Protocol for cross-platform characterization of human and murine extracellular vesicles and particles.

Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by ExoELISA and western blot analysis. This allows for EVP cross-validation across different platforms. For complete details on the use and execution of this protocol, please refer to Hoshino et al.1.

Breast adipose tissue-derived extracellular vesicles from obese women alter tumor cell metabolism.

Breast adipose tissue is an important contributor to the obesity-breast cancer link. Extracellular vesicles (EVs) are nanosized particles containing selective cargo, such as miRNAs, that act locally or circulate to distant sites to modulate target cell functions. Here, we find that long-term education of breast cancer cells with EVs obtained from breast adipose tissue of women who are overweight or obese (O-EVs) results in increased proliferation. RNA-seq analysis of O-EV-educated cells demonstrates increased expression of genes involved in oxidative phosphorylation, such as ATP synthase and NADH: ubiquinone oxidoreductase. O-EVs increase respiratory complex protein expression, mitochondrial density, and mitochondrial respiration in tumor cells. The mitochondrial complex I inhibitor metformin reverses O-EV-induced cell proliferation. Several miRNAs-miR-155-5p, miR-10a-3p, and miR-30a-3p-which promote mitochondrial respiration and proliferation, are enriched in O-EVs relative to EVs from lean women. O-EV-induced proliferation and mitochondrial activity are associated with stimulation of the Akt/mTOR/P70S6K pathway, and are reversed upon silencing of P70S6K. This study reveals a new facet of the obesity-breast cancer link with human breast adipose tissue-derived EVs causing metabolic reprogramming of breast cancer cells.

Advancement in Cellular Topographic and Nanoparticle Capture Imaging by High Resolution Microscopy Incorporating a Freeze-Drying and Gaseous Nitrogen-based Approach.

Scanning electron microscopy (SEM) offers an unparalleled view of the membrane topography of mammalian cells by using a conventional osmium (OsO4) and ethanol-based tissue preparation. However, conventional SEM methods limit optimal resolution due to ethanol and lipid interactions and interfere with visualization of fluorescent reporter proteins. Therefore, SEM correlative light and electron microscopy (CLEM) has been hindered by the adverse effects of ethanol and OsO4 on retention of fluorescence signals. To overcome this technological gap in achieving high-resolution SEM and retain fluorescent reporter signals, we developed a freeze-drying method with gaseous nitrogen (FDGN). We demonstrate that FDGN preserves cyto-architecture to allow visualization of detailed membrane topography while retaining fluorescent signals and that FDGN processing can be used in conjunction with a variety of high-resolution imaging systems to enable collection and validation of unique, high-quality data from these approaches. In particular, we show that FDGN coupled with high resolution microscopy provided detailed insight into viral or tumor-derived extracellular vesicle (TEV)-host cell interactions and may aid in designing new approaches to intervene during viral infection or to harness TEVs as therapeutic agents.

Updates on radiotherapy-immunotherapy combinations: Proceedings of 6th annual ImmunoRad conference.

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference.

The capture of extracellular vesicles endogenously released by xenotransplanted tumours induces an inflammatory reaction in the premetastatic niche.

The capture of tumour-derived extracellular vesicles (TEVs) by cells in the tumour microenvironment (TME) contributes to metastasis and notably to the formation of the pre-metastatic niche (PMN). However, due to the challenges associated with modelling release of small EVs in vivo, the kinetics of PMN formation in response to endogenously released TEVs have not been examined. Here, we have studied the endogenous release of TEVs in mice orthotopically implanted with metastatic human melanoma (MEL) and neuroblastoma (NB) cells releasing GFP-tagged EVs (GFTEVs) and their capture by host cells to demonstrate the active contribution of TEVs to metastasis. Human GFTEVs captured by mouse macrophages in vitro resulted in transfer of GFP vesicles and the human exosomal miR-1246. Mice orthotopically implanted with MEL or NB cells showed the presence of TEVs in the blood between 5 and 28 days after implantation. Moreover, kinetic analysis of TEV capture by resident cells relative to the arrival and outgrowth of TEV-producing tumour cells in metastatic organs demonstrated that the capture of TEVs by lung and liver cells precedes the homing of metastatic tumour cells, consistent with the critical roles of TEVs in PMN formation. Importantly, TEV capture at future sites of metastasis was associated with the transfer of miR-1246 to lung macrophages, liver macrophages, and stellate cells. This is the first demonstration that the capture of endogenously released TEVs is organotropic as demonstrated by the presence of TEV-capturing cells only in metastatic organs and their absence in non-metastatic organs. The capture of TEVs in the PMN induced dynamic changes in inflammatory gene expression which evolved to a pro-tumorigenic reaction as the niche progressed to the metastatic state. Thus, our work describes a novel approach to TEV tracking in vivo that provides additional insights into their role in the earliest stages of metastatic progression.

Tumor-derived nanoseeds condition the soil for metastatic organotropism.

Primary tumors secrete a variety of factors to turn distant microenvironments into favorable and fertile 'soil' for subsequent metastases. Among these 'seeding' factors that initiate pre-metastatic niche (PMN) formation, tumor-derived extracellular vesicles (EVs) are of particular interest as tumor EVs can direct organotropism depending on their surface integrin profiles. In addition, EVs also contain versatile, bioactive cargo, which include proteins, metabolites, lipids, RNA, and DNA fragments. The cargo incorporated into EVs is collectively shed from cancer cells and cancer-associated stromal cells. Increased understanding of how tumor EVs promote PMN establishment and detection of EVs in bodily fluids highlight how tumor EVs could serve as potential diagnostic and prognostic biomarkers, as well as provide a therapeutic target for metastasis prevention. This review focuses on tumor-derived EVs and how they direct organotropism and subsequently modulate stromal and immune microenvironments at distal sites to facilitate PMN formation. We also outline the progress made thus far towards clinical applications of tumor EVs.

Collection of Biospecimens from the Inspiration4 Mission Establishes the Standards for the Space Omics and Medical Atlas (SOMA).

The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from the crew at different stages of the mission, including before (L-92, L-44, L-3 days), during (FD1, FD2, FD3), and after (R+1, R+45, R+82, R+194 days) spaceflight, creating a longitudinal sample set. The collection process included samples such as venous blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies, which were processed to obtain aliquots of serum, plasma, extracellular vesicles, and peripheral blood mononuclear cells. All samples were then processed in clinical and research laboratories for optimal isolation and testing of DNA, RNA, proteins, metabolites, and other biomolecules. This paper describes the complete set of collected biospecimens, their processing steps, and long-term biobanking methods, which enable future molecular assays and testing. As such, this study details a robust framework for obtaining and preserving high-quality human, microbial, and environmental samples for aerospace medicine in the Space Omics and Medical Atlas (SOMA) initiative, which can also aid future experiments in human spaceflight and space biology.

Tumour extracellular vesicles and particles induce liver metabolic dysfunction.

Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.

Genomic mapping of metastatic organotropism in lung adenocarcinoma.

We analyzed 2,532 lung adenocarcinomas (LUAD) to identify the clinicopathological and genomic features associated with metastasis, metastatic burden, organotropism, and metastasis-free survival. Patients who develop metastasis are younger and male, with primary tumors enriched in micropapillary or solid histological subtypes and with a higher mutational burden, chromosomal instability, and fraction of genome doublings. Inactivation of TP53, SMARCA4, and CDKN2A are correlated with a site-specific shorter time to metastasis. The APOBEC mutational signature is more prevalent among metastases, particularly liver lesions. Analyses of matched specimens show that oncogenic and actionable alterations are frequently shared between primary tumors and metastases, whereas copy number alterations of unknown significance are more often private to metastases. Only 4% of metastases harbor therapeutically actionable alterations undetected in their matched primaries. Key clinicopathological and genomic alterations in our cohort were externally validated. In summary, our analysis highlights the complexity of clinicopathological features and tumor genomics in LUAD organotropism.

Immune determinants of the pre-metastatic niche.

Primary tumors actively and specifically prime pre-metastatic niches (PMNs), the future sites of organotropic metastasis, preparing these distant microenvironments for disseminated tumor cell arrival. While initial studies of the PMN focused on extracellular matrix alterations and stromal reprogramming, it is increasingly clear that the far-reaching effects of tumors are in great part achieved through systemic and local PMN immunosuppression. Here, we discuss recent advances in our understanding of the tumor immune microenvironment and provide a comprehensive overview of the immune determinants of the PMN's spatiotemporal evolution. Moreover, we depict the PMN immune landscape, based on functional pre-clinical studies as well as mounting clinical evidence, and the dynamic, reciprocal crosstalk with systemic changes imposed by cancer progression. Finally, we outline emerging therapeutic approaches that alter the dynamics of the interactions driving PMN formation and reverse immunosuppression programs in the PMN ensuring early anti-tumor immune responses.

Protocol for Plasma Extracellular Vesicle and Particle Isolation and Mass Spectrometry-Based Proteomic Identification.

Plasma extracellular vesicles and particles (EVPs) are enriched in biomolecules that reflect individuals' physiological and pathological states. Several studies have demonstrated the potential of human plasma EVPs as a novel liquid biopsy. Here we describe a protocol for human plasma EVPs isolation and proteomic characterization. We isolated human plasma EVPs by the classical ultracentrifugation method and performed mass spectrometry-based proteomic profiling. Using this protocol, researchers can reveal the plasma EVPs proteome and explore the clinical application of plasma EVPs proteins for developing disease biomarkers.

Breast adipose tissue-derived extracellular vesicles from women with obesity stimulate mitochondrial-induced dysregulated tumor cell metabolism.

Breast adipose tissue is an important contributor to the obesity-breast cancer link. Dysregulated cell metabolism is now an accepted hallmark of cancer. Extracellular vesicles (EVs) are nanosized particles containing selective cargo, such as miRNAs, that act locally or circulate to distant sites to modulate target cell functions. Here, we found that long-term education of breast cancer cells (MCF7, T47D) with EVs from breast adipose tissue of women who are overweight or obese (O-EVs) leads to sustained increased proliferative potential. RNA-Seq of O-EV-educated cells demonstrates increased expression of genes, such as ATP synthase and NADH: ubiquinone oxidoreductase, involved in oxidative phosphorylation. O-EVs increase respiratory complex protein expression, mitochondrial density, and mitochondrial respiration in tumor cells. Mitochondrial complex I inhibitor, metformin, reverses O-EV-induced cell proliferation. Several miRNAs, miR-155-5p, miR-10a-3p, and miR-30a-3p, which promote mitochondrial respiration and proliferation, are enriched in O-EVs relative to EVs from lean women. O-EV-induced proliferation and mitochondrial activity are associated with stimulation of the Akt/mTOR/P70S6K pathway, and are reversed upon silencing of P70S6K. This study reveals a new facet of the obesity-breast cancer link with human breast adipose tissue-derived EVs causing the metabolic reprogramming of ER+ breast cancer cells.

Extracellular vesicles and particles as mediators of long-range communication in cancer: connecting biological function to clinical applications.

Over the past decade, extracellular vesicles and particles (EVPs) have emerged as critical mediators of intercellular communication, participating in numerous physiological and pathological processes. In the context of cancer, EVPs exert local effects, such as increased invasiveness, motility, and reprogramming of tumor stroma, as well as systemic effects, including pre-metastatic niche formation, determining organotropism, promoting metastasis and altering the homeostasis of various organs and systems, such as the liver, muscle, and circulatory system. This review provides an overview of the critical advances in EVP research during the past decade, highlighting the heterogeneity of EVPs, their roles in intercellular communication, cancer progression, and metastasis. Moreover, the clinical potential of systemic EVPs as useful cancer biomarkers and therapeutic agents is explored. Last but not least, the progress in EVP analysis technologies that have facilitated these discoveries is discussed, which may further propel EVP research in the future.

BRCA mutational status shapes the stromal microenvironment of pancreatic cancer linking clusterin expression in cancer associated fibroblasts with HSF1 signaling.

Tumors initiate by mutations in cancer cells, and progress through interactions of the cancer cells with non-malignant cells of the tumor microenvironment. Major players in the tumor microenvironment are cancer-associated fibroblasts (CAFs), which support tumor malignancy, and comprise up to 90% of the tumor mass in pancreatic cancer. CAFs are transcriptionally rewired by cancer cells. Whether this rewiring is differentially affected by different mutations in cancer cells is largely unknown. Here we address this question by dissecting the stromal landscape of BRCA-mutated and BRCA Wild-type pancreatic ductal adenocarcinoma. We comprehensively analyze pancreatic cancer samples from 42 patients, revealing different CAF subtype compositions in germline BRCA-mutated vs. BRCA Wild-type tumors. In particular, we detect an increase in a subset of immune-regulatory clusterin-positive CAFs in BRCA-mutated tumors. Using cancer organoids and mouse models we show that this process is mediated through activation of heat-shock factor 1, the transcriptional regulator of clusterin. Our findings unravel a dimension of stromal heterogeneity influenced by germline mutations in cancer cells, with direct implications for clinical research.

Tumor-produced and aging-associated oncometabolite methylmalonic acid promotes cancer-associated fibroblast activation to drive metastatic progression.

The systemic metabolic shifts that occur during aging and the local metabolic alterations of a tumor, its stroma and their communication cooperate to establish a unique tumor microenvironment (TME) fostering cancer progression. Here, we show that methylmalonic acid (MMA), an aging-increased oncometabolite also produced by aggressive cancer cells, activates fibroblasts in the TME, which reciprocally secrete IL-6 loaded extracellular vesicles (EVs) that drive cancer progression, drug resistance and metastasis. The cancer-associated fibroblast (CAF)-released EV cargo is modified as a result of reactive oxygen species (ROS) generation and activation of the canonical and noncanonical TGFß signaling pathways. EV-associated IL-6 functions as a stroma-tumor messenger, activating the JAK/STAT3 and TGFß signaling pathways in tumor cells and promoting pro-aggressive behaviors. Our findings define the role of MMA in CAF activation to drive metastatic reprogramming, unveiling potential therapeutic avenues to target MMA at the nexus of aging, the tumor microenvironment and metastasis.

Extracellular vesicles and particles impact the systemic landscape of cancer.

Intercellular cross talk between cancer cells and stromal and immune cells is essential for tumor progression and metastasis. Extracellular vesicles and particles (EVPs) are a heterogeneous class of secreted messengers that carry bioactive molecules and that have been shown to be crucial for this cell-cell communication. Here, we highlight the multifaceted roles of EVPs in cancer. Functionally, transfer of EVP cargo between cells influences tumor cell growth and invasion, alters immune cell composition and function, and contributes to stromal cell activation. These EVP-mediated changes impact local tumor progression, foster cultivation of pre-metastatic niches at distant organ-specific sites, and mediate systemic effects of cancer. Furthermore, we discuss how exploiting the highly selective enrichment of molecules within EVPs has profound implications for advancing diagnostic and prognostic biomarker development and for improving therapy delivery in cancer patients. Altogether, these investigations into the role of EVPs in cancer have led to discoveries that hold great promise for improving cancer patient care and outcome.

Metastasis suppressor NME1 in exosomes or liposomes conveys motility and migration inhibition in breast cancer model systems.

Tumor-derived exosomes have documented roles in accelerating the initiation and outgrowth of metastases, as well as in therapy resistance. Little information supports the converse, that exosomes or similar vesicles can suppress metastasis. We investigated the NME1 (Nm23-H1) metastasis suppressor as a candidate for metastasis suppression by extracellular vesicles. Exosomes derived from two cancer cell lines (MDA-MB-231T and MDA-MB-435), when transfected with the NME1 (Nm23-H1) metastasis suppressor, secreted exosomes with NME1 as the predominant constituent. These exosomes entered recipient tumor cells, altered their endocytic patterns in agreement with NME1 function, and suppressed in vitro tumor cell motility and migration compared to exosomes from control transfectants. Proteomic analysis of exosomes revealed multiple differentially expressed proteins that could exert biological functions. Therefore, we also prepared and investigated liposomes, empty or containing partially purified rNME1. rNME1 containing liposomes recapitulated the effects of exosomes from NME1 transfectants in vitro. In an experimental lung metastasis assay the median lung metastases per histologic section was 158 using control liposomes and 15 in the rNME1 liposome group, 90.5% lower than the control liposome group (P = 0.016). The data expand the exosome/liposome field to include metastasis suppressive functions and describe a new translational approach to prevent metastasis.

ERα-LBD, an isoform of estrogen receptor alpha, promotes breast cancer proliferation and endocrine resistance.

Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of an ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple-negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(-) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.

Metastasis.

Metastasis, the major cause of cancer death, represents one of the major challenges in oncology. Scientists are still trying to understand the biological basis underlying the dissemination and outgrowth of tumor cells, why these cells can remain dormant for years, how they become resistant to the immune system or cytotoxic effects of systemic therapy, and how they interact with their new microenvironment. We asked experts to discuss some of the unknowns, advances, and areas of opportunity related to cancer metastasis.

Astrocytic laminin-211 drives disseminated breast tumor cell dormancy in brain.

Although dormancy is thought to play a key role in the metastasis of breast tumor cells to the brain, our knowledge of the molecular mechanisms regulating disseminated tumor cell (DTC) dormancy in this organ is limited. Here using serial intravital imaging of dormant and metastatic triple-negative breast cancer lines, we identify escape from the single-cell or micrometastatic state as the rate-limiting step towards brain metastasis. We show that every DTC occupies a vascular niche, with quiescent DTCs residing on astrocyte endfeet. At these sites, astrocyte-deposited laminin-211 drives DTC quiescence by inducing the dystroglycan receptor to associate with yes-associated protein, thereby sequestering it from the nucleus and preventing its prometastatic functions. These findings identify a brain-specific mechanism of DTC dormancy and highlight the need for a more thorough understanding of tumor dormancy to develop therapeutic approaches that prevent brain metastasis.