Breast Cancer-derived Exosomes Research Articles

Peptide-based biosensing approaches for targeting breast cancer-derived exosomes

Exosomes are nanovesicles present in all the biological fluids, making them attractive as non-invasive biomarkers for diseases like cancer, among many others. However, exosomes are complex to separate and detect, requiring comprehensive molecular characterization for their routine use in diagnostics. This study explores the use of peptides as cost-effective and stable alternatives to antibodies for exosome binding. To achieve that, phage display technology was employed to select peptides with high specificity for target molecules in exosomes. Specifically, a selected peptide was evaluated for its ability to selectively bind breast cancer-derived exosomes. Proteomic analysis identified 38 protein candidates targeted by the peptide on exosome membranes. The binding of the peptide to breast cancer-derived exosomes was successfully demonstrated by flow cytometry and magneto-actuated immunoassays. Furthermore, an electrochemical biosensor was also tested for breast cancer-derived exosome detection and quantification. The peptide demonstrated effective binding to exosomes from aggressive cancer cell lines, offering promising results in terms of specificity and recovery. This research shows potential for developing rapid, accessible diagnostic tools for breast cancer, especially in low-resource healthcare settings.

A highly sensitive and selective fluorescent biosensor for breast cancer derived exosomes using click reaction of azide-CD63 aptamer and alkyne-polymer dots.

Exosomes have gained recognition as valuable reservoirs of biomarkers, holding immense potential for early cancer detection. Consequently, there is a pressing need for the development of an economical and highly sensitive exosome detection methodology. In this work, we present a fluorescence method for breast cancer-derived exosome detection based on Cu-triggered click reaction of azide-modified CD63 aptamer and alkyne functionalized Pdots. The detection threshold for the exosomes obtained from the breast cancer serum was determined to be 6.09 × 107 particles per μL, while the measurable range spanned from 6.50 × 107 to 1.30 × 109 particles per μL. The employed methodology achieved notable success in accurately distinguishing breast cancer patients from healthy individuals through serum analysis. The application of this method showcases the significant potential for early exosome analysis in the clinical diagnosis of breast cancer patients.

CRISPR/Cas12a-based electrochemical aptasensor for determination of breast cancer-derived exosomes

Tumor-derived exosomes take an important role in the onset and progression of cancer and are promising biomarkers for early tumor diagnosis. However, detecting exosomes with ultra-sensitivity and high specific still remains a challenge. Herein, we developed an ultra-sensitivity and simple electrochemical method based on the subtly combination of CRISPR/Cas12a, aptamer and magnetic nanoparticles (MNPs) for the determination of breast cancer (BC)-derived exosomes. The complex of the aptamer and its partially complementary DNA (probe 1, P1) were conjugated to magnetic nanoparticles. In the presence of the exosome, the surface proteins of exosomes can specifically bind with the aptamer resulting in the release of P1. The freed P1 combines with crRNA, triggering the trans-cleavage activity of CRISPR/Cas12a, leading to the methylene blue labeled single-stranded DNA reporter (probe 2, P2) modified on the gold electrode being cut. Benefiting from the outstanding trans-cleavage activity of Cas12a, the high specific binding between the target and aptamer, and the excellent separation ability of the MNPs, our platform achieved an ultrasensitive and highly specific detection of exosomes with the limit of detection as low as 280 particles/mL. In addition, our platform has demonstrated the capability to accurately discriminate the healthy individuals from breast cancer patients, thereby expanding the avenues and insights of exosomes detection and providing a novel and attractive tool for early diagnosis BC.

Exosomal circRNA RHOT1 promotes breast cancer progression by targeting miR-204-5p/ PRMT5 axis

BackgroundCircular RNA RHOT1 (circRHOT1) plays crucial roles in tumorigenesis by competing with microRNAs. It is largely abundant in tumor cell-derived exosomes. Meanwhile, cancer-derived exosomes participate in diverse biological processes. However, the expression patterns and functions of exosomal circRHOT1 in breast cancer remain unknown. This study is aimed to investigate and elucidate the exosomal circRHOT1/miR-204-5p/PRMT5 axis in breast cancer.MethodsThe exosomes derived from serum samples of breast cancer patients and breast cancer cell lines were characterized using transmission electron microscopy and Western blot. MTT, colony formation, wound healing, and transwell assays were utilized to analyze cell proliferation, migration, and invasion of breast cancer cells. Flow cytometry was used for apoptosis analysis. The bioinformatics method was employed to screen differentially expressed novel circRNAs and predict the microRNA targets of circRHOT1. Dual-luciferase reporter gene assays were performed to verify their direct interaction. Finally, Xenograft experiments were used to investigate the effect of exosomal circRHOT1 on tumor growth in vivo.ResultsCircRHOT1 exhibited significantly high expression in exosomes derived from the serum of breast cancer patients and breast cancer cell lines, which suggested its potential diagnostic value. Breast cancer-derived exosomes promoted the cell proliferation, migration, invasion, and epithelial-mesenchymal transition of breast cancer cells while inhibiting apoptosis. However, exosomes with downregulated circRHOT1 inhibited the growth of co-cultured cells. Mechanistically, circRHOT1 acted as a sponge of miR-204-5p and promoted protein arginine methyltransferase 5 (PRMT5) expression. Moreover, miR-204-5p inhibitor and pcPRMT5 could reverse the tumor suppressive effects mediated by circRHOT1-knockdown. Furthermore, treatment with exosomes derived from breast cancer cells with circRHOT1 knockdown attenuated tumor growth in tumor-bearing nude mice, which was accompanied by a reduction in PRMT5 expression and an enhancement of miR-204-5p expression.ConclusionThe exosomal circRHOT1 may promote breast cancer progression by regulating the miR-204-5p/PRMT5 axis. The current study strengthens the role of circRHOT1, miR-204-5p, and PRMT5 in breast cancer development and provides a potential treatment strategy for breast cancer.

Metastatic breast cancer-derived exosomes and osteoclast-mediated bone metastasis

Metastatic breast cancer-derived exosomes and osteoclast-mediated bone metastasis

Tumor-derived exosomes RNA expression profiling identifies the prognosis, immune characteristics, and treatment in HR+/HER2-breast cancer.

Exosomes play crucial roles in intercellular communication and are involved in the onset and progression of various types of cancers, including breast cancer. However, the RNA composition of breast cancer-derived exosomes has not been comprehensively explored. We conducted microarray assays on exosomes isolated from breast cancer and healthy breast epithelial cells from three patients with hormone receptor (HR) +/ human epidermal growth factor receptor (HER2) - breast cancer and identified 817 differentially expressed genes (DEGs). Among these, 315 upregulated tumor-derived exosome genes (UTEGs) were used to classify HR+/HER2- breast cancers into two categories, revealing a difference in survival rates between the groups. We developed and validated a novel prognostic exosome score (ES) model consisting of four UTEGs that provides a refined prognosis prediction in HR+/HER2-breast cancer. ES reflects various immune-related features, including somatic variation, immunogenicity, and tumor immune infiltrate composition. Our findings indicate a considerable positive correlation between the ES and drug sensitivity values for vincristine, paclitaxel, and docetaxel. However, ES was remarkably higher in the endocrine therapy non-responder group than in the responder group. Immunohistochemistry confirmed the remarkable expression of the four model genes in tumor tissues, and their expression in MCF-7 cell exosomes was higher than that in MCF10A cells, as verified via qPCR. In summary, tumor-derived exosome genes provide novel insights into the subtyping, prognosis, and treatment of HR+/HER2-breast cancer.

Construction of a cleavable linker chemistry-based HBEXO-Chip to isolate circulating exosomes for breast cancer diagnosis.

Breast cancer is presently the most common form of malignant tumour globally, and its precise diagnosis is vital for enhancing patient survival rates and their quality of life. Exosomes, which are small extracellular vesicles containing proteins and nucleic acid molecules, have emerged as ideal cancer markers for liquid biopsy-based diagnostics. Nevertheless, the current methods for isolating exosomes present challenges for clinical implementation. Although immunoaffinity-based microfluidics hold potential for exosome-based cancer diagnostics, existing microfluidic chips struggle to capture and release intact, high-purity, and highly specific exosomes effectively. To surmount these obstacles, we developed the HBEXO-Chip, an innovative immunoaffinity microfluidic device that employs cleavable linker chemistry technology. This chip enables rapid isolation and detection of breast cancer-derived exosomes in peripheral blood. The fishbone-like microfluidic chip design of the HBEXO-Chip heightens the binding likelihood between specific exosomes and antibodies, significantly augmenting capture efficiency. Furthermore, the gentle reaction conditions of the cleavable linker chemistry retain the exosomes' membrane structure's integrity during the release process, which is advantageous for downstream experimental analysis. Our study demonstrated the effectiveness of the HBEXO-Chip in distinguishing breast cancer patients, patients with benign breast tumours, and healthy controls. By quantitatively analysing Epcam+ exosomes in clinical plasma samples, this technology platform provides a quick, user-friendly, highly sensitive, and specific assay for detecting tumour exosomes in peripheral blood, making it a valuable liquid biopsy tool for clinicians to diagnose breast cancer.

Tumor-Derived Exosomes and Their Role in Breast Cancer Metastasis.

Breast cancer has been the most common cancer in women worldwide, and metastasis is the leading cause of death from breast cancer. Even though the study of breast cancer metastasis has been extensively carried out, the molecular mechanism is still not fully understood, and diagnosis and prognosis need to be improved. Breast cancer metastasis is a complicated process involving multiple physiological changes, and lung, brain, bone and liver are the main metastatic targets. Exosomes are membrane-bound extracellular vesicles that contain secreted cellular constitutes. The biogenesis and functions of exosomes in cancer have been intensively studied, and mounting studies have indicated that exosomes play a crucial role in cancer metastasis. In this review, we summarize recent findings on the role of breast cancer-derived exosomes in metastasis organotropism and discuss the potential promising clinical applications of targeting exosomes as novel strategies for breast cancer diagnosis and therapy.

Novel secretion modification region (SMR) peptide exhibits anti-metastatic properties in human breast cancer cells

Breast cancer is the second leading cause of cancer-related mortality in women worldwide, with nearly 90% attributed to metastatic progression. Exosomes containing epithelial–mesenchymal transition (EMT) ‘programs’ transmit pro-metastatic phenotypes. Our group discovered and developed a novel anti-cancer SMR peptide that antagonizes breast cancer cell exosome release resulting in cell cycle arrest and tumor growth suppression. This study aims to evaluate the anti-metastatic capabilities of the SMR peptide, focusing on exosomes and EMT. Breast cancer cell lines MDA-MB-231 and MCF-7 were treated with the SMRwt peptide, and the following assays were performed: cell wound-healing, migration, invasion. The SMRwt peptide consists of the following amino acid sequence VGFPVAAVGFPVDYKDDDDK and contains the SMR domain (66VGFPV70) of the HIV-1 Nef protein. Western blot analysis detected epithelial and mesenchymal markers to evaluate EMT progression. Extracellular vesicle type and quantity were assessed through NanoSight analysis. Mortalin and Vimentin knockdown was achieved through antibody targeting and miRNAs. Data gathered demonstrated that the SMR peptide interacts with Mortalin and Vimentin to inhibit pro-EMT exosome release and induce EMT tumor suppressor protein expression. Specifically, SMRwt treatment reduced mesenchymal markers Mortalin and Vimentin expression, while the epithelial marker E-cadherin expression was increased in breast cancer cells and breast cancer-derived exosomes. The SMR peptide specificity was identified as no effect was observed for MCF-10A exosome release or function. Direct Mortalin knockdown paralleled the results of SMR peptide treatment with an effective blockade of breast cancer cell migration. Conversely, the invasion assay differed between breast cancer cell lines with invasion blocked for in MCF-7 but not in MDA-MB-231. These results reinforce the therapeutic value of targeting breast cancer exosome release and reinforce Mortalin and Vimentin as critical regulators and therapeutic targets in breast cancer cell progression, EMT, and metastatic potential. A greater understanding of the SMR peptide mechanism of action will benefit the therapeutic design of anti-metastatic agents.

A Washing-Free and Easy-to-Operate Fluorescent Biosensor for Highly Efficient Detection of Breast Cancer-Derived Exosomes.

Traditional detection methods for protein tumor markers in the early screening of breast cancer are restricted by complicated operation procedures and unstable reproducibility. As one of alternative emerging tumor markers, exosomes play an important role in diagnosing and treating cancers at the early stage due to traceability of their origins and great involvement in occurrence and development of cancers. Herein, a washing-free and efficient fluorescent biosensor has been proposed to realize simple and straightforward analysis of breast cancer cell-derived exosomes based on high affinity aptamers and G quadruplex-hemin (G4-hemin). The whole reaction process can be completed by several simple steps, which realizes washing-free and labor-saving. With simplified operation procedures and high repeatability, the linear detection range for this developed fluorescent biosensing strategy to breast cancer cell-derived exosomes is from 2.5 × 105 to 1.00 × 107 particles/ml, and the limit of detection is down to 0.54 × 105 particles/ml.

Evaluation of miRNA-21-5p and miRNA-10b-5p levels in serum-derived exosomes of breast cancer patients in different grades

Background & objectivesTumor cells have various effects and dominance over other healthy cells. Cancer cells alter the cell program in healthy cells by secreting exosomes containing microRNAs involved in epithelial-mesenchymal transition (EMT). They can migrate to distant organs and establish a pre-metastatic niche. The purpose of this study was to determine the expression of miRNA-21–5p and miRNA-10b-5p, both of which are involved in EMT, in breast cancer-derived exosomes of various grades in order to identify new biomarkers involved in breast cancer progression. MethodsIn this study, a blood sample was taken from 60 patients with grades I, II, or III breast cancer, as well as twenty healthy individuals as a control group. The exosomes were then purified from serum samples, and their relative expression of miRNA-21–5p and miRNA-10b-5p was determined using the real-time PCR method. ResultsmiRNA-21–5p expression was significantly increased in patients with breast cancer grades I, II, and III compared to the control group (p < 0.01), (p < 0.0001) and (p < 0.0001), respectively, as was miRNA-10b-5p expression in patients with breast cancer grades I, II, and III compared to the control group (p < 0.0001), (p < 0.0001) and (p < 0.0001), respectively. ConclusionOur results show that both microRNAs increase as cells lose their differentiation and become more invasive, which is evidence of cancer progression. Hence, both microRNAs may have the potential to be used alone or in combination with other biomarkers for the diagnosis and prognosis of breast cancer.

Engineered exosomes as an in situ DC-primed vaccine to boost antitumor immunity in breast cancer

BackgroundDendritic cells (DCs) are central for the initiation and regulation of innate and adaptive immunity in the tumor microenvironment. As such, many kinds of DC-targeted vaccines have been developed to improve cancer immunotherapy in numerous clinical trials. Targeted delivery of antigens and adjuvants to DCs in vivo represents an important approach for the development of DC vaccines. However, nonspecific activation of systemic DCs and the preparation of optimal immunodominant tumor antigens still represent major challenges.MethodsWe loaded the immunogenic cell death (ICD) inducers human neutrophil elastase (ELANE) and Hiltonol (TLR3 agonist) into α-lactalbumin (α-LA)-engineered breast cancer-derived exosomes to form an in situ DC vaccine (HELA-Exos). HELA-Exos were identified by transmission electron microscopy, nanoscale flow cytometry, and Western blot analysis. The targeting, killing, and immune activation effects of HELA-Exos were evaluated in vitro. The tumor suppressor and immune-activating effects of HELA-Exos were explored in immunocompetent mice and patient-derived organoids.ResultsHELA-Exos possessed a profound ability to specifically induce ICD in breast cancer cells. Adequate exposure to tumor antigens and Hiltonol following HELA-Exo-induced ICD of cancer cells activated type one conventional DCs (cDC1s) in situ and cross-primed tumor-reactive CD8+ T cell responses, leading to potent tumor inhibition in a poorly immunogenic triple negative breast cancer (TNBC) mouse xenograft model and patient-derived tumor organoids.ConclusionsHELA-Exos exhibit potent antitumor activity in both a mouse model and human breast cancer organoids by promoting the activation of cDC1s in situ and thus improving the subsequent tumor-reactive CD8+ T cell responses. The strategy proposed here is promising for generating an in situ DC-primed vaccine and can be extended to various types of cancers.Graphic Scheme 1. Schematic illustration of HELA-Exos as an in situ DC-primed vaccine for breast cancer. (A) Allogenic breast cancer-derived exosomes isolated from MDA-MB-231 cells were genetically engineered to overexpress α-LA and simultaneously loaded with the ICD inducers ELANE and Hiltonol (TLR3 agonist) to generate HELA-Exos. (B) Mechanism by which HELA-Exos activate DCs in situ in a mouse xenograft model ofTNBC. HELA-Exos specifically homed to the TME and induced ICD in cancer cells, which resulted in the increased release of tumor antigens, Hiltonol, and DAMPs, as well as the uptake of dying tumor cells by cDC1s. The activated cDC1s then cross-primed tumor-reactive CD8+ T cell responses. (C) HELA-Exos activated DCs in situ in the breast cancer patient PBMC-autologous tumor organoid coculture system. Abbreviations: DCs: dendritic cells; α-LA: α-lactalbumin; HELA-Exos: Hiltonol-ELANE-α-LA-engineered exosomes; ICD: immunogenic cell death; ELANE: human neutrophil elastase; TLR3: Toll-like receptor 3; TNBC: triple-negative breast cancer; TME: tumor microenvironment; DAMPs: damage-associated molecular patterns; cDC1s: type 1 conventional dendritic cells; PBMCs: peripheral blood mononuclear cells

Role of breast cancer-derived exosomes in metabolism of immune cells through PD1-GLUT1-HK2 metabolic axis

Tumor cells modulate immune responses by secreting exosomes. Tumor exosomes can affect the metabolism of immune cells and increase immune inhibitory molecules such as programmed cell death protein 1 (PD-1). PD-1 inhibits the glycolysis pathway in immune cells. We investigated the role of tumor exosomes in how metabolic changes occur through the PD1-GLUT1-HK2 metabolic axisin peripheral blood mononuclear cells (PBMCs). The MDA-MB-231 cell line was cultured, serum samples from breast cancer patients were collected, and exosomes purified from serum samples and the MDA-MB-231 cell line. PBMCs were treated with purified exosomes for 72 h and, the expression of PD1-GLUT1-HK2 genes was measured by real-time PCR. Our study results showed relative expression of the HK2 gene in both groups treated with MDA-MB-231 cell line exosomes and serum exosomes of breast cancer patients was significantly increased compared to the control group (p < 0.0001). Also, the relative expression of the PD1 gene and GLUT1 gene showed a significant increase compared to the control group only in the group treated with MDA-MB-231 cell line exosomes (p < 0.0001). Therefore, Breast cancer exosomes increased the expression of key genes in the glycolysis pathway, increasing the glycolysis pathway in PBMCs. Increased expression of PD-1 could not prevent the expression of critical genes in the glycolysis pathway as in previous studies.

Macrophage balance fraction determines the degree of immunosuppression and metastatic ability of breast cancer

Macrophages are important immune cells in the tumor microenvironment and can be divided into two polarized subtypes, M1 and M2. M1 type macrophages have anti-tumor effects, while M2 type macrophages have pro-tumor effect. Most of the current researches are limited to the effect of M1 or M2 macrophages on tumors, while ignoring the overall balance of macrophages. Our research suggests that the macrophage balance fraction (MBF) can more effectively and comprehensively reflect the balance of tumor associated macrophages. Using bioinformatics analysis and in vitro experiments, we found that MBF is also an effective indicator of the degree of immunosuppression and metastatic ability of breast cancer, and different MBF environment can impact the migration and invasion ability of breast cancer cells. Finally, we also found that the mechanism of MBF changes in breast cancer may be affected by breast cancer-derived exosomes. In summary, MBF was proposed and validated as a novel indicator of macrophage balance state. Using this indicator, we found that the balance of macrophages can affect the degree of immunosuppression and metastatic ability of breast cancer.

An artificial enzyme cascade amplification strategy for highly sensitive and specific detection of breast cancer-derived exosomes.

Tumor-related exosomes, which are heterogeneous membrane-enclosed nanovesicles shed from cancer cells, have been widely recognized as potential noninvasive biomarkers for early cancer diagnosis. Herein, an artificial enzyme cascade amplification strategy based on a switchable DNA tetrahedral (SDT) scaffold was proposed for quantification of breast cancer-derived exosomes. The SDT scaffold is composed of G-quadruplex mimicking DNAzyme sequences on its two single-stranded edges and glucose oxidase (GOx) on the four termini of the complementary strands. In the initial state, the SDT scaffold is blocked by the switch strand which consists of partial complementary domains with the DNA tetrahedron and a MUC1 aptamer. MCF-7 exosomes could release the quadruplex-forming sequences through the recognition of the MUC1 aptamer. The newly formed DNAzyme brings GOx into spatial proximity and induces high-efficiency enzyme cascade catalytic reactions on the SDT. Consequently, high sensitivity toward MCF-7 exosome analysis was obtained with a wide linear range of 3.8 × 106 to 1.2 × 108 particles per mL and a limit of detection of 1.51 × 105 particles per mL. In addition, such a DNAzyme reconfiguration strategy was able to distinguish MCF-7 exosomes from other breast cancer cell derived exosomes, indicating its excellent method specificity. The proposed enzyme cascade strategy not only provides a novel signal transformation and amplification nanoplatform for quantifying the specific populations of exosomes, but also can be further expanded to the analysis of multiple cancer biomarkers.

Prune-1 drives polarization of tumor-associated macrophages (TAMs) within the lung metastatic niche in triple-negative breast cancer

SummaryM2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Development of a simple, sensitive and selective colorimetric aptasensor for the detection of cancer-derived exosomes

There is a growing need for cancerous exosome detection towards potential non-invasive cancer diagnosis. This study aims to develop a reliable colorimetric aptasensor for sensitive and specific detection of circulating cancer-derived exosomes. In this design, target exosomes were firstly captured by latex beads via aldimine condensation, followed by bio-recognition using a specific CD63 aptamer, which was conjugated to horseradish peroxidase (HRP) through biotin-streptavidin binding. Colorimetric detection was achieved in 10 min via enzymatic catalysis to produce dark coloured polydopamine (PDA) from colourless substrate dopamine (DA) in especially prepared H2O2 reaction solution. The sensitivity was enhanced by in situ deposition of PDA around exosome particles to strengthen the developed colorimetric signal, which could be directly observed by naked eye. Signal quantification was carried out by absorbance measurement. The colour intensity correlates to the CD63 amount and the limit of detection can be as low as 7.7 × 103 particle/mL, improved by 3-5 orders of magnitude from conventional Dot-blot methods. The aptasensor showed specificity to HER2 and integrin αvβ6 positive, cell culture-derived, breast and pancreatic cancer-derived exosomes, respectively, when the correct aptamer sequence was used. Overall, a sensitive and selective colorimetric aptasensor was successfully developed for detecting cancer-derived exosomes facilitated by HRP-accelerated DA polymerization and in situ PDA deposition. This versatile aptasensor holds great potential for future development of point-of-care detection kits for cancer diagnosis in a clinical setting.

Abstract 1536: MiRNA expression profile of exosomes derived from breast cancer cells following induction of the epithelial-to-mesenchymal transition

Abstract Breast cancer-derived exosomes are small extracellular vesicles that play a crucial role in the metastatic progression of breast cancer due to their ability to transport a variety of biologically active molecules (e.g., miRNAs) to local and distant sites in the body. The molecular cargo transported by breast cancer-derived exosomes alters the transcriptome of recipient cells at these sites to stimulate pro-metastatic events, including the epithelial-to-mesenchymal transition (EMT). Importantly, the molecular cargo loaded into exosomes can vary depending on cell state. However, it is unclear whether the expression of miRNAs in breast cancer-derived exosomes is altered following inducement of the EMT. Regulation of exosomal miRNAs following the EMT may enhance the ability of circulating tumor cells to target specific tissue sites or improve formation and maintenance of metastatic tumors compared to miRNAs packaged into exosomes secreted from cancer cells without induction of the EMT. To address this knowledge gap, we hypothesized that the EMT alters expression of miRNAs in exosomes derived from EMT-induced MDA-MB-468 breast cancer cells. To induce the EMT, MDA-MB-468 cells were treated with exogenous epidermal growth factor. MDA-MB-468 cells treated with epidermal growth factor exhibited greater mRNA expression of the mesenchymal marker Vimentin compared to non-treated MDA-MB-468 cells by qRT-PCR. Subsequently, exosomes were isolated from the conditioned cell culture medium of MDA-MB-468 cells, with and without induction of the EMT, using ExoQuick exosome isolation reagent. Molecular size analysis of isolated exosomes revealed that they were similar in size to the typical size range reported for exosomes (40 - 150nm). Moreover, a common exosomal marker protein, CD9, was enriched in the MDA-MB-468-derived exosome fractions compared to whole cell lysates by Western blot. Furthermore, next generation sequencing and bioinformatic analysis were performed to determine the miRNA expression profiles of these exosomes. Results from this study reveal expression of miRNAs in breast cancer cell-derived exosomes before and after activation of the EMT and may indicate how exosomes secreted from advanced stage breast cancer enhance metastatic tumor progression. Citation Format: Christian A. Showalter, Yinan Huang, Monica M. Burdick. MiRNA expression profile of exosomes derived from breast cancer cells following induction of the epithelial-to-mesenchymal transition [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 1536.

Mixed-Dimensional Heterostructure Material-Based SERS for Trace Level Identification of Breast Cancer-Derived Exosomes.

Raman spectroscopy has capability for fingerprint molecular identification with high sensitivity if weak Raman scattering signal can be enhanced by several orders of magnitudes. Herein, we report a heterostructure-based surface-enhanced Raman spectroscopy (SERS) platform using 2D graphene oxide (GO) and 0D plasmonic gold nanostar (GNS), with capability of Raman enhancement factor (EF) in the range of ∼1010 via light–matter and matter–matter interactions. The current manuscript reveals huge Raman enhancement for heterostructure materials occurring via both electromagnetic enhancement mechanism though plasmonic GNS nanoparticle (EF ∼107) and chemical enhancement mechanism through 2D-GO material (EF ∼102). Finite-difference time-domain (FDTD) simulation data and experimental investigation indicate that GNS allows light to be concentrated into nanoscale “hotspots” formed on the heterostructure surface, which significantly enhanced Raman efficiency via a plasmon–exciton light coupling process. Notably, we have shown that mixed-dimensional heterostructure-based SERS can be used for tracking of cancer-derived exosomes from triple-negative breast cancer and HER2(+) breast cancer with a limit of detection (LOD) of 3.8 × 102 exosomes/mL for TNBC-derived exosomes and 4.4 × 102 exosomes/mL for HER2(+) breast cancer-derived exosomes.

Breast cancer-derived exosomes transmit lncRNA SNHG16 to induce CD73+\u03b3\u03b41 Treg cells

γδT cells have been reported to exert immunosuppressive functions in multiple solid malignant diseases, but their immunosuppressive functional subpopulation in breast cancer (BC) is still undetermined. Here, we collected 40 paired BC and normal tissue samples from Chinese patients for analysis. First, we showed that γδT1 cells comprise the majority of CD3+ T cells in BC; next, we found that CD73+γδT1 cells were the predominant regulatory T-cell (Treg) population in BC, and that their prevalence in peripheral blood was also related to tumour burden. In addition, CD73+γδT1 cells exert an immunosuppressive effect via adenosine generation. We also found that BC could modulate CD73 expression on γδT cells in a non-contact manner. The microarray analysis and functional experiments indicated that breast tumour cell-derived exosomes (TDEs) could transmit lncRNA SNHG16, which upregulates CD73 expression, to Vδ1 T cells. Regarding the mechanism, SNHG16 served as a ceRNA by sponging miR-16–5p, which led to the derepression of its target gene SMAD5 and resulted in potentiation of the TGF-β1/SMAD5 pathway to upregulate CD73 expression in Vδ1 T cells. Our results showed that the BC-derived exosomal SNHG16/miR-16–5p/SMAD5-regulatory axis potentiates TGF-β1/SMAD5 pathway activation, thus inducing CD73 expression in Vδ1 T cells. Our results first identify the significance of CD73+Vδ1 Tregs in BC, and therapy targeting this subpopulation or blocking TDEs might have potential for BC treatment in the future.