Publications by authors named "David Lyden"

108 Publications

Calcium signaling induces a partial EMT.

EMBO Rep 2021 09 29;22(9):e51872. Epub 2021 Jul 29.

Abramson Family Cancer Research Institute and Department of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

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.
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http://dx.doi.org/10.15252/embr.202051872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8419705PMC
September 2021

Extracellular vesicle- and particle-mediated communication shapes innate and adaptive immune responses.

J Exp Med 2021 Aug 28;218(8). Epub 2021 Jun 28.

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY.

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.
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http://dx.doi.org/10.1084/jem.20202579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8241538PMC
August 2021

The PI3K/mTOR inhibitor Gedatolisib eliminates dormant breast cancer cells in organotypic culture, but fails to prevent metastasis in preclinical settings.

Mol Oncol 2021 May 31. Epub 2021 May 31.

Public Health Sciences Division/Translational Research Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

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.
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http://dx.doi.org/10.1002/1878-0261.13031DOI Listing
May 2021

Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids.

STAR Protoc 2021 Mar 22;2(1):100225. Epub 2020 Dec 22.

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA.

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).
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http://dx.doi.org/10.1016/j.xpro.2020.100225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988237PMC
March 2021

Mutation Is Associated with Increased Risk of Recurrence in Surgically Resected Lung Adenocarcinoma.

Clin Cancer Res 2021 May 16;27(9):2604-2612. Epub 2021 Feb 16.

Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York.

Purpose: is the most common mutation in primary lung adenocarcinoma. Phase I clinical trials have demonstrated encouraging clinical activity of inhibitors in the metastatic setting. We investigated disease-free survival (DFS) and tumor genomic features in patients with surgically resected -mutant lung adenocarcinoma.

Experimental Design: Patients who underwent resection of stage I-III lung adenocarcinoma and next-generation sequencing (NGS) were evaluated. Exclusion criteria were receipt of induction therapy, incomplete resection, and low-quality NGS. Mutations were classified as wild-type ( ), G12C ( ), or non-G12C ( ). DFS was compared between groups using the log-rank test; factors associated with DFS were assessed using Cox regression. Mutual exclusivity and cooccurrence, tumor clonality, and mutational signatures were assessed.

Results: In total, 604 patients were included: 374 (62%), 95 (16%), and 135 (22%). Three-year DFS was not different between -mutant and tumors. However, 3-year DFS was worse in patients with than tumors (log-rank = 0.029). tumors had more lymphovascular invasion (51% vs. 37%; = 0.032) and higher tumor mutation burden [median (interquartile range), 7.0 (5.3-10.8) vs. 6.1 (3.5-9.7); = 0.021], compared with tumors. mutation was independently associated with worse DFS on multivariable analysis. Our DFS findings were externally validated in an independent The Cancer Genome Atlas cohort.

Conclusions: mutations are associated with worse DFS after complete resection of stage I-III lung adenocarcinoma. These tumors harbor more aggressive clinicopathologic and genomic features than other -mutant tumors. We identified a high-risk group for whom inhibitors may be investigated to improve survival.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-4772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102372PMC
May 2021

Tumour-regulated anorexia preceding cachexia.

Nat Cell Biol 2021 02;23(2):111-113

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA.

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http://dx.doi.org/10.1038/s41556-021-00635-8DOI Listing
February 2021

Cancer-Associated Fibroblasts Promote Aggressive Gastric Cancer Phenotypes via Heat Shock Factor 1-Mediated Secretion of Extracellular Vesicles.

Cancer Res 2021 04 5;81(7):1639-1653. Epub 2021 Feb 5.

Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel.

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.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-2756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8337092PMC
April 2021

Cell-free DNA (cfDNA) and Exosome Profiling from a Year-Long Human Spaceflight Reveals Circulating Biomarkers.

iScience 2020 Dec 25;23(12):101844. Epub 2020 Nov 25.

Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.

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.
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http://dx.doi.org/10.1016/j.isci.2020.101844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756145PMC
December 2020

A Genomic-Pathologic Annotated Risk Model to Predict Recurrence in Early-Stage Lung Adenocarcinoma.

JAMA Surg 2021 Feb 10;156(2):e205601. Epub 2021 Feb 10.

Thoracic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York.

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.
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http://dx.doi.org/10.1001/jamasurg.2020.5601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7758824PMC
February 2021

An exosome pathway without an ESCRT.

Cell Res 2021 02;31(2):105-106

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, 10021, USA.

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http://dx.doi.org/10.1038/s41422-020-00418-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027877PMC
February 2021

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

Cell 2020 08 13;182(4):1044-1061.e18. Epub 2020 Aug 13.

School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Japan.

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.
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http://dx.doi.org/10.1016/j.cell.2020.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7522766PMC
August 2020

A Human Pluripotent Stem Cell-based Platform to Study SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids.

Cell Stem Cell 2020 07 19;27(1):125-136.e7. Epub 2020 Jun 19.

The Pritzker School of Molecular Engineering, the Ben May Department for Cancer Research, the University of Chicago, IL, USA.

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.
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http://dx.doi.org/10.1016/j.stem.2020.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303620PMC
July 2020

Temozolomide in secondary prevention of HER2-positive breast cancer brain metastases.

Future Oncol 2020 May 9;16(14):899-909. Epub 2020 Apr 9.

Women's Malignancies Branch, National Cancer Institute, NIH, Bethesda, MD 20814, USA.

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).
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http://dx.doi.org/10.2217/fon-2020-0094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270957PMC
May 2020

Non-reversible tissue fixation retains extracellular vesicles for in situ imaging.

Nat Methods 2019 12 11;16(12):1269-1273. Epub 2019 Nov 11.

Department of Ophthalmology and Margaret M. Dyson Vision Research Institute, Weill Cornell Medicine, New York, NY, USA.

Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues.
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http://dx.doi.org/10.1038/s41592-019-0623-4DOI Listing
December 2019

Tumour exosomal CEMIP protein promotes cancer cell colonization in brain metastasis.

Nat Cell Biol 2019 11 4;21(11):1403-1412. Epub 2019 Nov 4.

Department of Pathology and Laboratory Medicine, Weill Cornell Medicine, New York, NY, USA.

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.
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http://dx.doi.org/10.1038/s41556-019-0404-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354005PMC
November 2019

Tumour vesicular micromachinery uncovered.

Nat Cell Biol 2019 07;21(7):795-797

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA.

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http://dx.doi.org/10.1038/s41556-019-0351-0DOI Listing
July 2019

Tumor Lymphatic Function Regulates Tumor Inflammatory and Immunosuppressive Microenvironments.

Cancer Immunol Res 2019 08 11;7(8):1345-1358. Epub 2019 Jun 11.

Department of Surgery, Plastic and Reconstructive Surgery Service, Memorial Sloan Kettering Cancer Center, New York, New York.

Proliferation of aberrant, dysfunctional lymphatic vessels around solid tumors is a common histologic finding. Studies have shown that abnormalities in lymphatic function result in accumulation of inflammatory cells with an immunosuppressive profile. We tested the hypothesis that dysfunctional lymphatic vessels surrounding solid tumors regulate changes in the tumor microenvironment and tumor-specific immune responses. Using subcutaneously implanted mouse melanoma and breast cancer tumors in a lymphatic endothelial cell-specific diphtheria toxin receptor transgenic mouse, we found that local ablation of lymphatic vessels increased peritumoral edema, as compared with controls. Comparative analysis of the peritumoral fluid demonstrated increases in the number of macrophages, CD4 inflammatory cells, F4/80/Gr-1 (myeloid-derived suppressor cells), CD4/Foxp3 (Tregs) immunosuppressive cells, and expression of inflammatory cytokines such as TNFα, IFNγ, and IL1β following lymphatic ablation. Tumors grown in lymphatic ablated mice exhibited reduced intratumoral accumulation of cytotoxic T cells and increased tumor PD-L1 expression, causing rapid tumor growth, compared with tumors grown in nonlymphatic-ablated mice. Our study suggests that lymphatic dysfunction plays a role in regulating tumor microenvironments and may be therapeutically targeted in combination with immunotherapy to prevent tumor growth and progression.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677612PMC
August 2019

Ribosome biogenesis during cell cycle arrest fuels EMT in development and disease.

Nat Commun 2019 05 8;10(1):2110. Epub 2019 May 8.

Department of Physiology and Pharmacology, Karolinska Institutet, 171 77, Stockholm, Sweden.

Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ERα) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.
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http://dx.doi.org/10.1038/s41467-019-10100-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506521PMC
May 2019

Exosome-Mediated Metastasis: Communication from a Distance.

Dev Cell 2019 05;49(3):347-360

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA. Electronic address:

Metastasis, a critical phase of tumor progression, remains a primary challenge in treating cancer and a major cause of cancer mortality. Cell-cell communication via extracellular vesicles (exosomes and microvesicles) between primary tumor cells and the microenvironment of distant organs is crucial for pre-metastatic niche (PMN) formation and metastasis. Here, we review work on the contribution of exosome cargo to cancer progression, the role of exosomes in PMN establishment, and the function of exosomes in organotropic metastasis. We also describe the clinical utility of exosomes.
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http://dx.doi.org/10.1016/j.devcel.2019.04.011DOI Listing
May 2019

Engineered niches model the onset of metastasis.

Nat Biomed Eng 2018 12;2(12):885-887

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA.

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http://dx.doi.org/10.1038/s41551-018-0326-7DOI Listing
December 2018

Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization.

Nat Protoc 2019 04 4;14(4):1027-1053. Epub 2019 Mar 4.

Children's Cancer and Blood Foundation Laboratories, Department of Pediatrics and Department of Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA.

We describe the protocol development and optimization of asymmetric-flow field-flow fractionation (AF4) technology for separating and characterizing extracellular nanoparticles (ENPs), particularly small extracellular vesicles (sEVs), known as exosomes, and even smaller novel nanoparticles, known as exomeres. This technique fractionates ENPs on the basis of hydrodynamic size and demonstrates a unique capability to separate nanoparticles with sizes ranging from a few nanometers to an undefined level of micrometers. ENPs are resolved by two perpendicular flows-channel flow and cross-flow-in a thin, flat channel with a semi-permissive bottom wall membrane. The AF4 separation method offers several advantages over other isolation methods for ENP analysis, including being label-free, gentle, rapid (<1 h) and highly reproducible, as well as providing efficient recovery of analytes. Most importantly, in contrast to other available techniques, AF4 can separate ENPs at high resolution (1 nm) and provide a large dynamic range of size-based separation. In conjunction with real-time monitors, such as UV absorbance and dynamic light scattering (DLS), and an array of post-separation characterizations, AF4 facilitates the successful separation of distinct subsets of exosomes and the identification of exomeres. Although the whole procedure of cell culture and ENP isolation from the conditioned medium by ultracentrifugation (UC) can take ~3 d, the AF4 fractionation step takes only 1 h. Users of this technology will require expertise in the working principle of AF4 to operate and customize protocol applications. AF4 can contribute to the development of high-quality, exosome- and exomere-based molecular diagnostics and therapeutics.
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http://dx.doi.org/10.1038/s41596-019-0126-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733524PMC
April 2019

Tumor Extracellular Vesicles Impede Interferon Alert Responses.

Cancer Cell 2019 01;35(1):3-5

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA; Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Tumor-derived extracellular vesicles promote metastasis by inducing functional changes in cells at pre-metastatic sites conducive for tumor cell colonization. In this issue of Cancer Cell, Ortiz and colleagues show that type I interferon regulates extracellular vesicle uptake and that modulating this pathway holds promise for treating metastasis.
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http://dx.doi.org/10.1016/j.ccell.2018.12.006DOI Listing
January 2019

Complex polymorphisms in endocytosis genes suggest alpha-cyclodextrin as a treatment for breast cancer.

PLoS One 2018 2;13(7):e0199012. Epub 2018 Jul 2.

Department of Pediatrics, and Cell and Developmental Biology Weill Medical College of Cornell University, New York, New York, United States of America.

Most breast cancer deaths are caused by metastasis and treatment options beyond radiation and cytotoxic drugs, which have severe side effects, and hormonal treatments, which are or become ineffective for many patients, are urgently needed. This study reanalyzed existing data from three genome-wide association studies (GWAS) using a novel computational biostatistics approach (muGWAS), which had been validated in studies of 600-2000 subjects in epilepsy and autism. MuGWAS jointly analyzes several neighboring single nucleotide polymorphisms while incorporating knowledge about genetics of heritable diseases into the statistical method and about GWAS into the rules for determining adaptive genome-wide significance. Results from three independent GWAS of 1000-2000 subjects each, which were made available under the National Institute of Health's "Up For A Challenge" (U4C) project, not only confirmed cell-cycle control and receptor/AKT signaling, but, for the first time in breast cancer GWAS, also consistently identified many genes involved in endo-/exocytosis (EEC), most of which had already been observed in functional and expression studies of breast cancer. In particular, the findings include genes that translocate (ATP8A1, ATP8B1, ANO4, ABCA1) and metabolize (AGPAT3, AGPAT4, DGKQ, LPPR1) phospholipids entering the phosphatidylinositol cycle, which controls EEC. These novel findings suggest scavenging phospholipids as a novel intervention to control local spread of cancer, packaging of exosomes (which prepare distant microenvironment for organ-specific metastases), and endocytosis of β1 integrins (which are required for spread of metastatic phenotype and mesenchymal migration of tumor cells). Beta-cyclodextrins (βCD) have already been shown to be effective in in vitro and animal studies of breast cancer, but exhibits cholesterol-related ototoxicity. The smaller alpha-cyclodextrins (αCD) also scavenges phospholipids, but cannot fit cholesterol. An in-vitro study presented here confirms hydroxypropyl (HP)-αCD to be twice as effective as HPβCD against migration of human cells of both receptor negative and estrogen-receptor positive breast cancer. If the previous successful animal studies with βCDs are replicated with the safer and more effective αCDs, clinical trials of adjuvant treatment with αCDs are warranted. Ultimately, all breast cancer are expected to benefit from treatment with HPαCD, but women with triple-negative breast cancer (TNBC) will benefit most, because they have fewer treatment options and their cancer advances more aggressively.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199012PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6028090PMC
December 2018

Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression.

Sci Adv 2018 02 28;4(2):eaao0665. Epub 2018 Feb 28.

Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065, USA.

The ribosome, the integration point for protein synthesis in the cell, is conventionally considered a homogeneous molecular assembly that only passively contributes to gene expression. Yet, epigenetic features of the ribosomal DNA (rDNA) operon and changes in the ribosome's molecular composition have been associated with disease phenotypes, suggesting that the ribosome itself may possess inherent regulatory capacity. Analyzing whole-genome sequencing data from the 1000 Genomes Project and the Mouse Genomes Project, we find that rDNA copy number varies widely across individuals, and we identify pervasive intra- and interindividual nucleotide variation in the 5, 5.8, 18, and 28 ribosomal RNA (rRNA) genes of both human and mouse. Conserved rRNA sequence heterogeneities map to functional centers of the assembled ribosome, variant rRNA alleles exhibit tissue-specific expression, and ribosomes bearing variant rRNA alleles are present in the actively translating ribosome pool. These findings provide a critical framework for exploring the possibility that the expression of genomically encoded variant rRNA alleles gives rise to physically and functionally heterogeneous ribosomes that contribute to mammalian physiology and human disease.
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http://dx.doi.org/10.1126/sciadv.aao0665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829973PMC
February 2018

Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation.

Nat Cell Biol 2018 03 19;20(3):332-343. Epub 2018 Feb 19.

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA.

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.
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http://dx.doi.org/10.1038/s41556-018-0040-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931706PMC
March 2018

Extracellular matrix proteins and carcinoembryonic antigen-related cell adhesion molecules characterize pancreatic duct fluid exosomes in patients with pancreatic cancer.

HPB (Oxford) 2018 07 12;20(7):597-604. Epub 2018 Jan 12.

Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Electronic address:

Background: Exosomes are nanovesicles that have been shown to mediate carcinogenesis in pancreatic ductal adenocarcinoma (PDAC). Given the direct communication of pancreatic duct fluid with the tumor and its relative accessibility, we aimed to determine the feasibility of isolating and characterizing exosomes from pancreatic duct fluid.

Methods: Pancreatic duct fluid was collected from 26 patients with PDAC (n = 13), intraductal papillary mucinous neoplasm (IPMN) (n = 8) and other benign pancreatic diseases (n = 5) at resection. Exosomes were isolated by serial ultracentrifugation, proteins were identified by mass spectrometry, and their expression was evaluated by immunohistochemistry.

Results: Exosomes were isolated from all specimens with a mean concentration of 5.9 ± 1 × 10 particles/mL and most frequent size of 138 ± 9 nm. Among the top 35 proteins that were significantly associated with PDAC, multiple carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) and extracellular matrix (ECM) proteins were identified. Interestingly, CEACAM 1/5 expression by immunohistochemistry was seen only on tumor epithelia whereas tenascin C positivity was restricted to stroma, suggesting that both tumor and stromal cells contributed to exosomes.

Conclusion: This is the first study showing that exosome isolation is feasible from pancreatic duct fluid, and that exosomal proteins may be utilized to diagnose patients with PDAC.
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http://dx.doi.org/10.1016/j.hpb.2017.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779041PMC
July 2018

Bone voyage-Osteoblasts remotely control tumors.

Science 2017 12;358(6367):1127-1128

Children's Cancer and Blood Foundation Laboratories, Departments of Pediatrics, and Cell and Developmental Biology, Drukier Institute for Children's Health, Meyer Cancer Center, Weill Cornell Medicine, New York, NY 10021, USA.

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http://dx.doi.org/10.1126/science.aar2640DOI Listing
December 2017

Packaging and transfer of mitochondrial DNA via exosomes regulate escape from dormancy in hormonal therapy-resistant breast cancer.

Proc Natl Acad Sci U S A 2017 10 11;114(43):E9066-E9075. Epub 2017 Oct 11.

Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY 10021;

The horizontal transfer of mtDNA and its role in mediating resistance to therapy and an exit from dormancy have never been investigated. Here we identified the full mitochondrial genome in circulating extracellular vesicles (EVs) from patients with hormonal therapy-resistant (HTR) metastatic breast cancer. We generated xenograft models of HTR metastatic disease characterized by EVs in the peripheral circulation containing mtDNA. Moreover, these human HTR cells had acquired host-derived (murine) mtDNA promoting estrogen receptor-independent oxidative phosphorylation (OXPHOS). Functional studies identified cancer-associated fibroblast (CAF)-derived EVs (from patients and xenograft models) laden with whole genomic mtDNA as a mediator of this phenotype. Specifically, the treatment of hormone therapy (HT)-naive cells or HT-treated metabolically dormant populations with CAF-derived mtDNA EVs promoted an escape from metabolic quiescence and HTR disease both in vitro and in vivo. Moreover, this phenotype was associated with the acquisition of EV mtDNA, especially in cancer stem-like cells, expression of EV mtRNA, and restoration of OXPHOS. In summary, we have demonstrated that the horizontal transfer of mtDNA from EVs acts as an oncogenic signal promoting an exit from dormancy of therapy-induced cancer stem-like cells and leading to endocrine therapy resistance in OXPHOS-dependent breast cancer.
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http://dx.doi.org/10.1073/pnas.1704862114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5664494PMC
October 2017

A phase II study of radioimmunotherapy with intraventricular I-3F8 for medulloblastoma.

Pediatr Blood Cancer 2018 Jan 22;65(1). Epub 2017 Sep 22.

Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York.

Background: High-risk and recurrent medulloblastoma (MB) is associated with significant mortality. The murine monoclonal antibody 3F8 targets the cell-surface disialoganglioside GD2 on MB. We tested the efficacy, toxicity, and dosimetry of compartmental radioimmunotherapy (cRIT) with intraventricular I-labeled 3F8 in patients with MB on a phase II clinical trial.

Methods: Patients with histopathologically confirmed high-risk or recurrent MB were eligible for cRIT. After determining adequate cerebrospinal fluid (CSF) flow, patients received 2 mCi (where Ci is Curie) I-3F8 or I-3F8 with nuclear imaging for dosimetry, followed by up to four therapeutic (10 mCi/dose) I-3F8 injections. Dosimetry estimates were based on serial CSF and blood samplings over 48 hr plus region-of-interest analyses on serial imaging scans. Disease evaluation included pre- and posttherapy brain/spine magnetic resonance imaging approximately every 3 months for the first year after treatment, and every 6-12 months thereafter.

Results: Forty-three patients received a total of 167 injections; 42 patients were evaluable for outcome. No treatment-related deaths occurred. Toxicities related to drug administration included acute bradycardia with somnolence, headache, fatigue, and CSF pleocytosis consistent with chemical meningitis and dystonic reaction. Total CSF absorbed dose was 1,453 cGy (where Gy is Gray; 350.0-2,784). Median overall survival from first dose of cRIT was 24.9 months (95% confidence interval [CI]:16.3-55.8). Patients treated in radiographic and cytologic remission were at a lower risk of death compared to patients with radiographically measurable disease (hazard ratio: 0.40, 95% CI: 0.18-0.88, P = 0.024).

Conclusions: cRIT with I-3F8 is safe, has favorable dosimetry to CSF, and when added to salvage therapy using conventional modalities, may have clinical utility in maintaining remission in high-risk or recurrent MB.
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http://dx.doi.org/10.1002/pbc.26754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692907PMC
January 2018
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