RESUMO
Macrophages, which are abundant in the tumour microenvironment, enhance malignancy. At metastatic sites, a distinct population of metastasis-associated macrophages promotes the extravasation, seeding and persistent growth of tumour cells. Here we define the origin of these macrophages by showing that Gr1-positive inflammatory monocytes are preferentially recruited to pulmonary metastases but not to primary mammary tumours in mice. This process also occurs for human inflammatory monocytes in pulmonary metastases of human breast cancer cells. The recruitment of these inflammatory monocytes, which express CCR2 (the receptor for chemokine CCL2), as well as the subsequent recruitment of metastasis-associated macrophages and their interaction with metastasizing tumour cells, is dependent on CCL2 synthesized by both the tumour and the stroma. Inhibition of CCL2-CCR2 signalling blocks the recruitment of inflammatory monocytes, inhibits metastasis in vivo and prolongs the survival of tumour-bearing mice. Depletion of tumour-cell-derived CCL2 also inhibits metastatic seeding. Inflammatory monocytes promote the extravasation of tumour cells in a process that requires monocyte-derived vascular endothelial growth factor. CCL2 expression and macrophage infiltration are correlated with poor prognosis and metastatic disease in human breast cancer. Our data provide the mechanistic link between these two clinical associations and indicate new therapeutic targets for treating metastatic breast cancer.
Assuntos
Neoplasias da Mama/patologia , Quimiocina CCL2/metabolismo , Inflamação/patologia , Monócitos/patologia , Metástase Neoplásica , Animais , Antígeno CD11b/metabolismo , Quimiocina CCL2/antagonistas & inibidores , Feminino , Proteínas Ligadas por GPI/metabolismo , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Neoplasias Pulmonares/secundário , Macrófagos/patologia , Camundongos , Monócitos/metabolismo , Metástase Neoplásica/tratamento farmacológico , Transplante de Neoplasias , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Receptores CCR2/antagonistas & inibidores , Receptores CCR2/metabolismo , Receptores de IgG/metabolismo , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: The TMPRSS2-ERG gene fusion is detected in approximately half of primary prostate cancers (PCa) yet the prognostic significance remains unclear. We hypothesized that ERG promotes the expression of common genes in primary PCa and metastatic castration-resistant PCa (CRPC), with the objective of identifying ERG-associated pathways, which may promote the transition from primary PCa to CRPC. METHODS: We constructed tissue microarrays (TMA) from 127 radical prostatectomy specimens, 20 LuCaP patient-derived xenografts (PDX), and 152 CRPC metastases obtained immediately at time of death. Nuclear ERG was assessed by immunohistochemistry (IHC). To characterize the molecular features of ERG-expressing PCa, a subset of IHC confirmed ERG+ or ERG- specimens including 11 radical prostatectomies, 20 LuCaP PDXs, and 45 CRPC metastases underwent gene expression analysis. Genes were ranked based on expression in primary PCa and CRPC. Common genes of interest were targeted for IHC analysis and expression compared with biochemical recurrence (BCR) status. RESULTS: IHC revealed that 43% of primary PCa, 35% of the LuCaP PDXs, and 18% of the CRPC metastases were ERG+ (12 of 48 patients [25%] had at least one ERG+ metastasis). Based on gene expression data and previous literature, two proteins involved in calcium signaling (NCALD, CACNA1D), a protein involved in inflammation (HLA-DMB), CD3 positive immune cells, and a novel ERG-associated protein, DCLK1 were evaluated in primary PCa and CRPC metastases. In ERG+ primary PCa, a weak association was seen with NCALD and CACNA1D protein expression. HLA-DMB association with ERG was decreased and CD3 cell number association with ERG was changed from positive to negative in CRPC metastases compared to primary PCa. DCLK1 was upregulated at the protein level in unpaired ERG+ primary PCa and CRPC metastases (P = 0.0013 and P < 0.0001, respectively). In primary PCa, ERG status or expression of targeted proteins was not associated with BCR-free survival. However, for primary PCa, ERG+DCLK1+ patients exhibited shorter time to BCR (P = 0.06) compared with ERG+DCLK1- patients. CONCLUSIONS: This study examined ERG expression in primary PCa and CRPC. We have identified altered levels of inflammatory mediators associated with ERG expression. We determined expression of DCLK1 correlates with ERG expression and may play a role in primary PCa progression to metastatic CPRC. Prostate 76:810-822, 2016. © 2016 Wiley Periodicals, Inc.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/metabolismo , Próstata/metabolismo , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias da Próstata/metabolismo , Humanos , Masculino , Prognóstico , Próstata/patologia , Próstata/cirurgia , Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/cirurgia , Regulador Transcricional ERG/metabolismoRESUMO
The tumor microenvironment can promote tumor growth and reduce treatment efficacy. Tumors can occur in many sites in the body, but how surrounding normal tissues at different anatomical sites affect tumor microenvironments and their subsequent response to therapy is not known.We demonstrated that tumors from renal, colon, or prostate cell lines in orthotopic locations responded to immunotherapy consisting of three agonist antibodies, termed Tri-mAb, to a much lesser extent than the same tumor type located subcutaneously. A tissue-specific response to Tri-mAb was confirmed by ex vivo separation of subcutaneous (SC) or orthotopic tumor cells from stromal cells, followed by reinjection of tumor cells into the opposite site. Compared with SC tumors, orthotopic tumors had a microenvironment associated with a type 2 immune response, related to immunosuppression, and an involvement of alternatively activated macrophages in the kidney model. Orthotopic kidney tumors were more highly vascularized than SC tumors. Neutralizing the macrophage- and Th2-associated molecules chemokine (C-C motif) ligand 2 or interleukin-13 led to a significantly improved therapeutic effect. This study highlights the importance of the tissue of implantation in sculpting the tumor microenvironment. These are important fundamental issues in tumor biology and crucial factors to consider in the design of experimental models and treatment strategies.
Assuntos
Imunoterapia , Neoplasias/imunologia , Neoplasias/patologia , Microambiente Tumoral/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/imunologia , Linhagem Celular Tumoral , Quimiocina CCL2/imunologia , Neoplasias do Colo/imunologia , Modelos Animais de Doenças , Expressão Gênica , Interleucina-13/imunologia , Neoplasias Renais/imunologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Neoplasias/mortalidade , Neoplasias/terapia , Neovascularização Patológica/imunologia , Especificidade de Órgãos/imunologia , Próstata/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/antagonistas & inibidores , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Resultado do Tratamento , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/antagonistas & inibidores , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
The C-C chemokine ligand 2 (CCL2) stimulates migration, proliferation, and invasion of prostate cancer (PCa) cells, and its signaling also plays a role in the activation of osteoclasts. Therefore targeting CCL2 signaling in regulation of tumor progression in bone metastases is an area of intense research. The objective of our study was to investigate the efficacy of CCL2 blockade by neutralizing antibodies to inhibit the growth of PCa in bone. We used a preclinical model of cancer growth in the bone in which PCa C4-2B cells were injected directly into murine tibiae. Animals were treated for ten weeks with neutralizing anti-CCL2 antibodies, docetaxel, or a combination of both, and then followed an additional nine weeks. CCL2 blockade inhibited the growth of PCa in bone, with even more pronounced inhibition in combination with docetaxel. CCL2 blockade also resulted in increases in bone mineral density. Furthermore, our results showed that the tumor inhibition lasted even after discontinuation of the treatment. Our data provide compelling evidence that CCL2 blockade slows PCa growth in bone, both alone and in combination with docetaxel. These results support the continued investigations of CCL2 blockade as a treatment for advanced metastatic PCa.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Ósseas/prevenção & controle , Quimiocina CCL2/antagonistas & inibidores , Neoplasias da Próstata/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/imunologia , Peso Corporal/efeitos dos fármacos , Densidade Óssea/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Docetaxel , Humanos , Imuno-Histoquímica , Masculino , Camundongos SCID , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Análise de Sobrevida , Taxoides/administração & dosagem , Fatores de Tempo , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patients with prostate cancer whose tumors bear deleterious mutations in DNA-repair pathways often respond to PARP inhibitors. Studies were conducted to compare the activity of several PARP inhibitors in vitro and their tissue exposure and in vivo efficacy in mice bearing PC-3M-luc-C6 prostate tumors grown subcutaneously or in bone. Niraparib, olaparib, rucaparib, and talazoparib were compared in proliferation assays, using several prostate tumor cell lines and in a cell-free PARP-trapping assay. PC-3M-luc-C6 cells were approximately 12- to 20-fold more sensitive to PARP inhibition than other prostate tumor lines, suggesting that these cells bear a DNA damage repair defect. The tissue exposure and efficacy of these PARP inhibitors were evaluated in vivo in PC-3M-luc-C6 subcutaneous and bone metastasis tumor models. A steady-state pharmacokinetic study in PC-3M-luc-C6 tumor-bearing mice showed that all of the PARP inhibitors had favorable subcutaneous tumor exposure, but niraparib was differentiated by superior bone marrow exposure compared with the other drugs. In a PC-3M-luc-C6 subcutaneous tumor efficacy study, niraparib, olaparib, and talazoparib inhibited tumor growth and increased survival to a similar degree. In contrast, in the PC-3M-luc-C6 bone metastasis model, niraparib showed the most potent inhibition of bone tumor growth compared with the other therapies (67% vs. 40%-45% on day 17), and the best survival improvement over vehicle control [hazard ratio (HR), 0.28 vs. HR, 0.46-0.59] and over other therapies (HR, 1.68-2.16). These results show that niraparib has superior bone marrow exposure and greater inhibition of tumor growth in bone, compared with olaparib, rucaparib, and talazoparib.
Assuntos
Neoplasias Ósseas , Neoplasias da Próstata , Animais , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Humanos , Indazóis , Masculino , Camundongos , Piperidinas , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poli(ADP-Ribose) Polimerases/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Distribuição TecidualRESUMO
The role of chemokines in the pathogenesis of lung cancer has been increasingly appreciated. Monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) is secreted from tumor cells and associated tumor stromal cells. The blockade of CCL2, as mediated by neutralizing antibodies, was shown to reduce tumorigenesis in several solid tumors, but the role of CCL2 in lung cancer remains controversial, with evidence of both protumorigenic and antitumorigenic effects. We evaluated the effects and mechanisms of CCL2 blockade in several animal models of non-small-cell lung cancer (NSCLC). Anti-murine-CCL2 monoclonal antibodies were administered in syngeneic flank and orthotopic models of NSCLC. CCL2 blockade significantly slowed the growth of primary tumors in all models studied, and inhibited lung metastases in a model of spontaneous lung metastases of NSCLC. In contrast to expectations, no significant effect of treatment was evident in the number of tumor-associated macrophages recruited into the tumor after CCL2 blockade. However, a change occurred in the polarization of tumor-associated macrophages to a more antitumor phenotype after CCL2 blockade. This was associated with the activation of cytotoxic CD8(+) T lymphocytes (CTLs). The antitumor effects of CCL2 blockade were completely lost in CB-17 severe combined immunodeficient mice or after CD8 T-cell depletion. Our data from NSCLC models show that CCL2 blockade can inhibit the tumor growth of primary and metastatic disease. The mechanisms of CCL2 blockade include an alteration of the tumor macrophage phenotype and the activation of CTLs. Our work supports further evaluation of CCL2 blockade in thoracic malignancies.
Assuntos
Quimiocina CCL2/antagonistas & inibidores , Neoplasias Pulmonares/terapia , Macrófagos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/secundário , Carcinoma Pulmonar de Células não Pequenas/terapia , Linhagem Celular Tumoral , Quimiocina CCL2/imunologia , Feminino , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Ativação Linfocitária , Depleção Linfocítica , Mesotelioma/imunologia , Mesotelioma/patologia , Mesotelioma/terapia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , FenótipoRESUMO
Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) inhibit anti-tumor immune responses and facilitate tumor growth. Precursors for these immune cell populations migrate to the tumor site in response to tumor secretion of chemokines, such as monocyte chemoattractant protein-1 (MCP-1/CCL2), which was originally purified and identified from human gliomas. In syngeneic mouse GL261 glioma and human U87 glioma xenograft models, we evaluated the efficacy of systemic CCL2 blockade by monoclonal antibodies (mAb) targeting mouse and/or human CCL2. Intraperitoneal (i.p.) administration of anti-mouse CCL2 mAb as monotherapy (2 mg/kg/dose, twice a week) significantly, albeit modestly, prolonged the survival of C57BL/6 mice bearing intracranial GL261 glioma (P = 0.0033), which was concomitant with a decrease in TAMs and MDSCs in the tumor microenvironment. Similarly, survival was modestly prolonged in severe combined immunodeficiency mice bearing intracranial human U87 glioma xenografts treated with both anti-human CCL2 mAb and anti-mouse CCL2 antibodies (2 mg/kg/dose for each, twice a week) compared to mice treated with control IgG (P = 0.0159). Furthermore, i.p. administration of anti-mouse CCL2 antibody in combination with temozolomide (TMZ) significantly prolonged the survival of C57BL/6 mice bearing GL261 glioma with 8 of 10 treated mice surviving longer than 70 days, while only 3 of 10 mice treated with TMZ and isotype IgG survived longer than 70 days (P = 0.0359). These observations provide support for development of mAb-based CCL2 blockade strategies in combination with the current standard TMZ-based chemotherapy for treatment of malignant gliomas.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Quimiocina CCL2/imunologia , Glioma/tratamento farmacológico , Animais , Antineoplásicos Alquilantes/uso terapêutico , Linhagem Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Modelos Animais de Doenças , Vias de Administração de Medicamentos , Esquema de Medicação , Sistemas de Liberação de Medicamentos , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo/métodos , Glioma/patologia , Humanos , Imunoterapia Ativa , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Células Mieloides/efeitos dos fármacos , Transplante de Neoplasias , Poli I-C/uso terapêutico , Estatísticas não Paramétricas , TemozolomidaRESUMO
Despite advancements in targeting the immune checkpoints program cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) for cancer immunotherapy, a large number of patients and cancer types remain unresponsive. Current immunotherapies focus on modulating an antitumor immune response by directly or indirectly expanding antitumor CD8 T cells. A complementary strategy might involve inhibition of Tregs that otherwise suppress antitumor immune responses. Here, we sought to identify functional immune molecules preferentially expressed on tumor-infiltrating Tregs. Using genome-wide RNA-Seq analysis of purified Tregs sorted from multiple human cancer types, we identified a conserved Treg immune checkpoint signature. Using immunocompetent murine tumor models, we found that antibody-mediated depletion of 4-1BB-expressing cells (4-1BB is also known as TNFRSF9 or CD137) decreased tumor growth without negatively affecting CD8 T cell function. Furthermore, we found that the immune checkpoint 4-1BB had a high selectivity for human tumor Tregs and was associated with worse survival outcomes in patients with multiple tumor types. Thus, antibody-mediated depletion of 4-1BB-expressing Tregs represents a strategy with potential activity across cancer types.
Assuntos
Ligante 4-1BB/imunologia , Linfócitos do Interstício Tumoral/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Linfócitos T Reguladores/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Estudo de Associação Genômica Ampla , Humanos , Depleção Linfocítica , Linfócitos do Interstício Tumoral/patologia , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , RNA-Seq , Linfócitos T Reguladores/patologiaRESUMO
Metastatic prostate cancer continues to be the second leading cause of cancer death in American men with an estimated 28,660 deaths in 2008. Recently, monocyte chemoattractant protein-1 (MCP-1, CCL2) has been identified as an important factor in the regulation of prostate metastasis. CCL2, shown to attract macrophages to the tumor site, has a direct promotional effect on tumor cell proliferation, migration, and survival. Previous studies have shown that anti-CCL2 antibodies given in combination with docetaxel were able to induce tumor regression in a pre-clinical prostate cancer model. A limitation for evaluating new treatments for metastatic prostate cancer to bone is the inability of imaging to objectively assess response to treatment. Diffusion-weighted MRI (DW-MRI) assesses response to anticancer therapies by quantifying the random (i.e., Brownian) motion of water molecules within the tumor mass, thus identifying cells undergoing apoptosis. We sought to measure the treatment response of prostate cancer in an osseous site to docetaxel, an anti-CCL2 agent, and combination treatments using DW-MRI. Measurements of tumor apparent diffusion coefficient (ADC) values were accomplished over time during a 14-day treatment period and compared to response as measured by bioluminescence imaging and survival studies. The diffusion data provided early predictive evidence of the most effective therapy, with survival data results correlating with the DW-MRI findings. DW-MRI is under active investigation in the pre-clinical and clinical settings to provide a sensitive and quantifiable means for early assessment of cancer treatment outcome.
Assuntos
Anticorpos/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Quimiocina CCL2/antagonistas & inibidores , Taxoides/uso terapêutico , Animais , Neoplasias Ósseas/secundário , Quimiocina CCL2/imunologia , Imagem de Difusão por Ressonância Magnética , Docetaxel , Sinergismo Farmacológico , Humanos , Masculino , Camundongos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The identification of novel tumor-interactive chemokines and the associated insights into the molecular and cellular basis of tumor-microenvironment interactions have continued to stimulate the development of targeted cancer therapeutics. Recently, we have identified monocyte chemoattractant protein 1 (MCP-1; CCL2) as a prominent regulator of prostate cancer growth and metastasis. Using neutralizing antibodies to human CCL2 (CNTO888) and the mouse homologue CCL2/JE (C1142), we show that treatment with anti-CCL2/JE antibody (2 mg/kg, twice weekly i.p.) attenuated PC-3Luc-mediated overall tumor burden in our in vivo model of prostate cancer metastasis by 96% at 5 weeks postintracardiac injection. Anti-CCL2 inhibition was not as effective as docetaxel (40 mg/kg, every week for 3 weeks) as a single agent, but inhibition of CCL2 in combination with docetaxel significantly reduced overall tumor burden compared with docetaxel alone, and induced tumor regression relative to initial tumor burden. These data suggest an interaction between tumor-derived chemokines and host-derived chemokines acting in cooperation to promote tumor cell survival, proliferation, and metastasis.
Assuntos
Anticorpos/farmacologia , Quimiocina CCL2/antagonistas & inibidores , Imunização Passiva/métodos , Imunoglobulina G/farmacologia , Neoplasias da Próstata/terapia , Animais , Anticorpos/imunologia , Processos de Crescimento Celular , Linhagem Celular Tumoral , Movimento Celular , Quimiocina CCL2/imunologia , Docetaxel , Humanos , Imunoglobulina G/imunologia , Masculino , Camundongos , Camundongos SCID , Metástase Neoplásica , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Receptores CCR2 , Receptores de Quimiocinas/biossíntese , Taxoides/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN) is a cell surface glycoprotein overexpressed in many solid tumors. In addition to its ability to stimulate stromal MMP expression, tumor-associated EMMPRIN also induces vascular endothelial growth factor (VEGF) expression. To explore the underlying signaling pathways used by EMMPRIN, we studied the involvement of phosphoinositide 3-kinase (PI3K)-Akt, mitogen-activated protein kinase (MAPK), JUN, and p38 kinases in EMMPRIN-mediated VEGF regulation. Overexpression of EMMPRIN in MDA-MB-231 breast cancer cells stimulated the phosphorylation of only Akt and MAPKs but not that of JUN and p38 kinases. Conversely, inhibition of EMMPRIN expression resulted in suppressed Akt and MAPK phosphorylation. Furthermore, the PI3K-specific inhibitor LY294002 inhibited VEGF production by EMMPRIN-overexpressing cells in a dose- and time-dependent manner. On the other hand, the MAPK inhibitor U0126 did not affect VEGF production. In vivo, EMMPRIN-overexpressing tumors with elevated VEGF expression had a high level of phosphorylation of Akt and MAPK. Finally, when fibroblast cells were treated with recombinant EMMPRIN, Akt kinase but not MAPK was phosphorylated concomitant with an increase in VEGF production. Both the activation of Akt kinase and the induction of VEGF were specifically inhibited with a neutralizing antibody to EMMPRIN. Our results show that in both tumor and fibroblast cells EMMPRIN regulates VEGF production via the PI3K-Akt pathway but not via the MAPK, JUN, or p38 kinase pathways.
Assuntos
Basigina/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Basigina/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Humanos , Metaloproteinases da Matriz , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transplante de Neoplasias , Neoplasias/metabolismo , Neovascularização Patológica , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Recombinantes/farmacologia , Transplante Heterólogo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The human CCL2 chemokine is implicated in many chronic inflammatory conditions. In the mouse, there are two CCL2 homologues, CCL2 (MCP-1/JE) and CCL12 (MCP-5). Both are potent monocyte chemoattractants and bind to and activate the same receptor, CCR2. The overlapping activities of these chemokines complicate the design of mouse model studies that are intended to mimic human disease. To study the roles of CCL2 and CCL12, we generated neutralizing antibodies specific to each chemokine. Consistent with binding and affinity analyses, the antibodies specifically inhibited CCL2- or CCL12- mediated Ca(2+) mobilization in THP-1 cells. When tested in nude mice bearing human PANC-1 pancreatic tumor cells in Matrigel plugs, CCL2 and CCL12 antibodies potently inhibited tumor angiogenesis, indicating that both CCL2 and CCL12 may contribute to tumor angiogenesis.
Assuntos
Anticorpos/imunologia , Quimiocina CCL2/imunologia , Proteínas Quimioatraentes de Monócitos/imunologia , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Neovascularização Patológica/prevenção & controle , Testes de NeutralizaçãoRESUMO
Tumor progression is facilitated immunologically by mechanisms that include low antigen expression, an absence of coimmunostimulatory signals, and the presence of regulatory T cells (Tregs), all of which act to suppress and restrict effector T cells in the tumor. It may be possible to overcome these conditions by a combination of modulatory immunotherapy agents and tumor-antigen targeting to activate and drive effective antitumor T cell responses. Here, we demonstrated that co-administration of aGITR and aPD-1 monoclonal antibodies (mAb) in combination with a peptide vaccine (Vax) in mice bearing established tumors significantly delayed tumor growth and induced complete regression in 50% of the mice. This response was associated with increased expansion and functionality of potent Ag-specific polyfunctional CD8+ T cells, reduced Tregs, and the generation of memory T cells. Tumor regression correlated with the expansion of tumor-infiltrating antigen-specific CD8+ effector memory T cells, as depletion of this cell population significantly reduced the effectiveness of the triple combination Vax/aGITR/aPD-1 therapy. These findings support the concept that dual aGITR/aPD-1 combination with cancer vaccines may be a novel strategy against poorly immunogenic tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Proteína Relacionada a TNFR Induzida por Glucocorticoide/antagonistas & inibidores , Melanoma Experimental/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Linfócitos T Reguladores/imunologia , Animais , Apoptose , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Feminino , Imunoterapia , Melanoma Experimental/metabolismo , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Células Tumorais Cultivadas , VacinaçãoRESUMO
Mounting evidence demonstrates that CD8+CD122+ T cells have suppressive properties with the capacity to inhibit T cell responses. Therefore, these cells are rational targets for cancer immunotherapy. Here, we demonstrate that CD122 monoclonal antibody (mAb; aCD122) therapy significantly suppressed tumor growth and improved long-term survival in tumor-bearing mice. This therapeutic effect correlated with enhanced polyfunctional, cytolytic intratumoral CD8+ T cells and a decrease in granulocytic myeloid-derived suppressor cells (G-MDSCs). In addition, aCD122 treatment synergized with a vaccine to augment vaccine-induced antigen (Ag)-specific CD8+ T cell responses, reject established tumors and generate memory T cells. Furthermore, aCD122 mAb synergized with an anti-GITR (aGITR) mAb to confer significant control of tumor growth. These results suggest CD122 might be a promising target for cancer immunotherapy, either as a single agent or in combination with other forms of immunotherapy.
RESUMO
Hepatocellular carcinoma, a deadly disease, commonly arises in the setting of chronic inflammation. C-C motif chemokine ligand 2 (CCL2/MCP1), a chemokine that recruits CCR2-positive immune cells to promote inflammation, is highly upregulated in hepatocellular carcinoma patients. Here, we examined the therapeutic efficacy of CCL2-CCR2 axis inhibitors against hepatitis and hepatocellular carcinoma in the miR-122 knockout (a.k.a. KO) mouse model. This mouse model displays upregulation of hepatic CCL2 expression, which correlates with hepatitis that progress to hepatocellular carcinoma with age. Therapeutic potential of CCL2-CCR2 axis blockade was determined by treating KO mice with a CCL2-neutralizing antibody (nAb). This immunotherapy suppressed chronic liver inflammation in these mice by reducing the population of CD11highGr1+ inflammatory myeloid cells and inhibiting expression of IL6 and TNFα in KO livers. Furthermore, treatment of tumor-bearing KO mice with CCL2 nAb for 8 weeks significantly reduced liver damage, hepatocellular carcinoma incidence, and tumor burden. Phospho-STAT3 (Y705) and c-MYC, the downstream targets of IL6, as well as NF-κB, the downstream target of TNFα, were downregulated upon CCL2 inhibition, which correlated with suppression of tumor growth. In addition, CCL2 nAb enhanced hepatic NK-cell cytotoxicity and IFNγ production, which is likely to contribute to the inhibition of tumorigenesis. Collectively, these results demonstrate that CCL2 immunotherapy could be an effective therapeutic approach against inflammatory liver disease and hepatocellular carcinoma. Mol Cancer Ther; 16(2); 312-22. ©2016 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/metabolismo , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores CCR2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Neutralizantes/farmacologia , Biomarcadores , Biópsia , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/tratamento farmacológico , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Quimiocina CCL2/genética , Modelos Animais de Doenças , Citometria de Fluxo , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , Camundongos Knockout , MicroRNAs/genética , Receptores CCR2/genética , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Malignant pleural effusion (MPE) is the lethal consequence of various human cancers metastatic to the pleural cavity. However, the mechanisms responsible for the development of MPE are still obscure. Here we show that mutant KRAS is important for MPE induction in mice. Pleural disseminated, mutant KRAS bearing tumour cells upregulate and systemically release chemokine ligand 2 (CCL2) into the bloodstream to mobilize myeloid cells from the host bone marrow to the pleural space via the spleen. These cells promote MPE formation, as indicated by splenectomy and splenocyte restoration experiments. In addition, KRAS mutations are frequently detected in human MPE and cell lines isolated thereof, but are often lost during automated analyses, as indicated by manual versus automated examination of Sanger sequencing traces. Finally, the novel KRAS inhibitor deltarasin and a monoclonal antibody directed against CCL2 are equally effective against an experimental mouse model of MPE, a result that holds promise for future efficient therapies against the human condition.
Assuntos
Adenocarcinoma/genética , Antineoplásicos/farmacologia , Neoplasias Pulmonares/genética , Células Mieloides/patologia , Derrame Pleural Maligno/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Animais , Antineoplásicos/uso terapêutico , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/metabolismo , Galinhas , Membrana Corioalantoide , Feminino , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Cavidade Pleural/citologia , Cavidade Pleural/patologia , Derrame Pleural Maligno/tratamento farmacológico , Derrame Pleural Maligno/patologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Interferente Pequeno/metabolismo , Baço/citologia , Baço/patologia , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor-associated macrophages (TAM) contribute to all aspects of tumor progression. Use of CSF1R inhibitors to target TAM is therapeutically appealing, but has had very limited anti-tumor effects. Here, we have identified the mechanism that limited the effect of CSF1R targeted therapy. We demonstrated that carcinoma-associated fibroblasts (CAF) are major sources of chemokines that recruit granulocytes to tumors. CSF1 produced by tumor cells caused HDAC2-mediated downregulation of granulocyte-specific chemokine expression in CAF, which limited migration of these cells to tumors. Treatment with CSF1R inhibitors disrupted this crosstalk and triggered a profound increase in granulocyte recruitment to tumors. Combining CSF1R inhibitor with a CXCR2 antagonist blocked granulocyte infiltration of tumors and showed strong anti-tumor effects.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Monócitos/metabolismo , Células Supressoras Mieloides/metabolismo , Neoplasias Experimentais/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Animais , Fibroblastos Associados a Câncer/efeitos dos fármacos , Linhagem Celular Tumoral , Granulócitos/metabolismo , Histona Desacetilase 2 , Humanos , Imidazóis/farmacologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Compostos de Fenilureia/farmacologia , Piridinas/farmacologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo , Carga Tumoral/efeitos dos fármacosRESUMO
Dissemination of prostate cancer (PCa) cells to the bone marrow is an early event in the disease process. In some patients, disseminated tumor cells (DTC) proliferate to form active metastases after a prolonged period of undetectable disease known as tumor dormancy. Identifying mechanisms of PCa dormancy and reactivation remain a challenge partly due to the lack of in vitro models. Here, we characterized in vitro PCa dormancy-reactivation by inducing cells from three patient-derived xenograft (PDX) lines to proliferate through tumor cell contact with each other and with bone marrow stroma. Proliferating PCa cells demonstrated tumor cell-cell contact and integrin clustering by immunofluorescence. Global gene expression analyses on proliferating cells cultured on bone marrow stroma revealed a downregulation of TGFB2 in all of the three proliferating PCa PDX lines when compared to their non-proliferating counterparts. Furthermore, constitutive activation of myosin light chain kinase (MLCK), a downstream effector of integrin-beta1 and TGF-beta2, in non-proliferating cells promoted cell proliferation. This cell proliferation was associated with an upregulation of CDK6 and a downregulation of E2F4. Taken together, our data provide the first clinically relevant in vitro model to support cellular adhesion and downregulation of TGFB2 as a potential mechanism by which PCa cells may escape from dormancy. Targeting the TGF-beta2-associated mechanism could provide novel opportunities to prevent lethal PCa metastasis.
Assuntos
Adesão Celular , Comunicação Celular , Pontos de Checagem do Ciclo Celular , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Análise por Conglomerados , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Masculino , Camundongos , Neoplasias da Próstata/genética , Células Estromais/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismoRESUMO
Mast cells (MCs) have been identified in various tumors; however, the role of these cells in tumorigenesis remains controversial. Here, we quantified MCs in human and murine malignant pleural effusions (MPEs) and evaluated the fate and function of these cells in MPE development. Evaluation of murine MPE-competent lung and colon adenocarcinomas revealed that these tumors actively attract and subsequently degranulate MCs in the pleural space by elaborating CCL2 and osteopontin. MCs were required for effusion development, as MPEs did not form in mice lacking MCs, and pleural infusion of MCs with MPE-incompetent cells promoted MPE formation. Once homed to the pleural space, MCs released tryptase AB1 and IL-1ß, which in turn induced pleural vasculature leakiness and triggered NF-κB activation in pleural tumor cells, thereby fostering pleural fluid accumulation and tumor growth. Evaluation of human effusions revealed that MCs are elevated in MPEs compared with benign effusions. Moreover, MC abundance correlated with MPE formation in a human cancer cell-induced effusion model. Treatment of mice with the c-KIT inhibitor imatinib mesylate limited effusion precipitation by mouse and human adenocarcinoma cells. Together, the results of this study indicate that MCs are required for MPE formation and suggest that MC-dependent effusion formation is therapeutically addressable.
Assuntos
Mastócitos/metabolismo , Derrame Pleural Maligno/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Benzamidas/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Mesilato de Imatinib , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Neoplasias Pulmonares/dietoterapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Piperazinas/farmacologia , Cavidade Pleural/metabolismo , Cavidade Pleural/patologia , Derrame Pleural Maligno/tratamento farmacológico , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/farmacologia , Triptases/genética , Triptases/metabolismoRESUMO
Ovarian cancer is associated with a leukocyte infiltrate and high levels of chemokines such as CCL2. We tested the hypothesis that CCL2 inhibition can enhance chemotherapy with carboplatin and paclitaxel. Elevated CCL2 expression was found in three non-MDR paclitaxel resistant ovarian cancer lines ES-2/TP, MES-OV/TP and OVCAR-3/TP, compared to parental cells. Mice xenografted with these cells were treated with the anti-human CCL2 antibody CNTO 888 and the anti-mouse MCP-1 antibody C1142, with and without paclitaxel or carboplatin. Our results show an additive effect of CCL2 blockade on the efficacy of paclitaxel and carboplatin. This therapeutic effect was largely due to inhibition of mouse stromal CCL2. We show that inhibition of CCL2 can enhance paclitaxel and carboplatin therapy of ovarian cancer.