RESUMEN
Cancer cells have higher reactive oxygen species (ROS) than normal cells, due to genetic and metabolic alterations. An emerging scenario is that cancer cells increase ROS to activate protumorigenic signaling while activating antioxidant pathways to maintain redox homeostasis. Here we show that, in basal-like and BRCA1-related breast cancer (BC), ROS levels correlate with the expression and activity of the transcription factor aryl hydrocarbon receptor (AhR). Mechanistically, ROS triggers AhR nuclear accumulation and activation to promote the transcription of both antioxidant enzymes and the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). In a mouse model of BRCA1-related BC, cancer-associated AhR and AREG control tumor growth and production of chemokines to attract monocytes and activate proangiogenic function of macrophages in the tumor microenvironment. Interestingly, the expression of these chemokines as well as infiltration of monocyte-lineage cells (monocyte and macrophages) positively correlated with ROS levels in basal-like BC. These data support the existence of a coordinated link between cancer-intrinsic ROS regulation and the features of tumor microenvironment. Therapeutically, chemical inhibition of AhR activity sensitizes human BC models to Erlotinib, a selective EGFR tyrosine kinase inhibitor, suggesting a promising combinatorial anticancer effect of AhR and EGFR pathway inhibition. Thus, AhR represents an attractive target to inhibit redox homeostasis and modulate the tumor promoting microenvironment of basal-like and BRCA1-associated BC.
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Anfirregulina/genética , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Receptores de Hidrocarburo de Aril/genética , Adulto , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Receptores ErbB/genética , Clorhidrato de Erlotinib/administración & dosificación , Femenino , Regulación Neoplásica de la Expresión Génica , Homeostasis/genética , Humanos , Ratones , Persona de Mediana Edad , Oxidación-Reducción/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Microambiente Tumoral/genéticaRESUMEN
Transcript sequencing is a crucial tool for gaining a deep understanding of biological processes in diagnostic and clinical medicine. Given their potential to study novel complex eukaryotic transcriptomes, long-read sequencing technologies are able to overcome some limitations of short-read RNA-Seq approaches. Oxford Nanopore Technologies (ONT) offers the ability to generate long-read sequencing data in real time via portable protein nanopore USB devices. This work aimed to provide the user with the number of reads that should be sequenced, through the ONT MinION platform, to reach the desired accuracy level for a human cell RNA study. We sequenced three cDNA libraries prepared from poly-adenosine RNA of human primary cardiac fibroblasts. Since the runs were comparable, they were combined in a total dataset of 48 million reads. Synthetic datasets with different sizes were generated starting from the total and analyzed in terms of the number of identified genes and their expression levels. As expected, an improved sensitivity was obtained, increasing the sequencing depth, particularly for the non-coding genes. The reliability of expression levels was assayed by (i) comparison with PCR quantifications of selected genes and (ii) by the implementation of a user-friendly multiplexing method in a single run.
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Secuenciación de Nanoporos , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Sistemas de Lectura Abierta/genética , RNA-SeqRESUMEN
In cancer, infection and inflammation, the immune system's function can be dysregulated. Instead of fighting disease, immune cells may increase pathology and suppress host-protective immune responses. Myeloid cells show high plasticity and adapt to changing conditions and pathological challenges. Despite their relevance in disease pathophysiology, the identity, heterogeneity and biology of myeloid cells is still poorly understood. We will focus on phenotypical and functional markers of one of the key myeloid regulatory subtypes, the myeloid derived suppressor cells (MDSC), in humans, mice and non-human primates. Technical issues regarding the isolation of the cells from tissues and blood, timing and sample handling of MDSC will be detailed. Localization of MDSC in a tissue context is of crucial importance and immunohistochemistry approaches for this purpose are discussed. A minimal antibody panel for MDSC research is provided as part of the Mye-EUNITER COST action. Strategies for the identification of additional markers applying state of the art technologies such as mass cytometry will be highlighted. Such marker sets can be used to study MDSC phenotypes across tissues, diseases as well as species and will be crucial to accelerate MDSC research in health and disease.
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Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Animales , Biomarcadores , Separación Celular/métodos , Humanos , Inmunofenotipificación/métodos , Ratones , Neutrófilos/inmunología , Neutrófilos/metabolismo , PrimatesRESUMEN
Tumour-infiltrating immune cells regulate tumour development and progression either negatively or positively. For example, cytotoxic lymphocytes (CTL) such as CD8+ T and natural killer (NK) cells can recognize and eliminate cancer cells, and thereby restrict the tumour growth and metastasis, if they exert full cytotoxicity. In contrast, tumour-infiltrating myeloid cells such as tumour-associated macrophages (TAM) promote the expansion and dissemination of cancer cells depending on their functional states. Given the tumour-killing ability of CTL, the augmentation of CTL-induced antitumour immune reactions has been considered as an attractive therapeutic modality for lethal solid tumours and several promising strategies have emerged, which include immune checkpoint inhibitors, cancer vaccines and adoptive CTL transfer. These immunotherapies are now tested in clinical trials and have shown significant antitumour effects in patients with lymphoma and some solid tumours such as melanoma and lung cancer. Despite these encouraging results, these therapies are not efficient in a certain fraction of patients and tumour types with tumour cell-intrinsic mechanisms such as impaired antigen presentation and/or tumour cell-extrinsic mechanisms including the accumulation of immunosuppressive cells. Several animal studies suggest that tumour-infiltrating myeloid cells, especially TAM, are one of the key targets to improve the efficacy of immunotherapies as these cells can suppress the functions of CD8+ T and NK cells. In this review, we will summarize recent animal studies regarding the involvement of TAM in the immune checkpoint, cancer vaccination and adoptive CTL transfer therapies, and discuss the therapeutic potential of TAM targeting to improve the immunotherapies.
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Inmunoterapia , Neoplasias Pulmonares , Macrófagos/inmunología , Melanoma , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Macrófagos/patología , Melanoma/inmunología , Melanoma/patología , Melanoma/terapiaRESUMEN
The tumor microenvironment is a complex network of cells that support tumor progression and malignancy. It has been demonstrated that tumor cells can educate the immune system to promote a tumor-friendly environment. Among all these immune cells, tumor-associated macrophages (TAMs) are well represented and their presence in mouse models has been shown to promote tumor progression and metastasis. These effects are through the stimulation of angiogenesis, enhancement of tumor cell invasion and intravasation, immunosuppression, and at the metastatic site tumor cell extravasation and growth. However, the precise mechanisms are not fully understood. Furthermore there is limited information on TAMs derived from human cancers. For this reason it is important to be able to extract TAMs from tumors in order to compare their phenotypes, functions, and transcriptomes with normal resident tissue macrophages. Isolation of these cells is challenging due to the lack of markers and standardized protocols. Here we show an optimized protocol for the efficient isolation and extraction of resident macrophages and TAMs from human and mouse tissues by using multicolor flow cytometry. These protocols allow for the extraction of thousands of macrophages in less than 5 h from tissues as small as half a gram. The isolated macrophages can then be used for both "omics" and in vitro studies.
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Separación Celular/métodos , Macrófagos/patología , Neoplasias/patología , Animales , Neoplasias de la Mama/patología , Femenino , Citometría de Flujo , Hemólisis , Humanos , RatonesRESUMEN
Tumour progression is modulated by the local microenvironment. This environment is populated by many immune cells, of which macrophages are among the most abundant. Clinical correlative data and a plethora of preclinical studies in mouse models of cancers have shown that tumour-associated macrophages (TAMs) play a cancer-promoting role. Within the primary tumour, TAMs promote tumour cell invasion and intravasation and tumour stem cell viability and induce angiogenesis. At the metastatic site, metastasis-associated macrophages promote extravasation, tumour cell survival and persistent growth, as well as maintain tumour cell dormancy in some contexts. In both the primary and metastatic sites, TAMs are suppressive to the activities of cytotoxic T and natural killer cells that have the potential to eradicate tumours. Such activities suggest that TAMs will be a major target for therapeutic intervention. In this Perspective article, we chronologically explore the evolution of our understanding of TAM biology put into the context of major enabling advances in macrophage biology.
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Antineoplásicos , Neoplasias , Animales , Ratones , Humanos , Macrófagos Asociados a Tumores/patología , Neoplasias/patología , Macrófagos , Biología , Microambiente TumoralRESUMEN
There is a growing body of evidence that cancer causes systemic changes. These influences are most evident in the bone marrow and the blood, particularly in the myeloid compartment. Here, we show that there is an increase in the number of bone marrow, circulating and splenic monocytes by using mouse models of breast cancer caused by the mammary epithelial expression of the polyoma middle T antigen. Cancer does not affect ratios of classical to non-classical populations of monocytes in the circulation nor does it affect their half-lives. Single cell RNA sequencing also indicates that cancer does not induce any new monocyte populations. Cancer does not change the monocytic progenitor number in the bone marrow, but the proliferation rate of monocytes is higher, thus providing an explanation for the expansion of the circulating numbers. Deep RNA sequencing of these monocytic populations reveals that cancer causes changes in the classical monocyte compartment, with changes evident in bone marrow monocytes and even more so in the blood, suggesting influences in both compartments, with the down-regulation of interferon type 1 signaling and antigen presentation being the most prominent of these. Consistent with this analysis, down-regulated genes are enriched with STAT1/STAT2 binding sites in their promoter, which are transcription factors required for type 1 interferon signaling. However, these transcriptome changes in mice did not replicate those found in patients with breast cancer. Consequently, this mouse model of breast cancer may be insufficient to study the systemic influences of human cancer.
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The HIV-1 transactivating factor Tat accumulates on the surface of endothelium by interacting with heparan sulfate proteoglycans (HSPGs). Tat also interacts with B-lymphoid Namalwa cells but only when these overexpress HSPGs after syndecan-1 cDNA transfection (SYN-NCs). Accordingly, SYN-NCs, but not mock-transfected cells, adhere to endothelial cells (ECs) when Tat is bound to the surface of either one of the 2 cell types or when SYN-NCs are transfected with a Tat cDNA. Moreover, endogenously produced Tat bound to cell-surface HSPGs mediates cell adhesion of HIV(+) ACH-2 lymphocytes to the endothelium. This heterotypic lymphocyte-EC interaction is prevented by HSPG antagonist or heparinase treatment, but not by integrin antagonists and requires the homodimerization of Tat protein. Tat tethered to the surface of SYN-NCs or of peripheral blood monocytes from healthy donors promotes their transendothelial migration in vitro in response to CXCL12 or CCL5, respectively, and SYN-NC extravasation in vivo in a zebrafish embryo model of inflammation. In conclusion, Tat homodimers bind simultaneously to HSPGs expressed on lymphoid and EC surfaces, leading to HSPG/Tat-Tat/HSPG quaternary complexes that physically link HSPG-bearing lymphoid cells to the endothelium, promoting their extravasation. These data provide new insights about how lymphoid cells extravasate during HIV infection.
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Movimiento Celular , Endotelio Vascular/metabolismo , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Linfocitos/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Bovinos , Adhesión Celular , Línea Celular Tumoral , Quimiocina CCL5/farmacología , Quimiocina CXCL12/farmacología , Embrión no Mamífero/metabolismo , Infecciones por VIH/genética , Proteoglicanos de Heparán Sulfato , Humanos , Multimerización de Proteína , Sindecano-1 , Transfección , Pez Cebra/genética , Pez Cebra/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The capacity of macrophages to support productive HIV-1 infection is known to be modulated by cytokines and other extracellular stimuli. In this study, we demonstrate that cytokine-induced polarization of human monocyte-derived macrophage (MDM) into either classical (M1) or alternatively activated (M2a) MDM is associated with a reduced capacity to support productive CCR5-dependent (R5) HIV-1 infection. M1 polarization was associated with a significant down-regulation of CD4 receptors, increased secretion of CCR5-binding chemokines (CCL3, CCL4, and CCL5), and a >90% decrease in HIV-1 DNA levels 48-h postinfection, suggesting that the inhibition occurred at an early preintegration step in the viral life cycle. In contrast, M2a polarization had no effect on either HIV-1 DNA or protein expression levels, indicating that inhibition occurred at a late/postintegration level in the viral life cycle. M2a inhibition was sustained for up to 72-h postinfection, whereas M1-effects were more short-lived. Most phenotypic and functional changes were fully reversible 7 days after removal of the polarizing stimulus, and a reciprocal down-regulation of M1-related chemokines and cytokines was observed in M2a MDM and vice versa. Since reversion to a nonpolarized MDM state was associated with a renewed capacity to support HIV replication to control levels, M1/M2a polarization may represent a mechanism that allows macrophages to cycle between latent and productive HIV-1 infection.
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Diferenciación Celular/inmunología , VIH-1/fisiología , Activación de Macrófagos/inmunología , Macrófagos/citología , Macrófagos/virología , Replicación Viral/fisiología , Antígenos CD4/biosíntesis , Antígenos CD4/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Interferón gamma/inmunología , Macrófagos/inmunología , Receptores CCR5/biosíntesis , Receptores CCR5/inmunología , Receptores CXCR4/biosíntesis , Receptores CXCR4/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Macrophages are terminally differentiated cells of the mononuclear phagocyte system that also encompasses dendritic cells, circulating blood monocytes, and committed myeloid progenitor cells in the bone marrow. Both macrophages and their monocytic precursors can change their functional state in response to microenvironmental cues exhibiting a marked heterogeneity. However, there are still uncertainties regarding distinct expression patterns of surface markers that clearly define macrophage subsets, particularly in the case of human macrophages. In addition to their tissue distribution, macrophages can be functionally polarized into M1 (proinflammatory) and M2 (alternatively activated) as well as regulatory cells in response to both exogenous infections and solid tumors as well as by systems biology approaches.
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Polaridad Celular , Activación de Macrófagos , Macrófagos/inmunología , Animales , Antígenos de Superficie/inmunología , Diferenciación Celular , Microambiente Celular , Citocinas/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Humanos , Inmunidad Innata , Macrófagos/patología , Macrófagos/virología , Neoplasias/inmunología , Neoplasias/patología , Biología de SistemasRESUMEN
Tumorigenesis is not only determined by the intrinsic properties of cancer cells but also by their interactions with components of the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are among the most abundant immune cells in the TME. During initial stages of tumor development, macrophages can either directly promote antitumor responses by killing tumor cells or indirectly recruit and activate other immune cells. As genetic changes occur within the tumor or T helper 2 (TH 2) cells begin to dominate the TME, TAMs begin to exhibit an immunosuppressive protumor phenotype that promotes tumor progression, metastasis, and resistance to therapy. Thus, targeting TAMs has emerged as a strategy for cancer therapy. To date, TAM targeting strategies have focused on macrophage depletion and inhibition of their recruitment into the TME. However, these strategies have shown limited therapeutic efficacy, although trials are still underway with combination therapies. The fact that macrophages have the potential for antitumor activity has moved the TAM targeting field toward the development of TAM-reprogramming strategies to support this antitumor immune response. Here, we discuss the various roles of TAMs in cancer therapy and their immunosuppressive properties, as well as implications for emerging checkpoint inhibitor-based immunotherapies. We review state-of-the-art TAM-targeting strategies, focusing on current ones at the preclinical and clinical trial stages that aim to reprogram TAMs as an oncological therapy.
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Macrófagos/inmunología , Macrófagos/metabolismo , Neoplasias/etiología , Neoplasias/metabolismo , Animales , Biomarcadores de Tumor , Citotoxicidad Inmunológica , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunidad , Terapia Molecular Dirigida , Neoplasias/patología , Neoplasias/terapia , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
Cassetta and Pollard introduce tumor-associated macrophages and discuss their origin, diversity, function and plasticity.
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Macrófagos Asociados a Tumores/fisiología , Animales , Humanos , Macrófagos Asociados a Tumores/citología , Macrófagos Asociados a Tumores/inmunologíaRESUMEN
Tumor-associated macrophages (TAMs) are becoming a promising target for cancer immunotherapy. Significant efforts have been made to study the detrimental role of TAMs both in vivo and in vitro. However, it remains challenging to isolate these macrophages to study their function in human cancers and there is the need to seek alternatives to address these limitations. In this review, we will focus on the three most relevant approaches to obtain in vitro fully differentiated macrophages i.e. peripheral blood, immortalized cell lines such as THP-1 or human induced pluripotent stem cells. We will also provide protocols for the polarization of human macrophages to a TAM-like cells in vitro.
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Macrófagos Asociados a Tumores/citología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Línea Celular , Separación Celular/métodos , Humanos , Inmunofenotipificación/métodos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/inmunología , Monocitos/citología , Monocitos/inmunología , Macrófagos Asociados a Tumores/inmunologíaRESUMEN
Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.
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Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/citología , Macrófagos/citología , Biomarcadores/metabolismo , Recuento de Células , Diferenciación Celular , Membrana Celular/metabolismo , Polaridad Celular , Forma de la Célula , Células Cultivadas , Cuerpos Embrioides/citología , Humanos , Macrófagos/metabolismo , Fagocitosis , FenotipoRESUMEN
BACKGROUND: Myeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells. METHODS: We developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders. RESULTS: We observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC. CONCLUSIONS: This study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation.
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Inflamación/inmunología , Células Supresoras de Origen Mieloide/inmunología , Neoplasias/inmunología , Femenino , Humanos , MasculinoRESUMEN
Red blood cells mature within the erythroblastic island (EI) niche that consists of specialized macrophages surrounded by differentiating erythroblasts. Here we establish an in vitro system to model the human EI niche using macrophages that are derived from human induced pluripotent stem cells (iPSCs), and are also genetically programmed to an EI-like phenotype by inducible activation of the transcription factor, KLF1. These EI-like macrophages increase the production of mature, enucleated erythroid cells from umbilical cord blood derived CD34+ haematopoietic progenitor cells and iPSCs; this enhanced production is partially retained even when the contact between progenitor cells and macrophages is inhibited, suggesting that KLF1-induced secreted proteins may be involved in this enhancement. Lastly, we find that the addition of three secreted factors, ANGPTL7, IL-33 and SERPINB2, significantly enhances the production of mature enucleated red blood cells. Our study thus contributes to the ultimate goal of replacing blood transfusion with a manufactured product.
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Eritroblastos/citología , Eritrocitos/citología , Eritropoyesis/fisiología , Células Madre Pluripotentes Inducidas/citología , Factores de Transcripción de Tipo Kruppel/metabolismo , Macrófagos/citología , Proteína 7 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina/metabolismo , Antígenos CD34/metabolismo , Sustitutos Sanguíneos/uso terapéutico , Transfusión Sanguínea , Células Madre Hematopoyéticas/citología , Humanos , Interleucina-33/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Inhibidor 2 de Activador Plasminogénico/metabolismoRESUMEN
The roles of tumor-associated macrophages (TAMs) and circulating monocytes in human cancer are poorly understood. Here, we show that monocyte subpopulation distribution and transcriptomes are significantly altered by the presence of endometrial and breast cancer. Furthermore, TAMs from endometrial and breast cancers are transcriptionally distinct from monocytes and their respective tissue-resident macrophages. We identified a breast TAM signature that is highly enriched in aggressive breast cancer subtypes and associated with shorter disease-specific survival. We also identified an auto-regulatory loop between TAMs and cancer cells driven by tumor necrosis factor alpha involving SIGLEC1 and CCL8, which is self-reinforcing through the production of CSF1. Together these data provide direct evidence that monocyte and macrophage transcriptional landscapes are perturbed by cancer, reflecting patient outcomes.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Reprogramación Celular , Macrófagos/metabolismo , Monocitos/metabolismo , Comunicación Paracrina , Transcripción Genética , Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Quimiocina CCL8/genética , Quimiocina CCL8/metabolismo , Neoplasias Endometriales/genética , Neoplasias Endometriales/metabolismo , Neoplasias Endometriales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor Estimulante de Colonias de Macrófagos/genética , Macrófagos/patología , Terapia Molecular Dirigida , Monocitos/patología , Lectina 1 Similar a Ig de Unión al Ácido Siálico/genética , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Transducción de Señal , Células THP-1 , Microambiente TumoralRESUMEN
BACKGROUND: Human monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective.Human cytomegalovirus (HCMV) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. The available therapeutic armamentarium (e.g. HCMV hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mAb may be a decisive alternative in the prevention of primary and re-activated HCMV infections in these patients. RESULTS: The purpose of this study was to generate neutralizing mAb against HCMV from the immunological repertoire of immune donors. To this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human B-lymphocytes are stimulated with TLR9-agonists and IL-2; second, after both additives are removed, the cells are infected with EBV. Using this strategy we obtained 29 clones secreting IgG neutralizing the HCMV infectivity; four among these were further characterized. All of the mAbs neutralize the infection in different combinations of HCMV strains and target cells, with a potency approximately 20 fold higher than that of the HCMV hyperimmune globulins, currently used in transplant recipients. Recombinant human monoclonal IgG1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated. CONCLUSION: The technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mAbs for immunotherapeutic uses.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Infecciones por Citomegalovirus/terapia , Citomegalovirus/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antivirales/biosíntesis , Anticuerpos Antivirales/uso terapéutico , Linfocitos B/virología , Secuencia de Bases , Línea Celular , Células Cultivadas , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/prevención & control , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Humanos , Interleucina-2/metabolismo , Datos de Secuencia Molecular , Pruebas de Neutralización , Receptores de IgE/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Reproducibilidad de los Resultados , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/metabolismo , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Inhibition of immune checkpoint pathways in CD8+ T cell is a promising therapeutic strategy for the treatment of solid tumors that has shown significant anti-tumor effects and is now approved by the FDA to treat patients with melanoma and lung cancer. However the response to this therapy is limited to a certain fraction of patients and tumor types, for reasons still unknown. To ensure success of this treatment, CD8+ T cells, the main target of the checkpoint inhibitors, should exert full cytotoxicity against tumor cells. However recent studies show that tumor-associated macrophages (TAM) can impede this process by different mechanisms. In this mini-review we will summarize recent studies showing the effect of TAM targeting on immune checkpoint inhibitors efficacy. We will also discuss on the limitations of the current strategies as well on the future scientific challenges for the progress of the tumor immunology field.
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Infiltration of macrophages in solid tumours is associated with poor prognosis and correlates with chemotherapy resistance in most cancers. In mouse models of cancer, macrophages promote cancer initiation and malignant progression by stimulating angiogenesis, increasing tumour cell migration, invasion and intravasation and suppressing antitumour immunity. At metastatic sites, macrophages promote tumour cell extravasation, survival and subsequent growth. Each of these pro-tumoural activities is promoted by a subpopulation of macrophages that express canonical markers but have unique transcriptional profiles, which makes tumour-associated macrophages (TAMs) good targets for anticancer therapy in humans through either their ablation or their re-differentiation away from pro-tumoural towards antitumoural states. In this Review, we evaluate the state of the art of TAM-targeting strategies, focusing on the limitations and potential side effects of the different therapies such as toxicity, rebound effects and compensatory mechanisms. We provide an extensive overview of the different types of therapy used in the clinic and their limitations in light of known macrophage biology and propose new strategies for targeting TAMs.