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PURPOSE: Clinical efficacy of CAR T cells against pediatric osteosarcoma (OS) has been limited. One strategy to improve efficacy may be to drive chemokine-mediated homing of CAR T cells to tumors. We investigated the primary chemokines secreted by OS and evaluated efficacy of B7-H3.CAR T cells expressing the cognate receptors. EXPERIMENTAL DESIGN: We developed a pipeline to identify chemokines secreted by OS by correlating RNA-seq data with chemokines detected in media from fresh surgical specimens. We identified CXCR2 and CXCR6 as promising receptors for enhancing CAR T cell homing against OS. We evaluated the homing kinetics and efficiency of CXCR2- and CXCR6.T cells and homing, cytokine production, and antitumor activity of CXCR2- and CXCR6.B7-H3.CAR T cells in vitro and in vivo. RESULTS: T cells transgenically expressing CXCR2 or CXCR6 exhibited ligand-specific enhanced migration over T cells modified with nonfunctional receptors. Differential homing kinetics were observed, with CXCR2.T cells homing quickly and plateauing early, while CXCR6.T cells homed more slowly but achieved a similar plateau. When expressed in B7-H3.CAR T cells, CXCR2- and CXCR6 modification conferred enhanced homing towards OS in vitro and in vivo. CXCR2- and CXCR6-B7-H3.CAR treated mice experienced prolonged survival in a metastatic model compared to B7-H3.CAR T cell treated mice. CONCLUSIONS: Our patient-based pipeline identified targets for chemokine receptor modification of CAR T cells targeting OS. CXCR2 and CXCR6 expression enhanced homing and anti-OS activity of B7-H3.CAR T cells. These findings support clinical evaluation of CXCR-modified CAR T cells to improve adoptive cell therapy for OS patients.
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ABSTRACT: Hematopoietic stem cells (HSCs) can generate all blood cells. This ability is exploited in HSC transplantation (HSCT) to treat hematologic disease. A clear understanding of the molecular mechanisms that regulate HSCT is necessary to continue improving transplant protocols. We identified the Beige and Chediak-Higashi domain-containing protein (BDCP), Neurobeachin (NBEA), as a putative regulator of HSCT. Here, we demonstrated that NBEA and related BDCPs, including LPS Responsive Beige-Like Anchor Protein (LRBA), Neurobeachin Like 1 (NBEAL1) and Lysosomal Trafficking Regulator (LYST), are required during HSCT to efficiently reconstitute the hematopoietic system of lethally irradiated mice. Nbea knockdown in mouse HSCs induced apoptosis and a differentiation block after transplantation. Nbea deficiency in hematopoietic progenitor cells perturbed the expression of genes implicated in vesicle trafficking and led to changes in NOTCH receptor localization. This resulted in perturbation of the NOTCH transcriptional program, which is required for efficient HSC engraftment. In summary, our findings reveal a novel role for NBEA in the control of NOTCH receptor turnover in hematopoietic cells and supports a model in which BDCP-regulated vesicle trafficking is required for efficient HSCT.
Assuntos
Diferenciação Celular , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Receptores Notch , Animais , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos , Receptores Notch/metabolismo , Sobrevivência Celular , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , ApoptoseRESUMO
Current dogma asserts that the foetal liver (FL) is an expansion niche for recently specified haematopoietic stem cells (HSCs) during ontogeny. Indeed, between embryonic day of development (E)12.5 and E14.5, the number of transplantable HSCs in the murine FL expands from 50 to about 1,000. Here we used a non-invasive, multi-colour lineage tracing strategy to interrogate the embryonic expansion of murine haematopoietic progenitors destined to contribute to the adult HSC pool. Our data show that this pool of fated progenitors expands only two-fold during FL ontogeny. Although Histone2B-GFP retention in vivo experiments confirmed substantial proliferation of phenotypic FL-HSC between E12.5 and E14.5, paired-daughter cell assays revealed that many mid-gestation phenotypic FL-HSCs are biased to differentiate, rather than self-renew, relative to phenotypic neonatal and adult bone marrow HSCs. In total, these data support a model in which the FL-HSC pool fated to contribute to adult blood expands only modestly during ontogeny.
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Células-Tronco Hematopoéticas , Fígado , Camundongos , AnimaisRESUMO
The outcome for metastatic pediatric osteosarcoma (OS) remains poor. Thus, there is an urgent need to develop novel therapies, and immunotherapy with CAR T cells has the potential to meet this challenge. However, there is a lack of preclinical models that mimic salient features of human disease including reliable development of metastatic disease post orthotopic OS cell injection. To overcome this roadblock, and also enable real-time imaging of metastatic disease, we took advantage of LM7 OS cells expressing firefly luciferase (LM7.ffLuc). LM7.ffLuc were implanted in a collagen mesh into the tibia of mice, and mice reliably developed orthotopic tumors and lung metastases as judged by bioluminescence imaging and histopathological analysis. Intratibial implantation also enabled surgical removal by lower leg amputation and monitoring for metastases development post-surgery. We then used this model to evaluate the antitumor activity of CAR T cells targeting B7-H3, an antigen that is expressed in a broad range of solid tumors including OS. B7-H3-CAR T cells had potent antitumor activity in a dose-dependent manner and inhibited the development of pulmonary metastases resulting in a significant survival advantage. In contrast T cells expressing an inactive B7-H3-CAR had no antitumor activity. Using unmodified LM7 cells also enabled us to demonstrate that B7-H3-CAR T cells traffic to orthotopic tumor sites. Hence, we have developed an orthotopic, spontaneously metastasizing OS model. This model may improve our ability not only to predict the safety and efficacy of current and next generation CAR T cell therapies but also other treatment modalities for metastatic OS.
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Antígenos B7/imunologia , Neoplasias Ósseas/terapia , Imunoterapia Adotiva , Osteossarcoma/terapia , Receptores de Antígenos Quiméricos/imunologia , Animais , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Camundongos , Osteossarcoma/patologia , Tíbia/patologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hematopoietic stem cell (HSC) transplantation (HSCT) is often exploited to treat hematologic disease. Donor HSCs must survive, proliferate, and differentiate in the damaged environment of the reconstituting niche. Illuminating molecular mechanisms regulating the activity of transplanted HSCs will inform efforts to improve HSCT. Here, we report that G-protein-coupled receptor-associated sorting proteins (GPRASPs) function as negative regulators of HSCT. Silencing of Gprasp1 or Gprasp2 increased the survival, quiescence, migration, niche retention, and hematopoietic repopulating activity of hematopoietic stem and progenitor cells (HSPCs) posttransplant. We further show that GPRASP1 and GPRASP2 promote the degradation of CXCR4, a master regulator of HSC function during transplantation. CXCR4 accumulates in Gprasp-deficient HSPCs, boosting their function posttransplant. Thus, GPRASPs negatively regulate CXCR4 stability in HSCs. Our work reveals GPRASP proteins as negative regulators of HSCT and CXCR4 activity. Disruption of GPRASP/CXCR4 interactions could be exploited in the future to enhance the efficiency of HSCT.
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Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Proteínas de Transporte , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Deleção de Genes , Inativação Gênica , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , Receptores CXCR4/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Although many recent studies describe the emergence and prevalence of "clonal hematopoiesis of indeterminate potential" in aged human populations, a systematic analysis of the numbers of clones supporting steady-state hematopoiesis throughout mammalian life is lacking. Previous efforts relied on transplantation of "barcoded" hematopoietic stem cells (HSCs) to track the contribution of HSC clones to reconstituted blood. However, ex vivo manipulation and transplantation alter HSC function and thus may not reflect the biology of steady-state hematopoiesis. Using a noninvasive in vivo color-labeling system, we report the first comprehensive analysis of the changing global clonal complexity of steady-state hematopoiesis during the natural murine lifespan. We observed that the number of clones (ie, clonal complexity) supporting the major blood and bone marrow hematopoietic compartments decline with age by â¼30% and â¼60%, respectively. Aging dramatically reduced HSC in vivo-repopulating activity and lymphoid potential while increasing functional heterogeneity. Continuous challenge of the hematopoietic system by serial transplantation provoked the clonal collapse of both young and aged hematopoietic systems. Whole-exome sequencing of serially transplanted aged and young hematopoietic clones confirmed oligoclonal hematopoiesis and revealed mutations in at least 27 genes, including nonsense, missense, and deletion mutations in Bcl11b, Hist1h2ac, Npy2r, Notch3, Ptprr, and Top2b.
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Envelhecimento/fisiologia , Células Clonais/citologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Animais , Transplante de Células-Tronco Hematopoéticas , CamundongosRESUMO
The embryonic site of definitive hematopoietic stem cell (dHSC) origination has been debated for decades. Although an intra-embryonic origin is well supported, the yolk sac (YS) contribution to adult hematopoiesis remains controversial. The same developmental origin makes it difficult to identify specific markers that discern between an intraembryonic versus YS-origin using a lineage trace approach. Additionally, the highly migratory nature of blood cells and the inability of pre-circulatory embryonic cells (i.e., 5-7 somite pairs (sp)) to robustly engraft in transplantation, even after culture, has precluded scientists from properly answering these questions. Here we report robust, multi-lineage and serially transplantable dHSC activity from cultured 2-7sp murine embryonic explants (Em-Ex). dHSC are undetectable in 2-7sp YS explants. Additionally, the engraftment from Em-Ex is confined to an emerging CD31+CD45+c-Kit+CD41- population. In sum, our work supports a model in which the embryo, not the YS, is the major source of lifelong definitive hematopoiesis.
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Linhagem da Célula , Embrião de Mamíferos/citologia , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Saco Vitelino/citologia , Animais , Movimento Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
C57BL/6N (N) and C57BL/6J (J) mice possess key genetic differences, including a deletion in the Nicotinamide nucleotide transhydrogenase (Nnt) gene that results in a non-functional protein in J mice. NNT regulates mitochondrial oxidative stress. Although elevated oxidative stress can compromise hematopoietic stem and progenitor cell (HSPC) function, it is unknown whether N- and J-HSPCs are functionally equivalent. Here, we report that J-HSPCs display compromised short-term hematopoietic repopulating activity relative to N-HSPCs that is defined by a delay in lymphoid reconstitution and impaired function of specific multi-potent progenitor populations post transplant. J-HSPCs also displayed elevated reactive oxygen species (ROS) relative to N-HSPCs post transplant and upregulate ROS levels more in response to hematopoietic stress. Nnt knockdown in N-HSPCs recapitulated J-HSPCs' short-term repopulating defect, indicating that NNT loss contributes to this defect. In summary, C57BL/6N and C57BL/6J HSPCs are not functionally equivalent, which should be considered when determining the substrain most appropriate for investigations of HSPC biology.
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Células-Tronco Hematopoéticas/metabolismo , Estresse Oxidativo , Animais , Diferenciação Celular , Técnicas de Silenciamento de Genes , Sobrevivência de Enxerto , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/genética , NADP Trans-Hidrogenase Específica para A ou B/genética , Espécies Reativas de Oxigênio/metabolismo , Especificidade da Espécie , Fatores de TempoRESUMO
Current dogma asserts that mammalian lifelong blood production is established by a small number of blood progenitors. However, this model is based on assays that require the disruption, transplantation and/or culture of embryonic tissues. Here, we used the sample-to-sample variance of a multicoloured lineage trace reporter to assess the frequency of emerging lifelong blood progenitors while avoiding the disruption, culture or transplantation of embryos. We find that approximately 719 Flk1+ mesodermal precursors, 633 VE-cadherin+ endothelial precursors and 545 Vav1+ nascent blood stem and progenitor cells emerge to establish the haematopoietic system at embryonic days (E)7-E8.5, E8.5-E11.5 and E11.5-E14.5, respectively. We also determined that the spatio-temporal recruitment of endothelial blood precursors begins at E8.5 and ends by E10.5, and that many c-Kit+ clusters of newly specified blood progenitors in the aorta are polyclonal in origin. Our work illuminates the dynamics of the developing mammalian blood system during homeostasis.
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Células Endoteliais/metabolismo , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular , Linhagem da Célula , Rastreamento de Células/métodos , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/transplante , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Idade Gestacional , Transplante de Células-Tronco Hematopoéticas , Integrases/genética , Integrases/metabolismo , Modelos Lineares , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Modelos Biológicos , Fenótipo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Transdução de Sinais , Fatores de Tempo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Hematopoietic stem and progenitor cells (HSPCs) sustain life-long hematopoiesis and are first detected in the embryo by transplantation at embryonic day 10.5 (E10.5). HSPCs are mesodermal in origin and ultimately emerge from a subset of arterial endothelium (i.e., hemogenic endothelium [HE]), which is highly concentrated in the aorta-gonad-mesonephros region of the midgestation embryo. Here, we used clonal ex vivo assays, in which endothelial cells isolated from the midgestation aorta and vitelline and umbilical arteries are co-cultured on supportive stroma, to show that only about 0.1%, 1.3%, and 0.29% of E9.5, E10.5, and E11.5 endothelium are functional HE, respectively. We further show high phenotypic and functional variability in the hematopoietic potential of individual hemogenic endothelial precursors. Using unique niche stroma capable of providing the signals necessary for definitive hematopoietic stem cell (dHSC) induction, we demonstrate that this variability in HE includes their potential to support phenotypic dHSCs. These data suggest the presence of a continuum of maturing HE with distinct hematopoietic potential or HE representative of a heterogeneous pool of precursors that give rise to HSPCs with disparate hematopoietic potential.