RESUMO
BACKGROUND: The tumor microenvironment plays a major tumor-supportive role in glioma. In particular, tumor-associated macrophages (TAMs), which can make up to one-third of the tumor mass, actively support tumor growth, invasion, and angiogenesis. Predominantly alternatively activated (M2-polarized) TAMs are found in late-stage glioma in both human and mouse tumors, as well as in relapse samples from patients. However, whether tumor-educated M2 TAMs can actively contribute to the emergence and growth of relapse is currently debated. METHODS: To investigate whether tumor-educated stromal cells remaining in the brain after surgical removal of the primary tumor can be long-lived and retain their tumor-supporting function, we developed a transplantation mouse model and performed lineage-tracing. RESULTS: We discovered that macrophages can survive transplantation and stay present in the tumor much longer than previously suggested, while sustaining an M2-polarized protumorigenic phenotype. Transplanted tumors showed a more aggressive growth and faster polarization of the TAMs toward an M2 phenotype compared with primary tumors, a process dependent on the presence of few cotransplanted macrophages. CONCLUSIONS: Overall, we propose a new way for tumor-educated TAMs to contribute to glioma aggressiveness by long survival and stable protumorigenic features. These properties could have a relapse-supporting effect.
RESUMO
Glioma growth and progression are characterized by abundant development of blood vessels that are highly aberrant and poorly functional, with detrimental consequences for drug delivery efficacy. The mechanisms driving this vessel dysmorphia during tumor progression are poorly understood. Using longitudinal intravital imaging in a mouse glioma model, we identify that dynamic sprouting and functional morphogenesis of a highly branched vessel network characterize the initial tumor growth, dramatically changing to vessel expansion, leakage, and loss of branching complexity in the later stages. This vascular phenotype transition was accompanied by recruitment of predominantly pro-inflammatory M1-like macrophages in the early stages, followed by in situ repolarization to M2-like macrophages, which produced VEGF-A and relocate to perivascular areas. A similar enrichment and perivascular accumulation of M2 versus M1 macrophages correlated with vessel dilation and malignancy in human glioma samples of different WHO malignancy grade. Targeting macrophages using anti-CSF1 treatment restored normal blood vessel patterning and function. Combination treatment with chemotherapy showed survival benefit, suggesting that targeting macrophages as the key driver of blood vessel dysmorphia in glioma progression presents opportunities to improve efficacy of chemotherapeutic agents. We propose that vessel dysfunction is not simply a general feature of tumor vessel formation, but rather an emergent property resulting from a dynamic and functional reorganization of the tumor stroma and its angiogenic influences.
Assuntos
Vasos Sanguíneos/patologia , Neoplasias Encefálicas/patologia , Glioma/patologia , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Alquilantes/uso terapêutico , Vasos Sanguíneos/anormalidades , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Modelos Animais de Doenças , Feminino , Glioma/irrigação sanguínea , Glioma/tratamento farmacológico , Glioma/mortalidade , Humanos , Fator Estimulador de Colônias de Macrófagos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neovascularização Patológica/patologia , Fenótipo , Proteínas Proto-Oncogênicas c-sis/genética , Temozolomida , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Both brain-resident microglia and peripheral macrophages/monocytes infiltrate into glioma and promote glioma growth. In the present study we analyzed coupling and membrane currents in glioma-associated microglia and macrophages/monocytes and compared this to control and stab wound-associated microglia. Using the Cx3cr1(GFP/wt)Ccr2(RFP/wt) knock-in mouse line, we distinguished membrane currents of glioma-associated microglia and macrophages/monocytes in acute brain slices prepared 14-16 days after inoculation of GL261 glioma cells. The current profile of microglia showed inward rectifying currents reminiscent of an intermediate activation state when compared to other disease models or cell culture. Macrophages/monocytes showed a higher specific outward conductance and a significantly lower capacitance indicative of a smaller membrane area than microglia. As controls, we also recorded currents from control microglia and stab wound-associated microglia. Since there are reports of microglial coupling in vitro, we injected biocytin into these cells and analyzed for cell coupling after fixing the slices and processed for biocytin labeling with Cy3-conjugated-Streptavidin. Neither control microglia nor glioma-associated microglia and macrophages/monocytes nor stab wound-associated microglia showed any sign of coupling. Moreover, performing qRT-PCR revealed that no connexin43 was detectable on isolated and sorted glioma-associated microglia and macrophages/monocytes, indicating that these cells are not part of a coupled network.
Assuntos
Junções Comunicantes/fisiologia , Glioma/patologia , Macrófagos/fisiologia , Microglia/fisiologia , Monócitos/fisiologia , Animais , Linhagem Celular Tumoral , Conexina 43/metabolismo , Potenciais da Membrana , Camundongos , Transplante de Neoplasias , Técnicas de Patch-ClampRESUMO
BACKGROUND: Glioblastomas are the most aggressive primary brain tumors in humans. Microglia/brain macrophage accumulation in and around the tumor correlates with malignancy and poor clinical prognosis of these tumors. We have previously shown that microglia promote glioma expansion through upregulation of membrane type 1 matrix metalloprotease (MT1-MMP). This upregulation depends on signaling via the Toll-like receptor (TLR) adaptor molecule myeloid differentiation primary response gene 88 (MyD88). METHODS: Using in vitro, ex vivo, and in vivo techniques, we identified TLR2 as the main TLR controlling microglial MT1-MMP expression and promoting microglia-assisted glioma expansion. RESULTS: The implantation of mouse GL261 glioma cells into TLR2 knockout mice resulted in significantly smaller tumors, reduced MT1-MMP expression, and enhanced survival rates compared with wild-type control mice. Tumor expansion studied in organotypic brain slices depended on both parenchymal TLR2 expression and the presence of microglia. Glioma-derived soluble factors and synthetic TLR2 specific ligands induced MT1-MMP expression in microglia from wild-type mice, but no such change in MT1-MMP gene expression was observed in microglia from TLR2 knockout mice. We also found evidence that TLR1 and TLR6 cofunction with TLR2 as heterodimers in regulating MT1-MMP expression in vitro. CONCLUSIONS: Our results thus show that activation of TLR2 along with TLRs 1 and/or 6 converts microglia into a glioma supportive phenotype.