Melanoma Exosomes Research Articles

Harnessing Spatiotemporal-Specific Tumorous Exosome Dynamics: Ultra-Sensitive Lanthanide Luminescence Detection Strategy Enabled by Exosomal Membrane Engineering for Melanoma Immunotherapy Monitoring.

Focused on the newly secreted tumorous exosomes during melanoma immunotherapy, this work has pioneered an ultra-sensitive spatiotemporal-specific exosome detection strategy, leveraging advanced exosomal membrane engineering techniques. The proposed strategy harnesses the power of amplified lanthanide luminescence signals on these exosomes, enabling precise and real-time monitoring of the efficacy of melanoma immunotherapy. The methodology comprises two pivotal steps. Initially, Ac4ManNAz-associated metabolic labeling is employed to evolve azide groups onto the membranes of newly secreted exosomes with remarkable selectivity. These azide groups serve as versatile clickable artificial tags, enabling the precise identification of melanoma exosomes emerging during immunotherapy. Subsequently, lanthanide-nanoparticle-functionalized polymer chains are controllably grafted onto the exosome surfaces through click chemistry and in situ Fenton-RAFT polymerization, serving as robust signal amplifiers. When integrated with time-resolved fluorescence detection, this strategy yields detection signals with an exceptionally high signal-to-noise ratio, enabling ultra-sensitive detection of PD-L1 antigen expression levels on the spatiotemporal-specific exosomes. The detection strategy boasts a wide linear concentration range spanning from 1.7 × 104 to 1.7 × 109 particles/mL, with a remarkable theoretical detection limit of 1.28 × 103 particles/mL. The remarkable enhancements in detection sensitivity and accuracy facilitate the evaluation of the efficacy of immunotherapeutic interventions in the mouse B16 melanoma model, notably revealing a substantial disparity in PD-L1 levels between immunotherapy-treated and untreated groups (P < 0.01) and further emphasizing the cumulative therapeutic effect that intensifies with repeated treatments (P < 0.001).

Protocol for Testing Human Melanoma Exosomes that Shift the Healthy Phenotype of Human Dermal Cells.

Exosomes are small membrane-derived vesicles that transmit DNA constituents, mRNAs, microRNAs, and proteins from donor cells to a receiver cell. Various cells comprising of mesenchymal, immune, and cancer cells discharge exosomes. Cancer cell exosomes form the entry and reprogramming of essentials connected to a tumor environment. Melanoma-derived exosomes transport diverse proteins such as c-MET and RAB27a, which leave a melanoma mark. Increased mesenchymal epithelial transition (MET) expressions in serum exosomes have been considered an indicator of disease progression. Meanwhile, RAB27a has been identified as being involved in exosome discharge and trafficking. Decreased expressions of RAB27a in human melanoma cells have shown to diminish exosome release.We examined the effects of the downregulation and upregulation of RAB27a and c-MET in human dermal fibroblasts by utilizing the isolated exosomes of malignant melanoma cell lines. Melanoma exosomes derived from cancer cells conveyed information to healthy dermal fibroblasts and stem cells while inducing phenotypic change. In this chapter, we show optimized protocols that were used by our group for in vitro analysis with melanoma exosomes.

Similarities and Differences in the Protein Composition of Cutaneous Melanoma Cells and Their Exosomes Identified by Mass Spectrometry

Intercellular transport of proteins mediated by extracellular vesicles (EVs)-exosomes and ectosomes-is one of the factors facilitating carcinogenesis. Therefore, the research on protein cargo of melanoma-derived EVs may provide a better understanding of the mechanisms involved in melanoma progression and contribute to the development of alternative biomarkers. Proteomic data on melanoma-derived EVs are very limited. The shotgun nanoLC-MS/MS approach was applied to analyze the protein composition of primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) cutaneous melanoma cells and exosomes released by them. All cells secreted homogeneous populations of exosomes that shared a characteristic set of proteins. In total, 3514 and 1234 unique proteins were identified in melanoma cells and exosomes, respectively. Gene ontology analysis showed enrichment in several cancer-related categories, including cell proliferation, migration, negative regulation of apoptosis, and angiogenesis. The obtained results broaden our knowledge on the role of selected proteins in exosome biology, as well as their functional role in the development and progression of cutaneous melanoma. The results may also inspire future studies on the clinical potential of exosomes.

Uveal Melanoma Exosomes Induce a Prometastatic Microenvironment through Macrophage Migration Inhibitory Factor.

This study provides the first in vivo evidence that MIF inhibition may serve as a novel adjuvant drug therapy to prevent metastasis in uveal melanoma.

The Role of Melanoma Cell-Derived Exosomes (MTEX) and Photodynamic Therapy (PDT) within a Tumor Microenvironment

Photodynamic Therapy (PDT), an unconventional cancer therapy with optimistic desirable effects, utilizes the delivery of a photosensitizer (PS) that is activated by light at a particular wavelength and inducing oxidative cytotoxic damage of a tumor and its surrounding vasculature. Deeper seated tumors such as internally metastasized melanomas are more difficult to treat with PDT as the penetration of laser light to those sites is less. Limitations in targeting melanomas can also be attributed to melanin pigments that hinder laser light from reaching targeted sites. Exosomes serve as naturally occurring nanoparticles that can be re-assembled with PSs, improving targeted cellular absorption of photosensitizing agents during PDT. Additionally, studies indicate that exosomes released from PDT-treated tumor cells play a critical role in mediating anti-tumor immune responses. This review collates the role of Melanoma Cell-Derived Exosomes (MTEX) in immune response mediation and metastasis. Tumor Cell-Derived Exosomes (TEX) post PDT treatment are also reviewed, as well as the effects of exosomes as carriers of photosensitizers and delivery systems for PDT. The understanding and research on the role of melanoma exosomes induced by Photodynamic Therapy and their tumor microenvironment will assist in future research in treatment prospects and implications.

UPAR-expressing melanoma exosomes promote angiogenesis by VE-Cadherin, EGFR and uPAR overexpression and rise of ERK1,2 signaling in endothelial cells

Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis. We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs). Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration. Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos. The CRISPR–Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells. A similar effect was obtained with a peptide that inhibits uPAR–EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation. This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos. We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.

Abstract LB-095: Synergistic cancer immunotherapy using tumor tissue derived exosomes and artificially produced bacterial outer membrane vesicles

Abstract Checkpoint inhibitors work only in cancers that host inflammatory cells, and “cold” tumors normally do not respond. Therefore, making “cold” tumors “hot” is required to increase the response rate to immunooncology therapies in general. Bacteria and bacterial products have been utilized for cancer immunotherapy for more than 100 years, but currently no such treatment is available because of the severe side effects that are observed. In this study, we produced artificial outer membrane vesicles (aOMVs) from Escherichia coli outer membrane, and injected them together with cancer tissue-derived exosomes to booster an immune response to the malignancy. Firstly, outer membranes were obtained from E. coli by chemical means, followed by ionic stress and applied mild energy to generate aOMVs. The yield and purity of aOMVs were analyzed by nanoparticle tracking analysis and transmission electron microscopy. The protein and RNA contents were examined by label-free quantitative mass spectrometry and bioanalyzer. Inflammation was evaluated in macrophage cell line (RAW 264.7) and mice in vivo, and bone marrow-derived dendritic cells were used to assess the immunomodulatory functions of the aOMVs. For the study of antitumor activity, mice were subcutaneously inoculated with B16F10 cells and then subcutaneously immunized with aOMVs and melanoma exosomes five times at 3-day intervals. Also, anti-mouse PD-1 antibody was intraperitoneally injected into mice 1 day prior to immunization to investigate the effects of combination therapy. To elucidate the immunogenic mechanism, blood and spleen were obtained for antibody titer and splenocyte function study. As a result, bacterial aOMVs presented nanosized spherical shape with closed membranes and exhibited high yield and purity with very few cytosolic components. These aOMVs do not cause pro-inflammatory cytokine responses in RAW 264.7 cells and mice in vivo, despite high exposure levels. The aOMVs could be taken up by dendritic cells to stimulate cytokine and maturation marker expression. Co-immunization with aOMVs and melanoma tissue-derived exosomes elicited tumor regression in melanoma-bearing mice through Th-1 type T cell immunity and anti-tumor exosome IgG antibody production. Also, the immunotherapeutic effect of aOMVs was synergistically enhanced by anti-PD-1 inhibitor. Collectively, bacterial aOMVs can be produced in a large quantities with high purity, but are “detoxified" compared to naturally released OMVs. The non-toxic aOMVs are powerful adjuvants for eliciting specific anti-tumor response, suggesting that aOMVs may be novel bacterial vesicle-mimetics clinically applicable as cancer treatment. Citation Format: Kyong-su Park, Kristina Svennerholm, Rossella Crescitelli, Cecilia Lässer, Inta Gribonika, Jan Lötvall. Synergistic cancer immunotherapy using tumor tissue derived exosomes and artificially produced bacterial outer membrane vesicles [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 LB-095.

Abstract 6082: Uveal melanoma exosomes express MET and re-program the recipient cells

Abstract Uveal melanoma (UM) is a rare melanoma subtype with an estimated annual incidence of approximately 2000 in the United States. Despite excellent rates of local disease control with surgery or radiotherapy, nearly 50% of patients will develop metastatic disease, mostly to the liver, with a life expectancy of 12 months. The receptor tyrosine kinase MET is overexpressed in 60-86% of cases and, together with its ligand HGF, it plays a significant role in UM migration and metastasis. In recent years, it has been demonstrated that tumor cells communicate with the microenvironment at distant organs by secreting exosomes, extracellular small vesicles released into body fluids, to support cancer development, immune evasion and metastatic progression. Exosomes are able to stably transfer their unique cargos of proteins and RNA from the primary tumor to distant sites, such as the pre-metastatic “niche”. The functional role of UM exosomes in the tumor microenvironment cross-talk is largely unknown. To get deeper insight into UM exosome biology, we have isolated exosomes from UM cells before and after treatment with the MET inhibitor crizotinib and examined their content by proteomic analysis. Approximately 600 proteins were identified from each of these samples, with 444 proteins in common and 145 and 167 proteins uniquely detected in exosomes from untreated and treated cells, respectively. The expression of several exosome-associated proteins, including MET, were suppressed by crizotinib, suggesting that the exosomal cargo can be pharmacologically manipulated. Exposure of normal hepatocytes to UM-derived exosomes results in activation of MET in a dose dependent fashion, with induction of both the MAPK and AKT/PI3K pathways. Exosomes derived from serum of patients with advanced uveal melanoma also express MET and induced similar effects in hepatocytes. Furthermore, UM-derived exosomes activate the expression of pro-angiogenic and pro-inflammatory factors in normal hepatocytes, while exosomes isolated from crizotinib-treated UM cells reduced these effects. UM-derived exosomes also stimulated immune cells to release cytokines and chemokines, including IL-6, IL-8 CCL2, CCL3, CCL5 and CXCL1. Functionally, we demonstrated that conditioned media from exosome-treated hepatocyte highly stimulated UM cell migration, which could be repressed by the combination of MET and cytokine receptor inhibitors. These findings provide evidence that UM derived exosomes play a role in reprogramming the recipient cells to facilitate the establishment of metastasis, and the inhibition of exosome-mediated signaling may provide a potential therapeutic strategy in the early stages of metastasis. Citation Format: Grazia Ambrosini, Alex J. Rai, Blake Ebert, Gary K. Schwartz. Uveal melanoma exosomes express MET and re-program the recipient cells [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 6082.

Melanoma-Derived Exosomal miR-125b-5p Educates Tumor Associated Macrophages (TAMs) by Targeting Lysosomal Acid Lipase A (LIPA).

Tumor-associated macrophages (TAMs) are the most abundant immune cells in the tumor microenvironment, promoting tumor initiation, growth, progression, metastasis, and immune evasion. Recently it was shown that cancer cell-derived exosomes induce a tumor-promoting phenotype in TAMs. Exosome-loaded proteins, DNA, and RNAs may contribute to the macrophage reprogramming. However, the exact mediators and mechanisms, particularly in melanoma, are not known. In this study we examined the effects of cutaneous melanoma-derived exosomes on macrophage function and the underlying mechanisms. First, we showed that exposure to melanoma exosomes induces a tumor-promoting TAM phenotype in macrophages. Sequencing revealed enrichment for several miRNAs including miR-125b-5p in cutaneous melanoma exosomes. We showed that miR-125b-5p is delivered to macrophages by melanoma exosomes and partially induces the observed tumor-promoting TAM phenotype. Finally, we showed that miR-125b-5p targets the lysosomal acid lipase A (LIPA) in macrophages, which in turn contributes to their phenotype switch and promotes macrophage survival. Thus, our data show for the first time that miR-125b-5p transferred by cutaneous melanoma-derived exosomes induces a tumor-promoting TAM phenotype in macrophages.

MicroRNAs in Tumor Exosomes Drive Immune Escape in Melanoma.

MicroRNAs (miRNA), small noncoding RNAs that regulate gene expression, exist not only in cells but also in a variety of body fluids. These circulating miRNAs could enable intercellular communication. miRNAs are packaged in membrane-encapsulated vesicles, such as exosomes, or protected by RNA-binding proteins. Here, we report that miRNAs included in human melanoma exosomes regulate the tumor immune response. Using microscopy and flow cytometry, we demonstrate that CD8+ T cells internalize exosomes from different tumor types even if these cells do not internalize vesicles as readily as other immune cells. We explored the function of melanoma-derived exosomes in CD8+ T cells and showed that these exosomes downregulate T-cell responses through decreased T-cell receptor (TCR) signaling and diminished cytokine and granzyme B secretions. The result reduces the cells' cytotoxic activity. Using mimics, we found that miRNAs enriched in exosomes-such as Homo sapiens (hsa)-miR-3187-3p, hsa-miR-498, hsa-miR-122, hsa-miR149, and hsa-miR-181a/b-regulate TCR signaling and TNFα secretion. Our observations suggest that miRNAs in melanoma-derived exosomes aid tumor immune evasion and could be a therapeutic target.

MicroRNA-300: A Transcellular Mediator in Exosome Regulates Melanoma Progression.

Melanoma is a common and high-mortality skin cancer. Oxidative stress and DNA damage caused by ultraviolet light (UV) are major causative factors of melanoma formation. However, the specific molecular mechanism is still unclear. In this study, 218 dysregulated genes and 104 dysregulated miRNAs in response to UV were screened by analyzing sequencing datasets. Among them, 29 up-regulated miRNAs and 28 down-regulated miRNAs were involved in the melanoma pathway. As the only differential gene in the melanoma pathway, GADD45B severely affects the prognosis of melanoma patients. MiR-300 is the only differentially expressed miRNA that regulates GADD45B. In addition, compared to normal melanocytes, miR-300 was significantly down-regulated in melanoma cells (log FC = −1.63) and exosomes (log FC = −1.34). Among the transcription factors predicted to regulate miR-300, MYC, PPARG, and ZIC2 were significantly up-regulated in melanoma cells, and TP53, JUN, JUNB, FOS, and FOSB interacted with GADD45B. We attempted to reveal the pathogenesis of melanoma and screen new biomarkers by constructing a TF-mRNA-miRNA axis in turn to provide a view for further research.

Natural melanoma-derived extracellular vesicles.

Melanoma cells produce a variety of extracellular vesicle (EV) types including shedding vesicles and exosomes (EXOs). These EVs are defined by their mechanism of cellular production. To date, the majority of EV investigations has centered around melanoma EXOs or small EVs (sEVs). Natural melanoma sEVs mediate pro-tumor processes including angiogenesis, immune regulation and modification of tissue microenvironments. A thorough examination of these processes reveals that they are interdependent. They work in concert to support tumor growth and survival. Pro-tumor functions attributed to melanoma cells are reproduced by melanoma sEVs. This ensures a certain degree of redundancy within the melanoma pathogenic process. It also allows for rapid adaptation of melanoma cells to changing microenvironments, anti-tumor immune responses, and therapeutic challenges. Further, as a result of their composition and inherent ability to engage the immune system, natural melanoma EVs possess excellent biomarker potential and might be used therapeutically as tumor vaccines.

Abstract 1983: Cancer exosomes immunosuppressive activity in the absence of contaminating soluble factors is multifactorial

Abstract Cancer derived exosomes, along with soluble factors such as growth factors, cytokines and chemokines have been identified as integral immunosuppressive pathways employed by cancer cells. Using melanoma cell lines as a model for generating human melanoma exosomes (HMEX), we studied the soluble factors associated with exosome purification methods. A Luminex 13-plex T cell activation kit was used to detect the presence of soluble factors in exosome isolates. We have demonstrated that combining Ultrafiltration and Size Exclusion Chromatography (REIUS method) can yield up to 58-fold more exosomes than ultracentrifugation (UC) with greatly reduced concentrations of contaminating soluble factors (IL-8: 4.6 pg/ml by REIUS versus 489 pg/ml by UC | IL-10: 1,052 pg/ml by REIUS versus 34,500 pg/ml by UC). A novel exosome-based Compact Surface Plasmon Resonance Biosensor demonstrated that REIUS-isolated exosomes abundantly express PD-L1 on their surface compared to UC-purified exosomes. These HMEX significantly inhibited a cloned NY-ESO tumor antigen-specific patient derived T cell function when co-cultured with a NY-ESO positive melanoma cell line for 4 h (IFN-γ positive T cell population: 78.5 ± 0.03 % versus 49.05 ± 0.07 %). Blocking with anti-PDL1 or anti-IL-10 failed to restore T cell function (IFN-γ production), while both anti-PD-L1 and anti-IL-10 antibodies partially reverse this suppression, thereby identifying IL-10 and PD-L1 as a novel collaborating inhibitory pathway. Taken together, we report that different exosome isolation methods impact the nature of the exosomes and contaminating soluble factors and should be chosen with caution. In addition, our data suggests a novel role for tumor-derived exosomes in the IL-10 and PD1-PDL1 axis crosstalk that impacts functional activity of T cells. Citation Format: Shin La Shu, Junko Matsuzaki, Cheryl Allen, Yunchen Yang, Edward Hurley, Kunle Odunsi, Yun Wu, Marc S. Ernstoff. Cancer exosomes immunosuppressive activity in the absence of contaminating soluble factors is multifactorial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1983.

Abstract 1334: Dual-isolation of CTCs and cancer exosomes from blood samples with melanoma using immunoaffinity based OncoBean microfluidic devices

Abstract Introduction: Melanoma is among the most aggressive cancers, and its incidence continues to grow. Due to the lack of promising markers to predict the disease and onset of metastasis, early detection and evaluation of treatment efficacy have been hampered. Recent advances in liquid biopsy have proposed alternatives for diagnosing disease with the merits of enabling continuous monitoring and non-invasiveness. CTCs and cancer exosomes are evolving as promising biomarkers due to their innate capability of having cancer-associated molecules and signatures. However, simultaneous isolation of CTCs and exosomes using the same methods from the identical samples has not been studied yet. Here, we propose the use of the OncoBean microfluidic device conjugated with melanoma specific antibodies, MCAM and MCSP for the simultaneous isolation. Using whole blood samples from patients, CTCs and exosomes are specifically isolated from the same samples and then undergo molecular profiling for comprehensive studies. Methods: OncoBean devices were fabricated by soft lithography using polydimethylsiloxane (PDMS). The fabricated devices were conjugated with biotinylated MCSP and MCAM, using Neutralavidin-biotin chemistry. Using 6ml blood samples, 3ml of whole blood and 3ml of plasma were used for CTC isolation and melanoma-associated exosome isolation, respectively. In order to remove cellular debris, the plasma sample was filtered using a 200nm filter. After CTC isolation, captured cells were stained with fluorescent antibodies for melanoma specific (Melan-A and S100), leukocyte (CD45), and nucleus (DAPI) markers. The captured and stained CTCs were enumerated with consideration of size, nucleus to cytoplasm ratio and melanoma marker expression. The isolated exosomes were examined by scanning electron microscopy (SEM) to evaluate their abundance and size. The protein concentration and exosomal protein expression were evaluated by protein extraction using RIPA buffer. Results: All melanoma patient samples showed 1-35 CTCs per 3ml of blood. Healthy donors had no CTCs. The abundance of isolated exosomes was evaluated by SEM and showed sizes ranging from 117-143nm. The average exosomal protein from melanoma was 28.6±19.2μg/ml, considerably higher than that of healthy donor, 11.0±3.1μg/ml. The exosomes isolated by OncoBean tested positively for the common exosomal protein, CD9, implying that the present device isolated melanoma exosomes specifically. Discussion and conclusion: We showed that melanoma patients have both circulating tumor cells and cancerous exosomes in their blood samples. Even though their absolute CTC number and exosomal protein concentration showed no high correlation, simultaneous isolation using the identical microfluidic devices will be helpful for further verification of disease and cross validation according to their roles in disease. Citation Format: Yoon-Tae Kang, Ting-Wen Lo, Thomas Hadlock, Emma Purcell, Aaron Kramer, Monica De Reguera, Scott Alan McLean, Sunitha Nagrath. Dual-isolation of CTCs and cancer exosomes from blood samples with melanoma using immunoaffinity based OncoBean microfluidic devices [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1334.

Abstract 3091: Effects of melanoma cell-derived exosomes in melanoma patients’ plasma on immune cell functions

Abstract Background: Exosomes are a subset of small (30-150nm) extracellular vesicles (EVs) of the endocytic origin that mediate intercellular communication in health and disease. Exosomes are actively produced by tumor cells, circulate freely in body fluids and carry messages that alter functions of normal cells in the tumor microenvironment (TME), including immune cell functions. The ability of melanoma exosomes to reprogram immune cell functions might contribute to disease progression. Methods: To evaluate effects of melanoma cell-derived exosomes (MTEX) on human immune cell subsets, we isolated exosomes from plasma of 12 patients with melanoma and separated MTEX from non-MTEX by immune capture using anti-CSPG4 mAb on beads. These exosome fractions, and exosomes from plasma of 6 normal donors used as controls (NC), were studied by on-bead flow cytometry to quantify their surface protein profiles and in co-incubation assays with immune cell subsets for functional attributes. Phenotypic and functional profiles of MTEX and non-MTEX were compared. Results &amp; Conclusion: MTEX were enriched (p&amp;lt;0.005)( in FasL and TRAIL, induced apoptosis of CD8+T cells, down-regulated CD69 expression levels on T cells and inhibited their proliferation (all p&amp;lt;0.0005). MTEX also interfered with NK cell functions (p&amp;lt;0.001). Non-MTEX were enriched (P&amp;lt;0.005) in co-stimulatory proteins, CD40L, OX40L, OX40 and, similar to exosomes of NC, were not immunosuppressive. Thus, MTEX, present in excess in plasma of patients with metastatic melanoma, mediate immune suppression and contribute to immune escape of the tumor. Citation Format: Priyanka Sharma, Brenda Diergaarde, Soldano Ferrone, John M. Kirkwood, Theresa L. Whiteside. Effects of melanoma cell-derived exosomes in melanoma patients’ plasma on immune cell functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3091.

813 Melanoma exosomes mediates the dysfunction of interferon signaling pathway in lymphocytes in tumor microenvironment

813 Melanoma exosomes mediates the dysfunction of interferon signaling pathway in lymphocytes in tumor microenvironment

Replication study: Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET.

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Lesnik et al., 2016) that described how we intended to replicate selected experiments from the paper 'Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET' (Peinado et al., 2012). Here we report the results. We regenerated tumor cells stably expressing a short hairpin to reduce Met expression (shMet) using the same highly metastatic mouse melanoma cell line (B16-F10) as the original study, which efficiently downregulated Met in B16F10 cells similar to the original study (Supplementary Figure 5A; Peinado et al., 2012). Exosomes from control cells expressed Met, which was reduced in exosomes from shMet cells; however, we were unable to reliably detect phosphorylated Met in exosomes. We tested the effect of exosome-dependent Met signaling on primary tumor growth and metastasis. Similar to the results in the original study, we did not find a statistically significant change in primary tumor growth. Measuring lung and femur metastases, we found a small increase in metastatic burden with exosomes from control cells that was diminished when Met expression was reduced; however, while the effects were in the same direction as the original study (Figure 4E; Peinado et al., 2012), they were not statistically significant. Differences between the original study and this replication attempt, such as level of knockdown efficiency, cell line genetic drift, sample sizes, study endpoints, and variability of observed metastatic burden, are factors that might have influenced the outcomes. Finally, we report meta-analyses for each result.

Exosome Isolation: Cyclical Electrical Field Flow Fractionation in Low-Ionic-Strength Fluids.

The influence of buffer substitution and dilution effects on exosome size and electrophoretic mobility were shown for the first time. Cyclical electrical field flow fractionation (Cy-El-FFF) in various substituted fluids was applied to exosomes and other particles. Tested carrier fluids of deionized (DI) water, 1× phosphate buffered saline (PBS), 0.308 M trehalose, and 2% isopropyl alcohol (IPA) influenced Cy-El-FFF-mediated isolation of A375 melanoma exosomes. All fractograms revealed a crescent-shaped trend in retention times with increasing voltage with the maximum retention time at ∼1.3 V AC. A375 melanoma exosome recovery was approximately 70-80% after each buffer substitution, and recovery was independent of whether the sample was substituted into 1× PBS or DI water. Exosome dilution in deionized water produced a U-shaped dependence on electrophoretic mobility. The effect of dilution using 1× PBS buffer revealed a very gradual change in electrophoretic mobility of exosomes from ∼-1.6 to -0.1 μm cm/s V, as exosome concentration was decreased. This differed from the use of DI water, where a large change from ∼-5.5 to -0.1 μm cm/s V over the same dilution range was observed. Fractograms of separated A375 melanoma exosomes in two substituted low-ionic-strength buffers were compared with synthetic particle fractograms. Overall, the ability of Cy-El-FFF to separate exosomes based on their size and charge is a highly promising, label-free approach to initially catalogue and purify exosome subtypes for biobanking as well as to enable further exosome subtype interrogations.

Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment

Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear. We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification. HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC). Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation. Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification. Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX. These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells. Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF. The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.

Abstract 5087: Human melanoma exosomes induce metabolic reprogramming in human adult dermal fibroblasts

Abstract The “seed and soil” hypothesis of cancer metastasis has led us to examine the role of tumor-derived exosomes on reprogramming stromal cells composing the tumor microenvironment. We used human adult dermal fibroblasts (HADF) in vitro as a model of the premetastatic niche. Specifically, we determined the role of human melanoma-derived exosomes (HMEX) on governing metabolic reprogramming of healthy HADF. HMEX from six melanoma cell lines (3 BRAF V600E mutant cell lines (526-mel, 888-mel and Hs 294T) and 3 BRAF wild-type cell lines (1300-mel, 2183-Her4 and HMCB)) were purified by size-exclusion chromatography. Using the fluorescent tag CellVue (Maroon) we demonstrated via ImageStream that HMEXs are rapidly taken up by HADF. Following 18 hours of exposure to HMEX, HADF increased aerobic glycolysis and reduced oxidative phosphorylation (OXPHOS) in vitro, as measured by Agilent Seahorse XFe96. We have also developed an immuno-biochip that captures exosomes via anti-CD63 antibodies and simultaneously detects exosomal microRNAs by cationic lipoplex nanoparticles (tCLN) containing molecular beacons. With this immuno-biochip, we demonstrated the presence of microRNAs miR-155 and miR-210 in HMEX, both of which work in concert to promote glycolysis and inhibit OXPHOS. The result has been consistent across all HMEXs from the six different melanoma cell lines. Our findings reveal a correlation between metabolic function and the presence of metabolically active microRNAs in HMEX that control aerobic glycolysis and OXPHOS of normal fibroblasts. Melanoma exosomes are potentially key players in promoting tumor progression by modulating normal stromal cell metabolism and creating an environment that is conducive to metastasis. Oxygen Consumption Rate (OCR) pmol/minHADF onlyHADF + HMEX 17.5 μgHADF + HMEX 25 μgHADF + HMEX 50μgHADF + HMEX100μgBasal OCR52.6753.5850.9638.2031.56 Citation Format: Shin La Shu, Cheryl L. Allen, Yunchen Yang, Orla Maguire, Hans Minderman, Arindam Sen, Michael J. Ciesielski, Katherine A. Collins, Peter J. Bush, Martin Morgan, Yun Wu, Richard B. Bankert, Marc S. Ernstoff. Human melanoma exosomes induce metabolic reprogramming in human adult dermal fibroblasts [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 5087.