RESUMEN
Vitamin A is an essential anti-infective agent with pleiotropic effects on cells of the immune system. The goal of the present study was to unravel the impact of the vitamin A metabolite retinoic acid (RA) on B-cell survival related both to normal B-cell homeostasis and to the detrimental effects imposed by DNA-damaging agents. By combining RA with Toll-like receptor 9 (TLR9) ligands, we show that RA prevents spontaneous, irradiation- and doxorubicin-induced apoptosis of human B cells in an RA receptor-dependent manner. RA-mediated survival involved up-regulation of the anti-apoptotic protein myeloid cell leukemia 1 (MCL1) at the transcriptional level, and knock down of MCL1 by small interfering RNA partially reversed the effects of RA. To ensure that the combination of TLR9-ligands and RA would not promote the survival of malignant B cells, the combined effects of stimulation with RA and TLR9 ligands was assessed on cells from patients with B-cell malignancies. In contrast to the effects on normal B cells, the combination of TLR9 stimulation and RA neither enhanced the MCL1 levels nor inhibited the death of malignant B cells challenged by DNA-damaging agents. Taken together, the present results reveal a vital role of MCL1 in RA-mediated survival of normal B cells. Moreover, the findings suggest that RA in combination with TLR9 ligands might be useful adjuvants in the treatment of B-cell malignancies by selectively protecting normal and not malignant B cells from DNA-damage-induced cell death.
Asunto(s)
Linfocitos B/efectos de los fármacos , Leucemia de Células B/tratamiento farmacológico , Tretinoina/farmacología , Apoptosis/efectos de los fármacos , Linfocitos B/fisiología , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Doxorrubicina/toxicidad , Femenino , Homeostasis/efectos de los fármacos , Humanos , Leucemia de Células B/patología , Activación de Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Radiación , Receptor Toll-Like 9/metabolismo , Tretinoina/química , Células Tumorales Cultivadas , Vitamina A/químicaRESUMEN
The tumour microenvironment influences outcome in patients with follicular lymphoma (FL), but its impact on transformation is less studied. We investigated the prognostic significance of the tumour microenvironment on transformation and survival in FL patients treated in the rituximab era. We examined diagnostic and transformed biopsies from 52 FL patients using antibodies against CD3, CD4, CD8, CD21 (CR2), CD57 (B3GAT1), CD68, FOXP3, TIA1, PD-1 (PDCD1), PD-L1 (CD274) and PAX5. Results were compared with a second cohort of 40 FL patients without signs of transformation during a minimum of five years observation time. Cell numbers and localization were semi-quantitatively assessed. Better developed CD21+ follicular dendritic cell (FDC) meshworks at diagnosis was a negative prognostic factor for overall survival (OS), progression-free survival (PFS) and time to transformation (TTT) in patients with subsequently transformed FL. Remnants of FDC meshworks at transformation were associated with shorter OS and PFS from transformation. High degrees of intrafollicular CD68+ and PD-L1+ macrophage infiltration, high total area scores and an extrafollicular/diffuse pattern of FOXP3+ T cells and high intrafollicular scores of CD4+ T cells at diagnosis were associated with shorter TTT. Scores of several T-cell subset markers from the combined patient cohorts were predictive for transformation, especially CD4 and CD57.
Asunto(s)
Transformación Celular Neoplásica , Linfoma Folicular/mortalidad , Linfoma Folicular/patología , Microambiente Tumoral , Adulto , Anciano , Antineoplásicos/uso terapéutico , Biomarcadores , Biopsia , Femenino , Estudios de Seguimiento , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfoma Folicular/tratamiento farmacológico , Linfoma Folicular/metabolismo , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Pronóstico , Modelos de Riesgos Proporcionales , Rituximab/uso terapéutico , Factores de Tiempo , Microambiente Tumoral/inmunología , Adulto JovenRESUMEN
Follicular lymphoma (FL) is the most common indolent type of B-cell non-Hodgkin lymphoma. Advances in treatment have improved overall survival, but early relapse or transformation to aggressive disease is associated with inferior outcome. To identify early genetic events and track tumor clonal evolution, we performed multi-omics analysis of 94 longitudinal biopsies from 44 FL patients; 22 with transformation (tFL) and 22 with relapse without transformation (nFL). Deep whole-exome sequencing confirmed recurrent mutations in genes encoding epigenetic regulators (CREBBP, KMT2D, EZH2, EP300), with similar mutational landscape in nFL and tFL patients. Calculation of genomic distances between longitudinal samples revealed complex evolutionary patterns in both subgroups. CREBBP and KMT2D mutations were identified as genetic events that occur early in the disease course, and cases with CREBBP KAT domain mutations had low risk of transformation. Gains in chromosomes 12 and 18 (TCF4), and loss in 6q were identified as early and stable copy number alterations. Identification of such early and stable genetic events may provide opportunities for early disease detection and disease monitoring. Integrative analysis revealed that tumors with EZH2 mutations exhibited reduced gene expression of numerous histone genes, including histone linker genes. This might contribute to the epigenetic dysregulation in FL.
Asunto(s)
Genómica , Linfoma Folicular , Mutación , Humanos , Linfoma Folicular/genética , Linfoma Folicular/patología , Femenino , Masculino , Persona de Mediana Edad , Anciano , Genómica/métodos , Adulto , Secuenciación del Exoma , Variaciones en el Número de Copia de ADN , Proteína de Unión a CREB/genética , Estudios Longitudinales , Anciano de 80 o más Años , MultiómicaRESUMEN
Genome editing in eukaryotes has greatly improved through the application of targeted editing tools. The development of the CRISPR/Cas9 technology has facilitated genome editing in mammalian cells. However, efficient delivery of CRISPR components into cells growing in suspension remains a challenge. Here, we present a strategy for sequential delivery of the two essential components, Cas9 and sgRNA, into B-lymphoid cell lines. Stable Cas9 expression is obtained by retroviral transduction, before sgRNA is transiently delivered into the Cas9+ cells. This method improves the on-target efficiency of genome editing and, through the transient presence of sgRNA, reduces the potential off-target sites. The current method can be easily applied to other cell types that are difficult to edit with CRISPR/Cas9.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Linfoma de Células B/genética , Linfocitos B/metabolismo , Proteína 9 Asociada a CRISPR/genética , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Electroporación/métodos , Técnicas de Transferencia de Gen , Humanos , Linfoma de Células B/terapia , ARN Guía de Kinetoplastida/genética , Transducción Genética/métodosRESUMEN
BACKGROUND: Adoptive T-cell transfer of therapeutic TCR holds great promise to specifically kill cancer cells, but relies on modifying the patient's own T cells ex vivo before injection. The manufacturing of T cells in a tailor-made setting is a long and expensive process which could be resolved by the use of universal cells. Currently, only the Natural Killer (NK) cell line NK-92 is FDA approved for universal use. In order to expand their recognition ability, they were equipped with Chimeric Antigen Receptors (CARs). However, unlike CARs, T-cell receptors (TCRs) can recognize all cellular proteins, which expand NK-92 recognition to the whole proteome. METHODS: We herein genetically engineered NK-92 to express the CD3 signaling complex, and showed that it rendered them able to express a functional TCR. Functional assays and in vivo efficacy were used to validate these cells. FINDINGS: This is the first demonstration that a non-T cell can exploit TCRs. This TCR-redirected cell line, termed TCR-NK-92, mimicked primary T cells phenotypically, metabolically and functionally, but retained its NK cell effector functions. Our results demonstrate a unique manner to indefinitely produce TCR-redirected lymphocytes at lower cost and with similar therapeutic efficacy as redirected T cells. INTERPRETATION: These results suggest that an NK cell line could be the basis for an off-the-shelf TCR-based cancer immunotherapy solution. FUND: This work was supported by the Research Council of Norway (#254817), South-Eastern Norway Regional Health Authority (#14/00500-79), by OUS-Radiumhospitalet (Gene Therapy program) and the department of Oncology at the University of Lausanne.
Asunto(s)
Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Animales , Biomarcadores , Línea Celular Tumoral , Respiración de la Célula , Citotoxicidad Inmunológica/genética , Modelos Animales de Enfermedad , Metabolismo Energético , Perfilación de la Expresión Génica , Humanos , Inmunofenotipificación , Ratones , Mitocondrias/metabolismo , Neoplasias/patología , Neoplasias/terapia , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transcriptoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Although chemo-immunotherapy has led to an improved overall survival for most B-cell lymphoma types, relapsed and refractory disease remains a challenge. The malaria drug artesunate has previously been identified as a growth suppressor in some cancer types and was tested as a new treatment option in B-cell lymphoma. METHODS: We included artesunate in a cancer sensitivity drug screen in B lymphoma cell lines. The preclinical properties of artesunate was tested as single agent in vitro in 18 B-cell lymphoma cell lines representing different histologies and in vivo in an aggressive B-cell lymphoma xenograft model, using NSG mice. Artesunate-treated B lymphoma cell lines were analyzed by functional assays, gene expression profiling, and protein expression to identify the mechanism of action. RESULTS: Drug screening identified artesunate as a highly potent anti-lymphoma drug. Artesunate induced potent growth suppression in most B lymphoma cells with an IC50 comparable to concentrations measured in serum from artesunate-treated malaria patients, while leaving normal B-cells unaffected. Artesunate markedly inhibited highly aggressive tumor growth in a xenograft model. Gene expression analysis identified endoplasmic reticulum (ER) stress and the unfolded protein response as the most affected pathways and artesunate-induced expression of the ER stress markers ATF-4 and DDIT3 was specifically upregulated in malignant B-cells, but not in normal B-cells. In addition, artesunate significantly suppressed the overall cell metabolism, affecting both respiration and glycolysis. CONCLUSIONS: Artesunate demonstrated potent apoptosis-inducing effects across a broad range of B-cell lymphoma cell lines in vitro, and a prominent anti-lymphoma activity in vivo, suggesting it to be a relevant drug for treatment of B-cell lymphoma.
Asunto(s)
Antineoplásicos/farmacología , Artesunato/farmacología , Linfoma de Células B/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Artesunato/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Glucólisis/efectos de los fármacos , Humanos , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Ratones , Transcriptoma/efectos de los fármacos , Respuesta de Proteína Desplegada/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Follicular lymphoma (FL) is an indolent non-Hodgkin lymphoma that has a risk of transformation to more aggressive lymphoma. Relatively little is known about the nonmalignant B-cell and T-cell subset composition within the tumor microenvironment and whether altered phenotypes are associated with patterns of lymphoma B-cell heterogeneity. METHODS: Two mass cytometry (CyTOF) panels were designed to immunophenotype B and T cells in FL tumors. Populations of malignant B cells, nonmalignant B cells, and T cells from each FL tumor were identified and their phenotypes compared to B and T cells from healthy human tonsillar tissue. RESULTS: Diversity in cellular phenotype between tumors was greater for the malignant B cells than for nonmalignant B or T cells. The malignant B-cell population bore little phenotypic similarity to any healthy B-cell subset, and unexpectedly clustered closer to naïve B-cell populations than GC B-cell populations. Among the nonmalignant B cells within FL tumors, a significant lack of GC and plasmablast B cells was observed relative to tonsil controls. In contrast, nonmalignant T cells in FL tumors were present at levels similar to their cognate tonsillar T-cell subsets. CONCLUSION: Mass cytometry revealed that diverse HLA-DR expression on FL cells within individual tumors contributed greatly to tumor heterogeneity. Both malignant and nonmalignant B cells in the tumor bore little phenotypic resemblance to healthy GC B cells despite the presence of T follicular helper cells in the tumor. These findings suggest that ongoing signaling interactions between malignant B cells and intra-tumor T cells shape the tumor microenvironment. © 2016 International Clinical Cytometry Society.
Asunto(s)
Linfocitos B/inmunología , Citometría de Flujo , Centro Germinal/patología , Antígenos HLA-DR/inmunología , Linfoma Folicular/inmunología , Centro Germinal/inmunología , Humanos , Inmunofenotipificación/métodos , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Linfoma Folicular/diagnóstico , Células Plasmáticas/citología , Células Plasmáticas/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
OBJECTIVE: Several transducing molecules, including JAK2, STAT5, MAP kinases, phosphatidylinositol 3-kinase (PI3K), phospholipase C-gamma1, and PKC are activated by interaction between erythropoietin (EPO) and the EPO receptor. The aim of this was to examine the relative involvement of PI3K in the development of glycophorin A (GPA)(+) erythroid cells from normal hematopoietic progenitor cells. MATERIALS AND METHODS: CD34(+) hematopoietic progenitor cells or subpopulations obtained by FACS sorting were cultured in serum-free medium containing EPO with or without inhibitors for PI3K, p38, MEK, or PKC for various time periods before phenotypic analysis or detection of apoptosis by flow cytometry, cell cycle analysis, high-resolution tracking of cell division, Western blot analysis, or Akt kinase assay were performed. RESULTS: The PI3K inhibitor LY294002 completely counteracted the EPO-induced proliferation of CD34(+) progenitor cells and CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 also highly suppressed the expanded erythropoiesis induced by the combined action of EPO and stem cell factor. The profound inhibitory effect of LY294002 on proliferation was caused by its induction of cell cycle arrest in the G(0)/G(1) phase of the cell cycle. Some cells acquired GPA expression before they went through cell division. This was completely blocked by LY294002, implying an inhibitory effect on maturation. In addition, LY294002 completely blocked the viability-enhancing effect of EPO in CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 and various inhibitors of PKC completely suppressed the EPO-induced increase in the activity of Akt kinase, a direct downstream target of PI3K. CONCLUSIONS: Our results point to an important role for PI3K in mediating EPO-induced survival, proliferation, and possibly maturation of early erythroid progenitors.
Asunto(s)
Células Precursoras Eritroides/efectos de los fármacos , Eritropoyesis/fisiología , Eritropoyetina/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas , Adulto , Antígenos CD34/análisis , Antígenos de Diferenciación/análisis , Butadienos/farmacología , Carbazoles/farmacología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Cromonas/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Precursoras Eritroides/citología , Eritropoyesis/efectos de los fármacos , Eritropoyetina/antagonistas & inhibidores , Flavonoides/farmacología , Glicoforinas/análisis , Células Madre Hematopoyéticas/citología , Humanos , Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/fisiología , Morfolinas/farmacología , Naftalenos/farmacología , Nitrilos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Proteína Quinasa C-alfa , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/farmacología , Estaurosporina/farmacología , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/farmacologíaRESUMEN
The S100A4 protein, which is involved in the metastasis process, is a member of the S100 superfamily of Ca-binding proteins. Members of this family are multifunctional signaling proteins with dual extra and intracellular functions involved in the regulation of diverse cellular processes. Several studies have established a correlation between S100A4 protein expression and worse prognosis for patients with various malignancies including breast cancer. In this article, we have used specific antibodies in combination with immunohistochemistry (IHC) to identify the cell types that express S100A4 in human breast cancer biopsies obtained from high-risk patients. IHC analysis of 68 tumor biopsies showed that the protein is expressed preferentially by various cell types present in the tumor microenvironment (macrophages, fibroblasts, activated lymphocytes), rather than by the tumor cells themselves. Moreover, we show that the protein is externalized by the stroma cells to the fluid that bathes the tumor microenvironment, where it is found in several forms that most likely correspond to charge variants. Using a specific ELISA test, we detected a significant higher concentration of S100A4 in the tumor interstitial fluid (TIF) as compared to their corresponding normal counterparts (NIF).
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas S100/biosíntesis , Adulto , Anciano de 80 o más Años , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Western Blotting , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibroblastos/química , Fibroblastos/patología , Humanos , Inmunohistoquímica/métodos , Linfocitos/química , Linfocitos/patología , Macrófagos/química , Macrófagos/patología , Persona de Mediana Edad , Proteína de Unión al Calcio S100A4 , Proteínas S100/sangre , Proteínas S100/inmunologíaRESUMEN
Erythropoietin (Epo) is the major regulator of differentiation, proliferation and survival of erythroid progenitors, but the Epo-induced changes in gene expression that lead to these effects are not fully understood. The aim of this study was to examine how Epo, via activation of phosphatidylinositol 3-kinase (PI3K)/Akt, exerts its role in the development of erythroid progenitors from CD34+ cells, and to identify early Epo target genes in human erythroid progenitors. In CD34+ progenitor cells, Epo alone was able to induce cell cycle progression as demonstrated by upregulation of cyclin D3, E and A leading to hyperphosphorylation of the retinoblastoma protein (RB). These effects were completely counteracted by the PI3K inhibitor LY294002. Furthermore, enforced expression of an activated form of Akt kinase highly augmented Epo-induced erythropoiesis. Fluorescent-activated cell sorting (FACS)-sorted CD34+CD71+CD45RA-GPA- erythroid progenitors stimulated with Epo in the presence or absence of LY294002 were subjected to gene expression profiling. Several novel target genes of Epo were identified, and the majority were regulated in a PI3K-dependent manner, including KIT (CD117) and CDH1 (E-cadherin). FACS analysis of Epo-stimulated erythroid progenitors showed that the increased mRNA expression of KIT and CDH1 was accompanied by an induction of the corresponding proteins CD117 and E-cadherin.