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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.

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., Dormant disseminated tumor cells (DTCs) are a potential source of late distant breast cancer recurrences. Previously, we identified integrin‐β1 as a promising target for DTC chemosensitization and metastasis prevention. Here, we explore downstream kinases, and find that despite promising initial results, targeting PI3K lacks efficacy on its own or in combination with chemotherapy in two different pre‐clinical models.

Mast cells; Melanoma; Metastasis Mastòcits; Melanoma; Metàstasi Mastocitos; Melanoma; Metástasis Metastatic disease is the major cause of death from cancer. From the primary tumour, cells remotely prepare the environment of the future metastatic sites by secreted factors and extracellular vesicles. During this process, known as pre-metastatic niche formation, immune cells play a crucial role. Mast cells are haematopoietic bone marrow-derived innate immune cells whose function in lung immune response to invading tumours remains to be defined. We found reduced melanoma lung metastasis in mast cell-deficient mouse models (Wsh and MCTP5-Cre-RDTR), supporting a pro-metastatic role for mast cells in vivo. However, due to evidence pointing to their antitumorigenic role, we studied the impact of mast cells in melanoma cell function in vitro. Surprisingly, in vitro co-culture of bone-marrow-derived mast cells with melanoma cells showed that they have an intrinsic anti-metastatic activity. Mass spectrometry analysis of melanoma-mast cell co-cultures secretome showed that HMGA1 secretion by melanoma cells was significantly impaired. Consistently, HMGA1 knockdown in B16-F10 cells reduced their metastatic capacity in vivo. Importantly, analysis of HMGA1 expression in human melanoma tumours showed that metastatic tumours with high HMGA1 expression are associated with reduced overall and disease-free survival. Moreover, we show that HMGA1 is reduced in the nuclei and enriched in the cytoplasm of melanoma metastatic lesions when compared to primary tumours. These data suggest that high HMGA1 expression and secretion from melanoma cells promote metastatic behaviour. Targeting HMGA1 expression intrinsically or extrinsically by mast cells actions reduce melanoma metastasis. Our results pave the way to the use of HMGA1 as anti-metastatic target in melanoma as previously suggested in other cancer types. US NIH (R01-CA169416); Children's Cancer and Blood Foundation; Feldestein Foundation; Melanoma Research Alliance; Nancy C. and Daniel P. Paduano Foundation; Starr Foundation; Translational NeTwork for the CLinical application of Extracellular VesicleS

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 (NGF) 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. we apologize to those authors whose work could not be cited due to size restrictions. We thank M. S. Soengas and the members of her laboratory for melanoma cells, primary melanocyte preparations and helpful discussions. We thank M. Detmar and S. Proulx for the mouse B16-F1R2 cell line. We are grateful to M. Yañez-Mo and M. Valés for antibodies against sEV markers. We thank D. Grela and A. Escobar from IESMAT for their support with the Zetasizer analysis. We thank G. Roncador, L. Maestre and J. L. Martinez Torrecuadrada for their help with the development and characterization of anti-NGFR antibodies and C. Villarroya Beltri for her help in flow cytometry analysis. This work was funded by the Starr Cancer Consortium (B.J.M., D.L. and H.P.), the US NIH (R01-CA169416), the Nancy C. and Daniel P. Paduano Foundation, the Children’s Cancer and Blood Foundation (H.P. and D.L.), the Melanoma Research Alliance, the Feldstein Foundation, RETOS SAF2017-82924-R (AEI/10.13039/501100011033/FEDER-UE), the Fundación Ramón Areces, the Fundación Bancaria ‘la Caixa’ (HR18-00256), ATRES-MEDIA AXA Foundation (CONSTANTES Y VITALES, una iniciativa de laSexta y Fundación AXA) and the Fundación Científica AECC (LABAE19027PEIN, GCB15152978SOEN-HP) (H.P.), the Malcolm Hewitt Wiener Foundation, the AHEPA Fifth District Cancer Research Foundation, the Hartwell Foundation and the Manning Foundation (D.L.). We are also grateful for the support of the Translational Network for the Clinical Application of Extracellular Vesicles (TeNTaCLES), RED2018-102411-T (AEI/10.13039/501100011033), the Ramón y Cajal Programme, the FERO Foundation, Comunidad of Madrid 2017-T2/BMD6026 (L.N.) and La Caixa Foundation (ID100010434, fellowship LCF/BQ/ES17/11600007) (A.H.-B.). The CNIO, certified as a Severo Ochoa Excellence Centre, is supported by the Spanish government through the ISCIII. No

Thromboembolism (TE) is a common complication in cancer patients and the second leading cause of cancer-related deaths. The incidence of TE varies in different cancer types, with the highest risk in pancreatic ductal adenocarcinoma (PDAC) and in advanced-stage and metastatic cancers. Despite the benefits associated with anti-coagulant therapy for symptomatic TE, the prevention of TE still remains an unmet clinical need due to lack of biomarkers predictive of TE risk and the bleeding risk associated with the routine use of anti-coagulants. Small extracellular vesicles (sEV) mediate cell-to-cell communication. Cancer cells and the tumor microenvironment release large numbers of sEV into the blood circulation and sEVs have displayed a therapeutic and predictive value in systemic diseases. However, the role of sEVs in cancer-associated TE remains to be investigated. Here we show that sEVs from (pre)metastatic lungs of mice with melanoma, breast, lung, and PDAC induce TE in mice and express high levels of integrin beta 2 (ITGB2), while sEVs from tumor cell lines, primary tumors, or other metastasis-bearing organs did not show any pro-thrombotic properties. A specific subtype of interstitial macrophages infiltrating (pre-)metastatic lungs were the main source of ITGB2+ pro-thrombotic sEVs. Blockade of ITGB2 on lung-derived sEVs, or systemically in mice, prevented EV-induced platelet aggregation and TE, and reduced metastasis. Examination of the mechanisms of ITGB2-induced TE showed that EV-associated ITGB2 interacts directly or through fibrin with different binding partners on platelets, and induce their activation and aggregation. Importantly, we found that levels of ITGB2 on sEVs are elevated in the plasma of PDAC patients prior ( Citation Format: Serena Lucotti, Yusuke Ogitani, Candia M. Kenific, Linda Bojmar, Michele Cioffi, Pernille Lauritzen, Henrik Molina, Soeren Heissel, Henry B. Lengel, Xiaohong Jing, Haiying Zhang, Irina Matei, Eileen M. O'Reilly, William R. Jarnagin, David Jones, James B. Bussel, David Kelsen, Jacqueline F. Bromberg, Diane M. Simeone, David Lyden. The lung pro-thrombotic niche drives cancer-associated thromboembolism and metastasis via extracellular vesicle ITGB2 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C028.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

While tumor-derived extracellular vesicles and particles (EVPs) are well characterized, little is known about the cargo and function of immune cell–derived EVPs. Here, we review the functional roles of immune cell–derived EVPs in normal physiology and disease., 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.

Summary 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)., Graphical abstract, Highlights • EVP isolation from human cell lines, tissues, and bodily fluids • First protocol describing EVP isolation from human tumor tissues and matched adjacent tissues • Improved EVP isolation and total EVP protein yield by dissecting tissues into pieces • AF4 fractionation to further analyze EVP samples, 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 necessitates 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.

Thromboembolism (TE) is a common complication in cancer patients and the second leading cause of cancer-related deaths. The incidence of TE varies in different cancer types, with the highest risk in lung cancer and pancreatic ductal adenocarcinoma (PDAC), and in advanced-stage and metastatic cancers. Despite the benefits associated with thromboprophylaxis for symptomatic TE, the prevention of TE still remains an unmet clinical need due to lack of biomarkers predictive of TE risk and the bleeding risk associated with the routine use of anti-coagulants. Exosomes are small circulating extracellular vesicles that mediate cell-to-cell communication. Cancer cells and the tumor microenvironment release large numbers of exosomes into the blood circulation and have displayed a therapeutic and predictive value in systemic diseases. Integrins expressed on the surface of exosomes drive their selective organotropism and prepare distant sites for metastatic seeding by establishing favorable pre-metastatic niches. Here we show that exosomes from metastasis-bearing lungs or pre-metastatic lungs of mice with melanoma, breast, lung and pancreatic cancer induce TE in mice and express high levels of integrin beta 2 (ITGB2). Instead, exosomes from tumor cell lines, primary tumors or other metastasis-bearing organs did not show any pro-thrombotic properties. Myeloid cells including monocytes/macrophages and neutrophils infiltrating pre- and post-metastatic lungs were the main source of ITGB2+ pro-thrombotic exosomes. Blockade of ITGB2 on lung-derived exosomes, or systemically in mice, prevented exosome-induced platelet aggregation and TE, and reduced metastasis. Examination of the mechanisms of ITGB2-induced TE showed that exosomal ITGB2 interact directly or through fibrin with different binding partners on platelets, and induce their activation and aggregation. Importantly, we found that exosomal ITGB2 levels are elevated in the plasma of PDAC patients prior to TE events in comparison to PDAC patients with no history of TE, and thus might serve as prognostic biomarker of TE. Together, our results provide the first evidence of the establishment of a pro-thrombotic lung niche in different cancer types. Moreover, we identify exosomal ITGB2 as a new target for the prevention and/or treatment of TE, as well as a potential “liquid biopsy” analyte for the early stratification of patients at high risk of TE. Citation Format: Serena Lucotti, Yusuke Ogitani, Candia M. Kenific, Linda Bojmar, Michele Cioffi, Pernille Lauritzen, Henrik Molina, Soren Heissel, Harry B. Lengel, Xiaohong Jing, Haiying Zhang, Irina Matei, Eileen M. O'Reilly, William R. Jarnagin, David R. Jones, James B. Bussel, David Kelsen, Jacqueline F. Bromberg, Diane M. Simeone, David Lyden. The lung pro-thrombotic niche drives cancer-associated thromboembolism via exosomal ITGB2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3138.

The health impact of prolonged space flight on the human body is not well understood. Liquid biopsies based on cell-free DNA (cfDNA) or exosome analysis provide a noninvasive approach to monitor the dynamics of genomic, epigenomic and proteomic biomarkers, and the occurrence of DNA damage, physiological stress, and immune responses. To study the molecular consequences of spaceflight we profiled cfDNA isolated from plasma of an astronaut (TW) during a year-long mission on the International Space Station (ISS), sampling before, during, and after spaceflight, and compared the results to cfDNA profiling of the subject’s identical twin (HR) who remained on Earth, as well as healthy donors. We characterized cfDNA concentration and fragment size, and the positioning of nucleosomes on cfDNA, observing a significant increase in the proportion of cell-free mitochondrial DNA inflight, suggesting that cf-mtDNA is a potential biomarker for space flight-associated stress, and that this result was robust to ambient transit from the International Space Station (ISS). Analysis of exosomes isolated from post-flight plasma revealed a 30-fold increase in circulating exosomes and distinct exosomal protein cargo, including brain-derived peptides, in TW compared to HR and all known controls. This study provides the first longitudinal analysis of astronaut cfDNA during spaceflight, as well as the first exosome profiles, and highlights cf-mtDNA levels as a potential biomarker for physiological stress or immune system responses related to microgravity, radiation exposure, and other unique environmental conditions on the ISS.

Summary Liquid biopsies based on cell-free DNA (cfDNA) or exosomes provide a noninvasive approach to monitor human health and disease but have not been utilized for astronauts. Here, we profile cfDNA characteristics, including fragment size, cellular deconvolution, and nucleosome positioning, in an astronaut during a year-long mission on the International Space Station, compared to his identical twin on Earth and healthy donors. We observed a significant increase in the proportion of cell-free mitochondrial DNA (cf-mtDNA) inflight, and analysis of post-flight exosomes in plasma revealed a 30-fold increase in circulating exosomes and patient-specific protein cargo (including brain-derived peptides) after the year-long mission. This longitudinal analysis of astronaut cfDNA during spaceflight and the exosome profiles highlights their utility for astronaut health monitoring, as well as cf-mtDNA levels as a potential biomarker for physiological stress or immune system responses related to microgravity, radiation exposure, and the other unique environmental conditions of spaceflight., Graphical Abstract, Highlights • Liquid biopsy can monitor the health conditions of astronauts during spaceflight • Increases in cell-free mitochondrial DNA were found during spaceflight • Post-flight astronaut blood had exosome increases and brain-derived peptides • Controls for sampling from the ISS can correct for technical noise, Space Medicine; Omics

A humanized biomaterial microenvironment that mimics the pre-metastatic niche captures disseminated tumour cells and recapitulates metastatic progression after implantation in xenografted mice.

In 1889, Stephen Paget first proposed that organ distribution of metastases is a non-random event, yet metastatic organotropism remains one of the greatest mysteries in cancer biology. A growing number of studies demonstrate that tumor-derived microvesicles, referred to as exosomes, may alter the tumor microenvironment at future sites of metastasis promoting pre-metastatic niche formation, and thus creating a favorable “soil” for incoming metastatic “seeds.” However, by what mechanism, and whether their role is significant in tumor progression remains unknown. We have recently demonstrated that exosomes released by lung-, liver- and brain-tropic tumor cells preferentially fuse with resident cells at their future metastatic sites, such as fibroblasts and epithelial cells in the lung, Kupffer cells in the liver, and endothelial cells in the brain. We found that tumor-derived exosome homing to organ-specific cell types prepares the pre-metastatic niche. Moreover, treatment with exosomes derived from lung-tropic models can redirect to the lung the metastatic distribution of cells that normally lack the capacity to form lung metastasis. Proteomic profiling of exosomes revealed distinct integrin expression patterns associated with each organ-specific metastatic site. Whereas exosomal α6β4 integrins were associated with lung metastasis, exosomal integrin αvβ5 was linked to liver metastasis and αvβ3 to brain metastasis. Targeting α6β4 and αvβ5 integrins decreased exosome uptake and metastasis in the lung and liver, respectively. Moreover, our clinical data indicate that integrin expression profiles in circulating plasma exosomes from cancer patients could be used to predict organ-specific metastasis. To gain a more comprehensive understanding of the relationship between exosomal protein cargo and tumor progression, we have built one of the largest exosome proteomics databases, including human and murine normal, pre-cancer and cancer exosomes from a variety sources, from cell lines to plasma and other bodily fluids. This dataset has allowed us to extend the list of markers that can be used to identify and isolate exosomes from human samples. When comparing the protein cargo of blood plasma exosomes isolated from normal subjects versus cancer patients with early stage pancreatic, colorectal, breast and lung cancer, we were able to define a cancer-specific proteomic signature that distinguishes cancer exosomes. Using paired samples from tumor tissue and plasma of the same cancer patients with breast, lung or pancreatic cancer, we were able to derive cancer type-specific exosomal signatures, which can be used as biomarkers to identify and classify tumors of unknown origin. Therefore, proteomic profiling of exosomes has great clinical potential as a non-invasive, rapid liquid biopsy that could aid in tumor detection classification. Citation Format: Ayuko Hoshino, Han Sang Kim, Linda Bojmar, Kofi Ennu Gyan, Haiying Zhang, Irina Matei, David Lyden. Tumor exosome and exomere biomarkers for early cancer detection [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA024.

Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer.

Summary 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.

Signaling via the pre-T cell antigen receptor (pre-TCR) and the receptor Notch1 induces transient self-renewal (β-selection) of TCRβ(+) CD4(-)CD8(-) double-negative stage 3 (DN3) and DN4 progenitor cells that differentiate into CD4(+)CD8(+) double-positive (DP) thymocytes, which then rearrange the locus encoding the TCR α-chain (Tcra). Interleukin 7 (IL-7) promotes the survival of TCRβ(-) DN thymocytes by inducing expression of the pro-survival molecule Bcl-2, but the functions of IL-7 during β-selection have remained unclear. Here we found that IL-7 signaled TCRβ(+) DN3 and DN4 thymocytes to upregulate genes encoding molecules involved in cell growth and repressed the gene encoding the transcriptional repressor Bcl-6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate but rearranged Tcra prematurely and differentiated rapidly. Deletion of Bcl6 partially restored the self-renewal of DN4 cells in the absence of IL-7, but overexpression of BCL2 did not. Thus, IL-7 critically acts cooperatively with signaling via the pre-TCR and Notch1 to coordinate proliferation, differentiation and Tcra recombination during β-selection.

© 2017 Macmillan Publishers Limited. All rights reserved. It is well established that organs of future metastasis are not passive receivers of circulating tumour cells, but are instead selectively and actively modified by the primary tumour before metastatic spread has even occurred. Sowing the 'seeds' of metastasis requires the action of tumour-secreted factors and tumour-shed extracellular vesicles that enable the 'soil' at distant metastatic sites to encourage the outgrowth of incoming cancer cells. In this Review, we summarize the main processes and new mechanisms involved in the formation of the pre-metastatic niche.

Tumors are capable of coopting hematopoietic cells to create a suitable microenvironment to support malignant growth. Here, we have demonstrated that upregulation of kinase insert domain receptor (KDR), also known as VEGFR2, in a myeloid cell sublineage is necessary for malignant progression of gliomas in transgenic murine models and is associated with high-grade tumors in patients. KDR expression increased in myeloid cells as myeloid-derived suppressor cells (MDSCs) accumulated, which was associated with the transformation and progression of low-grade fibrillary astrocytoma to high-grade anaplastic gliomas. KDR deficiency in murine BM-derived cells (BMDCs) suppressed the differentiation of myeloid lineages and reduced granulocytic/monocytic populations. The depletion of myeloid-derived KDR compromised its proangiogenic function, which inhibited the angiogenic switch necessary for malignant progression of low-grade to high-grade tumors. We also identified inhibitor of DNA binding protein 2 (ID2) as a key upstream regulator of KDR activation during myeloid differentiation. Deficiency of ID2 in BMDCs led to downregulation of KDR, suppression of proangiogenic myeloid cells, and prevention of low-grade to high-grade transition. Tumor-secreted TGF-β and granulocyte-macrophage CSF (GM-CSF) enhanced the KDR/ID2 signaling axis in BMDCs. Our results suggest that modulation of KDR/ID2 signaling may restrict tumor-associated myeloid cells and could potentially be a therapeutic strategy for preventing transformation of premalignant gliomas. This study was supported by the Department of Defense Con- gressionally Directed Medical Research Programs (DOD CDMRP, CA120318 to Y. Huang), Elizabeth’s Hope (J. Greenfield), the Starr Foundation, the Paduano Foundation, the Champalimaud Foun- dation, the Malcolm Hewitt Wiener Foundation, the POETIC Foundation, the Sohn Foundation, the Hartwell Foundation, and the Children’s Cancer and Blood Foundation (all to D. Lyden). Address correspondence to: David Lyden, Department of Pediatrics, Weill Medical Medicine, 413 E. 69th Street, Box 284, New York, New York 10021, USA. Phone: 646.962.6238; E-mail: dcl2001@med.cornell.edu. Or to: Jeffrey P. Greenfield, Department of Neurological Surgery, Weill Cornell Medicine, 525 E 68th Street, Box 99, New York, New York 10065, USA. Phone: 212.746.2363; E-mail: jpgreenf@med.cornell.edu. HP’s present address is: Microenvironment and Metastasis Group, Department of Molecular Oncology, Spanish National Cancer Research Center (CNIO), Madrid, Spain. Sí

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90–120 nm; small exosome vesicles, Exo-S, 60–80 nm) and discovered an abundant population of non-membranous nanoparticles termed ‘exomeres’ (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.

© 2015 Macmillan Publishers Limited. All rights reserved. Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

Fms-like tyrosine kinase-3 ligand (Flt3L) uniquely binds the Flt3 (CD135) receptor expressed on hematopoietic stem cells (HSCs), early progenitor cells, immature thymocytes and steady-state dendritic cells (DCs) and induces their proliferation, differentiation, development and mobilization in the bone marrow, peripheral blood and lymphoid organs. CDX-301 has an identical amino-acid sequence and comparable biological activity to the previously tested rhuFlt3L, which ceased clinical development over a decade ago. This Phase 1 trial assessed the safety, pharmacokinetic, pharmacodynamic and immunologic profile of CDX-301, explored alternate dosing regimens and examined the impact of rhuFlt3L on key immune cell subsets. Thirty healthy volunteers received CDX-301 (1-75 μg/kg/day) over 5-10 days. One event of Grade 3 community-acquired pneumonia occurred. There were no other infections, dose-limiting toxicities or serious adverse events. CDX-301 resulted in effective peripheral expansion of monocytes, hematopoietic stem and progenitor cells and key subsets of myeloid DCs and plasmacytoid DCs, with no clear effect on regulatory T cells. These data from healthy volunteers support the potential for CDX-301, as monotherapy or in combination with other agents, in various indications including allogeneic HSC transplantation and immunotherapy, but the effects of CDX-301 will need to be investigated in each of these patient populations.

A central mechanism of tumour progression and metastasis involves the generation of an immunosuppressive ‘macroenvironment' mediated in part through tumour-secreted factors. Here we demonstrate that upregulation of the Inhibitor of Differentiation 1 (Id1), in response to tumour-derived factors, such as TGFβ, is responsible for the switch from dendritic cell (DC) differentiation to myeloid-derived suppressor cell expansion during tumour progression. Genetic inactivation of Id1 largely corrects the myeloid imbalance, whereas Id1 overexpression in the absence of tumour-derived factors re-creates it. Id1 overexpression leads to systemic immunosuppression by downregulation of key molecules involved in DC differentiation and suppression of CD8 T-cell proliferation, thus promoting primary tumour growth and metastatic progression. Furthermore, advanced melanoma patients have increased plasma TGFβ levels and express higher levels of ID1 in myeloid peripheral blood cells. This study reveals a critical role for Id1 in suppressing the anti-tumour immune response during tumour progression and metastasis., Tumour progression is promoted by the generation of an immunosuppressive macroenvironment. Here, the authors demonstrate that the Inhibitor of Differentiation 1 promotes the switch from dendritic cell differentiation towards myeloid-derived suppressor cell expansion during tumour progression.

Mutations in ATM (ataxia-telangiectasia mutated) cause ataxia-telangiectasia (AT), a disease characterized by neurodegeneration, sterility, immunodeficiency, and T-cell leukemia. Defective ATM-mediated DNA damage responses underlie many aspects of the AT syndrome, but the basis for the immune deficiency has not been defined. ATM associates with DNA double-strand breaks (DSBs), and some evidence suggests that ATM may regulate V(D)J recombination. However, it remains unclear how ATM loss compromises lymphocyte development in vivo. Here, we show that T-cell receptor β (TCRβ)–dependent proliferation and production of TCRβlow CD4+CD8+ (DP) thymocytes occurred normally in Atm−/− mice. In striking contrast, the postmitotic maturation of TCRβlow DP precursors into TCRβint DP cells and TCRβhi mature thymocytes was profoundly impaired. Furthermore, Atm−/− thymocytes expressed abnormally low amounts of TCRα mRNA and protein. These defects were not attributable to the induction of a BCL-2–sensitive apoptotic pathway. Rather, they were associated with frequent biallelic loss of distal Va gene segments in DP thymocytes, revealing that ATM maintains Tcra locus integrity as it undergoes V(D)J recombination. Collectively, our data demonstrate that ATM loss increases the frequency of aberrant Tcra deletion events, which compromise DP thymocyte maturation and likely promote the generation of oncogenic TCR translocations.

The immune system is capable of recognizing and eliminating an enormous array of pathogens due to the extremely diverse antigen receptor repertoire of T and B lymphocytes. However, the development of lymphocytes bearing receptors with unique specificities requires the generation of programmed double strand breaks (DSBs) coupled with bursts of proliferation, rendering lymphocytes susceptible to mutations contributing to oncogenic transformation. Consequently, mechanisms responsible for monitoring global genomic integrity must be activated during lymphocyte development to limit the oncogenic potential of antigen receptor locus recombination. Mutations in ATM (ataxia-telangiectasia mutated), a kinase that coordinates DSB monitoring and the response to DNA damage, result in impaired T-cell development and predispose to T-cell leukemia. Here, we review recent evidence providing insight into the mechanisms by which ATM promotes normal lymphocyte development and protects from neoplastic transformation.

In the July issue of Nature Medicine, Massague and colleagues define a biphasic role for the extracellular matrix protein tenascin C as a metastatic niche component in lung colonization by breast cancer cells. These results provide a rationale for designing therapies targeting metastatic progression by disrupting its very foundations.

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

Intensified and central nervous system (CNS)-directed chemotherapy has improved outcomes for pediatric B cell acute lymphoblastic leukemia (B-ALL) but confers treatment-related morbidities. Moreover, many patients suffer relapses, underscoring the need to develop new molecular targeted B-ALL therapies. Using a mouse model, we show that leukemic B cells require pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) signaling in vivo for survival and proliferation. In diagnostic samples from human pediatric and adult B-ALL patients, SYK and downstream targets were phosphorylated regardless of pre-BCR expression or genetic subtype. Two small-molecule SYK inhibitors, fostamatinib and BAY61-3606, attenuated the growth of 69 B-ALL samples in vitro, including high-risk (HR) subtypes. Orally administered fostamatinib reduced heavy disease burden after xenotransplantation of HR B-ALL samples into immunodeficient mice and decreased leukemia dissemination into spleen, liver, kidneys, and the CNS of recipient mice. Thus, SYK activation sustains the growth of multiple HR B-ALL subtypes, suggesting that SYK inhibitors may improve outcomes for HR and relapsed B-ALL.

The present invention is directed to methods of prognosing, treating, or managing treatment of cancer in a subject. These methods involve selecting a subject having cancer, obtaining, from the selected subject, a sample containing exosomes, recovering the exosomes from the sample, and isolating the double-stranded DNA from within the exosomes. The isolated double-stranded DNA is then used to detect the presence or absence of one or more genetic mutations associated with cancer, quantify the amount of isolated double-stranded DNA from the recovered exosomes in the sample, detect the methylation status of the isolated double- stranded DNA, or quantify the amount isolated double-stranded DNA able to enter a recipient cell. The prognosing, treating, or managing treatment is carried out based on this information.

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

El pdf del artículo es el manuscrito de autor.-- et al., Tumor-derived exosomes are emerging mediators of tumorigenesis. We explored the function of melanoma-derived exosomes in the formation of primary tumors and metastases in mice and human subjects. Exosomes from highly metastatic melanomas increased the metastatic behavior of primary tumors by permanently 'educating' bone marrow progenitors through the receptor tyrosine kinase MET. Melanoma-derived exosomes also induced vascular leakiness at pre-metastatic sites and reprogrammed bone marrow progenitors toward a pro-vasculogenic phenotype that was positive for c-Kit, the receptor tyrosine kinase Tie2 and Met. Reducing Met expression in exosomes diminished the pro-metastatic behavior of bone marrow cells. Notably, MET expression was elevated in circulating CD45(-)C-KIT(low/+)TIE2(+) bone marrow progenitors from individuals with metastatic melanoma. RAB1A, RAB5B, RAB7 and RAB27A, regulators of membrane trafficking and exosome formation, were highly expressed in melanoma cells. Rab27A RNA interference decreased exosome production, preventing bone marrow education and reducing, tumor growth and metastasis. In addition, we identified an exosome-specific melanoma signature with prognostic and therapeutic potential comprised of TYRP2, VLA-4, HSP70, an HSP90 isoform and the MET oncoprotein. Our data show that exosome production, transfer and education of bone marrow cells supports tumor growth and metastasis, has prognostic value and offers promise for new therapeutic directions in the metastatic process.

Lunatic Fringe (Lfng) enhances Notch1 activation by Delta-like 4 (DL4) to promote Notch1-dependent T-lineage commitment of thymus-seeding progenitors. Subsequently, Notch1 and T-cell receptor-β (TCRβ)–containing pre-TCR complexes signal CD4/CD8 double-negative 3 (DN3) committed T-cell progenitors to survive, proliferate, and differentiate into CD4/CD8 double-positive (DP) αβ T-cell precursors. Few DP thymocytes develop without Notch1 or pre-TCR signals, whereas ectopic Notch1 activation causes T-cell leukemia. However, mechanisms of a Notch-pre-TCR collaboration during this “β-selection” process are poorly understood. We genetically manipulated Lfng to attenuate or enhance Notch1 activation in DN3 thymocytes without inducing leukemogenesis. We show that Lfng temporally sustains DL-induced Notch1 signaling to prolong proliferative self-renewal of pre-DP thymocytes. Pre-TCR signaling greatly augmented Notch trophic functions to promote robust proliferation of pre-DP progenitors. In contrast, in the absence of DL/Notch signaling, pre-TCR-expressing progenitors rapidly atrophied and differentiated into DP thymocytes. Thus, Lfng prolongs Notch1 signaling to promote self-renewal more than differentiation during the early stages of β-selection. Our data provide novel insights into the Notch-pre-TCR collaboration, and suggest that decreasing Lfng expression during the DN3-DP transition minimizes the potent leukemogenic potential of Notch1 signaling.

Abstract 3496 Notch1 signaling is required at multiple stages of normal T-lymphocyte development. Notch1 is a transmembrane receptor that is physiologically activated when Notch ligands induce conformational changes that allow Notch1 cleavage by the intramembranous γ-secretase complex, releasing active intracellular Notch1 (ICN1) fragment from the plasma membrane. Activating NOTCH1 mutations are very frequent in human and mouse T-cell lymphoblastic leukemia/lymphoma (T-LL). Typically, these mutations promote ligand-independent Notch1 cleavage by γ-secretase or increase ICN1 stability by truncating the C-terminal PEST domain. Understandably, much effort has focused on elucidating mechanisms of normal and oncogenic Notch1 signaling. However, some studies suggest that the absence of NOTCH1 mutations portends a worse prognosis for human T-LL. Therefore, we set out to define signals that promote proliferation and survival of T-LL cells lacking activated Notch1. We used Western blotting to detect γ-secretase cleaved ICN1 protein in a cohort of 35 primary T-LLs that developed spontaneously in mice lacking the Ataxia telangiectasia mutated (Atm) tumor suppressor. We identified 3 ICN1 subgroups: 63% expressed PEST-truncated ICN1 (T-ICN1); 17% expressed non-truncated ICN1 (NT-ICN1); and 20% had undetectable ICN1 (UD-ICN1), most lacked Notch1 mRNA. We confirmed the difference in Notch transcriptional activity and functional dependence between the UD-ICN1 and T-ICN1 subgroups and then compared their gene expression profiles to define pathways unique to the UD-ICN1 group. Gene set enrichment analyses revealed that UD-ICN1 T-LLs expressed higher levels of Klf9 and other transcription factors associated with a highly proliferative stage of normal T-cell development. siRNA knock-down studies demonstrated that Klf9 promoted proliferation of UD-ICN1 but not T-ICN1 T-LL cells. Collectively, these data demonstrate that Klf9 can regulate proliferation of Notch-independent T-LLs and suggest that Klf9 may provide a novel therapeutic target for human T-LLs lacking activating NOTCH1 mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract 2129 Objectives: T cell lineage is an independent high risk factor in acute lymphoblastic leukemia (ALL). T-ALL requires high dose multi-agent chemotherapy, conferring many toxic side effects. T-ALL treatment therefore needs new, targeted agents that preserve or improve current treatment efficacy, yet cause fewer side effects than existing chemotherapeutic regimens. To identify such drugs, we pioneered an in vivo screen using transgenic zebrafish with T cell-specific green fluorescent protein (GFP) expression. We reasoned that immature T cells in 5-day-old zebrafish larvae are developmentally analogous to T-ALL lymphoblasts, and likely rely upon similar signaling pathways. Hence, compounds that specifically eliminate T cells in zebrafish larvae might likewise selectively target T-ALL cells. An added benefit of our in vivo screen is that drugs added to the water housing fish larvae must cross an epithelial barrier, can be metabolized by the liver, and can be renally cleared, characteristics not assessed in in vitro-based screening strategies. In addition, by using early larvae as our subjects, drugs lacking T cell specificity will likely impair normal development and/or survival, which we postulate is a predictor of unwanted toxicities. In these ways, our screen mimicked the scenario encountered in patients. Our use of the transgenic p56lck::EGFP zebrafish line facilitated rapid visual assessment of efficacy of a large number of compounds in 96-well format. Materials and Results: In a proof-of-principle experiment, we identified several known anti-T-ALL drugs, most prominently glucocorticoids, from the “Spectrum” library (MicroSource Discovery Systems, Inc., Gaylordsville, CT) of well-characterized compounds. We then screened 39,200 small molecules from the “ChemBridge DIVERSet” combinatorial chemistry library (ChemBridge Corp., San Diego, CA) for those active against zebrafish larval T cells. Of 20 novel “hits” identified, one compound, dubbed Lenaldekar (“LDK”), met additional prioritization criteria. LDK does not impair the cell cycle of developing zebrafish, is well tolerated and orally bioavailable with favorable pharmacokinetic properties in mice. In addition, pilot studies with LDK indicate it is efficacious in treating T-ALL in juvenile and adult fish from an established transgenic rag2::ER-human cMyc T-ALL model. LDK kills all of several murine T-lymphocytic malignancies, induces apoptosis in all human T-ALL lines tested, and shows some activity in human B-ALL lines. However, glioblastoma, colon carcinoma, melanoma, or immortalized human embryonic kidney cell lines are not affected by LDK, even at high concentration (up to 25μM). Using the recently established “phosflow” technique we measured phosphorylation status of key signaling proteins in permeabilized T- and B-ALL lines. Regardless of PTEN status, PDK1, AKT and mTOR downstream targets p4EBP1 and p70S6kinase were dephosphorylated by LDK treatment, as was the p65 subunit of NFκB on serine 529. Results were corroborated by conventional Western blots. However, phosphorylation of STAT3, STAT5, pERK1-2, and p38 were not affected by LDK. LDK's action is distinguished from other AKT/mTOR inhibitors by its lack of activity against AKT-dependent glioblastoma and melanoma cell lines, and its lack of effect on cell size. Finally, LDK decreased tumor burden of human T-ALL in murine xenografts. Conclusions: In view of its apparent lymphocyte selectivity, we posit that LDK modulates a pathway relatively unique to ALL (and immature lymphoblast) cells. This suggests that LDK can serve as a novel molecular tool for studies of normal and malignant lymphocyte biology. Moreover, with its favorable pharmacokinetics, apparent lack of toxicity, and in vivo efficacy in two vertebrate ALL models, LDK is an attractive molecule for development into a targeted treatment for ALL and perhaps other lymphocytic malignancies. Disclosures: No relevant conflicts of interest to declare.