Tumor-derived Vesicles Research Articles

Extracellular vesicles as modulators of monocyte and macrophage function in tumors.

The tumor microenvironment (TME) harbors several cell types, such as tumor cells, immune cells, and non-immune cells. These cells communicate through several mechanisms, such as cell-cell contact, cytokines, chemokines, and extracellular vesicles (EVs). Tumor-derived vesicles are known to have the ability to modulate the immune response. Monocytes are a subset of circulating innate immune cells and play a crucial role in immune surveillance, being recruited to tissues where they differentiate into macrophages. In the context of tumors, it has been observed that tumor cells can attract monocytes to the TME and induce their differentiation into tumor-associated macrophages with a pro-tumor phenotype. Tumor-derived EVs have emerged as essential structures mediating this process. Through the transfer of specific molecules and signaling factors, tumor-derived EVs can shape the phenotype and function of monocytes, inducing the expression of cytokines and molecules by these cells, thus modulating the TME towards an immunosuppressive environment.

T-Lymphocytes Activated by Dendritic Cells Loaded by Tumor-Derived Vesicles Decrease Viability of Melanoma Cells In Vitro.

Immunotherapy represents an innovative approach to cancer treatment, based on activating the body's own immune system to combat tumor cells. Among various immunotherapy strategies, dendritic cell vaccines hold a special place due to their ability to activate T-lymphocytes, key players in cellular immunity, and direct them to tumor cells. In this study, the influence of dendritic cells processed with tumor-derived vesicles on the viability of melanoma cells in vitro was investigated. Dendritic cells were loaded with tumor-derived vesicles, after which they were used to activate T-cells. The study demonstrated that such modified T-cells exhibit high activity against melanoma cells, leading to a decrease in their viability. Our analysis highlights the potential efficacy of this approach in developing immunotherapy against melanoma. These results provide new prospects for further research and the development of antitumor strategies based on the mechanisms of T-lymphocyte activation using tumor-derived vesicles.

Mechanisms underlying focused ultrasound-induced immunogenicity in melanoma

Abstract Advanced melanoma is an aggressive skin cancer with low survival rates. While the anti-tumor response relies on dendritic cells (DCs) to process tumor antigens, migrate to lymph nodes, and activate T-cells, within the tumor microenvironment (TME), DC activation is impeded. Subsequent immature DCs are a major mechanism underlying the ineffective T-cell response. We have shown that low-intensity focused ultrasound (LOFU), a novel therapy that delivers mechanical and thermal stresses to the tumor site, may enhance DC function in a B16 melanoma model. We hypothesize that LOFU enhances the dendritic cell-mediated anti-tumor response by inducing changes in tumor cell and dendritic cell autophagy and increasing pro-immunogenic factors in the TME.Autophagy is a lysosome-dependent process of cellular content degradation also implicated in the regulation of multivesicular body formation/degradation, and thus in the secretion of extracellular vesicles. Autophagy may subsequently modify the cargo of tumor-derived vesicles in a pro-immunogenic manner. We have found that LOFU treatment leads to increased LC3-II flux in melanoma cells and identified 82 proteins within melanoma-derived vesicles that are significantly enriched/reduced with LOFU, with enriched proteins partaking in processes such as antigen processing and intracellular trafficking. DCs pulsed with vesicles from LOFU-treated melanoma cells show enhanced activation of CD4 +and CD8 +T-cells, suggesting that LOFU may promote a DC-mediated anti-tumor response. Further understanding of DC involvement in the anti-tumor response will provide novel targets for the development of immunotherapies to improve survival and quality of life in advanced melanoma patients. Supported by grant from the NIH (R01 CA226861)

Synergistic wound repair effects of a composite hydrogel for delivering tumor-derived vesicles and S-nitrosoglutathione.

Treating chronic wounds requires transition from proinflammatory M1 to anti-inflammatory M2 dominant macrophages. Based on the role of tumor extracellular vesicles (tEVs) in regulating the phenotypic switching from M1 to M2 macrophages, we propose that tEVs may have a beneficial impact on alleviating the overactive inflammatory microenvironment associated with refractory wounds. On the other hand, as a nitric oxide donor, S-nitrosoglutathione (GSNO) can regulate inflammation, promote angiogenesis, enhance matrix deposition, and facilitate wound healing. In this study, a guar gum-based hydrogel with tEVs and GSNO was designed for the treatment of diabetic refractory wounds. This hybrid hydrogel was formed through the phenyl borate bonds, which can automatically disintegrate in response to the high reactive oxygen species (ROS) level at the site of refractory diabetic wounds, releasing tEVs and GSNO. We conducted a comprehensive evaluation of this hydrogel in vitro, which demonstrated excellent performance. Meanwhile, using a full-thickness excision model in diabetic mice, the wounds exposed to the therapeutic hydrogel healed completely within 21 days. The increased closure rate was associated with macrophage polarization and collagen deposition, accelerated fibroblast proliferation, and increased angiogenesis in the regenerating tissues. Therefore, this multifunctional hybrid hydrogel appears to be promising for clinical applications.

Abstract IA020: Early cancer detection through the analyses of extracellular vesicles

Abstract Breakthroughs in deep molecular profiling of non-invasive clinical samples (“liquid biopsy”) promise to revolutionize how cancer is detected and monitored. Analyzing circulating biomarkers in patient blood has shown their diagnostic potential in advanced disease states. Early detection, however, remains challenging – most biomarkers either (i) arise in advanced disease and at low abundance or (ii) often suffer from poor specificity, thus creating false positives that are costly and stressful for patients. We have been exploring extracellular vesicles (EVs) as a new biomarker that may overcome these challenges. EVs are nanoscale membrane particles secreted by cells. Most types of cancer shed large numbers of EVs that carry molecular cargo from the parent tumor. Analyzing EVs thus can represent a new real-time window to detect, diagnose, and serially monitor tumor molecular status. This presentation will describe our recent advances that leverage EVs for early cancer detection. Specifically, we will discuss the single EV analysis (SEA) platform that characterizes individual EVs for multiple protein markers. This method helps us unveil EV heterogeneity, which maximizes biological information content (specificity and sensitivity) for tumor-derived vesicles. We will review the SEA’s development and pilot application to diagnose early cancers. Citation Format: Hakho Lee, Cesar M. Castro. Early cancer detection through the analyses of extracellular vesicles. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr IA020.

Tumor-Derived Membrane Vesicles from the IL-2 Overexpression Melanoma Cells Affect on the Expression of Surface Markers of Human Peripheral Blood Mononuclear Cells In Vitro

Nowadays, immunotherapy, in particular immunotherapeutic vaccines, is a promising approach in the treatment of cancer which has already demonstrated its effectiveness. Extracellular vesicles (EVs), which are capable of delivering biologically active agents to the cells, can be a promising candidate for such vaccines. M14 human melanoma cells were transduced with lentivirus encoding interleukin (IL)–2. Membrane vesicles from M14 cells expressing IL-2 were isolated using cytochalasin B. The interaction of membrane vesicles with human peripheral blood mononuclear cells has been analyzed. Activation of T cells was shown, as well as a decrease in the number of NK cells, after cultivation with tumor-derived vesicles. However, no cytotoxic activity of T cells after cultivation with tumor-derived vesicles was observed, which can be explained by their immunosuppressive properties. On the other hand, such vesicles can be a promising source of tumor-specific antigens for dendritic vaccines. Therefore, further studies are required in view of the possible use of tumor-derived vesicles as a target antigen for dendritic cells.

Bispecific antibodies targeting CD40 and tumor-associated antigens promote cross-priming of T cells resulting in an antitumor response superior to monospecific antibodies

BackgroundIndications with poor T-cell infiltration or deficiencies in T-cell priming and associated unresponsiveness to established immunotherapies represent an unmet medical need in oncology. CD40-targeting therapies designed to enhance antigen presentation,...

Advances in Breast Cancer Management and Extracellular Vesicle Research, a Bibliometric Analysis.

Extracellular vesicles transport variable content and have crucial functions in cell–cell communication. The role of extracellular vesicles in cancer is a current hot topic, and no bibliometric study has ever analyzed research production regarding their role in breast cancer and indicated the trends in the field. In this way, we aimed to investigate the trends in breast cancer management involved with extracellular vesicle research. Articles were retrieved from Scopus, including all the documents published concerning breast cancer and extracellular vesicles. We analyzed authors, journals, citations, affiliations, and keywords, besides other bibliometric analyses, using R Studio version 3.6.2. and VOSviewer version 1.6.0. A total of 1151 articles were retrieved, and as the main result, our analysis revealed trending topics on biomarkers of liquid biopsy, drug delivery, chemotherapy, autophagy, and microRNA. Additionally, research related to extracellular vesicles in breast cancer has been focused on diagnosis, treatment, and mechanisms of action of breast tumor-derived vesicles. Future studies are expected to explore the role of extracellular vesicles on autophagy and microRNA, besides investigating the application of extracellular vesicles from liquid biopsies for biomarkers and drug delivery, enabling the development and validation of therapeutic strategies for specific cancers.

High-throughput microfluidic 3D biomimetic model enabling quantitative description of the human breast tumor microenvironment

Cancer is driven by both genetic aberrations in the tumor cells and fundamental changes in the tumor microenvironment (TME). These changes offer potential targets for novel therapeutics, yet lack of in vitro 3D models recapitulating this complex microenvironment impedes such progress. Here, we generated several tumor-stroma scaffolds reflecting the dynamic in vivo breast TME, using a high throughput microfluidic system. Alginate (Alg) or alginate-alginate sulfate (Alg/Alg-S) hydrogels were used as ECM-mimics, enabling the encapsulation and culture of tumor cells, fibroblasts and immune cells (macrophages and T cells, of the innate and adaptive immune systems, respectively). Specifically, Alg/Alg-S was shown capable of capturing and presenting growth factors and cytokines with binding affinity that is comparable to heparin. Viability and cytotoxicity were shown to strongly correlate with the dynamics of cellular milieu, as well as hydrogel type. Using on-chip immunofluorescence, production of reactive oxygen species and apoptosis were imaged and quantitatively analyzed. We then show how macrophages in our microfluidic system were shifted from a proinflammatory to an immunosuppressive phenotype when encapsulated in Alg/Alg-S, reflecting in vivo TME dynamics. LC-MS proteomic profiling of tumor cells sorted from the TME scaffolds revealed upregulation of proteins involved in cell-cell interactions and immunomodulation in Alg/Alg-S scaffolds, correlating with in vivo findings and demonstrating the appropriateness of Alg/Alg-S as an ECM biomimetic. Finally, we show the formation of large tumor-derived vesicles, formed exclusively in Alg/Alg-S scaffolds. Altogether, our system offers a robust platform for quantitative description of the breast TME that successfully recapitulates in vivo patterns. Statement of significanceCancer progression is driven by profound changes in both tumor cells and surrounding stroma. Here, we present a high throughput microfluidic system for the generation and analysis of dynamic tumor-stroma scaffolds, that mimic the complex in vivo TME cell proportions and compositions, constructing robust in vitro models for the study of the TME. Utilizing Alg/Alg-S as a bioinspired ECM, mimicking heparin's in vivo capabilities of capturing and presenting signaling molecules, we show how Alg/Alg-S induces complex in vivo-like responses in our models. Alg/Alg-S is shown here to promote dynamic protein expression patterns, that can serve as potential therapeutic targets for breast cancer treatment. Formation of large tumor-derived vesicles, observed exclusively in the Alg/Alg-S scaffolds suggests a mechanism for tumor survival.

Chemotherapy-Induced Upregulation of Small Extracellular Vesicle-Associated PTX3 Accelerates Breast Cancer Metastasis.

Although neoadjuvant chemotherapy is a standard component of breast cancer treatment, recent evidence suggests that chemotherapeutic drugs can promote metastasis through poorly defined mechanisms. Here we utilize xenograft mouse models of triple-negative breast cancer to explore the importance of chemotherapy-induced tumor-derived small extracellular vesicles (sEV) in metastasis. Doxorubicin (DXR) enhanced tumor cell sEV secretion to accelerate pulmonary metastasis by priming the premetastatic niche. Proteomic analysis and CRISPR/Cas9 gene editing identified the inflammatory glycoprotein PTX3 enriched in DXR-elicited sEV as a critical regulator of chemotherapy-induced metastasis. Both genetic inhibition of sEV secretion from primary tumors and pharmacologic inhibition of sEV uptake in secondary organs suppressed metastasis following chemotherapy. Taken together, this research uncovers a mechanism of chemotherapy-mediated metastasis by which drug-induced upregulation of sEV secretion and PTX3 protein cargo primes the premetastatic niche and suggests that inhibition of either sEV uptake in secondary organs or secretion from primary tumor cells may be promising therapeutic strategies to suppress metastasis. SIGNIFICANCE: These findings show that chemotherapy-induced small extracellular vesicles accelerate breast cancer metastasis, and targeted inhibition of tumor-derived vesicles may be a promising therapeutic strategy to improve the efficacy of chemotherapy treatment.

Recent advances in microfluidic methods in cancer liquid biopsy.

Early cancer detection, its monitoring, and therapeutical prediction are highly valuable, though extremely challenging targets in oncology. Significant progress has been made recently, resulting in a group of devices and techniques that are now capable of successfully detecting, interpreting, and monitoring cancer biomarkers in body fluids. Precise information about malignancies can be obtained from liquid biopsies by isolating and analyzing circulating tumor cells (CTCs) or nucleic acids, tumor-derived vesicles or proteins, and metabolites. The current work provides a general overview of the latest on-chip technological developments for cancer liquid biopsy. Current challenges for their translation and their application in various clinical settings are discussed. Microfluidic solutions for each set of biomarkers are compared, and a global overview of the major trends and ongoing research challenges is given. A detailed analysis of the microfluidic isolation of CTCs with recent efforts that aimed at increasing purity and capture efficiency is provided as well. Although CTCs have been the focus of a vast microfluidic research effort as the key element for obtaining relevant information, important clinical insights can also be achieved from alternative biomarkers, such as classical protein biomarkers, exosomes, or circulating-free nucleic acids. Finally, while most work has been devoted to the analysis of blood-based biomarkers, we highlight the less explored potential of urine as an ideal source of molecular cancer biomarkers for point-of-care lab-on-chip devices.

The Role of Tumor-Derived Vesicles in the Regulation of Antitumor Immunity.

In this article, we present a comprehensive, updated, and elucidative review of the current knowledge on the function played by tumor-derived vesicles (TDVs) in the crosstalk between tumor and immune cells. Characterization of the structure, biogenesis, and the major functions of TDVs is reported. The review focuses on particular ways of suppression or activation of CD4+/CD8+ T cells by tumor-derived vesicles. Tumor-derived vesicles play an important role in the suppression of antitumor immunity. During the last 15 years, vesicle research has elucidated and improved our knowledge about the role of the vesicles in intercellular communication. Nevertheless, there are still blinds spots concerning vesicle heterogeneity and isolation methods, their uptake by target cells, and the role of mRNA in T-cell transformation or suppression. Along with the substantial progress in understanding of the role of tumor-derived vesicles in intercellular communication, novel antitumor therapy strategies based on vesicle inhibition in a tumor microenvironment are likely to appear very soon.

PO-243 Exosomes from plasma of HNSCC patients treated with photodynamic therapy are biomarkers for epithelial-mesenchymal transition

IntroductionPhotodynamic therapy (PDT) is a palliative treatment option for patients with head and neck squamous cell carcinoma (HNSCC). We and others have shown that PDT induces a local inflammatory reaction with the potential to initiate antitumor immune responses. Recent studies indicate that cancer cells can change their morphology after PDT due to cytoskeleton alterations and decreased cell adhesion. To see whether the cargo of exosomes released by these cancer cells reflects cellular alterations after PDT, plasma was collected from PDT-treated patients prior to and at time points after therapy for exosome isolation and molecular characterisation.Material and methodsHNSCC patients (n=9) were treated with PDT in a palliative setting. All patients had previously undergone several oncologic treatment regimens. Blood samples were taken before and after PDT. Exosomes were isolated from plasma by mini size exclusion chromatography and were co-incubated with cancer cells. Following co-incubation, tumour cell migration, proliferation and chemotaxis were measured. Expression on tumour cells of the EMT markers: Vimentin, EpCAM (by flow cytometry, PCR, immunofluorescence), Snail, Twist, ZEB1, Slug (by PCR), E-Cadherin, N-Cadherin (by flow and PCR) was determined. Total plasma exosomes were separated by immune capture on beads into CD3 +and CD3neg fractions. The CD3neg exosomes enriched in tumor-derived vesicles were tested by on-bead flow cytometry for the presence of E-Cadherin- and N-Cadherin.Results and discussionsExosomes harvested pre- and 24 hour after PDT contained high levels on N-Cadherin. In contrast exosomes isolated on day 7 and 4–6 weeks after PDT contained high levels of E-Cadherin. Exosomes collected before and 24 hour post PDT and co-incubated with tumour cells altered cell morphology and induced mesenchymal features, including co-expression of Vimentin, N-Cadherin, snail, slug, twist and ZEB1. In functional experiments, exosomes collected pre- and 24 hour after PDT significantly enhanced migration, proliferation and chemotaxis of tumour cells relative to exosomes harvested at two later time points.ConclusionWe have shown for the first time that PDT can change the mesenchymal character of tumour cells converting it into an epithelial phenotype, and that exosomes in plasma of the PDT –treated patients were responsible for mediating the conversion. In addition, plasma-derived exosomes served as biomarkers of response to PDT, as their molecular cargo reflected the phenotypic changes occurring in the tumour cells during therapy.

Abstract 2748: Liquid biopsy using extracellular vesicular DNA for EGFR mutation testing in the patients with NSCLC

Abstract Background Tumor-derived extracelluar vesicles (EV) have been proved to carry double-stranded DNA including oncogenic mutants and expected to be a novel biomarker in cancer detection. Liquid biopsy using cell-free (cf) DNA for EGFR mutation testing is being actively investigated. In this study, we investigated whether liquid biopsy using EV DNA is superior to cf DNA for EGFR mutation testing in the patients with NSCLC. Methods EV was isolated using ultracentrifugation and confirmed by transmission electron microscopy and zetasizer in 20 plasma samples (all tissue-proven EGFR mutated) and 32 bronchoalveolar lavage fluid (BALF) samples (23 EGFR-mutated including 14 TKI-naïve and 9 TKI resistant, and 9 wild-type). EV and cf DNA was separately extracted from each samples and EGFR mutation testing was performed by peptide nucleic acid (PNA)-clamping method. Results In 20 plasma samples, the concordance rate of EV DNA for tissue EGFR genotyping was 55% (11/20), while 30% (6/20) in cf DNA. In 32 BALF samples, however, the sensitivity, specificity and concordance rate of EV DNA for activating EGFR mutations was 100% (23/23), 100% (9/9), and 100% (32/32), while cf DNA showed 73.9% (17/23), 100% (9/9) and 81.3% (26/32), respectively. The kappa value (p-value) of EV DNA was 1.0 (<0.001), while it was 0.714 (p,0.001) in the case of cf DNA. As for 9 re-biopsy cases for T790M detection in which only 3 patients (33%) was proved as positive T790M, BALF EV DNA identified 2 more T790M patients (total 5 among 9 patients, 56%) without non-matching with tissue typing, while BALF cf DNA found 1 more T790M positive patient with missing 1 tissue proven case (total 3 among 9 patients, 33%) Conclusions Liquid biopsy using EV DNA is significantly superior to cf DNA in both plasma and BALF. A little low sensitivity in plasma EV DNA is supposed to be improved with much more sensitive method such as ddPCR or BEAMing. Liquid biopsy using BALF EV DNA showed almost 100% matching with tissue EGFR typing and seems to be highly promising for T790M detection. A large number of clinical trials should be prompted. Citation Format: Jae Young Hur, Hee Jung Kim, Chang-Min Choi, Jae Cheol Lee, Kye Young Lee. Liquid biopsy using extracellular vesicular DNA for EGFR mutation testing in the patients with NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2748. doi:10.1158/1538-7445.AM2017-2748

Abstract 5609: Macrophages in secondary lymphoid organs regulate t cell tolerance and immunosurveillance in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that shows striking resistance to standard therapies. Immunotherapy is an exciting new treatment strategy for PDAC, but has yet to reproducibly demonstrate clinical efficacy. This finding may reflect poor effector T cell infiltration into PDAC or mechanisms of peripheral T cell tolerance. We hypothesize that successful immunotherapy in PDAC will require strategies that reverse elements of innate immunity that regulate T cell tolerance. This hypothesis is based on our prior studies in the KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mouse model of PDAC where macrophages residing outside of the tumor microenvironment were found to regulate the efficacy of a vaccine strategy involving the combination of chemotherapy and a CD40 agonist (FGK45). Specifically, we found that depletion of peripheral phagocytes using clodronate-encapsulated liposomes (CEL) was necessary for gemcitabine chemotherapy and a CD40 agonist to induce productive anti-tumor T cell immunity against spontaneously arising PDAC tumors in the KPC model. We now investigate the mechanism by which CEL-depleted phagocytes regulate T cell tolerance in PDAC using the KPC mouse model and an orthotopic implantation model. We found that CEL depletes macrophages in the spleen and abdominal lymph nodes, thereby reversing the pathologic accumulation of macrophages in these secondary lymphoid organs that is induced by PDAC tumors. CEL targeted two macrophage subsets in the lymph node, CD11b+CD169+F4/80neg subcapsular sinus macrophages (SSM) and CD11b+CD169+F4/80+ medullary sinus macrophages (MSM). These macrophage populations may be exposed to tumor-derived antigens and apoptotic cell vesicles entering the lymph node, and have been previously identified as central orchestrators of tolerance in autoimmune models. We found that both fluorescently-labeled liposomes and OVA-FITC peptide injected into the peritoneal cavity were specifically targeted to SSM and MSM with little uptake by dendritic cells (DC). In contrast, depletion of these first-line phagocytes by CEL shifted the cellular uptake of these model antigens increasing uptake by CD11b+CD11c+ DC which are more efficient in antigen presentation and show increased expression of co-stimulatory molecules after FGK45 treatment. Using an orthotopic model of PDAC, we then determined that CEL can enhance the T cell stimulatory capacity of gemcitabine chemotherapy in combination with FGK45 as seen by a significant increase in CD38, CD44, CD69 and PD-1 expression on CD4 and CD8 T cells in secondary lymphoid organs. Together, our findings suggest that tumor-derived antigens may be sequestered by tolerogenic macrophages in peritumoral lymph nodes that act to impair optimal induction of T cell responses. Thus, strategies aimed at disrupting the tolerogenic functions of these macrophages may offer a novel approach for improving immunotherapy in PDAC. Citation Format: Fee Bengsch, Anni Liu, Kathleen Graham, Gregory L. Beatty. Macrophages in secondary lymphoid organs regulate t cell tolerance and immunosurveillance in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5609. doi:10.1158/1538-7445.AM2017-5609

Enhanced Detection of Cancer Biomarkers in Blood-Borne Extracellular Vesicles Using Nanodroplets and Focused Ultrasound.

The feasibility of personalized medicine approaches will be greatly improved by the development of noninvasive methods to interrogate tumor biology. Extracellular vesicles shed by solid tumors into the bloodstream have been under recent investigation as a source of tumor-derived biomarkers such as proteins and nucleic acids. We report here an approach using submicrometer perfluorobutane nanodroplets and focused ultrasound to enhance the release of extracellular vesicles from specific locations in tumors into the blood. The released extracellular vesicles were enumerated and characterized using micro flow cytometry. Only in the presence of nanodroplets could ultrasound release appreciable levels of tumor-derived vesicles into the blood. Sonication of HT1080-GFP tumors did not increase the number of circulating tumor cells or the metastatic burden in the tumor-bearing embryos. A variety of biological molecules were successfully detected in tumor-derived extracellular vesicles, including cancer-associated proteins, mRNAs, and miRNAs. Sonication of xenograft HT1080 fibrosarcoma tumors released extracellular vesicles that contained detectable RAC1 mRNA with the highly tumorigenic N92I mutation known to exist in HT1080 cells. Deep sequencing serum samples of embryos with sonicated tumors allowed the identification of an additional 13 known heterozygous mutations in HT1080 cells. Applying ultrasound to HT1080 tumors increased tumor-derived DNA in the serum by two orders of magnitude. This work is the first demonstration of enhanced extracellular vesicle release by ultrasound stimulation and suggests that nanodroplets/ultrasound offers promise for genetic profiling of tumor phenotype and aggressiveness by stimulating the release of extracellular vesicles. Cancer Res; 77(1); 3-13. ©2016 AACR.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

SCS macrophages suppress melanoma by restricting tumor-derived vesicle-B cell interactions.

Tumor-derived extracellular vesicles (tEVs) are important signals in tumor-host cell communication, yet it remains unclear how endogenously produced tEVs affect the host in different areas of the body. We combined imaging and genetic analysis to track melanoma-derived vesicles at organismal, cellular, and molecular scales to show that endogenous tEVs efficiently disseminate via lymphatics and preferentially bind subcapsular sinus (SCS) CD169(+) macrophages in tumor-draining lymph nodes (tdLNs) in mice and humans. The CD169(+) macrophage layer physically blocks tEV dissemination but is undermined during tumor progression and by therapeutic agents. A disrupted SCS macrophage barrier enables tEVs to enter the lymph node cortex, interact with B cells, and foster tumor-promoting humoral immunity. Thus, CD169(+) macrophages may act as tumor suppressors by containing tEV spread and ensuing cancer-enhancing immunity.

Abstract A70: Melanoma-derived nanovesicles as drivers of immunosuppression

Abstract Background: The metastatic advancement of melanoma is largely dependent on its ability to evade immunity and gain access to the lymphatics. Characterization of tumor-draining, sentinel lymph nodes in patients has identified the nodes to express an immunosuppressed, Th2-dominant immune profile. Importantly, this profile is observed in sentinel lymph nodes even prior to evidence of metastasis supporting a model in which melanoma-derived factors prime tumor-draining, sentinel lymph nodes for tumor progression. Recent work in our lab has characterized the cellular interactions and the contribution of soluble factors in facilitating this immunosuppressive immune profile in fresh sentinel lymph nodes of patients; however, an appreciation for the role of subcellular counterparts remains to be explored. In this study, we considered a novel role for melanoma-derived, membrane-bound nanoparticles as drivers in polarizing immunity towards an immunosuppressive, tumor-promoting state. Methods and Results: Utilizing recent technology advances in Nanoparticle Tracking Analysis (NTA), characterization of melanoma derived vesicles from human melanoma cell lines showed a size distribution of particles ranging from 30nm to 1um, representing both exosomes and microvesicles. Following hypoxic culture, vesicle production was amplified, suggesting the increasingly hypoxic tumor microenvironment may trigger vesicle secretion. To evaluate the immune regulating potential of melanoma-derived vesicles, proteomic analysis of vesicle cargo by mass spectrometry identified over 100 proteins including known immune regulators Galectin 3, MHC I, ICAM1 and MIF. Functional assessment revealed that dendritic cells matured in vitro in the presence of melanoma-derived vesicles have significantly reduced surface marker expression of co-stimulatory markers CD80 and CD86 compared to controls. In addition to an activation defect, microarray analysis of dendritic cells matured in the presence of melanoma vesicles displayed a unique RNA expression profile. Pathway analysis of the expression arrays identified numerous overlapping signaling pathways involving proteins present in the melanoma-derived vesicles by mass spectrometry suggesting tumor-derived vesicle cargo has the ability to modulate dendritic cell function. Conclusion: Taken together, this study demonstrates a critical function for melanoma-derived vesicles in driving immunosuppression by characterizing their generation, protein cargo and immune regulating properties. The enrichment of melanoma-derived vesicles for immune mediators and ability of the vesicles to suppress dendritic cell function suggests a mechanism by which regional immunosuppression precedes nodal involvement and may foster an environment for metastasis to occur. Moving forward, we are currently translating these findings into fresh sentinel lymph nodes obtained from patients in order to elucidate the role of melanoma-derived vesicles in metastasis. Understanding this mechanism may yield valuable insight as we seek to develop efficacious immunotherapies targeting nodal metastasis. Citation Format: Rachel Goldenstein, Wendy Nevala, Svetomir N. Markovic. Melanoma-derived nanovesicles as drivers of immunosuppression. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A70.

Serum exosomes and cytokines promote a T-helper cell type 2 environment in the peripheral blood of glioblastoma patients.

Glioblastoma (GBM) is an aggressive infiltrative brain tumor with a particularly poor prognosis that is characterized by microvascular proliferation, necrotic tissue, and significant infiltration of M2-like monocytes. Compromised barrier function in tumor vasculature might be expected to permit communication between the tumor microenvironment and peripheral blood. To ascertain whether tumor-derived vesicles and/or factors might reach the bloodstream and what effects these molecules have on the peripheral compartment, we analyzed blood samples collected from primary GBM patients. Notably, a significant number of patient sera samples contained tumor exosome-reactive immunoglobulin (Ig)G2 and IgG4 antibody isotypes, which are consistent with Th2 immunity. M2-like monocytes expressing CD14+ and CD163+, another indicator of Th2 bias, are elevated in GBM patient blood and associated with high serum concentrations of colony-stimulating factor 2 and 3, as well as interleukin-2, -4, and -13, the latter 2 cytokines being hallmarks of Th2 immunity. GBM patient sera samples induce high levels of CD163 expression when added to normal monocytes, providing mechanistic evidence of a basis for Th2 bias. Fractionation of GBM patient sera into samples enriched for exosomes or soluble factors proved that both fractions are capable of inducing CD163 expression in normal monocytes. The results of the current study indicate a Th2 bias in the periphery of GBM patients, likely as a result of products elaborated by the tumor. Consequentially, through immune modulation these brain tumors exert systemic effects beyond the confines of the CNS.