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Introduction: Tumor-associated macrophages (TAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype and function of these cells. The present study aims to characterize macrophages in high-grade serous ovarian cancer (HGSOC). Methods: Phenotype and expression of co-regulatory markers were assessed on TAMs derived from malignant ascites (MA) or peripheral blood (PB) by multiparametric flow cytometry. Samples were obtained from HGSOC patients (n=29) and healthy donors (HDs, n=16). Additional expression analysis was performed by RNAseq (n=192). Correlation with clinically relevant parameters was conducted and validated by a second patient cohort (n=517). Finally, the role of TIGIT in repolarization and phagocytosis was investigated in vitro. Results: Expression of the M2-associated receptors CD163, CD204, and CD206, as well as of the co-regulatory receptors TIGIT, CD226, TIM-3, and LAG-3 was significantly more frequent on macrophages in HGSOC than in HDs. CD39 and CD73 were broadly expressed on (mainly M2) macrophages, but without a clear clustering in HGSOC. CD163 mRNA levels were higher in TAMs from patients with residual tumor mass after surgery and associated with a shorter overall survival. In addition, TIGIT expression was associated with a higher tumor grading, indicating a prognostic relevance of M2 infiltration in HGSOC. TIGIT blockade significantly reduced the frequency of M2 macrophages. Moreover, combined blockade of TIGIT and CD47 significantly increased phagocytosis of ovarian cancer cells by TAMs in comparison to a single blockade of CD47. Conclusion: Combined blockade of TIGIT and CD47 represents a promising approach to enhance anti-CD47-facilitated phagocytosis.
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Antígeno CD47 , Neoplasias Ováricas , Humanos , Femenino , Antígeno CD47/genética , Antígeno CD47/metabolismo , Macrófagos Asociados a Tumores/metabolismo , Fagocitosis , Neoplasias Ováricas/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Microambiente TumoralRESUMEN
Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development.
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Leucemia Mieloide Aguda , Transcriptoma , Humanos , Transcriptoma/genética , Linfocitos T , Inmunoterapia Adoptiva , Línea Celular , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/metabolismo , Línea Celular TumoralRESUMEN
BACKGROUND: Leukemia-associated macrophages (LAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype, function, and plasticity of these cells. The present study provides an extensive characterization of macrophages in patients with acute myeloid leukemia (AML). METHODS: The phenotype and expression of coregulatory markers were assessed on bone marrow (BM)-derived LAM populations, using multiparametric flow cytometry. BM and blood aspirates were obtained from patients with newly diagnosed acute myeloid leukemia (pAML, n=59), patients in long-term remission (lrAML, n=8), patients with relapsed acute myeloid leukemia (rAML, n=7) and monocyte-derived macrophages of the blood from healthy donors (HD, n=17). LAM subpopulations were correlated with clinical parameters. Using a blocking anti-T-cell immunoreceptor with Ig and ITIM domains (TIGIT) antibody or mouse IgG2α isotype control, we investigated polarization, secretion of cytokines, and phagocytosis on LAMs and healthy monocyte-derived macrophages in vitro. RESULTS: In pAML and rAML, M1 LAMs were reduced and the predominant macrophage population consisted of immunosuppressive M2 LAMs defined by expression of CD163, CD204, CD206, and CD86. M2 LAMs in active AML highly expressed inhibitory receptors such as TIGIT, T-cell immunoglobulin and mucin-domain containing-3 protein (TIM-3), and lymphocyte-activation gene 3 (LAG-3). High expression of CD163 was associated with a poor overall survival (OS). In addition, increased frequencies of TIGIT+ M2 LAMs were associated with an intermediate or adverse risk according to the European Leukemia Network criteria and the FLT3 ITD mutation. In vitro blockade of TIGIT shifted the polarization of primary LAMs or peripheral blood-derived M2 macrophages toward the M1 phenotype and increased secretion of M1-associated cytokines and chemokines. Moreover, the blockade of TIGIT augmented the anti-CD47-mediated phagocytosis of AML cell lines and primary AML cells. CONCLUSION: Our findings suggest that immunosuppressive TIGIT+ M2 LAMs can be redirected into an efficient effector population that may be of direct clinical relevance in the near future.
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Leucemia Mieloide Aguda , Macrófagos , Animales , Ratones , Fagocitosis , Receptores Inmunológicos/metabolismo , Fenotipo , Citocinas/metabolismo , Microambiente TumoralRESUMEN
Recent experimental evidence suggests that mebendazole, a popular antiparasitic drug, binds to heat shock protein 90 (Hsp90) and inhibits acute myeloid leukemia cell growth. In this study we use quantum mechanics (QM), molecular similarity, and molecular dynamics (MD) calculations to predict possible binding poses of mebendazole to the adenosine triphosphate (ATP) binding site of Hsp90. Extensive conformational searches and minimization of the five mebendazole tautomers using the MP2/aug-cc-pVTZ theory level resulted in 152 minima. Mebendazole-Hsp90 complex models were subsequently created using the QM optimized conformations and protein coordinates obtained from experimental crystal structures that were chosen through similarity calculations. Nine different poses were identified from a total of 600 ns of explicit solvent, all-atom MD simulations using two different force fields. All simulations support the hypothesis that mebendazole is able to bind to the ATP binding site of Hsp90.
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Mebendazol , Simulación de Dinámica Molecular , Adenosina Trifosfato , Proteínas HSP90 de Choque Térmico/química , Humanos , Mebendazol/farmacología , Conformación Molecular , Unión Proteica , Conformación ProteicaRESUMEN
Phenotypic characterization of γδ T cells in the MALs (malignant ascites lymphocytes), TILs (tumor infiltrating lymphocytes), and PBLs (peripheral blood lymphocytes) of ovarian cancer (OvCA) patients is lacking. Therefore, we quantified γδ T cell prevalence in MAL, TIL, and PBL specimens from n = 18 OvCA patients and PBL from age-matched healthy donors (HD, n = 14). Multicolor flow cytometry was performed to evaluate the expression of inhibitory receptors (TIGIT, PD-1 and TIM-3), stimulatory receptors (Ox40), and purinergic ectoenzymes (CD39 and CD73) on γδ T cell subsets. We identified an abundant infiltration of Vδ1 T cells in the MALs and TILs. These cells varied in their differentiation: The majority of Vδ1 TILs displayed an effector memory (EM) phenotype, whereas Vδ1 MALs had a more mature phenotype of terminally differentiated effector memory cells (TEMRA) with high CD45RA expression. TIGIT and TIM-3 were abundantly expressed in both MALs and PBLs, whereas Vδ1 TILs exhibited the highest levels of PD-1, CD39, and Ox40. We also observed specific clusters on mature differentiation stages for the analyzed molecules. Regarding co-expression, Vδ1 TILs showed the highest levels of cells co-expressing TIGIT with PD-1 or CD39 compared to MALs and PBLs. In conclusion, the Vδ1 T cell population showed a high prevalence in the MALs and primary tumors of OvCA patients. Due to their (co-)expression of targetable immune receptors, in particular TIGIT with PD-1 and CD39 in TILs, Vδ1 T cell-based approaches combined with the inhibition of these targets might represent a promising strategy for OvCA.
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Neoplasias Ováricas , Receptor de Muerte Celular Programada 1 , Apirasa , Carcinoma Epitelial de Ovario/genética , Femenino , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Neoplasias Ováricas/patología , Receptor de Muerte Celular Programada 1/genética , Receptores InmunológicosRESUMEN
This study aimed to characterize different natural killer (NK) cell phenotypes on bone marrow and peripheral blood cells from acute myeloid leukemia (AML) patients and healthy donors (HDs). Our data show that CD56dimCD16- and CD56brightCD16- NK cells represent the predominant NK cell subpopulations in AML, while the CD56dimCD16+ NK cells are significantly reduced compared to HDs. Moreover, TIGIT+ and PVRIG+ cells cluster on the CD56dimCD16+ subset whereas CD39+ and CD38+ cells do so on CD56brightCD16- NK cells in AML. Furthermore, functional effects of (co-)blockade of TIGIT and CD39 or A2AR on NK cell functionality were analyzed. These experiments revealed that the single blockade of the TIGIT receptor results in an increased NK-92 cell-mediated killing of AML cells in vitro. Combined targeting of CD39 or A2AR significantly augments the anti-TIGIT-mediated lysis of AML cells. Our data indicate that distinct NK cell subsets in AML exhibit different immunosuppressive patterns (via the TIGIT/PVRIG receptors and the purinergic pathway). In summary, we conclude that TIGIT, CD39, and A2AR constitute relevant inhibitory checkpoints of NK cells in AML patients. A combinatorial blockade synergistically strengthens NK-92 cell-mediated cytotoxicity. As inhibitors of TIGIT, CD39, and A2AR are clinically available, studies on their combined use could be conducted in the near future.
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Apirasa/metabolismo , Células Asesinas Naturales/metabolismo , Leucemia Mieloide Aguda/inmunología , Receptor de Adenosina A2A/metabolismo , Receptores Inmunológicos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Apirasa/antagonistas & inhibidores , Estudios de Casos y Controles , Humanos , Inmunoterapia , Leucemia Mieloide Aguda/terapia , Persona de Mediana Edad , Receptores Inmunológicos/antagonistas & inhibidores , Adulto JovenRESUMEN
Background: γδ T cells represent a unique T cell subpopulation due to their ability to recognize cancer cells in a T cell receptor- (TCR) dependent manner, but also in a non-major histocompatibility complex- (MHC) restricted way via natural killer receptors (NKRs). Endowed with these features, they represent attractive effectors for immuno-therapeutic strategies with a better safety profile and a more favorable anti-tumor efficacy in comparison to conventional αß T cells. Also, remarkable progress has been achieved re-activating exhausted T lymphocytes with inhibitors of co-regulatory receptors e.g., programmed cell death protein 1 (PD-1), T cell immunoreceptor with Ig and ITIM domains (TIGIT) and of the adenosine pathway (CD39, CD73). Regarding γδ T cells, little evidence is available. This study aimed to immunophenotypically characterize γδ T cells from patients with diagnosed acute myeloid leukemia (AML) in comparison to patients with multiple myeloma (MM) and healthy donors (HD). Methods: The frequency, differentiation, activation, and exhaustion status of bone marrow- (BM) derived γδ T cells from patients with AML (n = 10) and MM (n = 11) were assessed in comparison to corresponding CD4+ and CD8+ T cells and peripheral blood- (PB) derived γδ T cells from HDs (n = 16) using multiparameter flow cytometry. Results: BM-infiltrating Vδ1 T cells showed an increased terminally differentiated cell population (TEMRAs) in AML and MM in comparison to HDs with an aberrant subpopulation of CD27-CD45RA++ cells. TIGIT, PD-1, TIM-3, and CD39 were more frequently expressed by γδ T cells in comparison to the corresponding CD4+ T cell population, with expression levels that were similar to that on CD8+ effector cells in both hematologic malignancies. In comparison to Vδ2 T cells, the increased frequency of PD-1+-, TIGIT+-, TIM-3+, and CD39+ cells was specifically observed on Vδ1 T cells and related to the TEMRA Vδ1 population with a significant co-expression of PD-1 and TIM-3 together with TIGIT. Conclusion: Our results revealed that BM-resident γδ T cells in AML and MM express TIGIT, PD-1, TIM-3 and CD39. As effector population for autologous and allogeneic strategies, inhibition of co-inhibitory receptors on especially Vδ1 γδ T cells may lead to re-invigoration that could further increase their cytotoxic potential.
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The prognosis of elderly AML patients is still poor due to chemotherapy resistance. The Hedgehog (HH) pathway is important for leukemic transformation because of aberrant activation of GLI transcription factors. MBZ is a well-tolerated anthelmintic that exhibits strong antitumor effects. Herein, we show that MBZ induced strong, dose-dependent anti-leukemic effects on AML cells, including the sensitization of AML cells to chemotherapy with cytarabine. MBZ strongly reduced intracellular protein levels of GLI1/GLI2 transcription factors. Consequently, MBZ reduced the GLI promoter activity as observed in luciferase-based reporter assays in AML cell lines. Further analysis revealed that MBZ mediates its anti-leukemic effects by promoting the proteasomal degradation of GLI transcription factors via inhibition of HSP70/90 chaperone activity. Extensive molecular dynamics simulations were performed on the MBZ-HSP90 complex, showing a stable binding interaction at the ATP binding site. Importantly, two patients with refractory AML were treated with MBZ in an off-label setting and MBZ effectively reduced the GLI signaling activity in a modified plasma inhibitory assay, resulting in a decrease in peripheral blood blast counts in one patient. Our data prove that MBZ is an effective GLI inhibitor that should be evaluated in combination to conventional chemotherapy in the clinical setting.
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Leucemia Mieloide Aguda/metabolismo , Mebendazol/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Moduladores de Tubulina/farmacología , Proteína con Dedos de Zinc GLI1/metabolismo , Estudios de Casos y Controles , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Humanos , Leucemia Mieloide Aguda/etiología , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Proteolisis , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteína con Dedos de Zinc GLI1/antagonistas & inhibidores , Proteína con Dedos de Zinc GLI1/químicaRESUMEN
Modern cancer therapies increased the survival rates of acute myeloid leukemia (AML) patients tremendously. However, the complexity of the disease and the identification of new targets require the adaptation of treatment protocols to reduce side effects and increase benefit for the patients. One key regulator of leukemogenesis and chemotherapy resistance in AML is the Hedgehog (HH) signaling pathway. It is deregulated in numerous cancer entities and inhibition of its downstream transcription factors GLI translates into anti-leukemic effects. One major regulator of GLI is BRD4, a BET family member with epigenetic functions. We investigated the effect of ZEN-3365, a novel BRD4 inhibitor, on AML cells in regard to the HH pathway. We show that ZEN-3365 alone or in combination with GANT-61 reduced GLI promoter activity, cell proliferation and colony formation in AML cell lines and primary cells. Our findings strongly support the evaluation of the BRD4 inhibitor ZEN-3365 as a new therapeutic option in AML.
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Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Factores de Transcripción/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
High mobility group box 1 (HMGB1) is a non-histone protein which is predominantly localised in the cell nucleus. However, stressed, dying, injured or dead cells can release this protein into the extracellular matrix passively. In addition, HMGB1 release was observed in cancer and immune cells where this process can be triggered by various endogenous as well as exogenous stimuli. Importantly, released HMGB1 acts as a so-called "danger signal" and could impact on the ability of cancer cells to escape host immune surveillance. However, the molecular mechanisms underlying the functional role of HMGB1 in determining the capability of human cancer cells to evade immune attack remain unclear. Here we report that the involvement of HMGB1 in anti-cancer immune evasion is determined by Toll-like receptor (TLR) 4, which recognises HMGB1 as a ligand. We found that HGMB1 induces TLR4-mediated production of transforming growth factor beta type 1 (TGF-ß), displaying autocrine/paracrine activities. TGF-ß induces production of the immunosuppressive protein galectin-9 in cancer cells. In TLR4-positive cancer cells, HMGB1 triggers the formation of an autocrine loop which induces galectin-9 expression. In malignant cells lacking TLR4, the same effect could be triggered by HMGB1 indirectly through TLR4-expressing myeloid cells present in the tumour microenvironment (e. g. tumour-associated macrophages).
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Galectinas/biosíntesis , Proteína HMGB1/fisiología , Neoplasias/inmunología , Receptor Toll-Like 4/fisiología , Humanos , Tolerancia Inmunológica , Células THP-1 , Factor de Crecimiento Transformador beta1/fisiologíaRESUMEN
Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.
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Antígenos B7/metabolismo , Galectinas/metabolismo , Linfocitos T Citotóxicos/inmunología , Antígenos de Neoplasias , Apoptosis , Citotoxicidad Inmunológica , Granzimas/metabolismo , Humanos , Terapia de Inmunosupresión , Ligandos , Potenciales de la Membrana , Unión Proteica , Multimerización de Proteína , Células THP-1 , Escape del TumorRESUMEN
Galectin-9 is one of the key proteins employed by a variety of human malignancies to suppress anti-cancer activities of cytotoxic lymphoid cells and thus escape immune surveillance. Human cancer cells in most cases express higher levels of galectin-9 compared to non-transformed cells. However, the biochemical mechanisms underlying this phenomenon remain unclear. Here we report for the first time that in human cancer as well as embryonic cells, the transcription factors hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) are involved in upregulation of transforming growth factor beta 1 (TGF-ß1) expression, leading to activation of the transcription factor Smad3 through autocrine action. This process triggers upregulation of galectin-9 expression in both malignant (mainly in breast and colorectal cancer as well as acute myeloid leukaemia (AML)) and embryonic cells. The effect, however, was not observed in mature non-transformed human cells. TGF-ß1-activated Smad3 therefore displays differential behaviour in human cancer and embryonic vs non-malignant cells. This study uncovered a self-supporting biochemical mechanism underlying high levels of galectin-9 expression operated by the human cancer and embryonic cells employed in our investigations. Our results suggest the possibility of using the TGF-ß1 signalling pathway as a potential highly efficient target for cancer immunotherapy.
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Galectinas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Comunicación Autocrina , Galectinas/genética , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HaCaT , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Células MCF-7 , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Transducción de Señal , Proteína smad3/genética , Proteína smad3/metabolismo , Células THP-1 , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Escape del Tumor , Hipoxia Tumoral , Microambiente TumoralRESUMEN
Aberrant activation of the hedgehog (HH) pathway is observed in many neoplasms, including acute myeloid leukemia (AML). The glioma-associated oncogene homolog (GLI) transcription factors are the main downstream effectors of the HH signaling cascade and are responsible for the proliferation and maintenance of leukemic stem cells, which support chemotherapy resistance and leukemia relapse. Cytarabine (Ara-C)-resistant variants of AML cell lines were established through long-term cultivation with successively increasing Ara-C concentrations. Subsequently, differences in GLI expression were analyzed by RT-qPCR. GLI3 mRNA levels were detectable in parental Kasumi-1, OCI-AML3, and OCI-AML5 cells, whereas GLI3 expression was completely silenced in all resistant counterparts. Therefore, we generated GLI3-knockdown cell lines using small hairpin RNAs (shRNA) and evaluated their sensitivity to Ara-C in vitro. The knockdown of GLI3 partly abolished the effect of Ara-C on colony formation and induction of apoptosis, indicating that GLI3 downregulation results in Ara-C resistance. Moreover, we analyzed the expression of several genes involved in Ara-C metabolism and transport. Knockdown of GLI3 resulted in the upregulation of SAM and HD domain-containing protein 1 (SAMHD1), cytidine deaminase (CDA), and ATP-binding cassette C11 (ABCC11)/multidrug resistance-associated protein 8 (MRP8), each of which has been identified as a predictive marker for Ara-C response in acute myeloid leukemia. Our results demonstrate that GLI3 downregulation is a potential mechanism to induce chemotherapy resistance in AML.
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Apoptosis/genética , Citarabina/farmacología , Resistencia a Antineoplásicos/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/genética , Proteína Gli3 con Dedos de Zinc/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Citarabina/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Leucemia Mieloide Aguda/genética , Proteínas del Tejido Nervioso/genética , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Gli3 con Dedos de Zinc/genéticaRESUMEN
Introduction: Myeloid malignancies are caused by uncontrolled proliferation of neoplastic cells and lack of mature hematopoietic cells. Beside intrinsic genetic and epigenetic alterations within the neoplastic population, abnormal function of the bone marrow stroma promotes the neoplastic process. To overcome the supportive action of the microenvironment, recent research focuses on the development of targeted therapies, inhibiting the interaction of malignant cells and niche cells.Areas covered: This review covers regulatory networks and potential druggable pathways within the hematopoietic stem cell niche. Recent insights into the cell-to-cell interactions in the bone marrow microenvironment are presented. We performed literature searches using PubMed Database from 2000 to the present.Expert opinion: Future therapy of myeloid malignancies must focus on targeted, personalized treatment addressing specific alterations within the malignant and the supporting niche cells. This includes treatments to overcome resistance mechanisms against chemotherapeutic agents mediated by supporting microenvironment. Novel techniques employing sequencing approaches, Crisp/Cas9, or transgenic mouse models are required to elucidate specific interactions between components of the bone marrow niche to identify new therapeutic targets.
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Neoplasias Hematológicas/terapia , Terapia Molecular Dirigida , Trastornos Mieloproliferativos/terapia , Animales , Antineoplásicos/farmacología , Células de la Médula Ósea/citología , Neoplasias Hematológicas/patología , Humanos , Ratones , Ratones Transgénicos , Trastornos Mieloproliferativos/patología , Nicho de Células Madre/fisiología , Células del Estroma/citología , Microambiente TumoralRESUMEN
During the last few years, diverse studies have shown that tumors can actively interact with the lymphatic system and promote metastases development. In order to examine the molecular mechanisms involved in this interaction, we co-cultured tumor and lymphatic endothelial cells (LEC) and subsequently analyzed the molecular alterations of LECs. Therefore, LECs were co-cultivated with either a highly or weakly metastatic breast cancer cell line using contact (mixture) and non-contact (transwell) co-cultures. mRNA profiles from LECs were subsequently analyzed for genes specifically induced by highly metastatic tumor cells ("metastatic specific"). Among the up-regulated "metastatic specific" genes, we found candidates involved in cell cycle, cell adhesion and motility (BST2, E-selectin, and HMMR), cytokines (CCL7, CXCL6, CXCL1, and CSF2) and factors of the complement system (C1R, C3, and CFB). Among the down-regulated genes, we detected the hyaluronan receptor STAB2, angiogenic factor apelin receptor (APLNR), and the glycosylation enzyme MAN1A1. In an additional prostate cancer co-culture model, we could confirm a "metastatic specific" upregulation of E-selectin and CCL7 in LECs after interaction with the prostate cancer cell lines LNCAP (highly metastatic) and DU145 (weakly metastatic). These data allowed us to identify a set of genes regulated in LECs during in vitro communication with cancer cells, which might subsequently facilitate lymphatic metastasis.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Linfangiogénesis , Vasos Linfáticos/patología , Neoplasias de la Próstata/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Adhesión Celular , Técnicas de Cocultivo , Femenino , Perfilación de la Expresión Génica , Humanos , Metástasis Linfática , Vasos Linfáticos/metabolismo , Masculino , Invasividad Neoplásica , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Células Tumorales CultivadasRESUMEN
INTRODUCTION: Prostate cancer has a special predilection to form bone metastases. Despite the known impact of the microvascular network on tumour growth and its dependence on the organ-specific microenvironment, the characteristics of the tumour vasculature in bone remain unknown. METHODS: The cell lines LNCaP, DU145, and PC3 were implanted into the femurs of NSG mice to examine the microvascular properties of prostate cancer in bone. Tumour growth and the functional and morphological alterations of the microvasculature were analysed for 21 days in vivo using a transparent bone chamber and fluorescence microscopy. RESULTS: Vascular density was significantly lower in tumour-bearing bone than in non-tumour-bearing bone, with a marked loss of small vessels. Accelerated blood flow velocity led to increased volumetric blood flow per vessel, but overall perfusion was not affected. All of the prostate cancer cell lines had similar vascular patterns, with more pronounced alterations in rapidly growing tumours. Despite minor differences between the prostate cancer cell lines associated with individual growth behaviours, the same overall pattern was observed and showed strong similarity to that of tumours growing in soft tissue. DISCUSSION: The increase in blood flow velocity could be a specific characteristic of prostate cancer or the bone microenvironment.
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Neoplasias Óseas/irrigación sanguínea , Neoplasias Óseas/secundario , Huesos/patología , Neoplasias de la Próstata/patología , Microambiente Tumoral , Animales , Humanos , Microscopía Intravital , Masculino , Ratones , Microcirculación , Células PC-3 , Distribución AleatoriaRESUMEN
Leukemia-initiating cells reside within the bone marrow in specialized niches where they undergo complex interactions with their surrounding stromal cells. We have identified the actin-binding protein Plastin-3 (PLS3) as potential player within the leukemic bone marrow niche and investigated its functional role in acute myeloid leukemia. High expression of PLS3 was associated with a poor overall and event-free survival for AML patients. These findings were supported by functional in vitro and in vivo experiments. AML cells with a PLS3 knockdown showed significantly reduced colony numbers in vitro while the PLS3 overexpression variants resulted in significantly enhanced colony numbers compared to their respective controls. Furthermore, the survival of NSG mice transplanted with the PLS3 knockdown cells showed a significantly prolonged survival in comparison to mice transplanted with the control AML cells. Further studies should focus on the underlying leukemia-promoting mechanisms and investigate PLS3 as therapeutic target.
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Immune checkpoints are intensively investigated as targets in cancer therapy. T-cell immunoreceptor with immunoglobulin (Ig) and ITIM domains (TIGIT) and its ligand poliovirus receptor (PVR) are recently emerging as novel promising targets in immunotherapy. Here, we show that high expression of PVR represents an independent prognostic marker being associated with poor outcome for breast cancer patients. Furthermore, PVR mRNA, as well as protein expression, is associated with more aggressive breast cancer subtypes such as HER2 positive and triple-negative breast cancer. In vitro, blocking TIGIT or PVR resulted in enhanced immune cell-mediated lysis of breast cancer cell lines SKBR-3, MDA-MB-231, MDA-MB-468, and BT549 and additionally increased the cytotoxic effects of a bispecific T cell engager BiTE® antibody construct targeting EGFR. Taken together, our data identify the immune checkpoint factor PVR as a novel prognostic marker in breast cancer and indicate that blocking the TIGIT-PVR axis might represent a novel therapeutic option for the treatment of breast cancer patients.
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Here we describe the establishment and characterization of an AR+, PSMA+, ERG+, PTEN-/-, CHD1+/- patient-derived xenograft (PDX) model termed 'C5', which has been developed from a 60 years old patient suffering from castration-resistant prostate cancer (CRPC). The patient underwent radical prostatectomy, showed early tumor marker PSA recurrence and, one year after surgery, abiraterone resistance. Subcutaneous C5 tumors can be serially transplanted between mice and grow within ~90 days to 1.5-2 cm³ tumors in SCID Balb/c mice (take rate 100%), NOD-scid IL2Rgnull (NSG) mice (100%) and C57BL/6 pfp-/-/rag2-/- mice (66%). In contrast, no tumor growth is observed in female mice. C5 tumors can be cryopreserved and show the same growth characteristics in vivo afterwards. C5 tumor cells do not grow stably in vitro, neither under two- nor three-dimensional cell culture conditions. Upon serial transplantation, some C5 tumors spontaneously disseminated to distant sites with an observable trend towards higher metastatic cell loads in scid compared to NSG mice. Lung metastases could be verified by histology by means of anti-PSMA immunohistochemistry, exclusively demonstrating single disseminated tumor cells (DTCs) and micro-metastases. Upon surgical resection of the primary tumors, such pulmonary foci rarely grew out to multi-cellular metastatic colonies despite doubled overall survival span. In the brain and bone marrow, the metastatic cell load present at surgery even disappeared during the post-surgical period. We provide shallow whole genome sequencing and whole exome sequencing data of C5 tumors demonstrating the copy number aberration/ mutation status of this PCa model and proving genomic stability over several passages. Moreover, we analyzed genomic and transcriptomic alterations during metastatic progression achieved by serial transplantation. This study describes a novel PCa PDX model that enables future research on several aspects of metastatic PCa, particularly for the AR+ , ERG+ , PTEN-/- PCa subtype.
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Trasplante de Neoplasias , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Animales , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones SCID , Persona de Mediana Edad , Metástasis de la NeoplasiaRESUMEN
The bone marrow is the home of hematopoiesis and is therefore a hotspot for the development of hematopoietic diseases. Complex interactions between the bone marrow microenvironment and hematopoietic stem cells must find a balance between proliferation, differentiation and homeostasis of the stem cell compartment. Changes in this tightly regulated network can provoke malignant transformation, leading to hematopoietic diseases. Here we focus on acute myeloid leukemia (AML), since this is the most frequent acute leukemia in adulthood with very poor overall survival rates and where relapse after chemotherapy continues to be a major challenge, driving demand for new therapeutic strategies. Current research is focusing on the identification of specific interactions between leukemic blasts and their niche components, which may be exploited as novel treatment targets along with induction chemotherapy. Significant progress has been gained over the last few years in the field of high-resolution imaging. Confocal ex vivo and intravital microscopy have revealed a detailed map of bone marrow structures and components; as well as identifying numerous alterations in the stem cell niche that correspond to disease progression. However, the underlying mechanisms are still not completely understood and due to the complexity, their elucidation remains a challenging. This review discusses the constitution of the AML niche in the bone marrow, the improvement in visualization of the complex three-dimensional niche structures and points out new therapeutic strategies to increase the overall survival of AML patients.