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Extracellular vesicles (EVs) mediate intercellular communication in many physiologic processes and can modulate immune responses in individuals with cancer. Most studies of EVs in cancer have focused on their tumour promoting properties. Whether and how EVs might mediate tumour regression besides carrying antigens has not been well characterized. Using a mouse model of highly immunogenic regressor versus poorly immunogenic progressor tumour cells, we have characterized the role of EVs in activating macrophages and promoting tumour rejection. We found that the signalling molecule MAP2K1 (MEK1) is enriched in EVs secreted by regressor relative to progressor cells. Progressor EVs engineered to have levels of MEK1 similar to regressor EVs could inhibit tumour growth by indirectly promoting adaptive immunity in both syngeneic and 3rd party tumours. This effect required MEK1 activity and could occur by activating macrophages to promote adaptive immune responses against the tumour via the cytokine interferon-gamma. Our results suggest that MEK inhibition may be deleterious to cancer treatment, since MEK1 plays an important cell-extrinsic, tumour-suppressive role within EVs. Moreover, the delivery of MEK1 to tumour-associated macrophages, either by EVs, nanoparticles, or some other means, could be a useful strategy to treat cancer via the activation of anti-tumour immunity., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (<6 months after resection) or late (>6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B + natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B + NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The lymphatic fluid is the conduit by which part of the tissue "omics" is transported to the draining lymph node for immunosurveillance. Following cannulation of the pre-nodal cervical and mesenteric afferent lymphatics, herein we investigate the lymph proteomic composition, uncovering that its composition varies according to the tissue of origin. Tissue specificity is also reflected in the dendritic cell-major histocompatibility complex class II-eluted immunopeptidome harvested from the cervical and mesenteric nodes. Following inflammatory disruption of the gut barrier, the lymph antigenic and inflammatory loads are analyzed in both mice and subjects with inflammatory bowel diseases. Gastrointestinal tissue damage reflects the lymph inflammatory and damage-associated molecular pattern signatures, microbiome-derived by-products, and immunomodulatory molecules, including metabolites of the gut-brain axis, mapped in the afferent mesenteric lymph. Our data point to the relevance of the lymphatic fluid to probe the tissue-specific antigenic and inflammatory load transported to the draining lymph node for immunosurveillance., Competing Interests: Declaration of interests The authors declare that they do not have any conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The SpaceX Inspiration4 mission provided a unique opportunity to study the impact of spaceflight on the human body. Biospecimen samples were collected from four crew members longitudinally before (Launch: L-92, L-44, L-3 days), during (Flight Day: FD1, FD2, FD3), and after (Return: R + 1, R + 45, R + 82, R + 194 days) spaceflight, spanning a total of 289 days across 2021-2022. The collection process included venous whole blood, capillary dried blood spot cards, saliva, urine, stool, body swabs, capsule swabs, SpaceX Dragon capsule HEPA filter, and skin biopsies. Venous whole blood was further processed to obtain aliquots of serum, plasma, extracellular vesicles and particles, and peripheral blood mononuclear cells. In total, 2,911 sample aliquots were shipped to our central lab at Weill Cornell Medicine for downstream assays and biobanking. This paper provides an overview of the extensive biospecimen collection and highlights their processing procedures and long-term biobanking techniques, facilitating future molecular tests and evaluations.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 aid future human spaceflight and space biology experiments., (© 2024. The Author(s).)

Spaceflight induces an immune response in astronauts. To better characterize this effect, we generated single-cell, multi-ome, cell-free RNA (cfRNA), biochemical, and hematology data for the SpaceX Inspiration4 (I4) mission crew. We found that 18 cytokines/chemokines related to inflammation, aging, and muscle homeostasis changed after spaceflight. In I4 single-cell multi-omics data, we identified a "spaceflight signature" of gene expression characterized by enrichment in oxidative phosphorylation, UV response, immune function, and TCF21 pathways. We confirmed the presence of this signature in independent datasets, including the NASA Twins Study, the I4 skin spatial transcriptomics, and 817 NASA GeneLab mouse transcriptomes. Finally, we observed that (1) T cells showed an up-regulation of FOXP3, (2) MHC class I genes exhibited long-term suppression, and (3) infection-related immune pathways were associated with microbiome shifts. In summary, this study reveals conserved and distinct immune disruptions occurring and details a roadmap for potential countermeasures to preserve astronaut health., (© 2024. The Author(s).)

Extracellular vesicles and particles (EVPs) are pivotal mediators of pre-metastatic niche formation and cancer progression, including induction of vascular permeability, which facilitates tumor cell extravasation and metastasis. However, the mechanisms through which EVPs exert this effect remain poorly understood. Here, we elucidate a novel mechanism by which tumor EVPs enhance endothelial cell permeability, tumor extravasation, and lung metastasis to different degrees, depending on tumor type. Strikingly, vascular leakiness is observed within 48h following tumor implantation and as early as one hour following intravenous injection of tumour-derived EVPs in naïve mice. Surprisingly, rather than acting directly on endothelial cells, EVPs first activate interstitial macrophages (IMs) leading to activation of JAK/STAT signaling and IL-6 secretion in IMs which subsequently promote endothelial permeability. Depletion of IMs significantly reduces tumour-derived EVP-dependent vascular leakiness and metastatic potential. Tumour EVPs that strongly induce vascular leakiness express high levels of ITGα5, and ITGα5 ablation impairs IM activation, cytokine secretion, and subsequently vascular permeability and metastasis. Importantly, IL-6 expression is elevated in IMs from non-involved tumor-adjacent lung tissue compared to distal lung tissue in lung cancer patients, highlight the clinical relevance of our discovery. Our findings identify a key role for IM activation as an initiating step in tumor type-specific EVP-driven vascular permeability and metastasis, offering promising targets for therapeutic intervention., Competing Interests: Competing interests The authors have no competing interests to declare.

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 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Conflicts of interest David Lyden is an advisor of Aufbau Inc. and receives research support from Atossa Inc.

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., Competing Interests: Declaration of Competing Interest D.L. is on the scientific advisory board of Aufbau Holdings. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., Competing Interests: MHBH is or has have been a recipient of research grants paid to UCSF or in-kind resources from Roche-Genentech, Varian Medical Systems, Eli Lilly, Pathway Innovations and has received fees for consulting from EMD-Serono, Varian Medical Systems, Genentech, Pathway Innovation, Scholar Rock. KHhas Honoraria: Arch Oncology (Inst), AstraZeneca (Inst), BMS (Inst), Boehringer Ingelheim (Inst), Codiak Biosciences (Inst), F-Star Therapeutics (Inst), Inzen Therapeutics (Inst), Merck Serono (Inst), MSD (Inst), Oncolys Biopharma (Inst), Pfizer (Inst), Replimune (Inst), VacV Biotherapeutics (Inst); Consulting or Advisory Role: Arch Oncology (Inst), AstraZeneca (Inst), BMS (Inst), Boehringer Ingelheim (Inst), Inzen Therapeutics (Inst), Merck Serono (Inst), MSD (Inst), Oncolys BioPharma (Inst), Replimune (Inst); Speakers’ Bureau: BMS (Inst), Merck Serono (Inst), MSD (Inst); Research Funding: AstraZeneca (Inst), Boehringer Ingelheim (Inst), Merck Sharp & Dohme (Inst), Replimune (Inst). FGH received Grant or Research Support Companies from Accuray inc, Bioprotect, Bristol-Myers Squibb, Roche-ImFlame/ImCore, Nanobiotix, AstraZeneca, Debio Pharmaceuticals, Seagen, Eisai, MSD; Grant or Research Support Foundations from Prostate Cancer Foundation, San Salvatore Foundation; Investigator or Co-Investigator Clinical Trials in Bristol-Myers Squibb; Consultations: Johnson & Johnson; Academic Collaborations: EORTC chairman Gynecology Cancer Group, ESMO Scientific Committee member for drug development, ASTRO Scientific Committee Annual Meeting. TH has Consulting: Synthetic Biologics, Novocure, Boston Scientific, Inivata, Merck, GSK; Scientific Advisory Board: PanTher Therapeutics (Equity), Lustgarten; Research Funding (Clinical Trials): Taiho, AstraZeneca, BMS, GSK, IntraOp, Ipsen. EJ reports other support from Abmeta and Adventris, personal fees from Achilles, Dragonfly, Mestag, The Medical Home Group, and Surgtx, other support from Parker Institute, grants and other support from the Lustgarten Foundation, Genentech, BMS, and Break Through Cancer outside the submitted work. SDK receives clinical funding from AstraZeneca, Genentech, and Ionis; she also receives preclinical research funding from Roche. KS is founder and consultant for Faeth Therapeutics and Transomic Technologies. CK is the co-recipient of a Sponsored Research Agreement from Ion Beam Applications (IBA). AM is funded by the Associazione Italiana per la Ricerca sul Cancro (AIRC IG 2018 Id.21763 and AIRC Programma di ricerca 5 per Mille 2019 Id.22737). MM declare grants from Boehringer Ingelheim, AC Biosciences and MSD outside the submitted work. ZSM has Scientific Advisory Board roles and equity options with Archeus Technologies and Seneca Therapeutics. JS owns stock and is a member of the Scientific Advisory Board of Surface Oncology, and is a member of the Scientific Advisory Board of Domain Therapeutics. RT has research grants to his institution from: Varian Medical Systems, Elekta Oncology, Accuray, Inc; scientific advisory board member for: Reflexion Medical, ImmuneSensor Therapeutics. AW acknowledge funding from AstraZeneca and imCORE. RW has stock and other ownership interests with Boost Therapeutics, Immvira LLC, Reflexion Pharmaceuticals, Coordination Pharmaceuticals Inc., Magi Therapeutics, Oncosenescence, Aqualung Therapeutics Corporation, and Cyntegron; he has served in a consulting or advisory role for Aettis Inc., AstraZeneca, Coordination Pharmaceuticals, Genus, Merck Serono S.A., Nano Proteagen, NKGen Biotech, Shuttle Pharmaceuticals, Highlight Therapeutics, S.L., Aqualung Therapeutics Corporation; he has research grants with Varian and Regeneron. RZ is scientific advisory board member of iTeos Therapeutics, receives research grant support from Bristol Myers Squibb and AstraZeneca, and is inventor on patent applications related to work on GITR, CTLA-4, and PD-1 (patent numbers: US20180244793A1; US10323091B2; WO2018106864A1; WO2019094352A1). SD has received compensation for consultant/advisory services from Lytix Biopharma, Mersana Therapeutics, EMD Serono, Ono Pharmaceutical, and Genentech, and research support from Lytix Biopharma and Boehringer-Ingelheim for unrelated projects. LG is/has been holding research contracts with Lytix Biopharma, Promontory and Onxeo, has received consulting/advisory honoraria from Boehringer Ingelheim, AstraZeneca, OmniSEQ, Onxeo, The Longevity Labs, Inzen, Imvax, Sotio, Promontory, Noxopharm, EduCom, and the Luke Heller TECPR2 Foundation, and holds Promontory stock options. ED reports grants and personal fees from Roche Genentech; grants from Servier; grants from AstraZeneca; grants and personal fees from Merck-Serono; grants from BMS; and grants from MSD outside the submitted work. SCF has Consultant: Bayer, Bristol Myers Squibb, Varian, ViewRay, Accuray, Elekta, Janssen, Regeneron, GlaxoSmithKline, Eisai, Astra Zeneca, MedImmune, Merck US, EMD Serono/Merck, Genentech/ROCHE, Boehringer Ingelheim, Nanobiotix and Grant/Research: support from: Bristol Myers Squibb, Varian, Regeneron, Merck, Celldex. All other authors have no conflict of interest to declare., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Cancer alters the function of multiple organs beyond those targeted by metastasis 1,2 . 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., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Extracellular vesicles secreted by tumor cells contain double-stranded DNA called extracellular vesicle DNA (evDNA). EvDNA is genomic DNA that reflects cancer driver mutations. However, the significance of evDNA analysis in the diagnosis and surveillance of colon cancer remains unclear. This study aimed to investigate the clinical utility of extracellular vesicles and evDNA isolated from the plasma of colon cancer patients harboring KRAS G12D and G13D mutations., Methods: Cell-free DNA (cfDNA) and evDNA were collected from the plasma of 30 patients with colon cancer. KRAS mutation status (G12D and G13D) was detected using a droplet digital polymerase chain reaction assay (ddPCR). Sensitivity and specificity were evaluated in patients with wild-type KRAS tumors. Mutation status was correlated with carcinoembryonic antigen (CEA) levels and overall survival (OS)., Results: Thirty cfDNA and evDNA pairs showed a KRAS fractional abundance (FA) ranging from 0 to 45.26% and 0 to 83.81%, respectively. When compared with eight wild-type KRAS samples, cfDNA exhibited 70% sensitivity and 100% specificity, whereas evDNA achieved 76.67% sensitivity and 100% specificity. The concentration of evDNA was significantly lower than that of cfDNA, but it obtained a higher FA than cfDNA, while showing a positive correlation with CEA., Conclusions: Our findings demonstrate the feasibility of evDNA as a complementary tool to aid current methods of patient evaluation in the diagnosis and surveillance of colon cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Choi, Cho, Jeon, Kim, Han, Ahn, Wortzel, Lyden and Kim.)

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., (© 2022. The Author(s).)

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 TGFβ signaling pathways. EV-associated IL-6 functions as a stroma-tumor messenger, activating the JAK/STAT3 and TGFβ 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., (© 2022. The Author(s).)

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., (© 2022 The Authors.)

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., (© 2022. The Author(s).)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Dormant, disseminated tumor cells (DTCs) are thought to be the source of breast cancer metastases several years or even decades after initial treatment. To date, a selective therapy that leads to their elimination has not been discovered. While dormant DTCs resist chemotherapy, evidence suggests that this resistance is driven not by their lack of proliferation, but by their engagement of the surrounding microenvironment, via integrin-β1-mediated interactions. Because integrin-β1-targeted agents have not been translated readily to the clinic, signaling nodes downstream of integrin-β1 could serve as attractive therapeutic targets in order to sensitize dormant DTCs to therapy. By probing a number of kinases downstream of integrin-β1, we determined that PI3K inhibition with either a tool compounds or a compound (PF-05212384; aka Gedatolisib) in clinical trials robustly sensitizes quiescent breast tumor cells seeded in organotypic bone marrow cultures to chemotherapy. These results motivated the preclinical study of whether Gedatolisib-with or without genotoxic therapy-would reduce DTC burden and prevent metastases. Despite promising results in organotypic culture, Gedatolisib failed to reduce DTC burden or delay, reduce or prevent metastasis in murine models of either triple-negative or estrogen receptor-positive breast cancer dissemination and metastasis. This result held true whether analyzing Gedatolisib on its own (vs. vehicle-treated animals) or in combination with dose-dense doxorubicin and cyclophosphamide (vs. animals treated only with dose-dense chemotherapies). These data suggest that PI3K is not the node downstream of integrin-β1 that confers chemotherapeutic resistance to DTCs. More broadly, they cast doubt on the strategy to target PI3K in order to eliminate DTCs and prevent breast cancer metastasis., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR + metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis., Competing Interests: Competing Interests Statement The authors have no conflict of interests.

Epithelial plasticity, or epithelial-to-mesenchymal transition (EMT), is a well-recognized form of cellular plasticity, which endows tumor cells with invasive properties and alters their sensitivity to various agents, thus representing a major challenge to cancer therapy. It is increasingly accepted that carcinoma cells exist along a continuum of hybrid epithelial-mesenchymal (E-M) states and that cells exhibiting such partial EMT (P-EMT) states have greater metastatic competence than those characterized by either extreme (E or M). We described recently a P-EMT program operating in vivo by which carcinoma cells lose their epithelial state through post-translational programs. Here, we investigate the underlying mechanisms and report that prolonged calcium signaling induces a P-EMT characterized by the internalization of membrane-associated E-cadherin (ECAD) and other epithelial proteins as well as an increase in cellular migration and invasion. Signaling through Gαq-associated G-protein-coupled receptors (GPCRs) recapitulates these effects, which operate through the downstream activation of calmodulin-Camk2b signaling. These results implicate calcium signaling as a trigger for the acquisition of hybrid/partial epithelial-mesenchymal states in carcinoma cells., (© 2021 The Authors.)

Intercellular communication among immune cells is vital for the coordination of proper immune responses. Extracellular vesicles and particles (EVPs) act as messengers in intercellular communication, with important consequences for target cell and organ physiology in both health and disease. Under normal physiological conditions, immune cell-derived EVPs participate in immune responses by regulating innate and adaptive immune responses. EVPs play a major role in antigen presentation and immune activation. On the other hand, immune cell-derived EVPs exert immunosuppressive and regulatory effects. Consequently, EVPs may contribute to pathological conditions, such as autoimmune and inflammatory diseases, graft rejection, and cancer progression and metastasis. Here, we provide an overview of the role of EVPs in immune homeostasis and pathophysiology, with a particular focus on their contribution to innate and adaptive immunity and their potential use for immunotherapies., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Pelissier Vatter et al.)

Gastric cancer is the third most lethal cancer worldwide, and evaluation of the genomic status of gastric cancer cells has not translated into effective prognostic or therapeutic strategies. We therefore hypothesize that outcomes may depend on the tumor microenvironment (TME), in particular, cancer-associated fibroblasts (CAF). However, very little is known about the role of CAFs in gastric cancer. To address this, we mapped the transcriptional landscape of human gastric cancer stroma by microdissection and RNA sequencing of CAFs from patients with gastric cancer. A stromal gene signature was associated with poor disease outcome, and the transcription factor heat shock factor 1 (HSF1) regulated the signature. HSF1 upregulated inhibin subunit beta A and thrombospondin 2, which were secreted in CAF-derived extracellular vesicles to the TME to promote cancer. Together, our work provides the first transcriptional map of human gastric cancer stroma and highlights HSF1 and its transcriptional targets as potential diagnostic and therapeutic targets in the genomically stable tumor microenvironment. SIGNIFICANCE: This study shows how HSF1 regulates a stromal transcriptional program associated with aggressive gastric cancer and identifies multiple proteins within this program as candidates for therapeutic intervention. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/7/1639/F1.large.jpg., (©2021 American Association for Cancer Research.)

Importance: Recommendations for adjuvant therapy after surgical resection of lung adenocarcinoma (LUAD) are based solely on TNM classification but are agnostic to genomic and high-risk clinicopathologic factors. Creation of a prediction model that integrates tumor genomic and clinicopathologic factors may better identify patients at risk for recurrence., Objective: To identify tumor genomic factors independently associated with recurrence, even in the presence of aggressive, high-risk clinicopathologic variables, in patients with completely resected stages I to III LUAD, and to develop a computational machine-learning prediction model (PRecur) to determine whether the integration of genomic and clinicopathologic features could better predict risk of recurrence, compared with the TNM system., Design, Setting, and Participants: This prospective cohort study included 426 patients treated from January 1, 2008, to December 31, 2017, at a single large cancer center and selected in consecutive samples. Eligibility criteria included complete surgical resection of stages I to III LUAD, broad-panel next-generation sequencing data with matched clinicopathologic data, and no neoadjuvant therapy. External validation of the PRecur prediction model was performed using The Cancer Genome Atlas (TCGA). Data were analyzed from 2014 to 2018., Main Outcomes and Measures: The study end point consisted of relapse-free survival (RFS), estimated using the Kaplan-Meier approach. Associations among clinicopathologic factors, genomic alterations, and RFS were established using Cox proportional hazards regression. The PRecur prediction model integrated genomic and clinicopathologic factors using gradient-boosting survival regression for risk group generation and prediction of RFS. A concordance probability estimate (CPE) was used to assess the predictive ability of the PRecur model., Results: Of the 426 patients included in the analysis (286 women [67%]; median age at surgery, 69 [interquartile range, 62-75] years), 318 (75%) had stage I cancer. Association analysis showed that alterations in SMARCA4 (clinicopathologic-adjusted hazard ratio [HR], 2.44; 95% CI, 1.03-5.77; P = .042) and TP53 (clinicopathologic-adjusted HR, 1.73; 95% CI, 1.09-2.73; P = .02) and the fraction of genome altered (clinicopathologic-adjusted HR, 1.03; 95% CI, 1.10-1.04; P = .005) were independently associated with RFS. The PRecur prediction model outperformed the TNM-based model (CPE, 0.73 vs 0.61; difference, 0.12 [95% CI, 0.05-0.19]; P < .001) for prediction of RFS. To validate the prediction model, PRecur was applied to the TCGA LUAD data set (n = 360), and a clear separation of risk groups was noted (log-rank statistic, 7.5; P = .02), confirming external validation., Conclusions and Relevance: The findings suggest that integration of tumor genomics and clinicopathologic features improves risk stratification and prediction of recurrence after surgical resection of early-stage LUAD. Improved identification of patients at risk for recurrence could enrich and enhance accrual to adjuvant therapy clinical trials.

We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020)., Competing Interests: D.L., A.H., H.S.K., and L.B. have filed a US patent application related to this work., (© 2020 The Author(s).)

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type., Competing Interests: Declaration of Interests D.L., A.H., H.S.K., and L.B. have filed a U.S. patent application related to this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

SARS-CoV-2 has caused the COVID-19 pandemic. There is an urgent need for physiological models to study SARS-CoV-2 infection using human disease-relevant cells. COVID-19 pathophysiology includes respiratory failure but involves other organ systems including gut, liver, heart, and pancreas. We present an experimental platform comprised of cell and organoid derivatives from human pluripotent stem cells (hPSCs). A Spike-enabled pseudo-entry virus infects pancreatic endocrine cells, liver organoids, cardiomyocytes, and dopaminergic neurons. Recent clinical studies show a strong association with COVID-19 and diabetes. We find that human pancreatic beta cells and liver organoids are highly permissive to SARS-CoV-2 infection, further validated using adult primary human islets and adult hepatocyte and cholangiocyte organoids. SARS-CoV-2 infection caused striking expression of chemokines, as also seen in primary human COVID-19 pulmonary autopsy samples. hPSC-derived cells/organoids provide valuable models for understanding the cellular responses of human tissues to SARS-CoV-2 infection and for disease modeling of COVID-19., Competing Interests: Declaration of Interests R.E.S. is on the scientific advisory board of Miromatrix Inc. The other authors have no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Brain metastases occur in up to 25-55% of patients with metastatic HER2-positive breast cancer. Standard treatment has high rates of recurrence or progression, limiting survival and quality of life in most patients. Temozolomide (TMZ) is known to penetrate the blood-brain barrier and is US FDA approved for treatment of glioblastoma. Our group has demonstrated that low doses of TMZ administered in a prophylactic, metronomic fashion can significantly prevent development of brain metastases in murine models of breast cancer. Based on these findings, we initiated a secondary-prevention clinical trial with oral TMZ given to HER2-positive breast cancer patients with brain metastases after recent local treatment in combination with T-DM1 for systemic control of disease. Primary end point is freedom from new brain metastases at 1 year. (NCT03190967).