RESUMO
Transforming growth factor beta (TGF-ß) signaling is involved in the regulation of proliferation, differentiation and survival/or apoptosis of many cells, including glioma cells. TGF-ß acts via specific receptors activating multiple intracellular pathways resulting in phosphorylation of receptor-regulated Smad2/3 proteins that associate with the common mediator, Smad4. Such complex translocates to the nucleus, binds to DNA and regulates transcription of many genes. Furthermore, TGF-ß-activated kinase-1 (TAK1) is a component of TGF-ß signaling and activates mitogen-activated protein kinase (MAPK) cascades. Negative regulation of TGF-ß/Smad signaling may occur through the inhibitory Smad6/7. While genetic alterations in genes related to TGF-ß signaling are relatively rare in gliomas, the altered expression of those genes is a frequent event. The increased expression of TGF-ß1-3 correlates with a degree of malignancy of human gliomas. TGF-ß may contribute to tumor pathogenesis in many ways: by direct support of tumor growth, by maintaining self-renewal of glioma initiating stem cells and inhibiting anti-tumor immunity. Glioma initiating cells are dedifferentiated cells that retain many stem cell-like properties, play a role in tumor initiation and contribute to its recurrence. TGF-ß1,2 stimulate expression of the vascular endothelial growth factor as well as the plasminogen activator inhibitor and some metalloproteinases that are involved in vascular remodeling, angiogenesis and degradation of the extracellular matrix. Inhibitors of TGF-ß signaling reduce viability and invasion of gliomas in animal models and show a great promise as novel, potential anti-tumor therapeutics.
Assuntos
Glioma/metabolismo , Glioma/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Carcinogênese , Glioma/tratamento farmacológico , Humanos , Fosforilação , Receptores de Fatores de Crescimento Transformadores beta/metabolismoRESUMO
Glioblastoma (GBM) is the most aggressive and prevalent primary brain malignancy in adults. Current treatments provide limited benefit, and thus, the median overall survival of GBM patients is only 15 months. GBM progression is highly dependent on its ability to evade immune response, so understanding the mechanisms behind GBM-driven immunosuppression seems crucial for designing more efficient therapies. Animal models of GBM constitute a convenient tool in glioma research, and several different approaches have been already developed to model this disease in vivo, including genetic and xenograft models. Here, we describe a murine syngeneic model of glioma which recapitulates many of the key features of human disease, including complex tumor microenvironment. We present an optimized protocol for stereotactic intracranial implantation of GL261 cells into C57BL/6 mice which results in tumor growth in the striatum. This model has been widely used to get insight into glioma biology, as well as in the studies aiming at the development and validation of new therapeutic approaches.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Adulto , Humanos , Camundongos , Animais , Glioblastoma/patologia , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BL , Glioma/patologia , Neoplasias Encefálicas/patologia , Modelos Animais de Doenças , Microambiente TumoralRESUMO
Malignant gliomas are aggressive, hard-to-treat brain tumors. Their tumor microenvironment is massively infiltrated by myeloid cells, mostly brain-resident microglia, bone marrow (BM)-derived monocytes/macrophages, and dendritic cells that support tumor progression. Single-cell omics studies significantly dissected immune cell heterogeneity, but dynamics and specific functions of individual subpopulations were poorly recognized. We use Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) to precisely dissect myeloid cell identities and functionalities in murine GL261 gliomas. We demonstrate that the diversity of myeloid cells infiltrating gliomas is dictated by cell type and cell state. Glioma-activated microglia are the major source of cytokines attracting other immune cells, whereas BM-derived cells show the monocyte-to-macrophage transition in the glioma microenvironment. This transition is coupled with a phenotypic switch from the IFN-related to antigen-presentation and tumor-supportive gene expression. Moreover, we found sex-dependent differences in transcriptional programs and composition of myeloid cells in murine and human glioblastomas.
Assuntos
Neoplasias Encefálicas , Glioma , Humanos , Camundongos , Animais , Caracteres Sexuais , Glioma/patologia , Macrófagos/metabolismo , Neoplasias Encefálicas/metabolismo , Monócitos/metabolismo , Microglia/metabolismo , Microambiente TumoralRESUMO
Chronic and acute myeloid leukemia evade immune system surveillance and induce immunosuppression by expanding proleukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse, and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector proleukemic Tregs. Using mouse model of leukemia-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, and IL21R and included 2 distinct effector Treg (eTreg) subsets: CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive Tregs and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms.
Assuntos
Ligante 4-1BB/imunologia , Vesículas Extracelulares , Leucemia Mieloide Aguda , Animais , Vesículas Extracelulares/metabolismo , Imunossupressores/uso terapêutico , Antígeno Ki-1/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Linfócitos T ReguladoresRESUMO
Microglia are resident myeloid cells in the central nervous system (CNS) that control homeostasis and protect CNS from damage and infections. Microglia and peripheral myeloid cells accumulate and adapt tumor supporting roles in human glioblastomas that show prevalence in men. Cell heterogeneity and functional phenotypes of myeloid subpopulations in gliomas remain elusive. Here we show single-cell RNA sequencing (scRNA-seq) of CD11b+ myeloid cells in naïve and GL261 glioma-bearing mice that reveal distinct profiles of microglia, infiltrating monocytes/macrophages and CNS border-associated macrophages. We demonstrate an unforeseen molecular heterogeneity among myeloid cells in naïve and glioma-bearing brains, validate selected marker proteins and show distinct spatial distribution of identified subsets in experimental gliomas. We find higher expression of MHCII encoding genes in glioma-activated male microglia, which was corroborated in bulk and scRNA-seq data from human diffuse gliomas. Our data suggest that sex-specific gene expression in glioma-activated microglia may be relevant to the incidence and outcomes of glioma patients.
Assuntos
Neoplasias Encefálicas/genética , Encéfalo/metabolismo , Glioma/genética , Glioma/metabolismo , Macrófagos/metabolismo , Análise de Sequência de RNA/métodos , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Antígeno CD11b/metabolismo , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Glioblastoma , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Masculino , Camundongos , Microglia/metabolismo , Células Mieloides/metabolismo , Fenótipo , Caracteres Sexuais , TranscriptomaRESUMO
Glioblastomas (GBM) are the common and aggressive primary brain tumors that are incurable by conventional therapies. Immunotherapy with immune checkpoint inhibitors is not effective in GBM patients due to the highly immunosuppressive tumor microenvironment (TME) restraining the infiltration and activation of cytotoxic T cells. Clinical and experimental studies showed the upregulation of expression of the arginase 1 and 2 (ARG1 and ARG2, respectively) in murine and human GBMs. The elevated arginase activity leads to the depletion of L-arginine, an amino-acid required for the proliferation of T lymphocytes and natural killer cells. Inhibition of ARG1/2 in the TME may unblock T cell proliferation and activate effective antitumor responses. To explore the antitumor potential of ARG1/2 inhibition, we analyzed bulk and single-cell RNA sequencing (scRNA-seq) data from human and murine gliomas. We found the upregulation of ARG1/2 expression in GBMs, both in tumor cells and in tumor infiltrating microglia and monocytes/macrophages. We employed selective arginase inhibitors to evaluate if ARG1/2 inhibition in vitro and in vivo exerts the antitumor effects. A novel, selective ARG1/2 inhibitor - OAT-1746 blocked microglia-dependent invasion of U87-MG and LN18 glioma cells in a Matrigel invasion assay better than reference compounds, without affecting the cell viability. OAT-1746 effectively crossed the blood brain barrier in mice and increased arginine levels in the brains of GL261 glioma bearing mice. We evaluated its antitumor efficacy against GL261 intracranial gliomas as a monotherapy and in combination with the PD-1 inhibition. The oral treatment with OAT-1746 did not affect the immune composition of TME, it induced profound transcriptomic changes in CD11b+ cells immunosorted from tumor-bearing brains as demonstrated by RNA sequencing analyses. Treatment with OAT-1746 modified the TME resulting in reduced glioma growth and increased antitumor effects of the anti-PD-1 antibody. Our findings provide the evidence that inhibition of ARG1/2 activity in tumor cells and myeloid cells in the TME unblocks antitumor responses in myeloid cells and NK cells, and improves the efficacy of the PD-1 inhibition.
RESUMO
Tumor-associated tertiary lymphoid structures (TA-TLS) are associated with enhanced patient survival and responsiveness to cancer therapies, but the mechanisms underlying their development are unknown. We show here that TA-TLS development in murine melanoma is orchestrated by cancer-associated fibroblasts (CAF) with characteristics of lymphoid tissue organizer cells that are induced by tumor necrosis factor receptor signaling. CAF organization into reticular networks is mediated by CD8 T cells, while CAF accumulation and TA-TLS expansion depend on CXCL13-mediated recruitment of B cells expressing lymphotoxin-α1ß2. Some of these elements are also overrepresented in human TA-TLS. Additionally, we demonstrate that immunotherapy induces more and larger TA-TLS that are more often organized with discrete T and B cell zones, and that TA-TLS presence, number, and size are correlated with reduced tumor size and overall response to checkpoint immunotherapy. This work provides a platform for manipulating TA-TLS development as a cancer immunotherapy strategy.
Assuntos
Fibroblastos Associados a Câncer/patologia , Neoplasias/imunologia , Neoplasias/patologia , Estruturas Linfoides Terciárias/imunologia , Animais , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Proliferação de Células , Humanos , Imunoterapia , Ativação Linfocitária/imunologia , Receptor beta de Linfotoxina/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias/terapia , Peritônio/patologia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de SinaisRESUMO
CD8+ T-cell infiltration and effector activity in tumors are correlated with better overall survival of patients, suggesting that the ability of T cells to enter and remain in contact with tumor cells supports tumor control. CD8+ T cells express the collagen-binding integrins CD49a and CD49b, but little is known about their function or how their expression is regulated in the tumor microenvironment (TME). Here, we found that tumor-infiltrating CD8+ T cells initially expressed CD49b, gained CD49a, and then lost CD49b over the course of tumor outgrowth. This differentiation sequence was driven by antigen-independent elements in the TME, although T-cell receptor (TCR) stimulation further increased CD49a expression. Expression of exhaustion markers and CD49a associated temporally but not mechanistically. Intratumoral CD49a-expressing CD8+ T cells failed to upregulate TCR-dependent Nur77 expression, whereas CD69 was constitutively expressed, consistent with both a lack of productive antigen engagement and a tissue-resident memory-like phenotype. Imaging T cells in live tumor slices revealed that CD49a increased their motility, especially of those in close proximity to tumor cells, suggesting that it may interfere with T-cell recognition of tumor cells by distracting them from productive engagement, although we were not able to augment productive engagement by short-term CD49a blockade. CD49b also promoted relocalization of T cells at a greater distance from tumor cells. Thus, our results demonstrate that expression of these integrins affects T-cell trafficking and localization in tumors via distinct mechanisms, and suggests a new way in which the TME, and likely collagen, could promote tumor-infiltrating CD8+ T-cell dysfunction.