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1.
Proc Natl Acad Sci U S A ; 118(45)2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34725151

RESUMO

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair-proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Reparo de Erro de Pareamento de DNA , Células Dendríticas , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Interferon gama/uso terapêutico , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas Experimentais/secundário , Masculino , Camundongos Endogâmicos C57BL
2.
Nat Mater ; 16(3): 379-389, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27798620

RESUMO

Expansion on stiff culture substrates activates pro-fibrotic cell programs that are retained by mechanical memory. Here, we show that priming on physiologically soft silicone substrates suppresses fibrogenesis and desensitizes mesenchymal stem cells (MSCs) against subsequent mechanical activation in vitro and in vivo, and identify the microRNA miR-21 as a long-term memory keeper of the fibrogenic program in MSCs. During stiff priming, miR-21 levels were gradually increased by continued regulation through the acutely mechanosensitive myocardin-related transcription factor-A (MRTF-A/MLK-1) and remained high over 2 weeks after removal of the mechanical stimulus. Knocking down miR-21 once by the end of the stiff-priming period was sufficient to erase the mechanical memory and sensitize MSCs to subsequent exposure to soft substrates. Soft priming and erasing mechanical memory following cell culture expansion protects MSCs from fibrogenesis in the host wound environment and increases the chances for success of MSC therapy in tissue-repair applications.


Assuntos
Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , Estresse Mecânico , Fatores de Transcrição/metabolismo , Animais , Fibrose , Células-Tronco Mesenquimais/patologia , MicroRNAs/genética , Ratos , Ratos Wistar , Fatores de Transcrição/genética
3.
Clin Cancer Res ; 29(8): 1605-1619, 2023 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-36749873

RESUMO

PURPOSE: Adding losartan (LOS) to FOLFIRINOX (FFX) chemotherapy followed by chemoradiation (CRT) resulted in 61% R0 surgical resection in our phase II trial in patients with locally advanced pancreatic cancer (LAPC). Here we identify potential mechanisms of benefit by assessing the effects of neoadjuvant LOS on the tumor microenvironment. EXPERIMENTAL DESIGN: We performed a gene expression and immunofluorescence (IF) analysis using archived surgical samples from patients treated with LOS+FFX+CRT (NCT01821729), FFX+CRT (NCT01591733), or surgery upfront, without any neoadjuvant therapy. We also conducted a longitudinal analysis of multiple biomarkers in the plasma of treated patients. RESULTS: In comparison with FFX+CRT, LOS+FFX+CRT downregulated immunosuppression and pro-invasion genes. Overall survival (OS) was associated with dendritic cell (DC) and antigen presentation genes for patients treated with FFX+CRT, and with immunosuppression and invasion genes or DC- and blood vessel-related genes for those treated with LOS+FFX+CRT. Furthermore, LOS induced specific changes in circulating levels of IL-8, sTie2, and TGF-ß. IF revealed significantly less residual disease in lesions treated with LOS+FFX+CRT. Finally, patients with a complete/near complete pathologic response in the LOS+FFX+CRT-treated group had reduced CD4+FOXP3+ regulatory T cells (Tregs), fewer immunosuppressive FOXP3+ cancer cells (C-FOXP3), and increased CD8+ T cells in pancreatic ductal adenocarcinoma lesions. CONCLUSIONS: Adding LOS to FFX+CRT reduced pro-invasion and immunosuppression-related genes, which were associated with improved OS in patients with LAPC. Lesions from responders in the LOS+FFX+CRT-treated group had reduced Tregs, decreased C-FOXP3 and increased CD8+ T cells. These findings suggest that LOS may potentiate the benefit of FFX+CRT by reducing immunosuppression.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias Pancreáticas , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Losartan/uso terapêutico , Fluoruracila , Leucovorina , Terapia Neoadjuvante/métodos , Terapia de Imunossupressão , Fatores de Transcrição Forkhead/genética , Microambiente Tumoral/genética
4.
Cancer Immunol Res ; 9(7): 765-778, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33839688

RESUMO

The mechanisms behind the antitumor effects of exercise training (ExTr) are not fully understood. Using mouse models of established breast cancer, we examined here the causal role of CD8+ T cells in the benefit acquired from ExTr in tumor control, as well as the ability of ExTr to improve immunotherapy responses. We implanted E0771, EMT6, MMTV-PyMT, and MCa-M3C breast cancer cells orthotopically in wild-type or Cxcr3-/- female mice and initiated intensity-controlled ExTr sessions when tumors reached approximately 100 mm3 We characterized the tumor microenvironment (TME) using flow cytometry, transcriptome analysis, proteome array, ELISA, and immunohistochemistry. We used antibodies against CD8+ T cells for cell depletion. Treatment with immune checkpoint blockade (ICB) consisted of anti-PD-1 alone or in combination with anti-CTLA-4. ExTr delayed tumor growth and induced vessel normalization, demonstrated by increased pericyte coverage and perfusion and by decreased hypoxia. ExTr boosted CD8+ T-cell infiltration, with enhanced effector function. CD8+ T-cell depletion prevented the antitumor effect of ExTr. The recruitment of CD8+ T cells and the antitumor effects of ExTr were abrogated in Cxcr3-/- mice, supporting the causal role of the CXCL9/CXCL11-CXCR3 pathway. ExTr also sensitized ICB-refractory breast cancers to treatment. Our results indicate that ExTr can normalize the tumor vasculature, reprogram the immune TME, and enhance the antitumor activity mediated by CD8+ T cells via CXCR3, boosting ICB responses. Our findings and mechanistic insights provide a rationale for the clinical translation of ExTr to improve immunotherapy of breast cancer.


Assuntos
Neoplasias da Mama/terapia , Linfócitos T CD8-Positivos/imunologia , Terapia por Exercício , Inibidores de Checkpoint Imunológico/farmacologia , Receptores CXCR3/metabolismo , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiocina CXCL11/metabolismo , Quimiocina CXCL9/metabolismo , Terapia Combinada/métodos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Camundongos , Camundongos Knockout , Condicionamento Físico Animal , Receptores CXCR3/genética , Transdução de Sinais/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia
5.
Sci Immunol ; 6(61)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215680

RESUMO

Immunotherapy is revolutionizing cancer treatment but is often restricted by toxicities. What distinguishes adverse events from concomitant antitumor reactions is poorly understood. Here, using anti-CD40 treatment in mice as a model of TH1-promoting immunotherapy, we showed that liver macrophages promoted local immune-related adverse events. Mechanistically, tissue-resident Kupffer cells mediated liver toxicity by sensing lymphocyte-derived IFN-γ and subsequently producing IL-12. Conversely, dendritic cells were dispensable for toxicity but drove tumor control. IL-12 and IFN-γ were not toxic themselves but prompted a neutrophil response that determined the severity of tissue damage. We observed activation of similar inflammatory pathways after anti-PD-1 and anti-CTLA-4 immunotherapies in mice and humans. These findings implicated macrophages and neutrophils as mediators and effectors of aberrant inflammation in TH1-promoting immunotherapy, suggesting distinct mechanisms of toxicity and antitumor immunity.


Assuntos
Inibidores de Checkpoint Imunológico/efeitos adversos , Imunoterapia/efeitos adversos , Células de Kupffer/efeitos dos fármacos , Fígado/efeitos dos fármacos , Neoplasias/terapia , Neutrófilos/efeitos dos fármacos , Animais , Antígenos CD40/antagonistas & inibidores , Antígenos CD40/imunologia , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Citocinas/imunologia , Humanos , Células de Kupffer/imunologia , Fígado/imunologia , Camundongos Transgênicos , Neoplasias/imunologia , Neutrófilos/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia
6.
JAMA Oncol ; 5(7): 1020-1027, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31145418

RESUMO

IMPORTANCE: Patients with locally advanced pancreatic cancer have historically poor outcomes. Evaluation of a total neoadjuvant approach is warranted. OBJECTIVE: To evaluate the margin-negative (R0) resection rate of neoadjuvant FOLFIRINOX (fluorouracil, leucovorin, oxaliplatin, and irinotecan) and losartan followed by chemoradiotherapy for locally advanced pancreatic cancer. DESIGN, SETTING, AND PARTICIPANTS: A single-arm phase 2 clinical trial was conducted at a large academic hospital from August 22, 2013, to May 22, 2018, among 49 patients with previously untreated locally advanced unresectable pancreatic cancer as determined by multidisciplinary review. Patients had Eastern Cooperative Oncology Group performance status 0 or 1 and adequate hematologic, renal, and hepatic function. Median follow-up for the analysis was 17.1 months (range, 5.0-53.7) among 27 patients still alive at study completion. INTERVENTIONS: Patients received FOLFIRINOX and losartan for 8 cycles. Patients with radiographically resectable tumor after chemotherapy received short-course chemoradiotherapy (5 GyE × 5 with protons) with capecitabine. Patients with persistent vascular involvement received long-course chemoradiotherapy (50.4 Gy with a vascular boost to 58.8 Gy) with fluorouracil or capecitabine. MAIN OUTCOMES AND MEASURES: R0 resection rate. RESULTS: Of the 49 patients (26 women and 23 men; median age 63 years [range, 42-78 years]), 39 completed 8 cycles of FOLFIRINOX and losartan; 10 patients had fewer than 8 cycles due to progression (5 patients), losartan intolerance (3 patients), and toxicity (2 patients). Seven patients (16%) had short-course chemoradiotherapy while 38 (84%) had long-course chemoradiotherapy. Forty-two (86%) patients underwent attempted surgery, with R0 resection achieved in 34 of 49 patients (69%; 95% CI, 55%-82%). Overall median progression-free survival was 17.5 months (95% CI: 13.9-22.7) and median overall survival was 31.4 months (95% CI, 18.1-38.5). Among patients who underwent resection, median progression-free survival was 21.3 months (95% CI, 16.6-28.2), and median overall survival was 33.0 months (95% CI, 31.4 to not reached). CONCLUSIONS AND RELEVANCE: Total neoadjuvant therapy with FOLFIRINOX, losartan, and chemoradiotherapy provides downstaging of locally advanced pancreatic ductal adenocarcinoma and is associated with an R0 resection rate of 61%. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01821729.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Quimiorradioterapia , Losartan/administração & dosagem , Terapia Neoadjuvante , Neoplasias Pancreáticas/terapia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Quimiorradioterapia/efeitos adversos , Feminino , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Humanos , Irinotecano/administração & dosagem , Irinotecano/efeitos adversos , Leucovorina/administração & dosagem , Leucovorina/efeitos adversos , Losartan/efeitos adversos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante/efeitos adversos , Oxaliplatina/administração & dosagem , Oxaliplatina/efeitos adversos , Neoplasias Pancreáticas/patologia , Resultado do Tratamento
7.
Mol Cancer Ther ; 17(1): 254-263, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29054988

RESUMO

Human tumor growth depends on rapidly dividing cancer cells driving population expansion. Even advanced tumors, however, contain slowly proliferating cancer cells for reasons that remain unclear. Here, we selectively disrupt the ability of rapidly proliferating cancer cells to spawn AKT1low daughter cells that are rare, slowly proliferating, tumor-initiating, and chemotherapy-resistant, using ß1-integrin activation and the AKT1-E17K-mutant oncoprotein as experimental tools in vivo Surprisingly, we find that selective depletion of AKT1low slow proliferators actually reduces the growth of a molecularly diverse panel of human cancer cell xenograft models without globally altering cell proliferation or survival in vivo Moreover, we find that unusual cancer patients with AKT1-E17K-mutant solid tumors also fail to produce AKT1low quiescent cancer cells and that this correlates with significantly prolonged survival after adjuvant treatment compared with other patients. These findings support a model whereby human solid tumor growth depends on not only rapidly proliferating cancer cells but also on the continuous production of AKT1low slow proliferators. Mol Cancer Ther; 17(1); 254-63. ©2017 AACR.


Assuntos
Neoplasias/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Transformação Celular Neoplásica , Feminino , Células HCT116 , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Neoplasias/patologia
8.
PLoS One ; 12(4): e0175300, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28448494

RESUMO

TGF-ß is a cytokine thought to function as a tumor promoter in advanced malignancies. In this setting, TGF-ß increases cancer cell proliferation, survival, and migration, and orchestrates complex, pro-tumorigenic changes in the tumor microenvironment. Here, we find that in melanoma, integrin ß1-mediated TGF-ß activation may also produce tumor suppression via an altered host response. In the A375 human melanoma cell nu/nu xenograft model, we demonstrate that cell surface integrin ß1-activation increases TGF-ß activity, resulting in stromal activation, neo-angiogenesis and, unexpectedly for this nude mouse model, increase in the number of intra-tumoral CD8+ T lymphocytes within the tumor microenvironment. This is associated with attenuation of tumor growth and long-term survival benefit. Correspondingly, in human melanomas, TGF-ß1 correlates with integrin ß1/TGF-ß1 activation and the expression of markers for vasculature and stromal activation. Surprisingly, this integrin ß1/TGF-ß1 transcriptional footprint also correlates with the expression of markers for tumor-infiltrating lymphocytes, multiple immune checkpoints and regulatory pathways, and, importantly, better long-term survival of patients. These correlations are unique to melanoma, in that we do not observe similar associations between ß1 integrin/TGF-ß1 activation and better long-term survival in other human tumor types. These results suggest that activation of TGF-ß1 in melanoma may be associated with the generation of an anti-tumor host response that warrants further study.


Assuntos
Anticorpos/imunologia , Integrina beta1/imunologia , Integrina beta1/metabolismo , Melanoma/imunologia , Melanoma/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Espaço Extracelular/metabolismo , Feminino , Melanoma/irrigação sanguínea , Melanoma/patologia , Camundongos , Camundongos Nus , Neovascularização Patológica/imunologia , Transdução de Sinais/imunologia , Análise de Sobrevida , Linfócitos T/citologia , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral/imunologia
9.
PLoS One ; 12(10): e0187348, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29088264

RESUMO

MSCs are widely applied to regenerate heart tissue in myocardial diseases but when grown in standard two-dimensional (2D) cultures exhibit limited potential for cardiac repair and develop fibrogenic features with increasing culture time. MSCs can undergo partial cardiomyogenic differentiation, which improves their cardiac repair capacity. When applied to collagen patches they may improve cardiac tissue regeneration but the mechanisms remain elusive. Here, we investigated the regenerative properties of MSCs grown in a collagen scaffold as a three-dimensional (3D) culture system, and performed functional analysis using an engineered heart tissue (EHT) model. We showed that the expression of cardiomyocyte-specific proteins by MSCs co-cultured with rat neonatal cardiomyocytes was increased in collagen patches versus conventional cultures. MSCs in 3D collagen patches were less fibrogenic, secreted more cardiotrophic factors, retained anti-apoptotic and immunomodulatory function, and responded less to TLR4 ligand lipopolysaccharide (LPS) stimulation. EHT analysis showed no effects by MSCs on cardiomyocyte function, whereas control dermal fibroblasts abrogated the beating of cardiac tissue constructs. We conclude that 3D collagen scaffold improves the cardioprotective effects of MSCs by enhancing the production of trophic factors and modifying their immune modulatory and fibrogenic phenotype. The improvement in myocardial function by MSCs after acquisition of a partial cardiac cell-like phenotype is not due to enhanced MSC contractility. A better understanding of the mechanisms of MSC-mediated tissue repair will help to further enhance the therapeutic potency of MSCs.


Assuntos
Colágeno/metabolismo , Células-Tronco Mesenquimais/citologia , Animais , Morte Celular , Diferenciação Celular , Proliferação de Células , Técnicas de Cocultura , Citometria de Fluxo , Humanos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley
10.
Stem Cell Reports ; 4(6): 1016-30, 2015 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-26028530

RESUMO

Pro-fibrotic microenvironments of scars and tumors characterized by increased stiffness stimulate mesenchymal stromal cells (MSCs) to express α-smooth muscle actin (α-SMA). We investigated whether incorporation of α-SMA into contractile stress fibers regulates human MSC fate. Sorted α-SMA-positive MSCs exhibited high contractile activity, low clonogenicity, and differentiation potential limited to osteogenesis. Knockdown of α-SMA was sufficient to restore clonogenicity and adipogenesis in MSCs. Conversely, α-SMA overexpression induced YAP translocation to the nucleus and reduced the high clonogenicity and adipogenic potential of α-SMA-negative MSCs. Inhibition of YAP rescued the decreased adipogenic differentiation potential induced by α-SMA, establishing a mechanistic link between matrix stiffness, α-SMA, YAP, and MSC differentiation. Consistent with in vitro findings, nuclear localization of YAP was positively correlated in α-SMA expressing stromal cells of adiposarcoma and osteosarcoma. We propose that α-SMA mediated contraction plays a critical role in mechanically regulating MSC fate by controlling YAP/TAZ activation.


Assuntos
Actinas/metabolismo , Células-Tronco Mesenquimais/citologia , Actinas/antagonistas & inibidores , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adipogenia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Porfirinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Sarcoma/metabolismo , Sarcoma/patologia , Neoplasias de Tecidos Moles/metabolismo , Neoplasias de Tecidos Moles/patologia , Fatores de Transcrição , Verteporfina , Proteínas de Sinalização YAP
11.
PLoS One ; 8(5): e64560, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23691248

RESUMO

Myofibroblast contraction is fundamental in the excessive tissue remodeling that is characteristic of fibrotic tissue contractures. Tissue remodeling during development of fibrosis leads to gradually increasing stiffness of the extracellular matrix. We propose that this increased stiffness positively feeds back on the contractile activities of myofibroblasts. We have previously shown that cycles of contraction directly correlate with periodic intracellular calcium oscillations in cultured myofibroblasts. We analyze cytosolic calcium dynamics using fluorescent calcium indicators to evaluate the possible impact of mechanical stress on myofibroblast contractile activity. To modulate extracellular mechanics, we seeded primary rat subcutaneous myofibroblasts on silicone substrates and into collagen gels of different elastic modulus. We modulated cell stress by cell growth on differently adhesive culture substrates, by restricting cell spreading area on micro-printed adhesive islands, and depolymerizing actin with Cytochalasin D. In general, calcium oscillation frequencies in myofibroblasts increased with increasing mechanical challenge. These results provide new insight on how changing mechanical conditions for myofibroblasts are encoded in calcium oscillations and possibly explain how reparative cells adapt their contractile behavior to the stresses occurring in normal and pathological tissue repair.


Assuntos
Sinalização do Cálcio , Espaço Intracelular/metabolismo , Fenômenos Mecânicos , Miofibroblastos/citologia , Actinas/química , Animais , Fenômenos Biomecânicos , Adesão Celular , Matriz Extracelular/metabolismo , Miofibroblastos/metabolismo , Ratos , Estresse Mecânico
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