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High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were â¼10 PE/â¼80 nm EV diameter for CytoFLEX and â¼10 PEs/â¼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.
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Vesículas Extracelulares , Citometria de Fluxo , Citometria de Fluxo/métodos , Citometria de Fluxo/instrumentação , Humanos , Vesículas Extracelulares/metabolismo , Neoplasias Colorretais/diagnóstico , Linhagem Celular Tumoral , Imagem Individual de Molécula/métodos , Imagem Individual de Molécula/instrumentaçãoRESUMO
Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.
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Endocitose , Exossomos , Tetraspanina 30 , Exossomos/metabolismo , Humanos , Tetraspanina 30/metabolismo , Biomarcadores/metabolismo , Sinteninas/metabolismo , Sinteninas/genética , Tetraspanina 28/metabolismo , Membrana Celular/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Tetraspanina 29/metabolismoRESUMO
Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.
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Exossomos , Vesículas Extracelulares , Vesículas Extracelulares/metabolismo , Exossomos/metabolismo , Transporte Biológico , Biomarcadores/metabolismo , FenótipoRESUMO
Trastuzumab therapy in HER2+ breast cancer patients has mixed success owing to acquired resistance to therapy. A detailed understanding of downstream molecular cascades resulting from trastuzumab resistance is yet to emerge. In this study, we investigate the cellular mechanisms underlying acquired resistance using trastuzumab-sensitive and -resistant cancer cells (BT474 and BT474R) treated with endogenous ligands EGF and HRG across time. We probe early receptor organization through microscopy and signaling events through multiomics measurements and assess the bioenergetic state through mitochondrial measurements. Integrative analyses of our measurements reveal significant alterations in EGF-treated BT474 HER2 membrane dynamics and robust downstream activation of PI3K/AKT/mTORC1 signaling. EGF-treated BT474R shows a sustained interferon-independent activation of the IRF1/STAT1 cascade, potentially contributing to trastuzumab resistance. Both cell lines exhibit temporally divergent metabolic demands and HIF1A-mediated stress responses. BT474R demonstrates inherently increased mitochondrial activity. HRG treatment in BT474R leads to a pronounced reduction in AR expression, affecting downstream lipid metabolism with implications for treatment response. Our results provide novel insights into mechanistic changes underlying ligand treatment in BT474 and BT474R and emphasize the pivotal role of endogenous ligands. These results can serve as a framework for furthering the understanding of trastuzumab resistance, with therapeutic implications for women with acquired resistance.
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Exosomes are small extracellular vesicles (sEVs) of ~30-150 nm in diameter that have the same topology as the cell, are enriched in selected exosome cargo proteins, and play important roles in health and disease. To address large unanswered questions regarding exosome biology in vivo, we created the exomap1 transgenic mouse model. In response to Cre recombinase, exomap1 mice express HsCD81mNG, a fusion protein between human CD81, the most highly enriched exosome protein yet described, and the bright green fluorescent protein mNeonGreen. As expected, cell type-specific expression of Cre induced the cell type-specific expression of HsCD81mNG in diverse cell types, correctly localized HsCD81mNG to the plasma membrane, and selectively loaded HsCD81mNG into secreted vesicles that have the size (~80 nm), topology (outside out), and content (presence of mouse exosome markers) of exosomes. Furthermore, mouse cells expressing HsCD81mNG released HsCD81mNG-marked exosomes into blood and other biofluids. Using high-resolution, single-exosome analysis by quantitative single molecule localization microscopy, we show here that that hepatocytes contribute ~15% of the blood exosome population whereas neurons contribute <1% of blood exosomes. These estimates of cell type-specific contributions to blood EV population are consistent with the porosity of liver sinusoidal endothelial cells to particles of ~50-300 nm in diameter, as well as with the impermeability of blood-brain and blood-neuron barriers to particles >5 nm in size. Taken together, these results establish the exomap1 mouse as a useful tool for in vivo studies of exosome biology, and for mapping cell type-specific contributions to biofluid exosome populations. In addition, our data confirm that CD81 is a highly-specific marker for exosomes and is not enriched in the larger microvesicle class of EVs.
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Naltrexone (NTX), a homologue of the opiate antidote naloxone, is an orally active long-acting mu-opioid receptor (MOP) antagonist used in the treatment of opiate dependence. NTX is also found to relieve craving for alcohol and is one of the few FDA-approved drugs for alcohol use disorder (AUD). Reports that NTX blocks the actions of endogenous opioids released by alcohol are not convincing, suggesting that NTX interferes with alcohol actions by affecting opioid receptors. MOP and kappa-opioid receptor (KOP) are structurally related but functionally different. MOP is mainly located in interneurons activated by enkephalins while KOP is located in longer projections activated by dynorphins. While the actions of NTX on MOP are well established, the interaction with KOP and addiction is not well understood. We used sensitive fluorescence-based methods to study the influence of alcohol on KOP and the interaction between KOP and NTX. Here we report that alcohol interacts with KOP and its environment in the plasma membrane. These interactions are affected by NTX and are exerted both on KOP directly and on the plasma membrane (lipid) structures ("off-target"). The actions of NTX are stereospecific. Selective KOP antagonists, recently in early clinical trials for major depressive disorder, block the receptor but do not show the full action profile of NTX. The therapeutic effect of NTX treatment in AUD may be due to direct actions on KOP and the receptor environment.
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Extracellular vesicles (EVs) and their cargo constitute novel biomarkers. EV subpopulations have been defined not only by abundant tetraspanins (e.g., CD9, CD63 and CD81) but also by specific markers derived from their source cells. However, it remains a challenge to robustly isolate and characterize EV subpopulations. Here, we combined affinity isolation with super-resolution imaging to comprehensively assess EV subpopulations from human plasma. Our Single Extracellular VEsicle Nanoscopy (SEVEN) assay successfully quantified the number of affinity-isolated EVs, their size, shape, molecular tetraspanin content, and heterogeneity. The number of detected tetraspanin-enriched EVs positively correlated with sample dilution in a 64-fold range (for SEC-enriched plasma) and a 50-fold range (for crude plasma). Importantly, SEVEN robustly detected EVs from as little as â¼0.1 µL of crude plasma. We further characterized the size, shape and molecular tetraspanin content (with corresponding heterogeneities) for CD9-, CD63- and CD81-enriched EV subpopulations. Finally, we assessed EVs from the plasma of four pancreatic ductal adenocarcinoma patients with resectable disease. Compared to healthy plasma, CD9-enriched EVs from patients were smaller while IGF1R-enriched EVs from patients were larger, rounder and contained more tetraspanin molecules, suggestive of a unique pancreatic cancer-enriched EV subpopulation. This study provides the method validation and demonstrates that SEVEN could be advanced into a platform for characterizing both disease-associated and organ-associated EV subpopulations.
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Vesículas Extracelulares , Humanos , Tetraspanina 29 , Tetraspaninas , BiomarcadoresRESUMO
Exosomes are small extracellular vesicles important in health and disease. Syntenin is thought to drive the biogenesis of CD63 exosomes by recruiting Alix and the ESCRT machinery to endosomes, initiating an endosome-mediated pathway of exosome biogenesis. Contrary to this model, we show here that syntenin drives the biogenesis of CD63 exosomes by blocking CD63 endocytosis, thereby allowing CD63 to accumulate at the plasma membrane, the primary site of exosome biogenesis. Consistent with these results, we find that inhibitors of endocytosis induce the exosomal secretion of CD63, that endocytosis inhibits the vesicular secretion of exosome cargo proteins, and that high-level expression of CD63 itself also inhibits endocytosis. These and other results indicate that exosomes bud primarily from the plasma membrane, that endocytosis inhibits their loading into exosomes, that syntenin and CD63 are expression-dependent regulators of exosome biogenesis, and that syntenin drives the biogenesis of CD63 exosomes even in Alix knockout cells.
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Introduction: The progression-free survival of patients with HER2-positive metastatic breast cancer is significantly extended by a combination of two monoclonal antibodies, trastuzumab and pertuzumab, which target independent epitopes of the extracellular domain of HER2. The improved efficacy of the combination over individual antibody therapies targeting HER2 is still being investigated, and several molecular mechanisms may be in play: the combination downregulates HER2, improves antibody-dependent cell mediated cytotoxicity, and/or affects the organization of surface-expressed antigens, which may attenuate downstream signaling. Methods: By combining protein engineering and quantitative single molecule localization microscopy (qSMLM), here we both assessed and optimized clustering of HER2 in cultured breast cancer cells. Results: We detected marked changes to the cellular membrane organization of HER2 when cells were treated with therapeutic antibodies. When we compared untreated samples to four treatment scenarios, we observed the following HER2 membrane features: (1) the monovalent Fab domain of trastuzumab did not significantly affect HER2 clustering; (2) individual therapy with either trastuzumab or (3) pertuzumab produced significantly higher levels of HER2 clustering; (4) a combination of trastuzumab plus pertuzumab produced the highest level of HER2 clustering. To further enhance this last effect, we created multivalent ligands using meditope technology. Treatment with a tetravalent meditope ligand combined with meditope-enabled trastuzumab resulted in pronounced HER2 clustering. Moreover, compared to pertuzumab plus trastuzumab, at early time points this meditope-based combination was more effective at inhibiting epidermal growth factor (EGF) dependent activation of several downstream protein kinases. Discussion: Collectively, mAbs and multivalent ligands can efficiently alter the organization and activation of the HER2 receptors. We expect this approach could be used in the future to develop new therapeutics.
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The proclivity of certain pre-malignant and pre-invasive breast lesions to progress while others do not continues to perplex clinicians. Clinicians remain at a crossroads with effectively managing the high-risk patient subpopulation owing to the paucity of biomarkers that can adequately risk-stratify and inform clinical decisions that circumvent unnecessary administration of cytotoxic and invasive treatments. The immune system mounts the most important line of defense against tumorigenesis and progression. Unfortunately, this defense declines or "ages" over time-a phenomenon known as immunosenescence. This results in "inflamm-aging" or the excessive infiltration of pro-inflammatory chemokines, which alters the leukocyte composition of the tissue microenvironment, and concomitant immunoediting of these leukocytes to diminish their antitumor immune functions. Collectively, these effects can foster the sequelae of neoplastic transformation and progression. The erythrocyte cell antigen, Duffy antigen receptor for chemokines(DARC/ACKR1), binds and internalizes chemokines to maintain homeostatic levels and modulate leukocyte trafficking. A negative DARC status is highly prevalent among subpopulations of West African genetic ancestry, who are at higher risk of developing breast cancer and disease progression at a younger age. However, the role of DARC in accelerated inflamm-aging and malignant transformation remains underexplored. Herein, we review compelling evidence suggesting that DARC may be protective against inflamm-aging and, therefore, reduce the risk of a high-risk lesion progressing to malignancy. We also discuss evidence supporting that immunotherapeutic intervention-based on DARC status-among high-risk subpopulations may evade malignant transformation and progression. A closer look into this unique role of DARC could glean deeper insight into the immune response profile of individual high-risk patients and their predisposition to progress as well as guide the administration of more "cyto-friendly" immunotherapeutic intervention to potentially "turn back the clock" on inflamm-aging-mediated oncogenesis and progression.
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Envelhecimento , Neoplasias da Mama , Sistema do Grupo Sanguíneo Duffy , Imunossenescência , Humanos , Quimiocinas/metabolismo , Sistema do Grupo Sanguíneo Duffy/genética , Genótipo , Leucócitos/metabolismo , Receptores de Antígenos , BiomarcadoresRESUMO
Black/African-American (AA) women, relative to their White/European-American (EA) counterparts, experience disproportionately high breast cancer mortality. Central to this survival disparity, Black/AA women have an unequal burden of aggressive breast cancer subtypes, such as triple-negative breast cancer (ER/PR-, HER2-wild type; TNBC). While TNBC has been well characterized, recent studies have identified a highly aggressive androgen receptor (AR)-negative subtype of TNBC, quadruple-negative breast cancer (ER/PR-, HER2-wildtype, AR-; QNBC). Similar to TNBC, QNBC disproportionately impacts Black/AA women and likely plays an important role in the breast cancer survival disparities experienced by Black/AA women. Here, we discuss the racial disparities of QNBC and molecular signaling pathways that may contribute to the aggressive biology of QNBC in Black/AA women. Our immediate goal is to spotlight potential prevention and therapeutic targets for Black/AA QNBC; ultimately our goal is to provide greater insight into reducing the breast cancer survival burden experienced by Black/AA women.
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Triple-negative breast cancer (TNBC) surpasses other BC subtypes as the most challenging to treat due to its lack of traditional BC biomarkers. Nearly 30% of TNBC patients express the androgen receptor (AR), and the blockade of androgen production and AR signaling have been the cornerstones of therapies for AR-positive TNBC. However, the majority of women are resistant to AR-targeted therapy, which is a major impediment to improving outcomes for the AR-positive TNBC subpopulation. The hypoxia signaling cascade is frequently activated in the tumor microenvironment in response to low oxygen levels; activation of the hypoxia signaling cascade allows tumors to survive despite hypoxia-mediated interference with cellular metabolism. The activation of hypoxia signaling networks in TNBC promotes resistance to most anticancer drugs including AR inhibitors. The activation of hypoxia network signaling occurs more frequently in TNBC compared to other BC subtypes. Herein, we examine the (1) interplay between hypoxia signaling networks and AR and (2) whether hypoxia and hypoxic stress adaptive pathways promote the emergence of resistance to therapies that target AR. We also pose the well-supported question, "Can the efficacy of androgen-/AR-targeted treatments be enhanced by co-targeting hypoxia?" By critically examining the evidence and the complex entwinement of these two oncogenic pathways, we argue that the simultaneous targeting of androgen biosynthesis/AR signaling and hypoxia may enhance the sensitivity of AR-positive TNBCs to AR-targeted treatments, derail the emergence of therapy resistance, and improve patient outcomes.
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Neoplasias de Mama Triplo Negativas , Androgênios/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Hipóxia , Receptores Androgênicos/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Microambiente TumoralRESUMO
The extracellular RNA communication consortium (ERCC) is an NIH-funded program aiming to promote the development of new technologies, resources, and knowledge about exRNAs and their carriers. After Phase 1 (2013-2018), Phase 2 of the program (ERCC2, 2019-2023) aims to fill critical gaps in knowledge and technology to enable rigorous and reproducible methods for separation and characterization of both bulk populations of exRNA carriers and single EVs. ERCC2 investigators are also developing new bioinformatic pipelines to promote data integration through the exRNA atlas database. ERCC2 has established several Working Groups (Resource Sharing, Reagent Development, Data Analysis and Coordination, Technology Development, nomenclature, and Scientific Outreach) to promote collaboration between ERCC2 members and the broader scientific community. We expect that ERCC2's current and future achievements will significantly improve our understanding of exRNA biology and the development of accurate and efficient exRNA-based diagnostic, prognostic, and theranostic biomarker assays.
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Trastuzumab, the prototype HER2-directed therapy, has markedly improved survival for women with HER2-positive breast cancers. However, only 40-60% of women with HER2-positive breast cancers achieve a complete pathological response to chemotherapy combined with HER2-directed therapy. The current diagnostic assays have poor positive-predictive accuracy in identifying therapy-responsive breast cancers. Here, we deployed quantitative single molecule localization microscopy to assess the molecular features of HER2 in a therapy-responsive setting. Using fluorescently labeled trastuzumab as a probe, we first compared the molecular features of HER2 in trastuzumab-sensitive (BT-474 and SK-BR-3) and trastuzumab-resistant (BT-474R and JIMT-1) cultured cell lines. Trastuzumab-sensitive cells had significantly higher detected HER2 densities and clustering. We then evaluated HER2 in pre-treatment core biopsies from women with breast cancer undergoing neoadjuvant therapy. A complete pathological response was associated with a high detected HER2 density and significant HER2 clustering. These results established the nano-organization of HER2 as a potential signature of therapy-responsive disease.
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Biomolecules can be investigated at the nanoscale with quantitative single molecule localization microscopy (qSMLM). This technique, which achieves single molecule sensitivity, can probe how membrane receptors are organized under both normal and pathological conditions. While a number of receptors have been extensively studied in cultured cells, technical challenges have largely impeded their robust quantification in tissue samples. To rigorously interrogate tissue samples, methodological advancements are needed in three areas: analytical preparation of the sample, proper characterization of fluorescent reporters, and rapid/unbiased data analysis. Towards these ends, we have combined qSMLM with a touch preparation technique (touch prep-qSMLM). In this new method, touch prep is first used to obtain monolayers of patient cells. Then, highly selective, fluorescently labeled probes are used to detect the receptors of interest on the plasma membranes of cells. Finally, quantitative algorithms are used to analyze the imaging data. Using this touch prep-qSMLM methodology, we interrogated the density and nano-organization of human epidermal growth factor receptor 2 (HER2) in fresh breast cancer tissues. Touch prep-qSMLM agreed well with current clinical methods. Importantly, touch prep-qSMLM can be easily extended to other pathological conditions and ultimately used in precision medicine.
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Neoplasias da Mama , Microscopia , Neoplasias da Mama/patologia , Feminino , Humanos , Receptor ErbB-2/metabolismo , Imagem Individual de Molécula/métodosRESUMO
The enigma of why some premalignant or pre-invasive breast lesions transform and progress while others do not remains poorly understood. Currently, no radiologic or molecular biomarkers exist in the clinic that can successfully risk-stratify high-risk lesions for malignant transformation or tumor progression as well as serve as a minimally cytotoxic actionable target for at-risk subpopulations. Breast carcinogenesis involves a series of key molecular deregulatory events that prompt normal cells to bypass tumor-suppressive senescence barriers. Kinesin family member C1 (KIFC1/HSET), which confers survival of cancer cells burdened with extra centrosomes, has been observed in premalignant and pre-invasive lesions, and its expression has been shown to correlate with increasing neoplastic progression. Additionally, KIFC1 has been associated with aggressive breast tumor molecular subtypes, such as basal-like and triple-negative breast cancers. However, the role of KIFC1 in malignant transformation and its potential as a predictive biomarker of neoplastic progression remain elusive. Herein, we review compelling evidence suggesting the involvement of KIFC1 in enabling pre-neoplastic cells to bypass senescence barriers necessary to become immortalized and malignant. We also discuss evidence inferring that KIFC1 levels may be higher in premalignant lesions with a greater inclination to transform and acquire aggressive tumor intrinsic subtypes. Collectively, this evidence provides a strong impetus for further investigation into KIFC1 as a potential risk-stratifying biomarker and minimally cytotoxic actionable target for high-risk patient subpopulations.
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Specific macromolecules are rapidly transported across the nuclear envelope via the nuclear pore complex (NPC). The selective transport process is facilitated when nuclear transport receptors (NTRs) weakly and transiently bind to intrinsically disordered constituents of the NPC, FG Nups. These two types of proteins help maintain the selective NPC barrier. To interrogate their binding interactions in vitro, we deployed an NPC barrier mimic. We created the stationary phase by covalently attaching fragments of a yeast FG Nup called Nsp1 to glass coverslips. We used a tunable mobile phase containing NTR, nuclear transport factor 2 (NTF2). In the stationary phase, three main factors affected binding: the number of FG repeats, the charge of fragments, and the fragment density. We also identified three main factors affecting binding in the mobile phase: the avidity of the NTF2 variant for Nsp1, the presence of nonspecific proteins, and the presence of additional NTRs. We used both experimentally determined binding parameters and molecular dynamics simulations of Nsp1FG fragments to create an agent-based model. The results suggest that NTF2 binding is negatively cooperative and dependent on the density of Nsp1FG molecules. Our results demonstrate the strengths of combining experimental and physical modeling approaches to study NPC-mediated transport.
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Núcleo Celular/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/fisiologia , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: During acute myeloid leukemia (AML) growth, the bone marrow (BM) niche acquires significant vascular changes that can be offset by therapeutic blast cytoreduction. The molecular mechanisms of this vascular plasticity remain to be fully elucidated. Herein, we report on the changes that occur in the vascular compartment of the FLT3-ITD+ AML BM niche pre and post treatment and their impact on leukemic stem cells (LSCs). METHODS: BM vasculature was evaluated in FLT3-ITD+ AML models (MllPTD/WT/Flt3ITD/ITD mouse and patient-derived xenograft) by 3D confocal imaging of long bones, calvarium vascular permeability assays, and flow cytometry analysis. Cytokine levels were measured by Luminex assay and miR-126 levels evaluated by Q-RT-PCR and miRNA staining. Wild-type (wt) and MllPTD/WT/Flt3ITD/ITD mice with endothelial cell (EC) miR-126 knockout or overexpression served as controls. The impact of treatment-induced BM vascular changes on LSC activity was evaluated by secondary transplantation of BM cells after administration of tyrosine kinase inhibitors (TKIs) to MllPTD/WT/Flt3ITD/ITD mice with/without either EC miR-126 KO or co-treatment with tumor necrosis factor alpha (TNFα) or anti-miR-126 miRisten. RESULTS: In the normal BM niche, CD31+Sca-1high ECs lining arterioles have miR-126 levels higher than CD31+Sca-1low ECs lining sinusoids. We noted that during FLT3-ITD+ AML growth, the BM niche lost arterioles and gained sinusoids. These changes were mediated by TNFα, a cytokine produced by AML blasts, which induced EC miR-126 downregulation and caused depletion of CD31+Sca-1high ECs and gain in CD31+Sca-1low ECs. Loss of miR-126high ECs led to a decreased EC miR-126 supply to LSCs, which then entered the cell cycle and promoted leukemia growth. Accordingly, antileukemic treatment with TKI decreased the BM blast-produced TNFα and increased miR-126high ECs and the EC miR-126 supply to LSCs. High miR-126 levels safeguarded LSCs, as shown by more severe disease in secondary transplanted mice. Conversely, EC miR-126 deprivation via genetic or pharmacological EC miR-126 knock-down prevented treatment-induced BM miR-126high EC expansion and in turn LSC protection. CONCLUSIONS: Treatment-induced CD31+Sca-1high EC re-vascularization of the leukemic BM niche may represent a LSC extrinsic mechanism of treatment resistance that can be overcome with therapeutic EC miR-126 deprivation.
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Medula Óssea/patologia , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Animais , Medula Óssea/irrigação sanguínea , Humanos , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/terapia , Camundongos , Camundongos Endogâmicos C57BL , Regulação para Cima , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Over 90% of breast cancer is cured; yet there remain highly aggressive breast cancers that develop rapidly and are extremely difficult to treat, much less prevent. Breast cancers that rapidly develop between breast image screening are called "interval cancers." The efforts of our team focus on identifying multiscale integrated strategies to identify biologically aggressive precancerous breast lesions. Our goal is to identify spatiotemporal changes that occur prior to development of interval breast cancers. To accomplish this requires integration of new technology. Our team has the ability to perform single cell in situ transcriptional profiling, noncontrast biological imaging, mathematical analysis, and nanoscale evaluation of receptor organization and signaling. These technological innovations allow us to start to identify multidimensional spatial and temporal relationships that drive the transition from biologically aggressive precancer to biologically aggressive interval breast cancer. This article is categorized under: Cancer > Computational Models Cancer > Molecular and Cellular Physiology Cancer > Genetics/Genomics/Epigenetics.