Effects Of Tumor-derived Exosomes Research Articles

Nanoscopic characterization of hepatocytes treated with normoxic and hypoxic tumor-derived exosomes

Hypoxia is a key factor in tumor microenvironments. Tumor-derived exosomes under hypoxia have their functions of communication between local and remote cells, and play an important role in tumor growth and metastasis. However, the effect of tumor-derived exosomes on cell structures and functions under hypoxia is unknown. In this work, the effects of exosomes derived from hepatocellular carcinoma cells (HCC-LM3) under normoxia (N-exos) and hypoxia (H-exos) environments on the biological and physical properties of target cells were stduied. The N-exos promoted the proliferation of hepatocytes (HL-7702) at 1.5 mg/mL, while the H-exos promoted the proliferation of hepatocytes at a lower concentration (1.0 mg/mL). After the cells cultured with the same concentrations of N-exos and H-exos for different time periods, the cell migration was enhanced. The stress fibers of the cells became loose and the cytoskeleton was rearranged, which were time dependent. The changes in morphological and mechanical parameters of the HL-7702 cells were detected by atomic force microscopy (AFM). The results showed that the cell edges became irregular and the filopodia were increased versus the exosomes treatment time. The heights and elastic moduli of cells were reduced. Compared with N-exos, H-exos had a more significant effect on the biological and physical properties of target cells. The results provide a method for studying how tumor-derived exosomes affect the interaction between tumor cells and their hypoxic microenvironment.

Gastric cancer-derived exosomes induce PD-L1 expression on human bone marrow mesenchymal stem cells through the AKT-c-Myc signal axis

Mesenchymal stem cells (MSCs) show high expression of PD-L1. Exosomes can transmit signals between tumor cells and stromal cells; however, the effect of tumor-derived exosomes on stromal cell signaling pathways is unclear. The expression of PD-L1 in bone marrow mesenchymal stem cells (BMMSCs) was detected by western blot and RT-PCR. The effects of gastric cancer cell line-derived exosomes on BMMSC migration and the effects of conditioned medium from educated BMMSCs on tumor cell migration were analyzed. Our results demonstrate that exosomes derived from gastric cancer cell lines activate the AKT/c-Myc pathway of BMMSCs and upregulate PD-L1 expression on BMMSCs. Moreover, the pro-migration effect of exosomes from gastric cancer cell lines on BMMSCs is dependent on PD-L1. The upregulation of PD-L1 on BMMSCs promoted the migration of gastric cancer cells. In conclusion, tumor-derived exosomes could upregulate the expression of PD-L1 on BMMSCs to promote tumor migration. Our results provide a new therapeutic avenue in gastric cancer.

A study on the effects of tumor-derived exosomes on hepatoma cells and hepatocytes by atomic force microscopy.

Tumor-derived exosomes (exos) are closely related to the occurrence, development and treatment of tumors. However, it is not clear how the exosomes affect the physical properties, which lead to the deterioration of the target cells. In this paper, atomic force microscopy (AFM) was used to study the effects of exosomes in HCC-LM3 cells and other cells (SMMC-7721 and HL-7702). The results showed that the HCC-LM3-exos (the exosomes secreted by HCC-LM3 cells, 50 μg mL-1) significantly promoted the proliferation and migration of HCC-LM3 cells. HCC-LM3-exos also promoted the proliferation and migration of SMMC-7721 and HL-7702 cells at 1000 and 1500 μg mL-1, respectively. With an increase in time and concentration, the proliferation effect was more significant. On comparing the mechanical properties of the three types of cells (HCC-LM3, SMMC-7721 and HL-7702 cells), the degradation degree and migration ability of the cells were from high to low in the above order. In turn, the surface roughness of the cells decreased, and adhesion and elastic modulus increased. With an increase in treatment time, surface roughness increased, while adhesion and elastic modulus decreased. These suggested that the HCC-LM3-exos could change the mechanical properties of cells, leading to their deterioration, and enhance their migration and invasion ability. In this paper, the effects of exosomes were analyzed from the perspective of the physical parameters of cells, which provide a new idea to study cancer metastasis and prognosis.

Isolation and characterization of exosomes from IDH mutant gliomas.

152 Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are found in > 70% of intermediate grade gliomas. Gain-of-function mutations in IDH1 promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Tumor-derived exosomes (TEX) accumulate in the tumor microenvironment (TME) and in body fluids of patients. TEX serve as a communication system between tumor and normal cells. Circulating TEX carry a complex molecular and genetic cargo and interfere with functions of immune cells. This study characterizes TEX produced by IDH mutant glioma spheres and TEX from plasma of patients with IDH mutant gliomas. Methods: TEX produced by patient-derived IDH1(mut) or IDH1(wt) glioma spheres or TEX in patient’s plasma were isolated by mini size exclusion chromatography (mini-SEC). TEX morphology, size, numbers and molecular profile were characterized and the crossing of the blood-brain barrier (BBB) and biodistribution was investigated by in vivo imaging of TEX. Interactions of TEX with endothelial cells, astrocytes and immune cells were demonstrated by confocal microscopy. Results: TEX isolated from supernatants of IDH1(mut) or IDH1(wt) glioma spheres by mini-SEC carried TSG101 and showed the typical size and vesicular morphology of exosomes. BCA protein assays and qNano analysis revealed an elevated exosome production by IDH mutant cells ( p< 0.01). TEX carried immunosuppressive proteins (FasL, CTLA-4 and TRAIL) and IDH mutant exosomes carried higher levels of these proteins and of adenosine pathway components CD39 and CD73. Labeled TEX injected in the white matter of mice crossed the BBB and were detectable in distant organs 24h after injection. TEX were rapidly internalized by endothelial cells and astrocytes, but not by T cells, which interacted with TEX by surface signaling. These results were confirmed by investigating TEX isolated from glioma patient’s plasma. Conclusions: The data suggest that TEX play an important role in IDH mutant gliomas and drive tumor progression in part by inducing suppression of immune cells. Future efforts will focus on characterizing immunosuppressive effects of TEX derived from IDH mutant cells vs gliomas without IDH mutations.

Tumor-derived exosomal microRNAs and proteins as modulators of macrophage function.

Tumor cells are able to modify their surrounding microenvironment by transmitting bioactive molecules via exosomes. In exosomes, proteins and nucleic acids that can be taken up by surrounding cells have been identified and modulate their functions. Tumor microenvironment consists of different cells such as macrophages. Tumors-associated macrophages (TAMs) express M2 phenotype and affect many processes including tumor initiation, angiogenesis, and metastasis. It has been demonstrated that a high number of TAMs is associated with poor prognosis of cancers. The contents of tumor-derived exosomes such as microRNAs and proteins induce macrophages to M2-like polarization to support tumor growth. Herein, we review the most recent studies on the effect of tumor-derived exosomes on macrophage polarization and function in different types of cancers.

Abstract 127: Exosomes derived from head and neck cancer enhance the cytotoxicities of NK cells by NAP1-IRF-3 pathway

Abstract Objective: To examine the effect of tumor-derived exosomes (TEXs) from oral cancer on natural killer (NK) cells and to explore the underlying mechanism. Materials and Methods: TEXs were isolated from the medium supernatant of oral cancer (OC) cells using ultrafiltration and affinity chromatography and were identified using electronic microscopy, nanoparticle tracking analysis and immunoblotting. The effects of TEXs on NK cells were analyzed using laser scanning confocal microscopy and several functional assays of NK cells. To explore the mechanism, antibody array, protein mass spectrometry and RNA interference were adopted. Results: The particles isolated from the OC cells were identified as exosomes with satisfactory morphology, concentration and purity. The TEXs were internalized by the NK cells and then promoted the biologic functions of these cells, including proliferation, cytotoxicity and the release of perforin and granzyme M. Furthermore, we found that the TEXs increased the expression of interferon regulatory factor 3 (IRF-3) and phosphorylated IRF-3 by antibody array, which drove the expression of the type I interferon (IFN) gene and the chemokine (C-X-C motif) ligand (CXCL) genes, thereby promoting the function of NK cells. We also found that NF-κB-activating kinase-associated protein 1 (NAP1), an upstream activator of IRF-3, was enriched in TEXs, and treatment with TEXs increased NAP1 in NK cells. Importantly, NAP1-depleted TEXs obtained from OC cells dramatically weakened the influence of the TEXs on NK cells. Conclusion: Our findings reveal a previously unrecognized function of exosome derived from oral cancer cells in enhancing the cytotoxicity of NK cells by NAP1- IRF-3 pathway. This work was supported by the National Program on Key Research Project of China (2016YFC0902700). Citation Format: Wantao Chen, Yingnan Wang, Jianjun Zhang. Exosomes derived from head and neck cancer enhance the cytotoxicities of NK cells by NAP1-IRF-3 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 127.

The role of tumor-derived exosomes in the formation of a pre-metastatic niche in cancer

Abstract Purpose Determine if tumor derived exosomes (TDE) can drive cancer progression through polarization of immunosuppressive cell phenotypes capable of restricting αβ T cell functioning. Preliminary data shows that exosome stimulation differentially upregulates PD-L1 (programmed-death-ligand-1) expression on peritoneal macrophages and lung resident γδ T cells while also upregulating PD-1 expression on αβ T cells. The ultimate goal is to assess whether elimination of TDE can synergize with current PD-1/PD-L1 treatments by removing antagonizing factors and improving clinical outcomes. Methods TDE were isolated using serial ultracentrifugation from Lewis Lung Carcinoma cells. Exosome stimulated peritoneal macrophages and γδ T cells were co-cultured with ova-transgenic –I (CD8+) T-cells to assay for suppressive function. Anti-PD-1 antibody was added to the culture to confirm that the PD-1/PDL-1 axis mediates the immune suppression. The effect of TDE on γδ T cell metabolism was also investigated. Lastly, human peripheral blood samples from lung cancer and anti-PD-1 treated lung cancer patients were compared via flow cytometry for γδ/PD-L1 expression profiling. Results The results suggest that TDE play a role in tumor metastasis by polarizing γδ T cells and macrophages to an immunosuppressive PD-L1+ phenotype capable of restricting αβ T cell effector functioning. Discussion/Conclusion TDE could be directly antagonizing the effects of immune checkpoint therapies. Therefore, future experiments will focus on eliminating exosome secretion by using siRNARab27 inhibition and the small drug compound, cambinol, concurrently with anti-PD-1 or anti-PD-L1 monoclonal antibody in efforts to increase progression free survival.

The effect of tumor-derived exosomes on immune regulation and cancer immunotherapy.

Tumor-derived exosomes (TEX) carry both immunosuppressive and immunostimulatory receptor/ligands that in part mimic the profiles of the parent tumor cells. Operating as an intercellular communication system, TEX deliver protumor or antitumor signals to immune and nonimmune cells reprogramming their functions. Mechanisms responsible for cellular reprogramming include cell surface signaling and/or uptake of TEX by recipient cells. Once internalized, TEX transfer mRNA, miRNA and proteins that promote transcriptional/translational activities. TEX-mediated signaling is contextual and, in the tumor microenvironment, TEX largely mediate suppression. TEX may interfere with immune therapies either by sequestration of therapeutic antibodies or elimination of vaccine-induced or adoptively-transferred immune effector cells. TEX are emerging as an ubiquitous subcellular system regulating immune responses in patients with cancer.

Tumor exosome-mediated promotion of adhesion to mesothelial cells in gastric cancer cells

BackgroundPeritoneal metastasis consists of a highly complex series of steps, and the details of the underlying molecular mechanism remain largely unclear. In this study, the effects of tumor-derived exosomes (TEX) on the progression of gastric cancers were investigated in peritoneal metastasis.ResultsTEX were internalized in both mesothelial and gastric cancer cells in a cellular origin non-specific manner. Internalization of TEX into mesothelial cells promoted significant adhesion between mesothelial and gastric cancer cells, and TEX internalization into gastric cancer cells significantly promoted migratory ability, while internalization of mesothelial cell-derived exosomes did not. Expression of adhesion-related molecules, such as fibronectin 1 (FN1) and laminin gamma 1 (LAMC1), were increased in mesothelial cells after internalization of TEX from gastric cancer cell line and malignant pleural effusion.MethodsTEX were extracted from cell-conditioned medium by ultracentrifugation. The effects of TEX on the malignant potential of gastric cancer were investigated in adhesion, invasion, and proliferation assays. PCR array as well as western blotting were performed to determine the underlying molecular mechanisms. The molecular changes in mesothelial cell after internalization of TEX derived from malignant pleural effusion were also confirmed.ConclusionsTEX may play a critical role in the development of peritoneal metastasis of gastric cancer, which may be partially due to inducing increased expression of adhesion molecules in mesothelial cells.

Pancreatic cancer derived exosomes regulate the expression of TLR4 in dendritic cells via miR-203

MicroRNAs (miRNAs) are aberrant in many human tumors which can be transferred to immune cells by tumor-derived exosomes. Dendritic cells (DCs) play an important role in activation of immune response. However, the effect of tumor-derived exosomes on toll-like receptor (TLR) in DCs remains unclear. We investigated the influence of pancreatic cancer derived exosomes on TLR4, and downstream cytokines via miR-203. Our results showed that miR-203 expressed in panc-1 cells and exosomes, and upregulated in exosomes-treated DCs. TLR4 decreased after treatment of exosomes and miR-203 mimics, while increased in exosomes-treated DCs by miR-203 inhibitors. But the mRNA level of TLR4 was not significantly different between DCs and exosomes-treated DCs. Tumor necrosis factor-α (TNF-α) and interleukin-12 (IL-12) also decreased under treatment of exosomes and miR-203 mimics, both of which increased in exosomes-treated DCs by miR-203 inhibitors. Collectively, pancreatic cancer derived exosomes downregulate TLR4 and downstream cytokines in DCs via miR-203.

Suppression of inflammation by tumor-derived exosomes: a kind of natural liposome packaged with multifunctional proteins

Exosomes are small-membrane vesicles secreted by hematopoietic and malignant epithelial cells as well as trophoblasts. The composition of cancerous exosomes has been proven to play pivotal roles in the maintenance of the microenvironment that is beneficial for the progression of cancer, such as Fas-ligand–triggered lymphocyte apoptosis. We supposed that the immunosuppressive effect of cancerous exosomes might be helpful in the treatment of diseases characterized by overactivation of the immune system and subsequent tissue injury. The aim of this study was to evaluate the protective effect of tumor-derived exosomes in the mice model of lipopolysaccharide (LPS)-induced inflammation. Tetrazolium (MTT) and DNA electrophoresis were used to measure the cytotoxicity of exosomes on lymphocytes. Pathologic observation of tissue sections, serologic analysis of aspartate aminotransferase/alanine aminotransferase (AST/ALT), and urinary analysis of protein were used to assess the protection effect of exosomes in LPS-induced multiorgan damage. In vitro outcomes of MTT and DNA electrophoresis showed the cytotoxicity of exosomes on lymphocytes. Together with the alleviation of organ damages evaluated by urine protein, serum AST/ALT, and pathologic analysis, we confirmed the possibility that pretreatment of mice with exosomes, produced by H22 hepatic tumor cells, resulted in protection against LPS-induced tissue damage, which is caused by overactivation of the immune system and inflammation response. This therapeutic strategy will raise an interesting way to search new therapeutics in pairs of diseases with complementarities in etiology and pathology, namely, a strategy of taking advantage of the mutual complementarities between diseases.