RESUMO
We recently reported that resistance to PD-1 blockade in a refractory lung cancer-derived model involved increased collagen deposition and the collagen-binding inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). Thus, we hypothesized that LAIR1 and collagen cooperated to suppress therapeutic response. In this study, we report that LAIR1 is associated with tumor stroma and is highly expressed by intratumoral myeloid cells in both human tumors and mouse models of cancer. Stroma-associated myeloid cells exhibit a suppressive phenotype and correlate with LAIR1 expression in human cancer. NGM438, a novel humanized LAIR1 antagonist mAb, elicits myeloid inflammation and allogeneic T-cell responses by binding to LAIR1 and blocking collagen engagement. Furthermore, a mouse-reactive NGM438 surrogate antibody sensitized refractory KP mouse lung tumors to anti-PD-1 therapy and resulted in increased intratumoral CD8+ T-cell content and inflammatory gene expression. These data place LAIR1 at the intersection of stroma and suppressive myeloid cells and support the notion that blockade of the LAIR1/collagen axis can potentially address resistance to checkpoint inhibitor therapy in the clinic.
Assuntos
Antineoplásicos , Colágeno , Inibidores de Checkpoint Imunológico , Receptores Imunológicos , Animais , Feminino , Humanos , Camundongos , Linhagem Celular Tumoral , Colágeno/metabolismo , Modelos Animais de Doenças , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/metabolismo , Antineoplásicos/uso terapêuticoRESUMO
Solid tumors are dense three-dimensional (3D) multicellular structures that enable efficient receptor-ligand trans interactions via close cell-cell contact. Immunoglobulin-like transcript (ILT)2 and ILT4 are related immune-suppressive receptors that play a role in the inhibition of myeloid cells within the tumor microenvironment. The relative contribution of ILT2 and ILT4 to immune inhibition in the context of solid tumor tissue has not been fully explored. We present evidence that both ILT2 and ILT4 contribute to myeloid inhibition. We found that although ILT2 inhibits myeloid cell activation in the context of trans-engagement by MHC-I, ILT4 efficiently inhibits myeloid cells in the presence of either cis- or trans-engagement. In a 3D spheroid tumor model, dual ILT2/ILT4 blockade was required for the optimal activation of myeloid cells, including the secretion of CXCL9 and CCL5, upregulation of CD86 on dendritic cells, and downregulation of CD163 on macrophages. Humanized mouse tumor models showed increased immune activation and cytolytic T-cell activity with combined ILT2 and ILT4 blockade, including evidence of the generation of immune niches, which have been shown to correlate with clinical response to immune-checkpoint blockade. In a human tumor explant histoculture system, dual ILT2/ILT4 blockade increased CXCL9 secretion, downregulated CD163 expression, and increased the expression of M1 macrophage, IFNγ, and cytolytic T-cell gene signatures. Thus, we have revealed distinct contributions of ILT2 and ILT4 to myeloid cell biology and provide proof-of-concept data supporting the combined blockade of ILT2 and ILT4 to therapeutically induce optimal myeloid cell reprogramming in the tumor microenvironment.
Assuntos
Antígenos CD , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Glicoproteínas de Membrana , Células Mieloides , Receptores Imunológicos , Microambiente Tumoral , Receptores Imunológicos/metabolismo , Animais , Humanos , Camundongos , Microambiente Tumoral/imunologia , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Glicoproteínas de Membrana/metabolismo , Linhagem Celular Tumoral , Neoplasias/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismoRESUMO
Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3-fibronectin interaction represents a "stromal checkpoint" through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.
Assuntos
Fibronectinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Mieloides/metabolismo , Receptores Imunológicos/metabolismo , Diferenciação Celular , Linhagem Celular , HumanosRESUMO
Inhibitors of VEGF (vascular endothelial growth factor)/VEGFR2 (vascular endothelial growth factor receptor 2) are commonly used in the clinic, but their beneficial effects are only observed in a subset of patients and limited by induction of diverse relapse mechanisms. We describe the up-regulation of an adaptive immunosuppressive pathway during antiangiogenic therapy, by which PD-L1 (programmed cell death ligand 1), the ligand of the negative immune checkpoint regulator PD-1 (programmed cell death protein 1), is enhanced by interferon-γ-expressing T cells in distinct intratumoral cell types in refractory pancreatic, breast, and brain tumor mouse models. Successful treatment with a combination of anti-VEGFR2 and anti-PD-L1 antibodies induced high endothelial venules (HEVs) in PyMT (polyoma middle T oncoprotein) breast cancer and RT2-PNET (Rip1-Tag2 pancreatic neuroendocrine tumors), but not in glioblastoma (GBM). These HEVs promoted lymphocyte infiltration and activity through activation of lymphotoxin ß receptor (LTßR) signaling. Further activation of LTßR signaling in tumor vessels using an agonistic antibody enhanced HEV formation, immunity, and subsequent apoptosis and necrosis in pancreatic and mammary tumors. Finally, LTßR agonists induced HEVs in recalcitrant GBM, enhanced cytotoxic T cell (CTL) activity, and thereby sensitized tumors to antiangiogenic/anti-PD-L1 therapy. Together, our preclinical studies provide evidence that anti-PD-L1 therapy can sensitize tumors to antiangiogenic therapy and prolong its efficacy, and conversely, antiangiogenic therapy can improve anti-PD-L1 treatment specifically when it generates intratumoral HEVs that facilitate enhanced CTL infiltration, activity, and tumor cell destruction.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/terapia , Feminino , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Recidiva Local de Neoplasia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Citotóxicos/fisiologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologiaAssuntos
Inibidores da Angiogênese/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Animais , Hipóxia Celular , Humanos , Recidiva Local de Neoplasia/irrigação sanguínea , Venenos de Serpentes/administração & dosagem , Venenos de Serpentes/uso terapêuticoRESUMO
Antiangiogenic therapy is commonly used in the clinic, but its beneficial effects are short-lived, leading to tumor relapse within months. Here, we found that the efficacy of angiogenic inhibitors targeting the VEGF/VEGFR pathway was dependent on induction of the angiostatic and immune-stimulatory chemokine CXCL14 in mouse models of pancreatic neuroendocrine and mammary tumors. In response, tumors reinitiated angiogenesis and immune suppression by activating PI3K signaling in all CD11b+ cells, rendering tumors nonresponsive to VEGF/VEGFR inhibition. Adaptive resistance was also associated with an increase in Gr1+CD11b+ cells, but targeting Gr1+ cells was not sufficient to further sensitize angiogenic blockade because tumor-associated macrophages (TAMs) would compensate for the lack of such cells and vice versa, leading to an oscillating pattern of distinct immune-cell populations. However, PI3K inhibition in CD11b+ myeloid cells generated an enduring angiostatic and immune-stimulatory environment in which antiangiogenic therapy remained efficient.
Assuntos
Inibidores da Angiogênese/farmacologia , Resistencia a Medicamentos Antineoplásicos , Células Mieloides/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Animais , Células Cultivadas , Quimiocinas CXC/metabolismo , Humanos , Glândulas Mamárias Humanas/irrigação sanguínea , Camundongos , Células Mieloides/metabolismo , Neovascularização Patológica/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Angiogenesis is a hallmark of cancer because its induction is indispensable to fuel an expanding tumor. The tumor microenvironment contributes to tumor vessel growth, and distinct myeloid cells recruited by the tumor have been shown not only to support angiogenesis but also to foster an immune suppressive environment that supports tumor expansion and progression. Recent findings suggest that the intertwined regulation of angiogenesis and immune modulation can offer therapeutic opportunities for the treatment of cancer. We review the mechanisms by which distinct myeloid cell populations contribute to tumor angiogenesis, discuss current approaches in the clinic that are targeting both angiogenic and immune suppressive pathways, and highlight important areas of future research.
Assuntos
Imunidade/imunologia , Células Mieloides/imunologia , Neoplasias/irrigação sanguínea , Neoplasias/imunologia , Neovascularização Patológica/imunologia , Humanos , Neoplasias/patologia , Neoplasias/terapiaRESUMO
The prognosis for patients with pancreatic cancer is extremely poor, as evidenced by the disease's five-year survival rate of ~5%. New approaches are therefore urgently needed to improve detection, treatment, and monitoring of pancreatic cancer. MRS-detectable metabolic changes provide useful biomarkers for tumor detection and response-monitoring in other cancers. The goal of this study was to identify MRS-detectable biomarkers of pancreatic cancer that could enhance currently available imaging approaches. We used (1) H high-resolution magic angle spinning MRS to probe metabolite levels in pancreatic tissue samples from mouse models and patients. In mice, the levels of lipids dropped significantly in pancreata with lipopolysaccharide-induced inflammation, in pancreata with pre-cancerous metaplasia (4 week old p48-Cre;LSL-Kras(G12D) mice), and in pancreata with pancreatic intraepithelial neoplasia, which precedes invasive pancreatic cancer (8 week old p48-Cre LSL-Kras(G12D) mice), to 26 ± 19% (p = 0.03), 19 ± 16% (p = 0.04), and 26 ± 10% (p = 0.05) of controls, respectively. Lactate and taurine remained unchanged in inflammation and in pre-cancerous metaplasia but increased significantly in pancreatic intraepithelial neoplasia to 266 ± 61% (p = 0.0001) and 999 ± 174% (p < 0.00001) of controls, respectively. Importantly, analysis of patient biopsies was consistent with the mouse findings. Lipids dropped in pancreatitis and in invasive cancer biopsies to 29 ± 15% (p = 0.01) and 26 ± 38% (p = 0.02) of normal tissue. In addition, lactate and taurine levels remained unchanged in inflammation but rose in tumor samples to 244 ± 155% (p = 0.02) and 188 ± 67% (p = 0.02), respectively, compared with normal tissue. Based on these findings, we propose that a drop in lipid levels could serve to inform on pancreatitis and cancer-associated inflammation, whereas elevated lactate and taurine could serve to identify the presence of pancreatic intraepithelial neoplasia and invasive tumor. Our findings may help enhance current imaging methods to improve early pancreatic cancer detection and monitoring.
Assuntos
Carcinoma Ductal Pancreático/química , Lactatos/análise , Lipídeos/análise , Espectroscopia de Ressonância Magnética/métodos , Pâncreas/química , Neoplasias Pancreáticas/química , Pancreatite/metabolismo , Taurina/análise , Animais , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Diagnóstico Precoce , Genes ras , Humanos , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Pâncreas/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Pancreatite/induzido quimicamente , Pancreatite/diagnóstico , Pancreatite/patologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologiaRESUMO
Elevated levels of TGFß are a negative prognostic indicator for patients diagnosed with pancreatic cancer; as a result, the TGFß pathway is an attractive target for therapy. However, clinical application of pharmacologic inhibition of TGFß remains challenging because TGFß has tumor suppressor functions in many epithelial malignancies, including pancreatic cancer. In fact, direct neutralization of TGFß promotes tumor progression of genetic murine models of pancreatic cancer. Here, we report that neutralizing the activity of murine TGFß receptor 2 using a monoclonal antibody (2G8) has potent antimetastatic activity in orthotopic human tumor xenografts, syngeneic tumors, and a genetic model of pancreatic cancer. 2G8 reduced activated fibroblasts, collagen deposition, microvessel density, and vascular function. These stromal-specific changes resulted in tumor cell epithelial differentiation and a potent reduction in metastases. We conclude that TGFß signaling within stromal cells participates directly in tumor cell phenotype and pancreatic cancer progression. Thus, strategies that inhibit TGFß-dependent effector functions of stromal cells could be efficacious for the therapy of pancreatic tumors. Cancer Res; 74(18); 4996-5007. ©2014 AACR.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Animais , Antimetabólitos Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Metástase Neoplásica , Neoplasias Pancreáticas/metabolismo , Distribuição Aleatória , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaAssuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Glicólise , Neovascularização Patológica/metabolismo , Animais , Proliferação de Células , Endotélio Vascular/citologia , Endotélio Vascular/crescimento & desenvolvimento , Humanos , Redes e Vias Metabólicas , Neovascularização Patológica/tratamento farmacológico , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosfofrutoquinase-2/antagonistas & inibidores , Fosfofrutoquinase-2/metabolismo , Receptores Notch/metabolismo , Fatores de Transcrição/metabolismoRESUMO
There is growing evidence that antiangiogenic therapy stimulates cancer cell invasion and metastasis. However, the underlying molecular mechanisms responsible for these changes have not been fully defined. Here, we report that anti-VEGF therapy promotes local invasion and metastasis by inducing collagen signaling in cancer cells. We show that chronic VEGF inhibition in a genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDA) induces hypoxia, a less differentiated mesenchymal-like tumor cell phenotype, TGF-ß expression, and collagen deposition and signaling. In addition, we show that collagen signaling is critical for protumorigenic activity of TGF-ß in vitro. To further model the impact of collagen signaling in tumors, we evaluated PDA in mice lacking Sparc, a protein that reduces collagen binding to cell surface receptors. Importantly, we show that loss of Sparc increases collagen signaling and tumor progression. Together, these findings suggest that collagen actively promotes PDA spread and that enhanced disease progression associated with anti-VEGF therapy can arise from elevated extracellular matrix-mediated signaling.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Colágeno/fisiologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Bevacizumab , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Transdução de Sinais/fisiologia , Falha de Tratamento , Fator A de Crescimento do Endotélio Vascular/imunologiaRESUMO
Macrophages infiltrate hypoxic tumor regions, where they promote angiogenesis and immunosuppression. In this issue of Cancer Cell, Casazza and colleagues report that tumor-associated macrophage (TAM) entry into avascular tumor areas is regulated by Semaphorin 3A/Neuropilin-1 signaling; interference with this pathway entraps TAMs in oxygenated areas, preventing their tumorigenic function.
Assuntos
Macrófagos/fisiologia , Neoplasias Experimentais/irrigação sanguínea , Neovascularização Patológica/prevenção & controle , Neuropilina-1/fisiologia , Semaforina-3A/fisiologia , Transdução de Sinais/fisiologia , AnimaisRESUMO
The TGF-ß pathway is under active consideration as a cancer drug target based on its capacity to promote cancer cell invasion and to create a protumorigenic microenvironment. However, the clinical application of TGF-ß inhibitors remains uncertain as genetic studies show a tumor suppressor function of TGF-ß in pancreatic cancer and other epithelial malignancies. Here, we used genetically engineered mouse models to investigate the therapeutic impact of global TGF-ß inhibition in pancreatic cancer in relation to tumor stage, genetic profile, and concurrent chemotherapy. We found that αvß6 integrin acted as a key upstream activator of TGF-ß in evolving pancreatic cancers. In addition, TGF-ß or αvß6 blockade increased tumor cell proliferation and accelerated both early and later disease stages. These effects were dependent on the presence of Smad4, a central mediator of TGF-ß signaling. Therefore, our findings indicate that αvß6 and TGF-ß act in a common tumor suppressor pathway whose pharmacologic inactivation promotes pancreatic cancer progression.
Assuntos
Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Modelos Animais de Doenças , Progressão da Doença , Imuno-Histoquímica , CamundongosRESUMO
Pancreatic adenocarcinoma, a desmoplastic disease, is the fourth leading cause of cancer-related death in the Western world due, in large part, to locally invasive primary tumor growth and ensuing metastasis. SPARC is a matricellular protein that governs extracellular matrix (ECM) deposition and maturation during tissue remodeling, particularly, during wound healing and tumorigenesis. In the present study, we sought to determine the mechanism by which lack of host SPARC alters the tumor microenvironment and enhances invasion and metastasis of an orthotopic model of pancreatic cancer. We identified that levels of active TGFß1 were increased significantly in tumors grown in SPARC-null mice. TGFß1 contributes to many aspects of tumor development including metastasis, endothelial cell permeability, inflammation and fibrosis, all of which are altered in the absence of stromal-derived SPARC. Given these results, we performed a survival study to assess the contribution of increased TGFß1 activity to tumor progression in SPARC-null mice using losartan, an angiotensin II type 1 receptor antagonist that diminishes TGFß1 expression and activation in vivo. Tumors grown in SPARC-null mice progressed more quickly than those grown in wild-type littermates leading to a significant reduction in median survival. However, median survival of SPARC-null animals treated with losartan was extended to that of losartan-treated wild-type controls. In addition, losartan abrogated TGFß induced gene expression, reduced local invasion and metastasis, decreased vascular permeability and altered the immune profile of tumors grown in SPARC-null mice. These data support the concept that aberrant TGFß1-activation in the absence of host SPARC contributes significantly to tumor progression and suggests that SPARC, by controlling ECM deposition and maturation, can regulate TGFß availability and activation.
Assuntos
Losartan/farmacologia , Osteonectina/deficiência , Neoplasias Pancreáticas/tratamento farmacológico , Fator de Crescimento Transformador beta/antagonistas & inibidores , Bloqueadores do Receptor Tipo 1 de Angiotensina II , Animais , Progressão da Doença , Matriz Extracelular/metabolismo , Losartan/uso terapêutico , Camundongos , Camundongos Knockout , Invasividade Neoplásica , Metástase Neoplásica , Taxa de Sobrevida , Fator de Crescimento Transformador beta/metabolismo , Resultado do TratamentoRESUMO
SPARC is a matricellular protein, able to modulate cell/ECM interactions and influence cell responses to growth factors, and therefore is particularly attuned to contribute to physiological processes involving changes in ECM and cell mobilization. Indeed, the list of biological processes affected by SPARC includes wound healing, tumor progression, bone formation, fibrosis, and angiogenesis. The process of angiogenesis is complex and involves a number of cellular processes such as endothelial cell proliferation, migration, ECM degradation, and synthesis, as well as pericyte recruitment to stabilize nascent vessels. In this review, we will summarize current results that explore the function of SPARC in the regulation of angiogenic events with a particular emphasis on the modulation of growth factor activity by SPARC in the context of blood vessel formation. The primary function of SPARC in angiogenesis remains unclear, as SPARC activity in some circumstances promotes angiogenesis and in others is more consistent with an anti-angiogenic activity. Undoubtedly, the mercurial nature of SPARC belies a redundancy of functional proteins in angiogenesis as well as cell-type-specific activities that alter signal transduction events in response to unique cellular milieus. Nonetheless, the investigation of cellular mechanisms that define functional activities of SPARC continue to contribute novel and exciting paradigms to vascular biology.
Assuntos
Proteínas da Matriz Extracelular/fisiologia , Neovascularização Fisiológica/fisiologia , Osteonectina/fisiologia , Animais , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/fisiologia , Humanos , Integrina alfaV/metabolismo , Camundongos , Neovascularização Fisiológica/genética , Osteonectina/química , Osteonectina/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Pericytes migrate to nascent vessels and promote vessel stability. Recently, we reported that secreted protein acidic and rich in cysteine (SPARC)-deficient mice exhibited decreased pericyte-associated vessels in an orthotopic model of pancreatic cancer, suggesting that SPARC influences pericyte behavior. In this paper, we report that SPARC promotes pericyte migration by regulating the function of endoglin, a TGF-ß1 accessory receptor. Primary SPARC-deficient pericytes exhibited increased basal TGF-ß1 activity and decreased cell migration, an effect blocked by inhibiting TGF-ß1. Furthermore, TGF-ß-mediated inhibition of pericyte migration was dependent on endoglin and αV integrin. SPARC interacted directly with endoglin and reduced endoglin interaction with αV integrin. SPARC deficiency resulted in endoglin-mediated blockade of pericyte migration, aberrant association of endoglin in focal complexes, an increase in αV integrins present in endoglin immunoprecipitates, and enhanced αV integrin-mediated activation of TGF-ß. These results demonstrate that SPARC promotes pericyte migration by diminishing TGF-ß activity and identify a novel function for endoglin in controlling pericyte behavior.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Osteonectina/fisiologia , Pericitos/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Movimento Celular/fisiologia , Endoglina , Integrina alfaV/metabolismo , Integrina alfaV/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/análise , Camundongos , Pericitos/citologia , Pericitos/metabolismoRESUMO
Cardiac tissue from mice that do not express secreted protein acidic and rich in cysteine (SPARC) have reduced amounts of insoluble collagen content at baseline and in response to pressure overload hypertrophy compared with wild-type (WT) mice. However, the cellular mechanism by which SPARC affects myocardial collagen is not clearly defined. Although expression of SPARC by cardiac myocytes has been detected in vitro, immunohistochemistry of hearts demonstrated SPARC staining primarily associated with interstitial fibroblastic cells. Primary cardiac fibroblasts isolated from SPARC-null and WT mice were assayed for collagen I synthesis by [(3)H]proline incorporation into procollagen and by immunoblot analysis of procollagen processing. Bacterial collagenase was used to discern intracellular from extracellular forms of collagen I. Increased amounts of collagen I were found associated with SPARC-null versus WT cells, and the proportion of total collagen I detected on SPARC-null fibroblasts without propeptides [collagen-α(1)(I)] was higher than in WT cells. In addition, the amount of total collagen sensitive to collagenase digestion (extracellular) was greater in SPARC-null cells than in WT cells, indicating an increase in cell surface-associated collagen in the absence of SPARC. Furthermore, higher levels of collagen type V, a fibrillar collagen implicated in collagen fibril initiation, were found in SPARC-null fibroblasts. The absence of SPARC did not result in significant differences in proliferation or in decreased production of procollagen I by cardiac fibroblasts. We conclude that SPARC regulates collagen in the heart by modulating procollagen processing and interactions with fibroblast cell surfaces. These results are consistent with decreased levels of interstitial collagen in the hearts of SPARC-null mice being due primarily to inefficient collagen deposition into the extracellular matrix rather than to differences in collagen production.
Assuntos
Membrana Celular/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo V/metabolismo , Fibroblastos/metabolismo , Miocárdio/metabolismo , Osteonectina/metabolismo , Pró-Colágeno/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Colagenases/metabolismo , Matriz Extracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteonectina/deficiência , Osteonectina/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismoRESUMO
Diabetes is characterized by the loss, or gradual dysfunction, of insulin-producing pancreatic beta-cells. Although beta-cells can replicate in younger adults, the available diabetes therapies do not specifically target beta-cell regeneration. Novel approaches are needed to discover new therapeutics and to understand the contributions of endocrine progenitors and beta-cell regeneration during islet expansion. Here, we show that the regulators of G protein signaling Rgs16 and Rgs8 are expressed in pancreatic progenitor and endocrine cells during development, then extinguished in adults, but reactivated in models of both type 1 and type 2 diabetes. Exendin-4, a glucagon-like peptide 1 (Glp-1)/incretin mimetic that stimulates beta-cell expansion, insulin secretion and normalization of blood glucose levels in diabetics, also promoted re-expression of Rgs16::GFP within a few days in pancreatic ductal-associated cells and islet beta-cells. These findings show that Rgs16::GFP and Rgs8::GFP are novel and early reporters of G protein-coupled receptor (GPCR)-stimulated beta-cell expansion after therapeutic treatment and in diabetes models. Rgs16 and Rgs8 are likely to control aspects of islet progenitor cell activation, differentiation and beta-cell expansion in embryos and metabolically stressed adults.
Assuntos
Diabetes Mellitus Tipo 1/embriologia , Diabetes Mellitus Tipo 1/patologia , Ilhotas Pancreáticas/embriologia , Ilhotas Pancreáticas/patologia , Proteínas RGS/metabolismo , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Animais , Animais Recém-Nascidos , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/genética , Modelos Animais de Doenças , Exenatida , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Hiperglicemia/complicações , Hiperglicemia/patologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Obesos , Peptídeos/farmacologia , Gravidez , Proteínas RGS/genética , Proteínas Recombinantes de Fusão/metabolismo , Regeneração/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Peçonhas/farmacologiaRESUMO
Utilizing subcutaneous tumor models, we previously validated SPARC (secreted protein acidic and rich in cysteine) as a key component of the stromal response, where it regulated tumor size, angiogenesis and extracellular matrix deposition. In the present study, we demonstrate that pancreatic tumors grown orthotopically in Sparc-null (Sparc(-/-)) mice are more metastatic than tumors grown in wild-type (Sparc(+/+)) littermates. Tumors grown in Sparc(-/-) mice display reduced deposition of fibrillar collagens I and III, basement membrane collagen IV and the collagen-associated proteoglycan decorin. In addition, microvessel density and pericyte recruitment are reduced in tumors grown in the absence of host SPARC. However, tumors from Sparc(-/-) mice display increased permeability and perfusion, and a subsequent decrease in hypoxia. Finally, we found that tumors grown in the absence of host SPARC exhibit an increase in alternatively activated macrophages. These results suggest that increased tumor burden in the absence of host SPARC is a consequence of reduced collagen deposition, a disrupted vascular basement membrane, enhanced vascular function and an immune-tolerant, pro-metastatic microenvironment.