Uptake Of Extracellular Vesicles Research Articles

Elevated extracellular vesicular Nm23-H1 subdues the pro-migratory potential of breast cancer cell-derived extracellular vesicles

Metastasis is a key determinant in cancer mortality which is often associated with decreased levels of Nm23-H1, a well-established metastasis suppressor. Despite lacking a secretion signal peptide, Nm23-H1 has been reported to be present in the extracellular space and enclosed within extracellular vesicles (EVs). While the presence of Nm23-H1 proteins in EVs released by cancer cells has been observed through proteomics profiling, the role of vesicular Nm23-H1 remains unclear. Here, we investigated the function of vesicular Nm23-H1 using MDA-MB-231 (highly metastatic, low Nm23-H1) and MCF-7 (low/non-metastatic, high Nm23-H1) breast cancer cell models. Our findings confirm that Nm23-H1 is indeed encapsulated within EVs, and its levels can be manipulated through overexpression and knockdown approaches. Functional assays revealed that EVs derived from MDA-MB-231 cells that contained high levels of Nm23-H1 exhibit impaired pro-migratory properties, suggesting that vesicular Nm23-H1 may act as a metastasis suppressor. Furthermore, EVs with increased levels of Nm23-H1 altered the transcript levels of multiple cancer-related genes in recipient cells and stimulated type I interferon signaling through STAT1 phosphorylation. These results suggest the existence of an unconventional signaling pathway mediated by the uptake of EVs enriched with Nm23-H1, which may contribute to the anti-metastatic effect of Nm23-H1 in the tumor microenvironment. Additionally, our study demonstrates that elevated Nm23-H1 levels can impact the abundance of various other proteins encapsulated within breast cancer cell-derived EVs, such as SUSD2 (Sushi Domain Containing 2) which can also modulate metastasis.

Abstract PO3-24-12: CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast cancers

Abstract Tumor progression involves the dynamic interaction between various cell types within the tumor microenvironment. Fibroblasts, which normally participate in wound healing, exhibit reciprocal communication with breast cancer cells that contributes to tumor cell plasticity and heterogeneity. Gaining insights into the molecular mechanisms underlying cell-to-cell communication, which drive cellular plasticity and play a pivotal role in breast cancer progression and metastasis, has the potential to unlock novel avenues for targeted therapeutic interventions. The small GPI-adaptor protein CRIPTO is a key player in this context. CRIPTO orchestrates the activity of TGF-β and growth factor-like signaling pathways that activate c-Src/MEK/AKT. CRIPTO is expressed during mammary development, wound healing, and breast cancer but is largely undetectable in homeostatic tissue, making it a promising therapeutic target. Our previous studies have demonstrated that the activity of CRIPTO is contingent upon cellular stress, with pronounced influence within pro-fibrotic, hypoxic domains of tumors. Importantly, we have shown that inhibition of CRIPTO using the soluble recombinant protein antagonist A4Fc effectively impedes the progression of triple negative breast cancer (TNBC) in murine xenograft models. Building upon these findings, we have been dissecting CRIPTO‘s role in coordinating fibroblast activation and cell-state reprogramming in TNBC. Notably, we have found that inhibition of CRIPTO with A4Fc suppresses cancer associated fibroblast (CAF) activation and collagen remodeling in vivo. And, by modeling tumor cell:fibroblast crosstalk using in vitro organoid co-cultures, we have demonstrated CRIPTO expression in tumor cells is necessary for extracellular vesicle (EV) uptake by fibroblasts, their SMAD pathway activation and their priming for reciprocal signaling back to tumor cells to for transcriptional reprogramming and cellular invasion. The significance of these findings lies in their potential to pave the way for the development of new therapies specifically targeting crucial tumor cell:fibroblast crosstalk mechanisms at primary and metastatic sites. Plasticity targeting therapies are urgently needed in the treatment of TNBC and other malignancies, and hold great promise for improving patient outcomes. Citation Format: David Freeman, Benjamin Spike, Brooke Gates. CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-12.

Food‐derived extracellular vesicles in the human gastrointestinal tract: Opportunities for personalised nutrition and targeted therapeutics

AbstractFood‐derived extracellular vesicles (FDEVs) such as those found in mammalian milk and plants are of great interest for both their health benefits and ability to act as biological nanocarriers. While the extracellular vesicle (EV) field is expanding rapidly to perform characterisation studies on FDEVs from plants, yeasts and bacteria, species‐specific differences in EV uptake and function in the human gastrointestinal (GI) tract are poorly understood. Moreover, the effects of food processing on the EV surfaceome and intraluminal content also raises questions surrounding biological viability once consumed. Here, I present a case for increasing community‐wide focus on understanding the cellular uptake of FDEVs from different animal, plant, yeast, and bacterial species and how this may impact their function in the human, which will have implications for human health and therapeutic strategies alike.

Thermotolerance in S. cerevisiae as a model to study extracellular vesicle biology.

The budding yeast Saccharomyces cerevisiae is a proven model organism for elucidating conserved eukaryotic biology, but to date its extracellular vesicle (EV) biology is understudied. Here, we show yeast transmit information through the extracellular medium that increases survival when confronted with heat stress and demonstrate the EV-enriched samples mediate this thermotolerance transfer. These samples contain vesicle-like particles that are exosome-sized and disrupting exosome biogenesis by targeting endosomal sorting complexes required for transport (ESCRT) machinery inhibits thermotolerance transfer. We find that Bro1, the yeast ortholog of the human exosome biomarker ALIX, is present in EV samples, and use Bro1 tagged with green fluorescent protein (GFP) to track EV release and uptake by endocytosis. Proteomics analysis reveals that heat shock protein 70 (HSP70) family proteins are enriched in EV samples that provide thermotolerance. We confirm the presence of the HSP70 ortholog stress-seventy subunit A2 (Ssa2) in EV samples and find that mutant yeast cells lacking SSA2 produce EVs but they fail to transfer thermotolerance. We conclude that Ssa2 within exosomes shared between yeast cells contributes to thermotolerance. Through this work, we advance Saccharomyces cerevisiae as an emerging model organism for elucidating molecular details of eukaryotic EV biology and establish a role for exosomes in heat stress and proteostasis that seems to be evolutionarily conserved.

Novel loading protocol combines highly efficient encapsulation of exogenous therapeutic toxin with preservation of extracellular vesicles properties, uptake and cargo activity

Extracellular vesicles (EVs) have mostly been investigated as carriers of biological therapeutics such as proteins and RNA. Nevertheless, small-molecule drugs of natural or synthetic origin have also been loaded into EVs, resulting in an improvement of their therapeutic properties. A few methods have been employed for EV cargo loading, but poor yield and drastic modifications of vesicles remain unsolved challenges. We tested a different strategy based on temporary pH alteration through incubation of EVs with alkaline sodium carbonate, which resulted in conspicuous exogenous molecule incorporation. In-depth characterization showed that vesicle size, morphology, composition, and uptake were not affected. Our method was more efficient than gold-standard electroporation, particularly for a potential therapeutic toxin: the plant Ribosome Inactivating Protein saporin. The encapsulated saporin resulted protected from degradation, and was efficiently conveyed to receiving cancer cells and triggered cell death. EV-delivered saporin was more cytotoxic compared to the free toxin. This approach allows both the structural preservation of vesicle properties and the transfer of protected cargo in the context of drug delivery.Graphic

Unraveling the Impact of Extracellular Vesicle-Depleted Serum on Endothelial Cell Characteristics over Time.

Extracellular vesicles (EVs) are produced by all kinds of cells, including endothelial cells. It has been observed that EVs present in fetal bovine serum (FBS), broadly used in cell culture, can be a confounding factor and lead to misinterpretation of results. To investigate this phenomenon, human brain microvascular endothelial cells (HBMECs) were cultured for 2 or 24 h in the presence of EV-depleted FBS (EVdS). Cell death, gene and protein expression, and the presence of EVs isolated from these cells were evaluated. The uptake of EVs, intercellular adhesion molecule 1 (ICAM-1) expression, and monocyte adhesion to endothelial cells exposed to EVs were also evaluated. Our results revealed higher apoptosis rates in cells cultured with EVdS for 2 and 24 h. There was an increase in interleukin 8 (IL8) expression after 2 h and a decrease in interleukin 6 (IL6) and IL8 expression after 24 h of culture. Among the proteins identified in EVs isolated from cells cultured for 2 h (EV2h), several were related to ribosomes and carbon metabolism. EVs from cells cultured for 24 h (EV24h) presented a protein profile associated with cell adhesion and platelet activation. Additionally, HBMECs exhibited increased uptake of EV2h. Treatment of endothelial cells with EV2h resulted in greater ICAM-1 expression and greater adherence to monocytes than did treatment with EV24h. According to our data, HBMEC cultivated with EVdS produce EVs with different physical characteristics and protein levels that vary over time.

β1-Integrin-Mediated Uptake of Chondrocyte Extracellular Vesicles Regulates Chondrocyte Homeostasis.

Osteoarthritis (OA) is the most prevalent age-related degenerative disorder, which severely reduces the quality of life of those affected. Whilst management strategies exist, no cures are currently available. Virtually all joint resident cells generate extracellular vesicles (EVs), and alterations in chondrocyte EVs during OA have previously been reported. Herein, we investigated factors influencing chondrocyte EV release and the functional role that these EVs exhibit. Both 2D and 3D models of culturing C28I/2 chondrocytes were used for generating chondrocyte EVs. We assessed the effect of these EVs on chondrogenic gene expression as well as their uptake by chondrocytes. Collectively, the data demonstrated that chondrocyte EVs are sequestered within the cartilage ECM and that a bi-directional relationship exists between chondrocyte EV release and changes in chondrogenic differentiation. Finally, we demonstrated that the uptake of chondrocyte EVs is at least partially dependent on β1-integrin. These results indicate that chondrocyte EVs have an autocrine homeostatic role that maintains chondrocyte phenotype. How this role is perturbed under OA conditions remains the subject of future work.

Bovine placental extracellular vesicles carry the fusogenic syncytin BERV-K1

Syncytins are endogenous retroviral envelope proteins which induce the fusion of membranes. A human representative of this group, endogenous retrovirus group W member 1 envelope (ERVW-1) or syncytin-1 is present in trophoblast-derived extracellular vesicles and supports the incorporation of these extracellular vesicles into recipient cells. During pregnancy, placenta-derived extracellular vesicles participate in feto-maternal communication. Bovine fetal binucleate trophoblast cells express the syncytin, bovine endogenous retroviral envelope protein K1 (BERV-K1). These cells release extracellular vesicles into the maternal stroma, but it is unclear whether BERV-K1 is included in these extracellular vesicles. Here, extracellular vesicles were isolated from bovine placental tissue using collagenase digestion, ultracentrifugation, and size exclusion chromatography. They were characterized with transmission electron microscopy, nanoparticle tracking analysis, immunoblotting and mass spectrometry. Immunohistochemistry and immunoelectron microscopy were used to localize BERV-K1 within the bovine placental tissue. The isolated extracellular vesicles range between 50 and 300 nm, carrying multiple extracellular vesicle biomarkers. Proteomic analysis and immunoelectron microscopy confirmed BERV-K1 presence on the isolated extracellular vesicles. Further, BERV-K1 was localized on intraluminal vesicles in secretory granules of binucleate trophoblast cells. The presence of BERV-K1 on bovine placental extracellular vesicles suggests their role in feto-maternal communication and potential involvement of BERV-K1 in uptake of extracellular vesicles by target cells.

Methods, Mechanisms, and Application Prospects for Enhancing Extracellular Vesicle Uptake.

Extracellular vesicles (EVs) are considered to be a new generation of bioinspired nanoscale drug delivery systems due to their low immunogenicity, natural functionality, and excellent biocompatibility. However, limitations such as low uptake efficiency, insufficient production, and inhomogeneous performance undermine their potential. To address these issues, numerous researchers have put forward various methods and applications for enhancing EV uptake in recent decades. In this review, we introduce various methods for the cellular uptake of EVs and summarize recent advances on the methods and mechanisms for enhancing EV uptake. In addition, we provide further understanding regarding enhancing EV uptake and put forward prospects and challenges for the development of EV-based therapy in the future.

Defining tropism and activity of natural and engineered extracellular vesicles.

Extracellular vesicles (EVs) have important roles as mediators of cell-to-cell communication, with physiological functions demonstrated in various in vivo models. Despite advances in our understanding of the biological function of EVs and their potential for use as therapeutics, there are limitations to the clinical approaches for which EVs would be effective. A primary determinant of the biodistribution of EVs is the profile of proteins and other factors on the surface of EVs that define the tropism of EVs in vivo. For example, proteins displayed on the surface of EVs can vary in composition by cell source of the EVs and the microenvironment into which EVs are delivered. In addition, interactions between EVs and recipient cells that determine uptake and endosomal escape in recipient cells affect overall systemic biodistribution. In this review, we discuss the contribution of the EV donor cell and the role of the microenvironment in determining EV tropism and thereby determining the uptake and biological activity of EVs.

Proton pump inhibitors enhance macropinocytosis-mediated extracellular vesicle endocytosis by inducing membrane v-ATPase assembly.

Besides participating in diverse pathological and physiological processes, extracellular vesicles (EVs) are also excellent drug-delivery vehicles. However, clinical drugs modulating EV levels are still lacking. Here, we show that proton pump inhibitors (PPIs) reduce EVs by enhancing macropinocytosis-mediated EV uptake. PPIs accelerate intestinal cell endocytosis of autocrine immunosuppressive EVs through macropinocytosis, thereby aggravating inflammatory bowel disease. PPI-induced macropinocytosis facilitates the clearance of immunosuppressive EVs from tumour cells, improving antitumor immunity. PPI-induced macropinocytosis also increases doxorubicin and antisense oligonucleotides of microRNA-155 delivery efficiency by EVs, leading to enhanced therapeutic effects of drug-loaded EVs on tumours and acute liver failure. Mechanistically, PPIs reduce cytosolic pH, promote ATP6V1A (v-ATPase subunit) disassembly from the vacuolar membrane and enhance the assembly of plasma membrane v-ATPases, thereby inducing macropinocytosis. Altogether, our results reveal a mechanism for macropinocytic regulation and PPIs as potential modulators of EV levels, thus regulating their functions.

Abstract 4122: Integrin avb3 and HSPG targeting agents prevent extracellular vesicles uptake and breast cancer metastasis

Abstract INTRODUCTION: Metastasis is the major cause of cancer-related deaths (90%). Intercellular communication between cancer cells and with cells in their environment plays a significant function in tumor progression. In fact, secreted factors like cytokines and extracellular vesicles (EVs) can either halt or stimulate tumorigenesis as well as metastasis. We have previously reported, that the integrin B3 (ITGB3) is required for metastasis of breast cancer cells in vivo (Sese et al. Oncotarget, 2017). We furthermore demonstrated, that EVs mediated colony growth is dependent on ITGB3 and that endocytosis mediated uptake of EVs relies on the interaction of ITGB3 with integrin av (ITGav) and heparan sulphated glycoproteins (HSPGs) (Fuentes et al. Nat. Commun., 2021). OBJECTIVE: The objective of this work is focused on identifying inhibitors targeting the ITGav/B3/HSPGs complex to prevent extracellular vesicle uptake in cancer cells and to test their ability to prevent metastasis. METHODOLOGY. Inhibitors targeting ITGB3/ITGAV or HSPGs were either commercially available or obtained through collaborations and single chain antibodies (scFv) were produced in house. Inhibition of fluorescent labeled EVs was determined by FACS analysis or Incucyte based measurements and the efficacy of inhibitors was determined in a dose dependent manner. Toxicity of inhibitors was studied by crystal violet-based assay in normal and tumor cell lines. Tail vein injection of luciferase labeled MDA-MB-231 cells into nude mice was used as model to study metastatic diseases under treatment with the identified inhibitors. Metastasis formation was monitored by bioluminescence imaging and lungs were examined pathologically at end point. Overall survival was determined. RESULTS: Among the tested Integrin and HSPGs targeting compounds, we identified an integrin-binding antibody, an integrin binding scFv, an RGD-mimetic compound and an HSPGs-binding peptide as inhibitors of EV uptake. Inhibition was validated in a dose dependent manner and toxicity studies did not show adverse effects. Three of those inhibitors, targeting either ITGB3/ITGAV or HSPGs were tested in mice models in vivo and all inhibitors could significantly reduce the formation of metastasis and strongly increase the overall survival. CONCLUSION: We have identified four drug candidates that target the ITGav/B3/HSPGs complex and are able to prevent EV uptake, demonstrating that EV uptake is a druggable process. Even more, the inhibitors tested in vivo could strongly reduce metastasis formation and increase overall survival. This data supports our hypothesis, that targeting EV mediated intercellular communication is a valuable approach to combat cancer metastasis. Citation Format: Elena Muro-Blanc, Rocío Bayona-Ramón y Cajal, Marta Cano-Galietero, Sara García-Ortega, Chiara Falciani, Luisa Bracci, Alberto J. Schuhmacher, Stefan Hümmer, Santiago Ramón y Cajal. Integrin avb3 and HSPG targeting agents prevent extracellular vesicles uptake and breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4122.

Abstract 540: Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development

Abstract Once cancer has metastasized, it is incurable because tumors evolve resistance to all systemically administered anti-cancer therapies. Resistance to therapy has classically been attributed to the presence of a cancer stem cell or genetic tumor cell heterogeneity. Extracellular vesicles (EVs) are nano-sized (40-1000 nm) vesicles that are released by all cells into the extracellular space. EVs carry diverse cargo dependent on the cell of origin and have been studied as a source of novel cancer-specific liquid biopsy biomarkers and contributors to tumor progression and metastasis. However, the paracrine function of EVs in promoting therapy resistance is under-studied, and the impact of EVs released from therapy-resistant cancer cells on surrounding therapy-sensitive cancer cells is unknown. In this study, we develop an innovative microfluidic device, TREVCo, to enable simultaneous single-cell trap, EV detection, and co-culture modules to evaluate EVs-mediated transfer of cellular information between therapy-resistant cancer cells and adjacent cells, and to characterize EVs released from resistant cells and determine their impact on the resistance phenotype of adjacent therapy-sensitive cancer cells. We utilize resistant cells from multiple prostate (PCa) cell lines that have been selected for therapy resistance by culture with increasing dosages of cancer drugs such as cisplatin, docetaxel. We use the matched untreated therapy-sensitive parental cell lines as the sensitive cells. We hypothesize a novel model of therapy resistance in lethal PCa: EVs released from resistant cells contain pro-resistance factors that are transferred to adjacent sensitive cells upon EV uptake, thus directly enabling therapeutic resistance. We will assess the impact of resistant cell-EVs at a single cell level via a novel device, TREVCo, that enables comprehensive downstream analysis. This single-cell approach enables dissection of critical EV-associated therapy-resistant factors within the large heterogeneity of resistant PCa. Co-culture of a single resistant cell and a hundred adjacent sensitive cells mimicking intra-tumoral interactions is also critical to demonstrate evidence of resistant cell secreting factors inducing a pro-resistance phenotype. These studies will be the first-in-field to assess the impact of resistant PCa cell-derived EVs at single-cell resolution. An understanding of resistant cell-derived EVs and the impact of that EV cargo on adjacent sensitive cells will provide critical insight into the acquisition of therapy resistance in lethal PCa. The novel approach to apply resistant cell-derived EVs in co-culture assay with sensitive cells will enable the screening of transmittable resistance-associated factors and provides critical opportunities for accelerating precision medicine for advanced PCa patients. Citation Format: Chi-Ju Kim, Sarah Amend, Kenneth Pienta. Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 540.

Abstract 1586: Unraveling the importance of pancreatic cancer extracellular signaling to endothelial cells within the tumor microenvironment

Abstract In many solid tumors, including pancreatic ductal adenocarcinoma (PDAC), the secretion of extracellular vesicles (EVs) has been shown to support tumor progression, chemotherapeutic resistance, and metastasis. Importantly, PDAC tumors are composed of up to 80% non-tumor cells and an extracellular matrix, yet no studies have been performed to determine which cells within PDAC tumors internalized PDAC EVs. Further, the RNA-binding protein HuR plays an important role in PDAC cells, supporting the stability and translation of transcripts that aid tumor cell stress responses. Our lab has recently found that EVs harbor distinct mRNA and phosphorylated protein cargoes from donor cells. Further, HuR knockout (KO) cells produce EVs with significantly different mRNAs and phosphorylated proteins that EVs isolated from HuR wildtype (WT) cells. Specifically, proteins relating to endothelial function and angiogenesis were significantly differentially phosphorylated. Considering the functional impact of EV cargoes on recipient cells, our study aims to identify the specific cell types in the tumor microenvironment that import PDAC EVs and investigate the role of PDAC cell HuR in this signaling process. We have generated human PDAC and mouse KrasG12D Trp53R172H driven (KPC) PDAC cell lines to express an EV reporter, PalmGRET. Utilizing these cells lines in an immunocompetent mouse model of PDAC, we found that endothelial cells are major importers of PDAC EVs. We next performed bulk RNA-sequencing on endothelial cells sorted from these tumors that have (GFP+) versus have not (GFP-) imported PDAC EVs to assess the functional impact of PDAC EVs. Subsequent studies will focus on delineating the importance of this signaling axis by ablating EV secretion in addition to assessing endothelial cell function when treated with EVs from HuR WT vs. KO cells. Collectively, our study sheds light on the preferential uptake of PDAC EVs by endothelial cells and highlights the potential role of tumor cell-intrinsic HuR in this signaling process. These findings expand our understanding of the interactions between PDAC and the tumor microenvironment, potentially paving the way for targeted therapeutic interventions in this devastating disease. Citation Format: Jennifer M. Finan, Yifei Guo, Alexandra Q. Bartlett, Kevin MacPherson, Valerie Calvert, Madeline D. Hedberg, Emanuel Petricoin, Rosalie Sears, Jonathan R. Brody. Unraveling the importance of pancreatic cancer extracellular signaling to endothelial cells within the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1586.

Abstract 6958: CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast cancers

Abstract Tumor progression involves dynamic interaction between various cell types within the tumor microenvironment. Fibroblasts, which normally participate in wound healing, reciprocally communicate with breast cancer cells to promote tumor cell plasticity and heterogeneity. Gaining insights into the molecular mechanisms underlying this cell-to-cell communication, could unlock novel therapeutic avenues for inhibiting cellular plasticity, breast cancer progression and metastasis. The small GPI-anchored adaptor protein CRIPTO is a key player in this context though important molecular details remain to be elucidated. CRIPTO orchestrates TGF-β pathway signaling as well as growth factor-like signaling pathways that activate c-Src/MEK/AKT, in an autocrine and paracrine manner. CRIPTO is expressed during mammary development, wound healing, and breast cancer but is largely undetectable in homeostatic tissue, making it a promising therapeutic target. Our previous studies have demonstrated that the activity of CRIPTO is contingent upon cellular stress, with pronounced influence within pro-fibrotic, hypoxic domains of tumors. Importantly, we have shown that inhibition of CRIPTO using the soluble recombinant protein antagonist A4Fc effectively impedes the progression of triple negative breast cancer (TNBC) in murine xenograft models. Building upon these findings, we have been dissecting CRIPTO’s role in coordinating fibroblast activation and cell-state reprogramming in TNBC. We find that inhibition of CRIPTO with A4Fc suppresses cancer associated fibroblast (CAF) activation and collagen remodeling in vivo. Further, by modeling tumor cell:fibroblast crosstalk in vitro, we have demonstrated mechanistically that CRIPTO expression in tumor cells mediates subsequent extracellular vesicle (EV) uptake by fibroblasts, as well as fibroblast SMAD pathway activation and priming for reciprocal signaling back to tumor cells to promote transcriptional reprogramming and cellular invasion.The significance of these findings lies in their elucidation of key EV control mechanisms that could be exploited to intervene in essential tumor cell:fibroblast crosstalk or to enhance targeting of therapeutic moieties based on EV architecture and function in cancer and/or normative regenerative processes. Citation Format: David W. Freeman, Brooke L. Gates, Mauri D. Spendlove, Benjamin T. Spike. CRIPTO-regulated extracellular vesicles mediate heterotypic cellular crosstalk in triple negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6958.

Abstract 5358: The influence of extracellular vesicles on immune cells in PDAC

Abstract Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) is bad. The tumor microenvironment consists of an abundance of immunosuppressive immune cells and anti-inflammatory cytokines that contribute to local and systemic immunosuppression in PDAC. Tumor cells can influence the tumor microenvironment and, with that, the present immune cells through the secretion of cytokines or chemokines or the secretion of extracellular vesicles (EVs). EVs are a media of communication among the tumor cells themselves, but also with other cells of the body, with the goal to promote tumor expansion and metastasis and decrease any anti-tumor-directed forces of the immune system. Our lab has shown that those EVs play a significant role in tumor growth and metastasis in PDAC. We have found MUC1 to be an important marker for tumor-derived EVs (tdEVs), associated with their activity and influence on cell proliferation and tumor growth in vitro and in vivo. Determining which immune cell populations are most targeted and impacted by tdEVs is essential to improving our understanding of the role of EVs in PDAC. Methods: TdEVs were isolated from murine pancreatic cancer cell lines through differential ultracentrifugation. EVs were then labeled with a membrane dye, which allows tracking of the EVs in the mouse. We designed an in vivo study to investigate the effects that tdEVs have on different immune cell populations, as well as tumor growth and metastasis. This study has two distinct phases: a 3-week “Pre-Education Phase”, with injection of either MUC1+ or MUC1KO EVs, is followed by orthotopic implantation of tumor cells in the pancreas. Data: We hypothesized that tdEVs impact effector immune cells in PDAC, leading to phenotypic, transcriptional, and functional changes that result in a decreased anti-tumor immune response. The highest uptake of EVs was found in macrophages and dendritic cells (DCs). Furthermore, most immune cells preferably take up MUC1+ as compared to MUC1KO EVs. After three weeks of EV education, RNA sequencing of the liver showed differential gene expression in a variety of genes related to processes associated with B cells. Treatment with MUC1+ EVs downregulated B cell-related processes in the liver, B cell differentiation, proliferation and activity are reduced. Several genes coding for immunoglobulin parts were affected as well. Immune cell profiling of the blood and spleen showed that MUC1+ EVs reduce NK and macrophage numbers in the spleen as compared to MUC1KO EV treatment after three weeks. Additionally, EV-treatment increased CD8+ T cell numbers in the blood but decreased CD8+ T cells in the spleen. Conclusion: We have shown that tdEVs induce phenotypic and transcriptional changes in immune cell populations and alter immune cell frequencies in vivo. Ongoing research in our lab involves analysis of phenotypic and transcriptional changes after tumor implantation as well as functional differences in EV-treated immune cells in vitro and in vivo. Citation Format: Clara S. Mundry, Ying Huang, Paul M. Grandgenett, Michael A. Hollingsworth. The influence of extracellular vesicles on immune cells in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5358.

Molecular Determinants Involved in the Docking and Uptake of Tumor-Derived Extracellular Vesicles: Implications in Cancer.

Extracellular vesicles produced by tumor cells (TEVs) influence all stages of cancer development and spread, including tumorigenesis, cancer progression, and metastasis. TEVs can trigger profound phenotypic and functional changes in target cells through three main general mechanisms: (i) docking of TEVs on target cells and triggering of intra-cellular signaling; (ii) fusion of TEVs and target cell membranes with release of TEVs molecular cargo in the cytoplasm of recipient cell; and (iii) uptake of TEVs by recipient cells. Though the overall tumor-promoting effects of TEVs as well as the general mechanisms involved in TEVs interactions with, and uptake by, recipient cells are relatively well established, current knowledge about the molecular determinants that mediate the docking and uptake of tumor-derived EVs by specific target cells is still rather deficient. These molecular determinants dictate the cell and organ tropism of TEVs and ultimately control the specificity of TEVs-promoted metastases. Here, we will review current knowledge on selected specific molecules that mediate the tropism of TEVs towards specific target cells and organs, including the integrins, ICAM-1 Inter-Cellular Adhesion Molecule), ALCAM (Activated Leukocyte Cell Adhesion Molecule), CD44, the metalloproteinases ADAM17 (A Disintegrin And Metalloproteinase member 17) and ADAM10 (A Disintegrin And Metalloproteinase member 10), and the tetraspanin CD9.

Proteomic characterization of extracellular vesicles in programmed cell death.

Programmed cell death (PCD) is a fundamental biological process that plays a critical role in cell development, differentiation, and homeostasis. The secretion and uptake of extracellular vesicles (EVs) is one of the important regulatory mechanisms for PCD. EVs are natural membrane structures secreted by cells that contain a variety of proteins, lipids, nucleic acids, and other bioactive molecules. Due to their important roles in intercellular communication and disease progression, there is great interest in studying EVs and their cargo. Different protein components are sorted and packaged in EVs, allowing EVs to perform their functions. The study of EV proteomics helps us understand the role of PCD in the development of diseases. Meanwhile, proteomics is a powerful tool for studying the composition and function of EVs, which assists in the identification, quantification, and profiling of protein components of EVs, and provides insight into the molecular mechanisms involved in PCD and related diseases. In this review, we summarize the characteristics of EV proteomics in different types of PCD, compare different proteomic profiling strategies for EVs, and discuss the impact of EV proteomics on cell function and regulation during PCD, to understand its role in the pathogenesis of related diseases.

Dynamics of extracellular vesicle uptake by mural granulosa cells in mice.

Extracellular vesicles (EVs) play an important role in the development and function of mammalian ovarian follicles. However, the mechanisms by which they are taken up by the follicular granulosa cells remain unclear. In addition, while oocytes play a pivotal role in follicular development, the possible interactions between oocyte-derived paracrine factors (ODPFs) and EV signals are unknown. Therefore, this study aimed to elucidate the mechanism of EV uptake and the effects of ODPFs on EV uptake by follicular somatic mural granulosa cells in mice. Fluorescence-labeled transferrin (TRF) and cholera toxin B (CTB), substrates for clathrin- and caveolae-mediated endocytosis, respectively, were taken up by mural granulosa cells in vitro. Their uptake was inhibited by Pitstop 2 and genistein, inhibitors of clathrin and caveolae pathways, respectively. Mural granulosa cells took up EVs, and this uptake was suppressed by Pitstop 2 and genistein. Moreover, ODPFs promoted the uptake of EVs and CTB, but not TRF, by mural granulosa cells. These results suggest that mural granulosa cells take up EVs through both clathrin- and caveolae-mediated endocytosis and that oocytes may promote caveolae-mediated endocytosis to facilitate the uptake of EVs.

Multiple Myeloma Derived Extracellular Vesicle Uptake by Monocyte Cells Stimulates IL-6 and MMP-9 Secretion and Promotes Cancer Cell Migration and Proliferation.

Multiple Myeloma (MM) is an incurable haematological malignancy caused by uncontrolled growth of plasma cells. MM pathogenesis is attributed to crosstalk between plasma cells and the bone marrow microenvironment, where extracellular vesicles (EVs) play a role. In this study, EVs secreted from a panel of MM cell lines were isolated from conditioned media by ultracentrifugation and fluorescently stained EVs were co-cultured with THP-1 monocyte cells. MM EVs from three cell lines displayed a differential yet dose-dependent uptake by THP-1 cells, with H929 EVs displaying the greatest EV uptake compared to MM.1s and U266 EVs suggesting that uptake efficiency is dependent on the cell line of origin. Furthermore, MM EVs increased the secretion of MMP-9 and IL-6 from monocytes, with H929 EVs inducing the greatest effect, consistent with the greatest uptake efficiency. Moreover, monocyte-conditioned media collected following H929 EV uptake significantly increased the migration and proliferation of MM cells. Finally, EV proteome analysis revealed differential cargo enrichment that correlates with disease progression including a significant enrichment of spliceosome-related proteins in H929 EVs compared to the U266 and MM.1s EVs. Overall, this study demonstrates that MM-derived EVs modulate monocyte function to promote tumour growth and metastasis and reveals possible molecular mechanisms involved.