RESUMO
The receptor tyrosine kinase VEGFR-3 plays a crucial role in cancer-induced angiogenesis and lymphangiogenesis, promoting tumor development and metastasis. Here, we report the novel VEGFR-3 inhibitor EVT801 that presents a more selective and less toxic profile than two major inhibitors of VEGFRs (i.e., sorafenib and pazopanib). As monotherapy, EVT801 showed a potent antitumor effect in VEGFR-3-positive tumors, and in tumors with VEGFR-3-positive microenvironments. EVT801 suppressed VEGF-C-induced human endothelial cell proliferation in vitro and tumor (lymph)angiogenesis in different tumor mouse models. In addition to reduced tumor growth, EVT801 decreased tumor hypoxia, favored sustained tumor blood vessel homogenization (i.e., leaving fewer and overall larger vessels), and reduced important immunosuppressive cytokines (CCL4, CCL5) and myeloid-derived suppressor cells (MDSC) in circulation. Furthermore, in carcinoma mouse models, the combination of EVT801 with immune checkpoint therapy (ICT) yielded superior outcomes to either single treatment. Moreover, tumor growth inhibition was inversely correlated with levels of CCL4, CCL5, and MDSCs after treatment with EVT801, either alone or combined with ICT. Taken together, EVT801 represents a promising anti(lymph)angiogenic drug for improving ICT response rates in patients with VEGFR-3 positive tumors. Significance: The VEGFR-3 inhibitor EVT801 demonstrates superior selectivity and toxicity profile than other VEGFR-3 tyrosine kinase inhibitors. EVT801 showed potent antitumor effects in VEGFR-3-positive tumors, and tumors with VEGFR-3-positive microenvironments through blood vessel homogenization, and reduction of tumor hypoxia and limited immunosuppression. EVT801 increases immune checkpoint inhibitors' antitumor effects.
Assuntos
Neoplasias , Receptor 3 de Fatores de Crescimento do Endotélio Vascular , Humanos , Camundongos , Animais , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Neoplasias/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Imunoterapia , Microambiente TumoralRESUMO
Dysregulation of lipid metabolism affects the behavior of cancer cells, but how this happens is not completely understood. Neutral sphingomyelinase 2 (nSMase2), encoded by SMPD3, catalyzes the breakdown of sphingomyelin to produce the anti-oncometabolite ceramide. We found that this enzyme was often downregulated in human metastatic melanoma, likely contributing to immune escape. Overexpression of nSMase2 in mouse melanoma reduced tumor growth in syngeneic wild-type but not CD8-deficient mice. In wild-type mice, nSMase2-overexpressing tumors showed accumulation of both ceramide and CD8+ tumor-infiltrating lymphocytes, and this was associated with increased level of transcripts encoding IFNγ and CXCL9. Overexpressing the catalytically inactive nSMase2 failed to alter tumor growth, indicating that the deleterious effect nSMase2 has on melanoma growth depends on its enzymatic activity. In vitro, small extracellular vesicles from melanoma cells overexpressing wild-type nSMase2 augmented the expression of IL12, CXCL9, and CCL19 by bone marrow-derived dendritic cells, suggesting that melanoma nSMase2 triggers T helper 1 (Th1) polarization in the earliest stages of the immune response. Most importantly, overexpression of wild-type nSMase2 increased anti-PD-1 efficacy in murine models of melanoma and breast cancer, and this was associated with an enhanced Th1 response. Therefore, increasing SMPD3 expression in melanoma may serve as an original therapeutic strategy to potentiate Th1 polarization and CD8+ T-cell-dependent immune responses and overcome resistance to anti-PD-1.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma/imunologia , Melanoma/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunidade , Imunoterapia , Melanoma/tratamento farmacológico , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Esfingomielina Fosfodiesterase/genética , Células Th1/imunologiaRESUMO
Immune checkpoint inhibitors (ICIs) have dramatically modified the prognosis of several advanced cancers, however many patients still do not respond to treatment. Optimal results might be obtained by targeting cancer cell metabolism to modulate the immunosuppressive tumor microenvironment. Here, we identify sphingosine kinase-1 (SK1) as a key regulator of anti-tumor immunity. Increased expression of SK1 in tumor cells is significantly associated with shorter survival in metastatic melanoma patients treated with anti-PD-1. Targeting SK1 markedly enhances the responses to ICI in murine models of melanoma, breast and colon cancer. Mechanistically, SK1 silencing decreases the expression of various immunosuppressive factors in the tumor microenvironment to limit regulatory T cell (Treg) infiltration. Accordingly, a SK1-dependent immunosuppressive signature is also observed in human melanoma biopsies. Altogether, this study identifies SK1 as a checkpoint lipid kinase that could be targeted to enhance immunotherapy.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Neoplasias Cutâneas/tratamento farmacológico , Idoso , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Linfócitos T CD8-Positivos/patologia , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Melanoma/imunologia , Melanoma/mortalidade , Melanoma/patologia , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Nivolumabe/uso terapêutico , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Taxa de Sobrevida , Linfócitos T Reguladores/patologia , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/fisiologiaRESUMO
Antibodies against programmed cell death-1 (PD-1) have considerably changed the treatment for melanoma. However, many patients do not display therapeutic response or eventually relapse. Moreover, patients treated with anti-PD-1 develop immune-related adverse events that can be cured with anti-tumor necrosis factor α (TNF) antibodies. Whether anti-TNF antibodies affect the anti-cancer immune response remains unknown. Our recent work has highlighted that TNFR1-dependent TNF signalling impairs the accumulation of CD8+ tumor-infiltrating T lymphocytes (CD8+ TILs) in mouse melanoma. Herein, our results indicate that TNF or TNFR1 blockade synergizes with anti-PD-1 on anti-cancer immune responses towards solid cancers. Mechanistically, TNF blockade prevents anti-PD-1-induced TIL cell death as well as PD-L1 and TIM-3 expression. TNF expression positively correlates with expression of PD-L1 and TIM-3 in human melanoma specimens. This study provides a strong rationale to develop a combination therapy based on the use of anti-PD-1 and anti-TNF in cancer patients.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores Tipo I de Fatores de Necrose Tumoral/antagonistas & inibidores , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/efeitos dos fármacos , Antígeno B7-H1/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Receptor Celular 2 do Vírus da Hepatite A/efeitos dos fármacos , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Ipilimumab/uso terapêutico , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Nivolumabe , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Tumor Necrosis Factor α (TNF) is a pleiotropic cytokine exhibiting a dual activity in oncoimmunology, either acting as a cytotoxic effector produced by leukocytes or behaving as an immunosuppressive molecule. We have just discovered that TNF signaling impairs the accumulation of tumor-infiltrating CD8+ T lymphocytes in experimental melanoma.
RESUMO
TNF plays a dual, still enigmatic role in melanoma, either acting as a cytotoxic cytokine or favoring a tumorigenic inflammatory microenvironment. Herein, the tumor growth of melanoma cell lines expressing major histocompatibility complex class I molecules at high levels (MHC-I(high)) was dramatically impaired in TNF-deficient mice, and this was associated with enhanced tumor-infiltrating CD8(+) T lymphocytes. Immunodepletion of CD8 T cells fully restored melanoma growth in TNF(-/-) mice. Systemic administration of Etanercept inhibited MHC-I(high) melanoma growth in immunocompetent but not in immunodeficient (IFNγ(-/-), nude, or CD8(-/-)) mice. MHC-I(high) melanoma growth was also reduced in mice lacking TNF-R1, but not TNF-R2. TNF(-/-) and TNF-R1(-/-) mice as well as Etanercept-treated WT mice displayed enhanced intratumor content of high endothelial venules surrounded by high CD8(+) T-cell density. Adoptive transfer of activated TNF-R1-deficient or -proficient CD8(+) T cells in CD8-deficient mice bearing B16K1 tumors demonstrated that TNF-R1 deficiency facilitates the accumulation of live CD8(+) T cells into the tumors. Moreover, in vitro experiments indicated that TNF triggered activated CD8(+) T cell death in a TNF-R1-dependent manner, likely limiting the accumulation of tumor-infiltrating CD8(+) T cells in TNF/TNF-R1-proficient animals. Collectively, our observations indicate that TNF-R1-dependent TNF signaling impairs tumor-infiltrating CD8(+) T-cell accumulation and may serve as a putative target to favor CD8(+) T-cell-dependent immune response in melanoma.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Melanoma Experimental/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Linhagem Celular Tumoral , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores Tipo I de Fatores de Necrose Tumoral/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Evasão Tumoral/imunologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
It is presently assumed that lethal hit delivery by cytotoxic T lymphocytes (CTLs) is mechanistically linked to centrosome polarization toward target cells, leading to dedicated release of lytic granules within a confined secretory domain. Here we provide three lines of evidence showing that this mechanism might not apply as a general paradigm for lethal hit delivery. First, in CTLs stimulated with immobilized peptide-MHC complexes, lytic granules and microtubule organizing center localization into synaptic areas are spatio-temporally dissociated, as detected by total internal reflection fluorescence microscopy. Second, in many CTL/target cell conjugates, lytic granule secretion precedes microtubule polarization and can be detected during the first minute after cell-cell contact. Third, inhibition of microtubule organizing center and centrosome polarization impairs neither lytic granule release at the CTL synapse nor killing efficiency. Our results broaden current views of CTL biology by revealing an extremely rapid step of lytic granule secretion and by showing that microtubule organizing center polarization is dispensable for efficient lethal hit delivery.
Assuntos
Centrossomo/ultraestrutura , Sinapses Imunológicas , Microtúbulos/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/citologia , Polaridade Celular , Centrossomo/metabolismo , Grânulos Citoplasmáticos/metabolismo , Citotoxicidade Imunológica , Citometria de Fluxo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Centro Organizador dos Microtúbulos/ultraestrutura , Proteína Quinase C/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Cognate interactions between T and B lymphocytes lead to the formation of the immunological synapse (IS) where bidirectional activation signals are exchanged. Although the molecular architecture and the function of the IS have been studied extensively on the T cell side, little is known about events occurring during synapse formation in Ag-presenting B cells. We investigated the impact of BCR and TLR signaling on human B cell activation and on the T and B cell side of the IS. On the T cell side, we observed that T cells polarized toward both naive and previously activated B cells. Nevertheless, when T cells interacted with different B cells simultaneously, T cells selectively polarized their secretory machinery toward preactivated B cells. Furthermore, both naive and preactivated B cells reoriented their microtubule-organizing center toward the synaptic T cell during cognate interactions. This phenomenon was rapid and not dependent on T cell secretory activity. Interestingly, not only the microtubule-organizing center but also the Golgi apparatus and Lamp-3(+) and MHC class II(+) vesicles all repositioned beneath the IS, suggesting that the entire endocytic/exocytic B cell compartment was reoriented toward the T cell. Taken together, our results show that the B cell activation status fine-tunes T cell polarization responses and reveal the capacity of naive and activated B cells to polarize toward T cells during cognate interactions.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Comunicação Celular/imunologia , Polaridade Celular/imunologia , Sinapses Imunológicas/metabolismo , Subpopulações de Linfócitos T/imunologia , Animais , Subpopulações de Linfócitos B/citologia , Comunicação Celular/genética , Polaridade Celular/genética , Células Cultivadas , Células Clonais , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Sinapses Imunológicas/genética , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
A key feature in T lymphocyte biology is that Th cells rapidly polarize their secretory machinery toward cognate APCs. The molecular mechanisms of these dynamic Th cell responses and their impact on APC biology remain to be elucidated. In this study, we demonstrate that protein kinase Czeta (PKCzeta) is rapidly activated at the immunological synapse (IS) in human Th cells interacting with cognate dendritic cells (DCs) and that a functional PKCzeta is required for the polarization of Th cell secretory machinery toward DCs. We also show that PKCzeta-dependent Th cell polarization allows dedicated delivery of IFN-gamma and CD40L at the IS and is required for the activation of cognate DCs to IL-12 production. PKCzeta synaptic activation is a low-threshold phenomenon and, in Th cells interacting with multiple DCs, selectively occurs at the IS formed with the DCs offering the strongest stimulus leading to dedicated Th cell polarization. Our results identify the PKCzeta signaling pathway as a key component of the Th cell polarization machinery and provide a molecular basis for T cell-dedicated activation of cognate DCs.