RESUMEN
Folate receptors can perform folate transport, cell adhesion, and/or transcription factor functions. The beta isoform of the folate receptor (FRß) has attracted considerable attention as a biomarker for immunosuppressive macrophages and myeloid-derived suppressor cells, however, its role in immunosuppression remains uncharacterized. We demonstrate here that FRß cannot bind folate on healthy tissue macrophages, but does bind folate after macrophage incubation in anti-inflammatory cytokines or cancer cell-conditioned media. We further show that FRß becomes functionally active following macrophage infiltration into solid tumors, and we exploit this tumor-induced activation to target a toll-like receptor 7 agonist specifically to immunosuppressive myeloid cells in solid tumors without altering myeloid cells in healthy tissues. We then use single-cell RNA-seq to characterize the changes in gene expression induced by the targeted repolarization of tumor-associated macrophages and finally show that their repolarization not only changes their own phenotype, but also induces a proinflammatory shift in all other immune cells of the same tumor mass, leading to potent suppression of tumor growth. Because this selective reprogramming of tumor myeloid cells is accompanied by no systemic toxicity, we propose that it should constitute a safe method to reprogram the tumor microenvironment.
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Receptor 2 de Folato , Neoplasias , Humanos , Microambiente Tumoral , Neoplasias/metabolismo , Macrófagos , Ácido Fólico/metabolismoRESUMEN
Non-invasive imaging modalities constitute an increasingly important tool in diagnostic and therapy response monitoring of patients with autoimmune diseases, including rheumatoid arthritis (RA). In particular, macrophage imaging with positron emission tomography (PET) using novel radiotracers based on differential expression of plasma membrane proteins and functioning of cellular processes may be suited for this. Over the past decade, selective expression of folate receptor ß (FRß), a glycosylphosphatidylinositol-anchored plasma membrane protein, on myeloid cells has emerged as an attractive target for macrophage imaging by exploiting the high binding affinity of folate-based PET tracers. This work discusses molecular, biochemical and functional properties of FRß, describes the preclinical development of a folate-PET tracer and the evaluation of this tracer in a translational model of arthritis for diagnostics and therapy-response monitoring, and finally the first clinical application of the folate-PET tracer in RA patients with active disease. Consequently, folate-based PET tracers hold great promise for macrophage imaging in a variety of (chronic) inflammatory (autoimmune) diseases beyond RA.
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Artritis Reumatoide/metabolismo , Receptor 2 de Folato/metabolismo , Macrófagos/metabolismo , Animales , Artritis Reumatoide/diagnóstico por imagen , Artritis Reumatoide/tratamiento farmacológico , Ácido Fólico/metabolismo , Antagonistas del Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/uso terapéutico , Humanos , Tomografía de Emisión de PositronesRESUMEN
Provoked by sterile/nonsterile insults, prolonged monocyte mobilization and uncontrolled monocyte/macrophage activation can pose imminent or impending harm to the affected organs. Curiously, folate receptor beta (FRß), with subnanomolar affinity for the vitamin folic acid (FA), is upregulated during immune activation in hematopoietic cells of the myeloid lineage. This phenomenon has inspired a strong interest in exploring FRß-directed diagnostics/therapeutics. Previously, we have reported that FA-targeted aminopterin (AMT) therapy can modulate macrophage function and effectively treat animal models of inflammation. Our current investigation of a lead compound (EC2319) leads to discovery of a highly FR-specific mechanism of action independent of the root causes against inflammatory monocytes. We further show that EC2319 suppresses interleukin-6/interleukin-1ß release by FRß+ monocytes in a triple co-culture leukemic model of cytokine release syndrome with anti-CD19 chimeric antigen receptor T cells. Because of its chemical stability and metabolically activated linker, EC2319 demonstrates favorable pharmacokinetic characteristics and cross-species translatability to support future pre-clinical and clinical development.
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Aminopterina/farmacología , Síndrome de Liberación de Citoquinas/prevención & control , Receptor 2 de Folato/genética , Antagonistas del Ácido Fólico/farmacología , Ácido Fólico/metabolismo , Macrófagos/efectos de los fármacos , Animales , Antígenos CD19/genética , Antígenos CD19/inmunología , Células CHO , Cricetulus , Síndrome de Liberación de Citoquinas/genética , Síndrome de Liberación de Citoquinas/inmunología , Síndrome de Liberación de Citoquinas/patología , Femenino , Receptor 1 de Folato/antagonistas & inhibidores , Receptor 1 de Folato/genética , Receptor 1 de Folato/inmunología , Receptor 2 de Folato/antagonistas & inhibidores , Receptor 2 de Folato/inmunología , Humanos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/patología , Ratones , Modelos Biológicos , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/patología , Células RAW 264.7 , Ratas , Ratas Endogámicas Lew , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Activated macrophages overexpress the folate receptor ß (FR-ß) that can be used for targeted delivery of drugs conjugated to folic acid. FR-expressing macrophages contribute to arthritis progression by secreting prostaglandin E2 (PGE2). Non-steroidal anti-inflammatory drugs (NSAIDs) block PGs and thromboxane by inhibiting the cyclooxygenase (COX) enzymes and are used for chronic pain and inflammation despite their well-known toxicity. New NSAIDs target an enzyme downstream of COXs, microsomal prostaglandin E synthase-1 (mPGES-1). Inhibition of mPGES-1 in inflammatory macrophages promises to retain NSAID efficacy while limiting toxicity. We conjugated a potent mPGES-1 inhibitor, MK-7285, to folate, but the construct released the drug inefficiently. Folate conjugation to the primary alcohol of MK-7285 improved the construct's stability and the release of free drug. Surprisingly, the drug-folate conjugate potentiated PGE2 in FR-positive KB cells, and reduced PGE2 in macrophages independently of the FR. Folate conjugation of NSAIDs is not an optimal strategy for targeting of macrophages.
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Receptor 2 de Folato/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Dolor/tratamiento farmacológico , Prostaglandina-E Sintasas/metabolismo , Animales , Sistemas de Liberación de Medicamentos , Receptor 2 de Folato/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/complicaciones , Ratones , Ratones Transgénicos , Dolor/etiología , Prostaglandina-E Sintasas/genéticaRESUMEN
Folate receptor beta (FRß) is a folate binding receptor expressed on myeloid lineage hematopoietic cells. FRß is commonly expressed at high levels on malignant blasts in patients with acute myeloid leukemia (AML), as well as on M2 polarized tumor-associated macrophages (TAMs) in the tumor microenvironment of many solid tumors. Therefore, FRß is a potential target for both direct and indirect cancer therapy. We demonstrate that FRß is expressed in both AML cell lines and patient-derived AML samples and that a high-affinity monoclonal antibody against FRß (m909) has the ability to cause dose- and expression-dependent ADCC against these cells in vitro. Importantly, we find that administration of m909 has a significant impact on tumor growth in a humanized mouse model of AML. Surprisingly, m909 functions in vivo with and without the infusion of human NK cells as mediators of ADCC, suggesting potential involvement of mouse macrophages as effector cells. We also found that TAMs from primary ovarian ascites samples expressed appreciable levels of FRß and that m909 has the ability to cause ADCC in these samples. These results indicate that the targeting of FRß using m909 has the potential to limit the outgrowth of AML in vitro and in vivo. Additionally, m909 causes cytotoxicity to TAMs in the tumor microenvironment of ovarian cancer warranting further investigation of m909 and its derivatives as therapeutic agents in patients with FRß-expressing cancers.
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Antineoplásicos Inmunológicos/farmacología , Receptor 2 de Folato , Inmunoterapia , Leucemia Mieloide Aguda , Proteínas de Neoplasias , Neoplasias Ováricas , Animales , Células CHO , Cricetulus , Femenino , Receptor 2 de Folato/antagonistas & inhibidores , Receptor 2 de Folato/inmunología , Células HL-60 , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/inmunología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/patología , Neoplasias Ováricas/terapia , Células THP-1 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Non-small cell lung cancer (NSCLC) is the leading cause of lung cancer-related deaths worldwide. Tumor-associated macrophages (TAMs), which can be polarized into tumor-promoting M2 phenotype, overexpress folate receptor beta (FRß) and are associated with poor prognosis in NSCLC. In addition, calpain-2 (CAPN2) is overexpressed in NSCLC and is involved in tumor growth. To improve the anticancer efficacy of drugs and reduce their side effects in the treatment of NSCLC, it is important to develop smart drug delivery systems with specific targeting ability and controlled release mechanisms. In this study, FRß-targeted pH-sensitive liposomes were designed as carriers to ensure efficient drug delivery and acid-responsive release in NSCLC cells. Folate-mediated targeting of FRß in M2 TAMs and NSCLC cells effectively inhibited tumor growth and the stimulus-responsive drug release reduced the toxic side effects of the drug. The combination of doxycycline (anti-CAPN2) and docetaxel (anticancer drug) showed a synergistic inhibitory effect on tumor growth by suppressing CAPN2 expression.
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Carcinoma de Pulmón de Células no Pequeñas , Receptor 2 de Folato , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , Liposomas , Neoplasias Pulmonares/tratamiento farmacológicoRESUMEN
Herein, we discuss the potential role of folic acid-based radiopharmaceuticals for macrophage imaging to support clinical decision-making in patients with COVID-19. Activated macrophages play an important role during coronavirus infections. Exuberant host responses, i.e., a cytokine storm with increase of macrophage-related cytokines, such as TNFα, IL-1ß, and IL-6 can lead to life-threatening complications, such as acute respiratory distress syndrome (ARDS), which develops in approximately 20% of the patients. Diverse immune modulating therapies are currently being tested in clinical trials. In a preclinical proof-of-concept study in experimental interstitial lung disease, we showed the potential of 18F-AzaFol, an 18F-labeled folic acid-based radiotracer, as a specific novel imaging tool for the visualization and monitoring of macrophage-driven lung diseases. 18F-AzaFol binds to the folate receptor-beta (FRß) that is expressed on activated macrophages involved in inflammatory conditions. In a recent multicenter cancer trial, 18F-AzaFol was successfully and safely applied (NCT03242993). It is supposed that the visualization of activated macrophage-related disease processes by folate radiotracer-based nuclear imaging can support clinical decision-making by identifying COVID-19 patients at risk of a severe disease progression with a potentially lethal outcome.
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OBJECTIVES: Most therapies for autoimmune and inflammatory diseases either neutralize or suppress production of inflammatory cytokines produced by activated macrophages (e.g., TNFα, IL-1, IL-6, IL-17, GM-CSF). However, no approved therapies directly target this activated subset of macrophages. METHODS: First, we undertook to examine whether the folate receptor beta (FR-ß) positive subpopulation of macrophages, which marks the inflammatory subset in animal models of rheumatoid arthritis, might constitute the prominent population of macrophages in inflamed lesions in humans. Next, we utilized anti-FR-ß monoclonal antibodies capable of mediating antibody-dependent cell cytotoxicity (ADCC) to treat animal models of rheumatoid arthritis and peritonitis. RESULTS: Human tissue samples of rheumatoid arthritis, Crohn's disease, ulcerative colitis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia, chronic obstructive pulmonary disease, systemic lupus erythematosus, psoriasis, and scleroderma are all characterized by dramatic accumulation of macrophages that express FR-ß, a protein not expressed on resting macrophages or any other healthy tissues. A monoclonal antibody to FR-ß accumulates specifically in inflamed lesions of murine inflammatory disease models and successfully treats such models of rheumatoid arthritis and peritonitis. More importantly, elimination of FR-ß-positive macrophages upon treatment with an anti-FR-ß monoclonal antibody promotes the departure of other immune cells, including T cells, B cells, neutrophils, and dendritic cells from the inflamed lesions. CONCLUSIONS: These data suggest that specific elimination of FR-ß-expressing macrophages may constitute a highly specific therapy for multiple autoimmune and inflammatory diseases and that a recently developed human anti-human FR-ß monoclonal antibody (m909) might contribute to suppression of this subpopulation of macrophages.
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Anticuerpos Monoclonales/uso terapéutico , Artritis Reumatoide/inmunología , Receptor 2 de Folato/inmunología , Inmunidad Celular , Activación de Macrófagos , Macrófagos/metabolismo , Animales , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/patología , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Macrófagos/inmunología , Macrófagos/patología , RatonesRESUMEN
INTRODUCTION: Nonalcoholic steatohepatitis (NASH) afflicts 20-36% of individuals with nonalcoholic fatty liver disease (NAFLD). A lipotoxic hepatic environment, altered innate immune signaling and inflammation are defining features of progression to NASH. Activated resident liver macrophages express folate receptor beta (FR-ß) which may be an indicator of progression from steatosis to NASH. The goals of this study were to characterize FR-ß protein expression in human NAFLD and rodent models of NASH, and demonstrate liver targeting of an FR-ß imaging agent to the liver of a rodent NASH model using FR-ß. METHODS: Rat liver lysates from methionine choline deficient (MCD) fed rats, high fat diet (HFD) and methionine choline sufficient (MC+) rat controls were analyzed for hepatic FR-ß protein. The FR-ß-targeted agent, Etarfolatide was injected into MCD and MCâ¯+â¯-fed C57BL/6 mice for efficient FastSPECT hepatic imaging. Additionally, FR-ß expression across the stages of human NAFLD from normal to NASH was assessed. RESULTS: FastSPECT images show targeting of Etarfolatide to the liver of mice fed 8â¯weeks of MCD diet but not control-fed mice. The MCD rat model exhibited significantly increased protein expression of hepatic FR-ß in contrast to HFD or normal samples. Similarly human liver samples categorized as NASH Fatty or NASH Not Fatty showed elevated FR-ß protein when compared to normal liver. FR-ß transcript expression levels were elevated across both NASH Fatty and NASH Not Fatty samples. CONCLUSION: The findings in this study indicate that FR-ß expression in NASH may be harnessed to target agents directly to the liver.
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Receptor 2 de Folato/metabolismo , Hígado/diagnóstico por imagen , Hígado/metabolismo , Macrófagos/metabolismo , Imagen Molecular/métodos , Enfermedad del Hígado Graso no Alcohólico/diagnóstico por imagen , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Tomografía Computarizada de Emisión de Fotón Único , Animales , Biomarcadores/metabolismo , Deficiencia de Colina/complicaciones , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Receptor 2 de Folato/genética , Ácido Fólico/administración & dosificación , Ácido Fólico/análogos & derivados , Humanos , Masculino , Metionina/deficiencia , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/genética , Compuestos de Organotecnecio/administración & dosificación , Valor Predictivo de las Pruebas , Radiofármacos/administración & dosificación , Ratas Sprague-DawleyRESUMEN
Receptor-targeted delivery of imaging and therapeutic agents has emerged as an attractive strategy to diagnosis and treat many diseases including cancer. One of the most well-studied receptors for targeted therapies is the folate receptor (FR) family. FR-α and FR-ß are present on many cancers with little expression in normal tissues; leading to the testing of at least six folate-targeted drugs in human clinical trials for various cancers. However, the expression of FR in blood cancers has not been fully explored with no reports of FR expression in myelomas. Herein, we report the expression of both FR-α and FR-ß on CD138 + plasma cells isolated from patients with multiple myeloma. In addition, all-trans retinoic acid was shown to increase the levels of FR-α and FR-ß in two myeloma cell lines. Altogether, this data suggests that folate-targeted therapies for the treatment of multiple myeloma warrants further investigation.
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Antineoplásicos/farmacología , Receptor 1 de Folato/metabolismo , Receptor 2 de Folato/metabolismo , Mieloma Múltiple/patología , Células Plasmáticas/efectos de los fármacos , Tretinoina/farmacología , Antineoplásicos/uso terapéutico , Médula Ósea/patología , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Mieloma Múltiple/tratamiento farmacológico , Células Plasmáticas/metabolismo , Sindecano-1/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
A number of folate receptor (FR) targeted small molecular drugs and monoclonal antibodies have been introduced into clinical trials to treat FR positive cancers. Because the therapeutic efficacy of these drugs depends prominently on the level of FR-α expression on the cancer cells, patients have been commonly selected for FR-targeted therapies based on the intensity of a folate-targeted radioimaging agent. Unfortunately, uptake of such imaging agents can be mediated by both major isoforms of the folate receptor, FR-α and FR-ß. Logically, if the FR positive cell population in a tumor mass is dominated by FR-ß positive macrophages, patients could be selected for therapy that have few FR-expressing cancer cells. Although several IHC studies have examined expression of either FR-α or FR-ß, no study to date has investigated expression of both FR-α and FR-ß in the same tumor mass. Herein, we utilize monoclonal antibodies specific for FR-α (mAb343) and FR-ß (m909) to query each isoform's expression in a range of cancers. We show that lung and pancreatic adenocarcinomas express the full spectrum of FR-α and FR-ß combinations with ~76% of lung adenocarcinomas expressing both FR-α and FR-ß while pancreatic cancers express primarily FR-ß. Thus, while folate-targeted imaging of lung cancer patients might accurately reflect the expression of FR-α on lung cancer cells, imaging of pancreatic cancer patients could mislead a physician into treating a nonresponding patient. Overall, these data suggest that an independent analysis of both FR-α and FR-ß should be obtained to predict the potential efficacy of a folate-targeted drug.
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Rheumatoid arthritis (RA) is the most common complex multifactorial joint related autoimmune inflammatory disease with unknown etiology accomplished with increased cardiovascular risks. RA is characterized by the clinical findings of synovial inflammation, autoantibody production, and cartilage/bone destruction, cardiovascular, pulmonary and skeletal disorders. Pro-inflammatory cytokines such as IL-1, IL-6, IL-8, and IL-10 were responsible for the induction of inflammation in RA patients. Drawbacks such as poor efficacy, higher doses, frequent administration, low responsiveness, and higher cost and serious side effects were associated with the conventional dosage forms for RA treatment. Nanomedicines were recently gaining more interest towards the treatment of RA, and researchers were also focusing towards the development of various anti-inflammatory drug loaded nanoformulations with an aid to both actively/passively targeting the inflamed site to afford an effective treatment regimen for RA. Alterations in the surface area and nanoscale size of the nanoformulations elicit beneficial physical and chemical properties for better pharmacological activities. These drug loaded nanoformulations may enhances the solubility of poorly water soluble drugs, improves the bioavailability, affords targetability and may improve the therapeutic activity. In this regimen, the present review focus towards the novel nanoparticulate formulations (nanoparticles, nanoemulsions, solid lipid nanoparticles, nanomicelles, and nanocapsules) utilized for the treatment of RA. The recent advancements such as siRNA, peptide and targeted based nanoparticulate systems for RA treatment were also discussed. Special emphasis was provided regarding the pathophysiology, prevalence and symptoms towards the development of RA.
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BACKGROUND: Giant cell arteritis (GCA) is a chronic vasculitis of large and medium vessels in which no targetable biomarkers exist to allow selective treatment, predict disease activity and monitor therapeutic responses. The accessibility of the temporal artery (TA) for biopsy allows morphologic studies to characterize macrophages and T cells in the microenvironment of the arterial wall. We evaluated the expression of folate receptor beta (FRB), a candidate diagnostic/therapeutic biomarker, compared its expression with key macrophage markers and correlated it with GCA severity. METHODS: Formalin-fixed paraffin-embedded tissue sections were examined from 6 patients with GCA and 2 controls. Immunohistochemistry was performed using FRB, ETB, CD68 and CD3 antibodies to evaluate for activated macrophages and T cells, assess FRB distribution along the intima, media and adventitial layers and composition of inflammatory infiltrates. We compared the expression of FRB, ETB and CD68 in GCA versus negative controls and in severe (with visual loss) versus mild (without visual loss) disease. RESULTS: In GCA, moderate to severe inflammation was accompanied by >90% destruction of the internal elastic lamina. Macrophages comprised 36.3 ± 4.1% while CD3+ lymphocytes accounted for 61.7 ± 4.1% of total leukocytes. FRB was selectively expressed in macrophages and localized to the adventitia. GCA patients had marginally increased median FRB (9.8 cells/hpf vs. 0; p=0.095), ETB (20.5 vs. 0; p=0.095) and CD68 (38.8 vs. 5; p=0.071) expression versus controls. ETB was found in endothelial cells, smooth muscle cells and macrophages in intima/media. FRB positively correlated with ETB (r=0.90; p-0.037) and CD68 levels (r=0.90; p=0.037). ETB expression positively correlated with CD68 (r=1.0; p<0.0001). There was no difference in FRB between severe and mild GCA. CONCLUSION: FRB is a potential diagnostic and therapeutic biomarker with restricted expression in GCA macrophages. FRB+ macrophages localized to the adventitia and their expression correlated with ETB and CD68 macrophages, suggesting that they contribute to GCA pathogenesis.
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Over-expression of folate receptor alpha on cancer cells has been frequently exploited for delivery of folate-targeted imaging and therapeutic agents to tumors. Because limited information exists on expression of the beta isoform of the folate receptor in human cancers (FR-ß), we have evaluated the immunohistochemical staining pattern of FR-ß in 992 tumor sections from 20 different human cancer types using a new anti-human FR-ß monoclonal antibody. FR-ß expression was shown to be more pronounced in cells within the stroma, primarily macrophages and macrophage-like cells than cancer cells in every cancer type studied. Moreover, FR-ß expression in both cancer and stromal cells was found to be statistically more prominent in females than males. A significant positive correlation was also observed between FR-ß expression on stromal cells and both the stage of the cancer and the presence of lymph node metastases. Based on these data we conclude FR-ß may constitute a good target for specific delivery of therapeutic agents to activated macrophages and that accumulation of FR-ß positive macrophages in the stroma could serve as a useful indicator of a tumor's metastatic potential.
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Receptor 2 de Folato/metabolismo , Macrófagos/metabolismo , Neoplasias/metabolismo , Femenino , Humanos , MasculinoRESUMEN
Vitamin B9, commonly known as folate, is an essential cofactor for one-carbon metabolism that enters cells through three major specialized transporter molecules (RFC, FR, and PCFT), which differ in expression pattern, affinity for substrate, and ligand-binding pH dependency. We now report that the expression of the folate transporters differs between macrophage subtypes and explains the higher accumulation of 5-MTHF-the major folate form found in serum-in M2 macrophages in vitro and in vivo. M1 macrophages display a higher expression of RFC, whereas FRß and PCFT are preferentially expressed by anti-inflammatory and homeostatic M2 macrophages. These differences are also seen in macrophages from normal tissues involved in folate transit (placenta, liver, colon) and inflamed tissues (ulcerative colitis, RA), as M2-like macrophages from normal tissues express FRß and PCFT, whereas TNF-α-expressing M1 macrophages from inflamed tissues are RFC+. Besides, we provide evidences that activin A is a critical factor controlling the set of folate transporters in macrophages, as it down-regulates FRß, up-regulates RFC expression, and modulates 5-MTHF uptake. All of these experiments support the notion that folate handling is dependent on the stage of macrophage polarization.