer progression and, thus, may provide novel diagnostic and therapeutic targets for lung cancer.

Drug Repurposing of Pantoprazole and Vitamin C Targeting Tumor Microenvironment Conditions Improves Anticancer Effect in Metastatic Castration-Resistant Prostate Cancer.

The effective and economical therapeutic strategy for metastatic castration-resistant prostate cancer (mCRPC) is still requested from patients, who are not available for Lu-177 or Ra-223 treatment. Drug repurposing as a cost-effective and time-saving alternative to traditional drug development has been increasingly discussed. Proton pump inhibitors (PPIs) such as pantroprazole, which are commonly used as antacids, have also been shown to be effective in cancer chemoprevention via induction of apoptosis in multiple cancer cell lines. Vitamin C is an essential micronutrient for human body, has been proposed as a potential anti-cancer agent. In this context, have we investigated the combination of vitamin C and pantoprazole for the management of metastatic castration-resistant prostate cancer (mCRPC). Six chosen human adenocarcinoma cell lines were used to investigate the influence of pantoprazole on the microenvironment of cancer cells (extracellular pH and production of exosomes). Tumor growth and tumor 18F-FDG uptake in PC3 xenografts were analyzed following varied treatment. Our in vitro Results have suggested that pantoprazole enhanced the cytotoxic activity of vitamin C by regulating pH values and production of exosomes in cancer cells. Moreover, the synergistic effect of pantoprazole and vitamin C was pH-dependent since pantoprazole was more effective at a slightly acidic pH. In vivo, the combined treatment using pantoprazole and vitamin C produced better therapeutic outcomes than treatment with vitamin C or pantoprazole alone, as demonstrated via tumor growth and uptake of 18F-FDG. Therefore, we suggest that pantoprazole combined with vitamin C could be as a possible strategy to manage mCRPC.

Circular RNA circ-MMP11 Contributes to Lapatinib Resistance of Breast Cancer Cells by Regulating the miR-153-3p/ANLN Axis.

BackgroundDrug-resistance is a major obstacle to the treatment of breast cancer. Circular RNA (circRNA) circ-MMP11 has been reported to be promoting the progression of breast cancer. This study is designed to explore the role and mechanism of circ-MMP11 in lapatinib resistance in breast cancer.MethodsCirc-MMP11, microRNA-153-3p (miR-153-3p), and Anillin (ANLN) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability, number of colonies, apoptosis, migration, and invasion were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation, flow cytometry, and transwell assays, respectively. Exosomes were exerted and detected by differential centrifugation and a transmission electron microscope. The protein levels of CD63, CD9, and ANLN were assessed by western blot assay. The binding relationship between miR-153-3p and circ-MMP11 or ANLN was predicted by circinteractome or starbase, and then verified by a dual-luciferase reporter assay and RNA pull-down assay. The biological role of circ-MMP11 on breast cancer tumor growth and drug resistance was detected by the xenograft tumor model in vivo. ResultsCirc-MMP11 and ANLN were highly expressed, and miR-153-3p was decreased in LR breast cancer tissues and cells. Circ-MMP11 could be transported by exosomes. Furthermore, circ-MMP11 knockdown promoted lapatinib sensitivity by repressing cell viability, colony number, migration, invasion, and boosting apoptosis in LR breast cancer cells. Circ-MMP11 deficiency improved the drug sensitivity of breast cancer in vivo. Mechanically, circ-MMP11 could regulate ANLN expression through sponging miR-153-3p.ConclusionCirc-MMP11 could be transferred by exosomes in breast cancer cells. And circ-MMP11 functioned as a sponge of miR-153-3p to regulate ANLN expression, thereby promoting lapatinib resistance in breast cancer cells, providing therapeutic targets for the treatment of breast cancer.

Secretion of Mutant DNA and mRNA by the Exosomes of Breast Cancer Cells.

Exosomes are the small vesicles that are secreted by different types of normal and tumour cells and can incorporate and transfer their cargo to the recipient cells. The main goal of the present work was to study the tumour exosomes’ ability to accumulate the parent mutant DNA or RNA transcripts with their following transfer to the surrounding cells. The experiments were performed on the MCF7 breast cancer cells that are characterized by the unique coding mutation in the PIK3CA gene. Using two independent methods, Sanger sequencing and allele-specific real-time PCR, we revealed the presence of the fragments of the mutant DNA and RNA transcripts in the exosomes secreted by the MCF7 cells. Furthermore, we demonstrated the MCF7 exosomes’ ability to incorporate into the heterologous MDA-MB-231 breast cancer cells supporting the possible transferring of the exosomal cargo into the recipient cells. Sanger sequencing of the DNA from MDA-MB-231 cells (originally bearing a wild type of PIK3CA) treated with MCF7 exosomes showed no detectable amount of mutant DNA or RNA; however, using allele-specific real-time PCR, we revealed a minor signal from amplification of a mutant allele, showing a slight increase of mutant DNA in the exosome-treated MDA-MB-231 cells. The results demonstrate the exosome-mediated secretion of the fragments of mutant DNA and mRNA by the cancer cells and the exosomes’ ability to transfer their cargo into the heterologous cells.

Nanogenomics and Artificial Intelligence: A Dynamic Duo for the Fight Against Breast Cancer.

Application software is utilized to aid in the diagnosis of breast cancer. Yet, recent advances in artificial intelligence (AI) are addressing challenges related to the detection, classification, and monitoring of different types of tumors. AI can apply deep learning algorithms to perform automated analysis on mammographic or histologic examinations. Large volume of data generated by digitalized mammogram or whole-slide images can be interoperated through advanced machine learning. This enables fast evaluation of every tissue patch on an image, resulting in a quicker more sensitivity, and more reproducible diagnoses compared to human performance. On the other hand, cancer cell-exosomes which are extracellular vesicles released by cancer cells into the blood circulation, are being explored as cancer biomarker. Recent studies on cancer-exosome-content revealed that the encapsulated miRNA and other biomolecules are indicative of tumor sub-type, possible metastasis and prognosis. Thus, theoretically, through nanogenomicas, a profile of each breast tumor sub-type, estrogen receptor status, and potential metastasis site can be constructed. Then, a laboratory instrument, fitted with an AI program, can be used to diagnose suspected patients by matching their sera miRNA and biomolecules composition with the available template profiles. In this paper, we discuss the advantages of establishing a nanogenomics-AI-based breast cancer diagnostic approach, compared to the gold standard radiology or histology based approaches that are currently being adapted to AI. Also, we discuss the advantages of building the diagnostic and prognostic biomolecular profiles for breast cancers based on the exosome encapsulated content, rather than the free circulating miRNA and other biomolecules.

12P DNMT1/3-targeting microRNAs are over-expressed in the exosomes of estrogen-resistant breast cancer cells

12P DNMT1/3-targeting microRNAs are over-expressed in the exosomes of estrogen-resistant breast cancer cells

Natural Killer Cell Phenotype and Functionality Affected by Exposure to Extracellular Survivin and Lymphoma-Derived Exosomes.

The inherent abilities of natural killer (NK) cells to recognize and kill target cells place them among the first cells with the ability to recognize and destroy infected or transformed cells. Cancer cells, however, have mechanisms by which they can inhibit the surveillance and cytotoxic abilities of NK cells with one believed mechanism for this: their ability to release exosomes. Exosomes are vesicles that are found in abundance in the tumor microenvironment that can modulate intercellular communication and thus enhance tumor malignancy. Recently, our lab has found cancer cell exosomes to contain the inhibitor of apoptosis (IAP) protein survivin to be associated with decreased immune response in lymphocytes and cellular death. The purpose of this study was to explore the effect of survivin and lymphoma-derived survivin-containing exosomes on the immune functions of NK cells. NK cells were obtained from the peripheral blood of healthy donors and treated with pure survivin protein or exosomes from two lymphoma cell lines, DLCL2 and FSCCL. RNA was isolated from NK cell samples for measurement by PCR, and intracellular flow cytometry was used to determine protein expression. Degranulation capacity, cytotoxicity, and natural killer group 2D receptor (NKG2D) levels were also assessed. Lymphoma exosomes were examined for size and protein content. This study established that these lymphoma exosomes contained survivin and FasL but were negative for MHC class I-related chains (MIC)/B (MICA/B) and TGF-β. Treatment with exosomes did not significantly alter NK cell functionality, but extracellular survivin was seen to decrease natural killer group 2D receptor (NKG2D) levels and the intracellular protein levels of perforin, granzyme B, TNF-α, and IFN-γ.

Changes in phospholipid metabolism in exosomes of hormone-sensitive and hormone-resistant prostate cancer cells.

Background: To explore the changes in lipids in exosomes of hormone-sensitive and hormone-resistant prostate cancer cells and develop an inexpensive and rapid technique for screening lipid-based biomarkers of prostate cancer.Methods: Exosomes were extracted from LnCap, PC3 and DU-145 cells, and their lipid composition was analyzed quantitatively using high-throughput mass spectrometry. Exosomes released by LnCap prostate cancer cells were also purified using a modified procedure based on polyethylene glycol (PEG) precipitation.Results: Exosomes extracted from LnCap cells contained higher proportions of phosphatidyl choline, phosphatidyl ethanolamine and phosphatidyl inositol lipids than whole LnCap cells. Lysophosphatidylcholine, a harmful intermediate product of phosphatidylcholine metabolism in vivo, was not found in LnCap cells but in exosomes. Phospholipids were different in exosomes from LnCap, PC3 and DU-145 prostate cancer cells. The main lipid pathways involved, i.e., glycerophospholipid metabolism, autophagy, and ferroptosis pathways, were also different in these cells. Exosomes isolated by this modified PEG precipitation technique were similar in purity to those obtained using a commercial kit.Conclusions: This study demonstrates that phosphatidylcholine and its harmful product lysophosphatidylcholine may play important roles in hormone-sensitive prostate cancer. Phospholipid exosome metabolism was changed in hormone-sensitive and hormone-resistant prostate cancer cells. The LPC, lipid pathway of autophagy and ferroptosis may act as therapeutic targets. The possibility of purifying prostate cancer cell exosomes using modified PEG precipitation is suitable for cancer screening.

Platinum Nanoparticles Enhance Exosome Release in Human Lung Epithelial Adenocarcinoma Cancer Cells (A549): Oxidative Stress and the Ceramide Pathway are Key Players.

BackgroundSeveral studies have demonstrated various molecular mechanisms involved in the biogenesis and release of exosomes. However, how external stimuli, such as platinum nanoparticles (PtNPs), induces the biogenesis and release of exosomes remains unclear. To address this, PtNPs were synthesized using lutein to examine their effect on the biogenesis and release of exosomes in human lung epithelial adenocarcinoma cancer cells (A549).MethodsThe size and concentration of isolated exosomes were characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis system (NTA). Morphology and structure of exosomes were examined using scanning electron microscopy and transmission electron microscopy (TEM), respectively. Quantification of exosomes were analyzed by EXOCETTM assay and fluorescence polarization (FP). The expression of typical markers of exosomes were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsA549 cells cultured with PtNPs enhance exosome secretion by altering various physiological processes. Interestingly, A549 cells treated with PtNPs increases total protein concentration, biogenesis and release of exosomes associated with PtNPs-induced oxidative stress. GW4869 inhibits PtNPs induced biogenesis and release of exosomes and also acetylcholinesterase (AChE), neutral sphingomyelinase activity (n-SMase), and exosome counts. A549 cells pre-treated with N-acetylcysteine (NAC) significantly inhibited PtNPs induced exosome biogenesis and release. These findings confirmed that PtNPs-induced exosome release was due to the induction of oxidative stress and the ceramide pathway. These factors enhanced exosome biogenesis and release and may be useful in understanding the mechanism of exosome formation, release, and function.ConclusionPtNPs provide a promising agent to increase exosome production in A549 cells. These findings offer novel strategies for enhancing exosome release, which can be applied in the treatment and prevention of cancer. Importantly, this is the first study, to our knowledge, showing that PtNPs stimulate exosome biogenesis by inducing oxidative stress and the ceramide pathway.

Flipping an exosome switch

Nutrient stress changes the biogenesis and biological activity of exosomes in cancer cells.