Colorectal Cancer Exosomes Research Articles

Tumor exosome promotes Th17 cell differentiation by transmitting the lncRNA CRNDE-h in colorectal cancer

The T helper 17 (Th17) cells in tumor microenvironment play an important role in colorectal cancer (CRC) progression. This study investigated the mechanism of Th17 cell differentiation in CRC with a focus on the role of tumor exosome-transmitted long noncoding RNA (lncRNA). Exosomes were isolated from the CRC cells and serum of CRC patients. The role and mechanism of the lncRNA CRNDE-h transmitted by CRC exosomes in Th17 cell differentiation were assessed by using various molecular biological methods. The serum exosomal CRNDE-h level was positively correlated with the proportion of Th17 cells in the tumor-infiltrating T cells in CRC patients. CRC exosomes contained abundant CRNDE-h and transmitted them to CD4+ T cells to increase the Th17 cell proportion, RORγt expression, and IL-17 promoter activity. The underlying mechanism is that, CRNDE-h bound to the PPXY motif of RORγt and impeded the ubiquitination and degradation of RORγt by inhibiting its binding with the E3 ubiquitin ligase Itch. The in vivo experiments confirmed that the targeted silence of CRNDE-h in CD4+ T cells attenuated the CRC tumor growth in mice. The present findings demonstrated that the tumor exosome transmitted CRNDE-h promoted Th17 cell differentiation by inhibiting the Itch-mediated ubiquitination and degradation of RORγt in CRC, expanding our understanding of Th17 cell differentiation in CRC.

Mesenchymal Stem Cell-Derived Exosomal microRNA-3940-5p Inhibits Colorectal Cancer Metastasis by Targeting Integrin α6.

Exosomes are potential tools for disease control by regulating intercellular communication through carrying proteins and RNAs between cells or remote organs. Exosome activities have aroused wide concerns in cancer biology and malignancy control. This study was performed to explore the roles of mesenchymal stem cell (MSC)-derived exosomes in colorectal cancer (CRC) progression. MSC-exosomal microRNAs (miRNAs) in CRC tissues were analyzed, and aberrantly expressed miRNAs in CRC tissues were obtained from the data available on the GEO database. Altered expression of miR-3940-5p was introduced to identify its role in CRC invasion and metastasis in both cell and animal models. The binding relationship between miR-3940-5p and Integrin alpha6 (ITGA6) was predicted on TargetScan and validated through a luciferase assay. The effects of ITGA6 on CRC were figured out. MSC-derived exosomes carried miR-3940-5p into CRC cells. Up-regulation of miR-3940-5p inhibited epithelial-mesenchymal transition (EMT) and invasion of CRC cells, and suppressed the tumor metastasis and growth in vivo. miR-3940-5p was found to directly bind to ITGA6. Overexpression of ITGA6 promoted CRC cell invasion and EMT and tumor progression through upregulating the transforming growth factor-beta1 (TGF-β1) signaling. A TGF-β1-specific antagonist, Disitertide, blocked the functions of ITGA6 both in vivo and in vitro. MSC-exosomal miR-3940-5p inhibits invasion and EMT of CRC cells as well as growth and metastasis of tumors through targeting ITGA6 and the following TGF-β1 inactivation. This study may provide novel insights into exosome-based treatment for CRC.

Terminal deoxynucleotidyl transferase based signal amplification for enzyme-linked aptamer-sorbent assay of colorectal cancer exosomes

Exosomes have received increasingly significant attention and have shown great clinical value as biomarkers for a number of diseases. However, there is still a lack of a highly sensitive and visualized method for the detection of exosomes in numerous samples simultaneously. Here, we developed a high-throughput, colorimetric and simple method to detect colorectal cancer (CRC) exosomes based on terminal deoxynucleotidyl transferase (TdT)-aided ultraviolet signal amplification. Anti-A33, a CRC exosomal protein marker, was selected as a capture probe, and a facility-prepared EpCAM (CRC exosomal protein) aptamer-Au-primer complex was used as a signal probe. After the CRC exosomes were captured onto the surface of 96-well plates, the primer was extended to the poly(biotin-adenine) chains with the help of TdT, resulting in an increase in the binding amount of avidin-modified horseradish peroxidase (Av-HRP) for H2O2-mediated oxidation of 3,3′,5,5′-tetramethyl benzidine (TMB) in enzyme-linked aptamer-sorbent assay (ELASA). The results showed that the incorporation of ploy(biotin-A) enabled approximately 10.4-fold signal amplification. This approach achieved a linear range of 9.75 × 103–1.95 × 106 particles/μL for CRC cell-derived exosomes. The feasibility of the developed assay was evaluated using clinical serum samples. CRC patients (n = 16) could be clearly and successfully distinguished from healthy individuals (n = 9). Furthermore, this proposed platform holds considerable potential for the detection of different targets, simply by changing the aptamer and antibody

Research progress on the functions of exosomes in colorectal cancer

Exosomes are a class of extracellular vesicles that secreted by a variety of cells, with the length of 40-100 nm and a lipid bilayer membrane structure. They are often observed in multiple body fluids and are important mediators in intercellular communication. Several researches demonstrated that exosomes are closely linked to the development, invasion, metastasis, and drug resistance of colorectal cancer (CRC). In this review, the role of CRC-derived exosomes in the pathogenesis, diagnosis, prognosis and treatment of CRC were briefly summarized to provide a strategy for new therapeutic targets in CRC. Key words: Colorectal neoplasms; Exosomes; Biomarkers, tumor

Common molecular markers between circulating tumor cells and blood exosomes in colorectal cancer: a systematic and analytical review.

Nearly half of patients with colorectal cancer (CRC), the third leading cause of cancer deaths worldwide, are diagnosed in the late stages of the disease. Appropriate treatment is not applied in a timely manner and nearly 90% of the patients who experience metastasis ultimately die. Timely detection of CRC can increase the five-year survival rate of patients. Existing histopathological and molecular classifications are insufficient for prediction of metastasis, which limits approaches to treatment. Detection of reliable cancer-related biomarkers can improve early diagnosis, prognosis, and treatment response prediction and recurrence risk. Circulating tumor cells (CTCs) and exosomes in peripheral blood can be used in a liquid biopsy to assess the status of a tumor. Exosomes are abundant and available in all fluids of the body, have a high half-life and are released by most cells. Tumor-derived exosomes are released from primary tumors or CTCs with selective cargo that represents the overall tumor. The current systematic review highlights new trends and approaches in the detection of CRC biomarkers to determine tumor signatures using CTC and exosomes. When these are combined, they could be used to guide molecular pathology and can revolutionize detection tools. Relevant observational studies published until July 24, 2019 which evaluated the expression of tumor markers in CTCs and exosomes were searched in PubMed, Scopus, Embase, and ISI Web of Science databases. The extracted biomarkers were analyzed using String and EnrichR tools.

Revisiting the Role of Exosomes in Colorectal Cancer: Where Are We Now?

Exosomes (Exos) are nano-sized extracellular vesicles constitutively released by both prokaryotic and eukaryotic cells. Their role as inter-cellular messengers involved in both physiological and pathological processes has overwhelmingly come to light in the last decade, and their contribution to cancerogenesis and tumor metastasis is under intensive investigation. Here we review the most recent information concerning Exos in colorectal cancer (CRC) and focus on their effects on tumor microenvironment and the immune system, as well as unravel their role in the formation of the pre-metastatic niche and in drug resistance. Such a recent knowledge on Exos depicts their potential translations into the clinical arena, either as an alternative tool of “liquid biopsy” or novel therapeutic approaches for CRC. However, due to the limited data available from clinical trials, they need further validations before addressing their putative application in oncology.

Exosome-transmitted miR-128-3p increase chemosensitivity of oxaliplatin-resistant colorectal cancer

BackgroundOxaliplatin resistance is a major challenge for treatment of advanced colorectal cancer (CRC). Both acquisition of epithelial-mesenchymal transition (EMT) and suppressed drug accumulation in cancer cells contributes to development of oxaliplatin resistance. Aberrant expression of small noncoding RNA, miR-128-3p, has been shown to be a key regulator in tumorigenesis and cancer development. However, its roles in the progression of CRC and oxaliplatin-resistance are largely unknown.MethodsOxaliplatin-resistant CRC and normal intestinal FHC cells were transfected with a miR-128-3p expression lentivirus. After transfection, FHC-derived exosomes were isolated and co-cultured with CRC cells. miR-128-3p expression in resistant CRC cells, FHC cells, and exosomes was quantified by quantitative real-time PCR (RT-qPCR). The mRNA and protein levels of miR-128-3p target genes in resistant CRC cells were quantified by RT-qPCR and western blot, respectively. The effects of miR-128-3p on CRC cell viability, apoptosis, EMT, motility and drug efflux were evaluated by CCK8, flow cytometry, Transwell and wound healing assays, immunofluorescence, and atomic absorption spectrophotometry. Xenograft models were used to determine whether miR-128-3p loaded exosomes can re-sensitize CRC cells to oxaliplatin in vivo.ResultsIn our established stable oxaliplatin-resistant CRC cell lines, in vitro and vivo studies revealed miR-128-3p suppressed EMT and increased intracellular oxaliplatin accumulation. Importantly, our results indicated that lower miR-128-3p expression was associated with poor oxaliplatin response in advanced human CRC patients. Moreover, data showed that miR-128-3p-transfected FHC cells effectively packaged miR-128-3p into secreted exosomes and mediated miR-128-3p delivery to oxaliplatin-resistant cells, improving oxaliplatin response in CRC cells both in vitro and in vivo. In addition, miR-128-3p overexpression up-regulated E-cadherin levels and inhibited oxaliplatin-induced EMT by suppressing Bmi1 expression in resistant cells. Meanwhile, it also decreased oxaliplatin efflux through suppressed expression of the drug transporter MRP5.ConclusionOur results demonstrate that miR-128-3p delivery via exosomes represents a novel strategy enhancing chemosensitivity in CRC through negative regulation of Bmi1 and MRP5. Moreover, miR-128-3p may be a promising diagnostic and prognostic marker for oxaliplatin-based chemotherapy.

Circulating long non-coding RNA colon cancer-associated transcript 2 protected by exosome as a potential biomarker for colorectal cancer

BackgroundColon cancer-associated transcript 2 (CCAT2) plays a crucial role in several cancers. However, the clinical significance of circulating CCAT2 expression in colorectal cancer (CRC) has not previously been elucidated. In this study, we aimed to elucidate the potential role of CCAT2 and its clinical significance of circulating expression level in CRC. MethodsWe detected the expression of CCAT2 in 75 pairs of tumorous and adjacent non-tumorous tissues derived from CRC patients by quantitative real-time polymerase chain reaction. The serum levels of CCAT2 expression were detected in an independent cohort of healthy controls and CRC patients. We analyzed the relationship between CCAT2 levels in serum and clinicopathological features of CRC patients. We also compared CCAT2 levels in paired pre-operative and post-operative serum samples. Furthermore, the existence of serum CCAT2 in exosomes was investigated. ResultsThe levels of CCAT2 expression were significantly over-expressed in tumor tissues (p < 0.05) compared to adjacent non-tumorous tissues. Higher CCAT2 expression was associated with advanced CRC patients. Moreover, the serum levels of CCAT2 expression were significantly over-expressed in CRC patients (p < 0.05) compared to those in healthy subjects. In addition, the CCAT2 levels were significantly decreased in post-operative samples than those in pre-operative ones (P = 0.01). We also found that CCAT2 expression was up-regulated in CRC exosomes (P < 0.001) and no significant differences of CCAT2 levels were found between in serum and in exosomes. ConclusionsOur data indicate that circulating CCAT2 which might protected by exosomes can serve as a novel potential predictor in CRC.

Exosomes and their importance in metastasis, diagnosis, and therapy of colorectal cancer.

Extracellular vesicles are known as actual intermediaries of intercellular communications, such as biological signals and cargo transfer between different cells. A variety of cells release the exosomes as nanovesicular bodies. Exosomes contain different compounds such as several types of nucleic acids and proteins. In this study, we focused on exosomes in colorectal cancer as good tools that can be involved in various cancer-related processes. Furthermore, we summarize the advantages and disadvantages of exosome extraction methods and review related studies on the role of exosomes in colorectal cancer. Finally, we focus on reports available on relations between mesenchymal stem cell-derived exosomes and colorectal cancer. Several cancer-related processes such as cancer progression, metastasis, and drug resistance of colorectal cancer are related to the cargoes of exosomes. A variety of molecules, especially proteins, microRNAs, and long noncoding RNAs, play important roles in these processes. The microenvironment features, such as hypoxia, also have very important effects on the properties of the origin cell-derived exosomes. On the other hand, exosomes derived from colorectal cancer cells also interfere with cancer chemoresistance. Furthermore, today it is known that exosomes and their contents can likely be very effective in noninvasive colorectal cancer diagnosis and therapy. Thus, exosomes, and especially their cargoes, play different key roles in various aspects of basic and clinical research related to both progression and therapy of colorectal cancer.

Abstract 5036: Visualization of cancer-derived exosome-mediated angiogenesis in a layered 3D stromal tissue model

Abstract Background: Exosomes are small vesicles of sizes ranging from 30 to 150 nm that originate from the endosomal luminal membrane and are constantly released by fusion with cell membranes. Several studies have revealed that exosomes play an important role in mediating local and systemic cellular communication through the transmission of nucleic acid materials and information in the form of proteins. Here, we demonstrated a unique layered 3D stromal tissue model of visualization of promoting angiogenesis by exosomes in colorectal cancer (CRC) patients. Method: The CRC specimens of exosome were obtained from 7 patients in stage IV. Isolation of total exosome was performed according to standard procedure by ultracentrifuge. Layered 3D stromal tissue model was constructed with human umbilical vein endothelial cells (HUVEC) and normal human dermal fibroblasts. Cells were aggregated by heparin and collagen to suspension under cationic condition. The cell suspension was seeded to transwell inserts membrane at a density of 2×106. Media were changed after 24h, and serum, exosome or supernatant after ultracentrifuge of serum (SAUS) was added to the 3D stromal tissue after 6 days. The isolated exosomes were diluted with an equal volume of PBS buffer to the serum sample. After cultivation, the tube formation of HUVEC was stained with Immunofluorescence, and the degree of hypervascularity was compared with healthy volunteers. Result: Except for one serum specimen, hypervascularization was observed in the 3D stromal tissue model in an additive-dependent manner. Especially when exosome was added, hypervascularization was observed in the layered 3D stromal tissue model more efficiently than serum in all specimens (see Table). Interestingly, the frequency of hypervascularization was drastically reduced in the SAUS. Conclusion: Our data encourage that this layered 3D stromal tissue model may be applicable to cancer diagnosis using exosomes from CRC patients. Table. Frequency of hypervascularization by exosomes derived from CRC5μl10μl50μlExosome5/7 (71.4%)7/7 (100.0%)7/7 (100.0%)Serum4/7 (57.1%)6/7 (85.7%)6/7 (85.7%)SAUS1/7 (14.3%)0/7 (0.0%)0/7 (0.0%) Citation Format: Shiro Kitano, Kei Tsukamoto, Rii Morimura, Shinji Irie, Eiji Shinozaki, Kensei Yamaguchi. Visualization of cancer-derived exosome-mediated angiogenesis in a layered 3D stromal tissue model [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 5036.

Identification of novel genes and pathways in colorectal cancer exosomes: a bioinformatics study

Background: Colorectal cancer (CRC) is a major public health problem, being the leading causes of mortality and morbidity worldwide. Exosomes are crucial to the molecular pathogenesis of CRC. It is essential to investigate the key differentially expressed genes (DEGs) in CRC exosomes. Methods: The DEGs between CRC patients and normal participants were screened using microarray data downloaded from the Gene Expression Omnibus (GEO) and exoRBase database. Gene ontology (GO), pathway enrichment analysis, protein-protein interaction (PPI) network, module analysis, overall survival (OS) analysis of hub genes and functional annotation of DEGs were performed. Results: A total of 149 DEGs in CRC exosomes and 1,507 DEGs in CRC tissues were identified with selecting criteria of P value <0.05. GO terms of positive regulation of gene expression, protein binding to Golgi, cellular protein metabolic process and endonuclease activity were significantly enriched in CRC exosomes. CRC exosomes were mainly enriched in pathways of RNA transport, alcoholism, viral carcinogenesis, spliceosome and Ras signaling pathway. We then selected UBC, H3F3A, HIST2H2AA3, AKT3, and HSPA1B as the hub protein in CRC exosomes according to the PPI network. OS analysis showed overexpression of HIST2H2AA3 was related to poor prognosis. Four oncogenes including H3F3A , U2AF1 , P2RY8 and APOBEC3B were identified in the exosomes. There was no significant difference in the ratio of tumor suppressor genes (TSGs) to oncogenes between tumor tissues and exosomes according to functional annotation of DEGs. Conclusions: Hub genes including UBC, H3F3A, HIST2H2AA3, AKT3 , and HSPA1B might serve as new diagnostic and therapeutic targets of CRC in the future.

Identification of novel genes and pathways in colorectal cancer exosomes: a bioinformatics study

Identification of novel genes and pathways in colorectal cancer exosomes: a bioinformatics study

DNase I enzyme-aided fluorescence signal amplification based on graphene oxide-DNA aptamer interactions for colorectal cancer exosome detection

Exosomes have proved to be an effective cancer biomarker with significant potential, and several cell-specific molecules have been found in colorectal cancer (CRC) exosomes. Nevertheless, it is challenging to use exosomes in clinical lab diagnostics due to their nanoscale and the lack of a convenient and effective detection platform. Here, we developed a DNase I enzyme-aided fluorescence amplification method for CRC exosome detection, based on graphene oxide (GO)-DNA aptamer (CD63 and EpCAM aptamers) interactions. The fluorescence of fluorophore-labeled aptamers quenched by GO, recovered after incubation with samples containing CRC exosomes. The DNase I enzyme digested the single-stranded DNA aptamers on the exosome surface and the exosomes were able to interact with more fluorescent aptamer probes, resulting in an increase of signal amplification. The limit of detection for CRC exosomes is 2.1 × 104 particles/μl. Consequently, a rapid and effective method with high sensitivity was established. The method was verified in 19 clinical blood serum samples to distinguish healthy and CRC patients, showing significant diagnostic power. Moreover, it can be expanded to other kinds of cancer exosomes, in addition to CRC.

The role of exosomes on colorectal cancer: A review.

Exosomes are extracellular microvesicles released from cells, which are involved in many biological and pathological processes, mainly because of their role in intercellular communication. Exosomes derived from colorectal cancer (CRC) cells are related to oncogenesis, tumor cell survival, chemo-resistance, and metastasis. The role of the exosomes in these processes involves the transfer of proteins, RNAs, or mutant versions of proto-oncogenes to the target cells. In recent years, great efforts have been made to identify useful biomarkers in CRC exosomes for diagnosis, prediction of prognosis, and treatment response. This review focuses on recent studies on CRC exosomes, considering isolation, cargo, biomarkers, and the effects of exosomes on the development and progression of CRC, including resistance to antitumor therapy.

Tumor-derived exosomes in colorectal cancer progression and their clinical applications.

Colorectal cancer (CRC) ranks as the third leading cause of cancer mortality in both of men and women worldwide due to its metastatic properties and resistance to current treatment. Recent studies have shown that tumor-derived exosomes play emerging roles in the development of cancer. Exosomes are nano-sized extracellular vesicles (EVs) that contain lipids, proteins, DNAs, and RNA species (mRNA, miRNA, long non-coding RNA). These exosomal cargos can be transferred locally and systemically, after taken by recipient cells, so exosomes represent a new form of intercellular communication. There is increasing evidence demonstrating that exosomes control a wide range of pathways bolstering tumor development, metastasis and drug resistance. This review provides an in-depth and timely summary of the role of exosomes in CRC. We first describe the common features and biogenesis of exosomes. We then highlight important findings that support the emerging roles of exosomes in CRC cell growth, invasion and metastasis, as well as resistance to treatment. Finally, we discuss the clinical application of exosomes as diagnostic biomarkers, in vivo drug delivery system and the potential of novel exosome-based immunotherapy for CRC.

Clinical and biological significance of circulating tumor cells, circulating tumor DNA, and exosomes as biomarkers in colorectal cancer

Colorectal cancer (CRC) has been the fourth leading cause of cancer-related mortality worldwide. Owing to clonal evolution and selection, CRC treatment needs multimodal therapeutic approaches and due monitoring of tumor progression and therapeutic efficacy. Liquid biopsy, involving the use of circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes, may offer a promising noninvasive alternative for diagnosis and for real-time monitoring of tumor evolution and therapeutic response compared to traditional tissue biopsy. Monitoring of the disease processes can enable clinicians to readily adopt a strategy based on optimal therapeutic decision-making. This article provides an overview of the significant advances and the current clinical and biological significance of CTCs, ctDNA, and exosomes in CRC, as well as a comparison of the main merits and demerits of these three components. The hurdles that need to be resolved and potential directions to be followed with respect to liquid biopsies for detection and therapy of CRC are also discussed.

Cytoskeleton-centric protein transportation by exosomes transforms tumor-favorable macrophages.

The exosome is a key initiator of pre-metastatic niche in numerous cancers, where macrophages serve as primary inducers of tumor microenvironment. However, the proteome that can be exosomally transported from cancer cells to macrophages has not been sufficiently characterized so far. Here, we used colorectal cancer (CRC) exosomes to educate tumor-favorable macrophages. With a SILAC-based mass spectrometry strategy, we successfully traced the proteome transported from CRC exosomes to macrophages. Such a proteome primarily focused on promoting cytoskeleton rearrangement, which was biologically validated with multiple cell lines. We reproduced the exosomal transportation of functional vimentin as a proof-of-concept example. In addition, we found that some CRC exosomes could be recognized by macrophages via Fc receptors. Therefore, we revealed the active and necessary role of exosomes secreted from CRC cells to transform cancer-favorable macrophages, with the cytoskeleton-centric proteins serving as the top functional unit.

PTH-320 Exosomes: extracellular vesicles which can immortalise cancer and stromal cells in the colorectal tumour microenvironment

<h3>Introduction</h3> Exosomes are nano-sized (40–100 nm) extracellular vesicles which are exchanged by cancer and stromal cells. They contain proteins and nucleotides (DNA, mRNA, microRNA) and are therefore capable of conveying genetic information. Exosome transfer affects cellular function but this is not well defined in the colorectal cancer (CRC) setting. We aimed to isolate, label and transfer exosomes from colorectal cancer cells to fibroblasts and vice versa. Moreover, we investigated the effect of exosome transfer at the cellular level, focussing on two key pathways: ERK and AKT. <h3>Method</h3> Exosomes were isolated from conditioned media from DLD-1 CRC cells or MRC5 fibroblasts by selective centrifugation and validated by transmission electron microscopy, western blotting and flow cytometry. The resulting exosome pellet was labelled with a lipophilic (green) fluorescent dye and co-cultured with the reciprocal target cell (MRC5 or DLD-1). The cells were then washed thoroughly to remove all extracellular particles and visualised by fluorescence microscopy. Cell lysate was analysed by western blotting for activation of key signalling pathways such as Ras (ERK) and AKT. <h3>Results</h3> Microscopy revealed presence of fluorescent labelled exosomes within both DLD-1 and MRC5 cells following exosome co-culture. There was marked increase in AKT pathway activation in MRC5 fibroblasts after co-culture with DLD-1 CRC exosomes. As a result, significant changes to anti-apoptotic stimuli have been observed. There was a marked increase in ERK pathway activation and moderate increase in AKT activation in DLD-1 CRC cells after co-culture with MRC5 fibroblast exosomes. Again, significant changes to downstream anti-apoptotic signals were observed. <h3>Conclusion</h3> We have shown that CRC exosomes can be transferred to fibroblasts and vice versa. This transfer induces selective phosphorylation of AKT and resistance to apoptosis in recipient fibroblasts and CRC cells. Stromal and tumour-derived exosomes promote resistance to apoptosis and can create an apoptosis resistant niche in the tumour microenvironment. <h3>Disclosure of interest</h3> None Declared.