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1.
Muscle Nerve ; 67(3): 244-251, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36533970

RESUMO

INTRODUCTION/AIMS: Although therapeutic electrical stimulation (TES) of injured peripheral nerve promotes axon regeneration and functional recovery, clinical applications of this therapy are limited to the intraoperative timeframe. Implantable, thin-film wireless nerve stimulators offer a potential solution to this problem by enabling delivery of electrical stimuli to an injured nerve over a period of several days post-surgery. The aim of this study was to determine the optimal time course of stimulation for maximizing functional recovery in a rat sciatic nerve isograft repair model. METHODS: Adult male Lewis rats underwent thin-film wireless nerve stimulator implantation following sciatic nerve transection and 40 mm nerve isograft repair. Immediately after surgery, animals began a daily regimen of TES for up to 12 consecutive days. Functional recovery was assessed by compound muscle action potential (CMAP), evoked muscle force, wet muscle mass, and axon counting. RESULTS: Serial CMAP measurements increased in amplitude over the course of the study, yet no significant difference between cohorts for serial or terminal CMAPs was observed. Axon counts and wet muscle mass measurements were greatest in the 6-day stimulation group, which correlated with a significant increase in evoked muscle force for the 6-day stimulation group at the terminal time point. DISCUSSION: Six daily sessions of TES were found to be most effective for augmenting functional recovery compared to other time courses of stimulation. Future studies should incorporate additional subjects and track axonal sprouting or measure neurotrophin levels during the therapeutic window to further elucidate the mechanisms behind, and ideal amount of, TES.


Assuntos
Terapia por Estimulação Elétrica , Músculo Esquelético , Ratos , Masculino , Animais , Músculo Esquelético/fisiologia , Axônios , Isoenxertos , Regeneração Nervosa/fisiologia , Ratos Endogâmicos Lew , Nervo Isquiático/cirurgia , Recuperação de Função Fisiológica/fisiologia , Estimulação Elétrica
2.
Front Immunol ; 13: 853349, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35757709

RESUMO

Islet transplantation to treat the late stage of type 1 diabetic patient (T1DM) has recently made inspiring success in clinical trials. However, most patients experience a decline in islet graft function in one to three years due to immune rejection. Although the mechanisms of immune cells, including macrophages, dendritic cells (DCs), neutrophils, natural killer cells (NKs), B cells, and T cells, that mediate immune rejection have been investigated, the overall characteristics of immune infiltrates in islet allografts and syngeneic grafts remain unclear. Single-cell RNA sequencing (scRNA-seq) has provided us with new opportunities to study the complexity of the immune microenvironment in islet transplants. In the present study, we used scRNA-seq to comprehensively analyze the immune heterogeneity in the mouse model of islet transplantation. Our data revealed T lymphocytes and myeloid cells as the main immune components of grafts 7 days post-islet transplantation, especially in allografts. Moreover, our results indicated that allogeneic islet cells were transformed into antigen-presenting cell-like cells with highly expressed MHC class I molecules and genes involved in MHC class I-mediated antigen presentation. This transformation may dramatically facilitate the interaction with cytotoxic CD8+ T cells and promote the destruction of islet allografts. Our study provides insight into the transcriptomics and diverse microenvironment of islet grafts and their impacts on immune rejection.


Assuntos
Linfócitos T CD8-Positivos , Transplante das Ilhotas Pancreáticas , Aloenxertos , Animais , Antígenos de Histocompatibilidade Classe I , Humanos , Isoenxertos , Camundongos , Transplante Homólogo
3.
Transpl Int ; 35: 10057, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35497886

RESUMO

Objectives: Cold ischemia and subsequent reperfusion injury are non-immunologic cornerstones in the development of graft injury after heart transplantation. The nitric oxide donor S-nitroso-human-serum-albumin (S-NO-HSA) is known to attenuate myocardial ischemia-reperfusion (I/R)-injury. We assessed whether donor preservation with S-NO-HSA affects isograft injury and myocardial expression of GATA2 as well as miR-126-3p, which are considered protective against vascular and endothelial injury. Methods: Donor C57BL/6 mice received intravenous (0.1 µmol/kg/h) S-NO-HSA (n = 12), or 0.9% saline (control, n = 11) for 20 min. Donor hearts were stored in cold histidine-tryptophan-α-ketoglutarate-N solution for 12 h and underwent heterotopic, isogenic transplantation, except 5 hearts of each group, which were analysed immediately after preservation. Fibrosis was quantified and expression of GATA2 and miR-126-3p assessed by RT-qPCR after 60 days or immediately after preservation. Results: Fibrosis was significantly reduced in the S-NO-HSA group (6.47% ± 1.76 vs. 11.52% ± 2.16; p = 0.0023; 12 h-S-NO-HSA-hHTX vs. 12 h-control-hHTX). Expression of miR-126-3p was downregulated in all hearts after ischemia compared to native myocardium, but the effect was significantly attenuated when donors received S-NO-HSA (1 ± 0.27 vs. 0.33 ± 0.31; p = 0.0187; 12 h-S-NO-HSA-hHTX vs. 12 h-control-hHTX; normalized expression to U6 snRNA). Conclusion: Donor pre-treatment with S-NO-HSA lead to reduced fibrosis and preservation of myocardial miR-126-3p and GATA2 levels in murine cardiac isografts 60 days after transplantation.


Assuntos
Transplante de Coração , MicroRNAs , Animais , Fibrose , Humanos , Isoenxertos , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio , Albumina Sérica Humana , Doadores de Tecidos
4.
Hand (N Y) ; 17(3): 540-548, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-32666827

RESUMO

Background: Therapeutic electrical stimulation (ES) applied to repaired nerve is a promising treatment option to improve regeneration. However, few studies address the impact of ES following nerve graft reconstruction. The purpose of this study was to determine if ES applied to a nerve repair using nerve isograft in a rodent model could improve nerve regeneration and functional recovery. Methods: Adult rats were randomized to 2 groups: "ES" and "Control." Rats received a tibial nerve transection that was repaired using a tibial nerve isograft (1.0 cm length), where ES was applied immediately after repair in the applicable group. Nerve was harvested 2 weeks postrepair for immunohistochemical analysis of axon growth and macrophage accumulation. Independently, rats were assessed using walking track and grid-walk analysis for up to 21 weeks. Results: At 2 weeks, more robust axon regeneration and greater macrophage accumulation was observed within the isografts for the ES compared to Control groups. Both walking track and grid-walk analysis revealed that return of functional recovery was accelerated by ES. The ES group demonstrated improved functional recovery over time, as well as improved recovery compared to the Control group at 21 weeks. Conclusions: ES improved early axon regeneration into a nerve isograft and was associated with increased macrophage and beneficial M2 macrophage accumulation within the isograft. ES ultimately improved functional recovery compared to isograft repair alone. This study supports the clinical potential of ES to improve the management of nerve injuries requiring a nerve graft repair.


Assuntos
Axônios , Regeneração Nervosa , Animais , Axônios/fisiologia , Estimulação Elétrica , Humanos , Isoenxertos , Regeneração Nervosa/fisiologia , Ratos , Recuperação de Função Fisiológica/fisiologia
5.
Front Immunol ; 12: 617316, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33737925

RESUMO

Background: Adenosine receptor type 2 (A2AR) inhibitor, AZD4635, has been shown to reduce immunosuppressive adenosine effects within the tumor microenvironment (TME) and to enhance the efficacy of checkpoint inhibitors across various syngeneic models. This study aims at investigating anti-tumor activity of AZD4635 alone and in combination with an anti-PD-L1-specific antibody (anti-PD-L1 mAb) across various TME conditions and at identifying, via mathematical quantitative modeling, a therapeutic combination strategy to further improve treatment efficacy. Methods: The model is represented by a set of ordinary differential equations capturing: 1) antigen-dependent T cell migration into the tumor, with subsequent proliferation and differentiation into effector T cells (Teff), leading to tumor cell lysis; 2) downregulation of processes mediated by A2AR or PD-L1, as well as other immunosuppressive mechanisms; 3) A2AR and PD-L1 inhibition by, respectively, AZD4635 and anti-PD-L1 mAb. Tumor size dynamics data from CT26, MC38, and MCA205 syngeneic mice treated with vehicle, anti-PD-L1 mAb, AZD4635, or their combination were used to inform model parameters. Between-animal and between-study variabilities (BAV, BSV) in treatment efficacy were quantified using a non-linear mixed-effects methodology. Results: The model reproduced individual and cohort trends in tumor size dynamics for all considered treatment regimens and experiments. BSV and BAV were explained by variability in T cell-to-immunosuppressive cell (ISC) ratio; BSV was additionally driven by differences in intratumoral adenosine content across the syngeneic models. Model sensitivity analysis and model-based preclinical study simulations revealed therapeutic options enabling a potential increase in AZD4635-driven efficacy; e.g., adoptive cell transfer or treatments affecting adenosine-independent immunosuppressive pathways. Conclusions: The proposed integrative modeling framework quantitatively characterized the mechanistic activity of AZD4635 and its potential added efficacy in therapy combinations, across various immune conditions prevailing in the TME. Such a model may enable further investigations, via simulations, of mechanisms of tumor resistance to treatment and of AZD4635 combination optimization strategies.


Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Antineoplásicos/farmacologia , Modelos Biológicos , Receptor A2A de Adenosina/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Algoritmos , Animais , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Linhagem Celular Tumoral , Suscetibilidade a Doenças , Resistencia a Medicamentos Antineoplásicos , Quimioterapia Combinada , Isoenxertos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Microsurgery ; 41(2): 157-164, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32949430

RESUMO

INTRODUCTION: In the last years, limb salvage has become the gold standard treatment over amputation. Today, 90% of extremity osteogenic sarcomas can be treated with limb salvage surgery. However, these reconstructions are not exempt from complications. Massive allografts have been associated to high risk of nonunion (12-57%), fracture (7-30%) and infection (5-21%). Association of vascularized periosteum flap to a massive bone allograft (MBA) has shown to halve the average time of allograft union in clinical series, even compared to vascularized fibular flap. Creeping substitution process has been reported in massive allograft when periosteum flap was associated. However, we have little data about whether it results into allograft revitalization. We hypothesize that the association of a periosteum flap to a bone isograft promotes isograft revitalization, defined as the colonization of the devitalized bone by new-form vessels and viable osteocytes, turning it vital. MATERIALS AND METHODS: Forty-four New Zealand white male rabbits underwent a 10 mm segmental radial bone defect. In 24 rabbits the bone excision included the periosteum (controls); in 20 rabbits (periosteum group) bone excision was performed carefully detaching periosteum in order to preserve it. Cryopreserved bone isograft from another rabbit was trimmed and placed to the defect gap and was fixed with a retrograde intramedullar 0.6 mm Kirschner wire. Rabbits were randomized and distributed in 3 subgroups depending on the follow-up (control group: 5 rabbits in 5-week follow up group, 8 rabbits in 10-week follow-up group, 7 rabbits in 20-week follow-up group; periosteum group: 5 rabbits in 5-week follow up group, 7 rabbits in 10-week follow-up group, 7 rabbits in 20-week follow-up group). Fluoroscopic images of rabbit forelimb were taken after sacrifice to address union. Each specimen was blindly evaluated in optical microscope (magnification, ×4) after hematoxylin and eosin staining to qualitative record: presence of new vessels and osteocytes in bone graft lacunae (yes/no) to address revitalization, presence of callus (yes/no) and woven bone and cartilage tissue area (mm2 ) to address remodeling (osteoclast resorption of old bone and substitution by osteoblastic new bone formation). RESULTS: No isograft revitalization occurred in any group, but it was observed bone graft resorption and substitution by new-formed bone in periosteum group. This phenomenon was accelerated in 5-week periosteum group (control group: 49.5 ± 9.6 mm2 vs. periosteum group: 34.9 ± 10.4 mm2 ; p = .07). Remodeled lamellar bone was observed in both 20-week groups (control group: 6.1 ± 6.3 mm2 vs. periosteum group: 5.8 ± 3.0 mm2 , p = .67). Periosteum group showed complete integration and graft substitution, whereas devitalized osteons were still observed in 20-week controls. All periosteum group samples showed radiographic union through a bone callus, whereas controls showed nonunion in eight specimens (Union rate: control group 60% vs. periosteum group 100%, p = .003). CONCLUSIONS: Association of vascularized periosteum to a massive bone isograft has shown to accelerate bone graft substitution into a newly formed bone, thus, no bone graft revitalization occurs.


Assuntos
Isoenxertos , Periósteo , Animais , Masculino , Coelhos , Transplante Ósseo , Osteogênese , Retalhos Cirúrgicos
7.
STAR Protoc ; 1(2): 100047, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-33111093

RESUMO

This protocol is a procedure for generating orthotopic isografts using mouse pancreatic cancer organoids. These isografts can be used to track the evolution of pancreatic ductal adenocarcinoma (PDA) from a preinvasive lesion to a metastatic disease and therefore represent a suitable model for identification of determinants of PDA progression. For complete details on the use and execution of this protocol, please refer to Boj et al. (2015) and Filippini et al. (2019).


Assuntos
Técnicas de Cultura de Células/métodos , Isoenxertos/citologia , Organoides/citologia , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas/citologia , Animais , Progressão da Doença , Camundongos
8.
Sci Rep ; 10(1): 17464, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060772

RESUMO

In this study we undertook a novel combination therapy using rAd-p53 in situ gene therapy and immunotherapy with immune checkpoint inhibitor (ICI) anti-PD-1 antibody for urogenital cancers. Three mouse syngeneic tumor cell lines, TRAMP-C2 (prostate cancer derived from C57BL/6 mice), MBT-2 (bladder cancer derived from C3H mice) and Renca (kidney cancer derived from BALB/c mice) were used in this study. The highest coxsackie and adenovirus receptor (CAR) mRNA expression was observed in TRAMP-C2 cells, followed by Renca and then MBT-2 cells. Consistent with the CAR expressions, rAd-p53 at 160 multiplicity of infection (MOI) significantly inhibited the cell proliferation of TRAMP-C2 and Renca cells, but not MBT-2 cells. In in vivo experiments, the combination of intratumoral injections of rAd-p53 (1 × 109 plaque-forming units) every other day and intraperitoneal injections of anti-mouse PD-1 antibody (200 µg) twice a week suppressed tumor growth and prolonged survival compared to rAd-p53 or anti-PD-1 antibody monotherapy in both the TRAMP-C2 and Renca models. Our results encourage the clinical development of combination therapy comprised of in situ gene therapy with rAd-p53 and immunotherapy with an ICI anti-PD-1 antibody for urogenital cancers.


Assuntos
Antineoplásicos/uso terapêutico , Genes p53 , Terapia Genética/métodos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Neoplasias Urogenitais/genética , Neoplasias Urogenitais/terapia , Adenoviridae , Animais , Linhagem Celular Tumoral , Isoenxertos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/imunologia , RNA Mensageiro/metabolismo
10.
Int J Mol Sci ; 21(18)2020 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-32899865

RESUMO

The immune checkpoint blockade represents a revolution in cancer therapy, with the potential to increase survival for many patients for whom current treatments are not effective. However, response rates to current immune checkpoint inhibitors vary widely between patients and different types of cancer, and the mechanisms underlying these varied responses are poorly understood. Insights into the antitumor activities of checkpoint inhibitors are often obtained using syngeneic mouse models, which provide an in vivo preclinical basis for predicting efficacy in human clinical trials. Efforts to establish in vitro syngeneic mouse equivalents, which could increase throughput and permit real-time evaluation of lymphocyte infiltration and tumor killing, have been hampered by difficulties in recapitulating the tumor microenvironment in laboratory systems. Here, we describe a multiplex in vitro system that overcomes many of the deficiencies seen in current static histocultures, which we applied to the evaluation of checkpoint blockade in tumors derived from syngeneic mouse models. Our system enables both precision-controlled perfusion across biopsied tumor fragments and the introduction of checkpoint-inhibited tumor-infiltrating lymphocytes in a single experiment. Through real-time high-resolution confocal imaging and analytics, we demonstrated excellent correlations between in vivo syngeneic mouse and in vitro tumor biopsy responses to checkpoint inhibitors, suggesting the use of this platform for higher throughput evaluation of checkpoint efficacy as a tool for drug development.


Assuntos
Inibidores de Checkpoint Imunológico/metabolismo , Inibidores de Checkpoint Imunológico/farmacologia , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Isoenxertos/imunologia , Isoenxertos/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Modelos Biológicos , Receptor de Morte Celular Programada 1/imunologia , Microambiente Tumoral/imunologia
11.
Nat Commun ; 11(1): 3912, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764562

RESUMO

Immunotherapy has emerged as a promising approach to treat cancer, however, its efficacy in highly malignant brain-tumors, glioblastomas (GBM), is limited. Here, we generate distinct imageable syngeneic mouse GBM-tumor models and utilize RNA-sequencing, CyTOF and correlative immunohistochemistry to assess immune-profiles in these models. We identify immunologically-inert and -active syngeneic-tumor types and show that inert tumors have an immune-suppressive phenotype with numerous exhausted CD8 T cells and resident macrophages; fewer eosinophils and SiglecF+ macrophages. To mimic the clinical-settings of first line of GBM-treatment, we show that tumor-resection invigorates an anti-tumor response via increasing T cells, activated microglia and SiglecF+ macrophages and decreasing resident macrophages. A comparative CyTOF analysis of resected-tumor samples from GBM-patients and mouse GBM-tumors show stark similarities in one of the mouse GBM-tumors tested. These findings guide informed choices for use of GBM models for immunotherapeutic interventions and offer a potential to facilitate immune-therapies in GBM patients.


Assuntos
Neoplasias Encefálicas/imunologia , Glioblastoma/imunologia , Animais , Encéfalo/imunologia , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Linhagem Celular Tumoral , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Tolerância Imunológica , Imunofenotipagem , Imunoterapia , Isoenxertos , Linfócitos do Interstício Tumoral/classificação , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Microambiente Tumoral/imunologia
12.
Int J Mol Sci ; 21(11)2020 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-32526987

RESUMO

Recent advances in the development of new methods of cancer immunotherapy require the production of complex cancer animal models that reliably reflect the complexity of the tumor and its microenvironment. Mice are good animals to create tumor models because they are low cost, have a short reproductive cycle, exhibit high tumor growth rates, and can be easily genetically modified. However, the obvious problem of these models is the high failure rate observed in human clinical trials after promising results obtained in mouse models. In order to increase the reliability of the results obtained in mice, the tumor model should reflect the heterogeneity of the tumor, contain components of the tumor microenvironment, in particular immune cells, to which the action of immunotherapeutic drugs are directed. This review discusses the current immunocompetent and immunocompromised mouse models of human tumors that are used to evaluate the effectiveness of immunotherapeutic agents, in particular chimeric antigen receptor (CAR) T-cells and immune checkpoint inhibitors.


Assuntos
Imunoterapia/métodos , Neoplasias Experimentais/etiologia , Neoplasias Experimentais/terapia , Animais , Carcinógenos/toxicidade , Humanos , Hospedeiro Imunocomprometido , Isoenxertos , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Exp Neurol ; 331: 113328, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32333909

RESUMO

In order to repair chronic nerve injuries (injuries repaired after a long delay), the damaged nerve segments are resected and stumps are bridged by grafts. Autografts remain the gold-standard, but outcomes are typically poor, even after long periods of recovery. In a recent study, we described the use of a nerve lengthening device to gradually elongate the proximal stump of a transected nerve towards the distal stump, enabling a tension-free end-to-end repair. This approach showed significantly improved outcomes in comparison to autografts in repairing acutely injured nerves. In this study, we compared the use of nerve lengthening/end-to-end repair (LETER) to isograft repair of chronically transected nerves in a rat model. Structural and functional regenerative outcomes following LETER were comparable to isograft-based repair, with no significant differences found in outcomes involving functional recovery or axon growth. These data demonstrate the feasibility of nerve lengthening as a viable graft-free strategy for repairing chronically injured nerves. Not unexpectedly, outcomes for chronic nerve injuries were less favorable in both groups compared to repair of acutely injured nerves. Nonetheless, the findings provide insight into barriers to restoring function after chronic nerve injury through novel comprehensive characterization of a diverse set of neuromuscular outcomes. This analysis revealed key parameters predicting functional recovery.


Assuntos
Expansão do Nervo/métodos , Traumatismos dos Nervos Periféricos/cirurgia , Recuperação de Função Fisiológica , Nervo Isquiático/transplante , Anastomose Cirúrgica , Animais , Axotomia , Doença Crônica , Isoenxertos , Ratos , Ratos Endogâmicos Lew , Nervo Isquiático/lesões
14.
Sci Rep ; 10(1): 4681, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170125

RESUMO

Triple-negative breast cancer (TNBC) has a more aggressive phenotype and higher metastasis and recurrence rates than other breast cancer subtypes. TNBC currently lacks a transplantation model that is suitable for clinical simulations of the tumor microenvironment. Intraductal injection of tumor cells into the mammary duct could mimic the occurrence and development of breast cancer. Herein, we injected 4T1 cells into the mammary ducts of BALB/C mice to build a preclinical model of TNBC and optimized the related construction method to observe the occurrence and spontaneous metastasis of tumors. We compared the effects of different cell numbers on tumorigenesis rates, times to tumorigenesis, and metastases to determine the optimal number of cells for modelling. We demonstrated that 4T1-MIND model mice injected with 20,000 cells revealed a suitable tumor formation rate and time, thus indicating a potential treatment time window after distant metastasis. We also injected 20,000 cells directly into the breast fat pad or breast duct for parallel comparison. The results still showed that the 4T1-MIND model provides sufficient treatment time for lung metastases in mice and that it is a more reliable model for early tumor development. The 4T1-MIND model requires continuous improvement and optimization. A suitable and optimized model for translational research and studies on the microenvironment in TNBC should be developed.


Assuntos
Modelos Animais de Doenças , Neoplasias Mamárias Experimentais/patologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Biópsia , Feminino , Imuno-Histoquímica , Isoenxertos , Camundongos , Modelos Biológicos , Metástase Neoplásica , Estadiamento de Neoplasias , Especificidade de Órgãos , Pesquisa Translacional Biomédica
15.
Nutrition ; 72: 110659, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31986320

RESUMO

OBJECTIVES: Chemotherapy is one of the major treatments of cancer. However, the emergence of resistance to chemotherapeutic agents is still a major obstacle in the successful management of resistant tumors. Therefore, development of new mechanisms to overcome drug resistance is essential and may be further developed into effective therapies that can flip the switch from drug resistance to susceptibility. The aim of this study was to evaluate a combination consisting of a ketogenic diet and melatonin to determine whether it would inhibit cisplatin- and vincristine-resistant breast cancer. METHODS: In the in vitro part of the study, drug-resistant cell lines were treated with melatonin and real-time polymerase chain reaction was used to measure levels of gene expression involved in apoptosis and resistance. On the protein level, the activity of caspase-3 and the level of vascular endothelin growth factor protein were determined. In the in vivo part, tumor-bearing mice received one of the following treatments: ketogenic diet, melatonin, combination of melatonin and ketogenic diet, vehicle, or chemotherapy. RESULTS: Successful inhibition of resistant cell lines was achieved by melatonin. This inhibition was mediated by induction of apoptosis, inhibition of angiogenesis, and downregulation of resistance genes. A synergistic anticancer effect was observed between melatonin and the ketogenic diet against resistant breast tumors inoculated in mice with a cure rate of 70%. CONCLUSIONS: The combination of melatonin and a ketogenic diet represents a promising option to overcome drug resistance in cancer chemotherapy. However, further testing on the protein level using flow cytometry is important to better understand the mechanisms of action.


Assuntos
Neoplasias da Mama/terapia , Cisplatino , Dieta Cetogênica/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melatonina/administração & dosagem , Vincristina , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Feminino , Isoenxertos , Camundongos , Neovascularização Patológica , Resultado do Tratamento , Vincristina/uso terapêutico
16.
Leukemia ; 34(4): 1017-1026, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31740809

RESUMO

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5'-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4+ and CD8+ T cells, but not on NK or B cells, and relied on intact IFN and mitochondrial antiviral signaling protein (MAVS) signaling in the host. Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3+ T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.


Assuntos
Anticorpos Neutralizantes/farmacologia , Proteína DEAD-box 58/imunologia , Imunoterapia/métodos , Leucemia Mieloide Aguda/terapia , RNA de Cadeia Dupla/farmacologia , Receptores Virais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Proteína DEAD-box 58/genética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica , Xenoenxertos , Humanos , Memória Imunológica/efeitos dos fármacos , Interferons/genética , Interferons/imunologia , Isoenxertos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/mortalidade , Camundongos , Receptores Virais/agonistas , Receptores Virais/genética , Indução de Remissão , Transdução de Sinais , Análise de Sobrevida , Resultado do Tratamento
17.
Theranostics ; 9(26): 8221-8238, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31754392

RESUMO

Predicting the outcome of immunotherapy is essential for efficient treatment. The recent clinical success of immunotherapy is increasingly changing the paradigm of cancer treatment. Accordingly, the development of immune-based agents is accelerating and the number of agents in the global immuno-oncology pipeline has grown 60-70% over the past year. However, despite remarkable clinical efficacy in some patients, only few achieve a lasting clinical response. Treatment failure can be attributed to poorly immunogenic tumors that do not attract tumor infiltrating lymphocytes (TILs). Therefore, we developed positron emission tomography (PET) radiotracers for non-invasive detection of CD4+ and CD8a+ TILs in syngeneic mouse tumor models for preclinical studies. Methods: Seven syngeneic mouse tumor models (B16F10, P815, CT26, MC38, Renca, 4T1, Sa1N) were quantified for CD4+ and CD8a+ TILs using flow cytometry and immunohistochemistry (IHC), as well as for tumor growth response to Sym021, a humanized PD-1 antibody cross-reactive with mouse PD-1. Radiotracers were generated from F(ab)'2 fragments of rat-anti-mouse CD4 and CD8a antibodies conjugated to the p-SCN-Bn-Desferrioxamine (SCN-Bn-DFO) chelator and radiolabeled with Zirconium-89 (89Zr-DFO-CD4/89Zr-DFO-CD8a). Tracers were optimized for in vivo PET/CT imaging in CT26 tumor-bearing mice and specificity was evaluated by depletion studies and isotype control imaging. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET/CT imaging was conducted in the panel of syngeneic mouse models prior to immunotherapy with Sym021. Results: Syngeneic tumor models were characterized as "hot" or "cold" according to number of TILs determined by flow cytometry and IHC. 89Zr-DFO-CD4 and 89Zr-DFO-CD8a were successfully generated with a radiochemical purity >99% and immunoreactivity >85%. The optimal imaging time-point was 24 hours post-injection of ~1 MBq tracer with 30 µg non-labeled co-dose. Reduced tumor and spleen uptake of 89Zr-DFO-CD8a was observed in CD8a+ depleted mice and the uptake was comparable with that of isotype control (89Zr-DFO-IgG2b) confirming specificity. PET imaging in syngeneic tumor models revealed a varying maximum tumor-to-heart ratio of 89Zr-DFO-CD4 and 89Zr-DFO-CD8a across tumor types and in-between subjects that correlated with individual response to Sym021 at day 10 relative to start of therapy (p=0.0002 and p=0.0354, respectively). The maximum 89Zr-DFO-CD4 tumor-to-heart ratio could be used to stratify mice according to Sym021 therapy response and overall survival was improved in mice with a 89Zr-DFO-CD4 ratio >9 (p=0.0018). Conclusion: We developed 89Zr-DFO-CD4 and 89Zr-DFO-CD8a PET radiotracers for specific detection and whole-body assessment of CD4+ and CD8a+ status. These radiotracers can be used to phenotype preclinical syngeneic mouse tumor models and to predict response to an immune checkpoint inhibitor. We foresee development of such non-invasive in vivo biomarkers for prediction and evaluation of clinical efficacy of immunotherapeutic agents, such as Sym021.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias/tratamento farmacológico , Tomografia por Emissão de Pósitrons/métodos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Técnicas Biossensoriais/métodos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Desferroxamina/química , Modelos Animais de Doenças , Imunoterapia , Isoenxertos/citologia , Isoenxertos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Imagem Molecular/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/imunologia , Radioisótopos/química , Zircônio/química
18.
Breast Cancer Res ; 21(1): 94, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409375

RESUMO

BACKGROUND: Human epidermal growth factor receptor-2 (HER2)-targeted therapies prolong survival in HER2-positive breast cancer patients. Benefit stems primarily from improved control of systemic disease, but up to 50% of patients progress to incurable brain metastases due to acquired resistance and/or limited permeability of inhibitors across the blood-brain barrier. Neratinib, a potent irreversible pan-tyrosine kinase inhibitor, prolongs disease-free survival in the extended adjuvant setting, and several trials evaluating its efficacy alone or combination with other inhibitors in early and advanced HER2-positive breast cancer patients are ongoing. However, its efficacy as a first-line therapy against HER2-positive breast cancer brain metastasis has not been fully explored, in part due to the lack of relevant pre-clinical models that faithfully recapitulate this disease. Here, we describe the development and characterisation of a novel syngeneic model of spontaneous HER2-positive breast cancer brain metastasis (TBCP-1) and its use to evaluate the efficacy and mechanism of action of neratinib. METHODS: TBCP-1 cells were derived from a spontaneous BALB/C mouse mammary tumour and characterised for hormone receptors and HER2 expression by flow cytometry, immunoblotting and immunohistochemistry. Neratinib was evaluated in vitro and in vivo in the metastatic and neoadjuvant setting. Its mechanism of action was examined by transcriptomic profiling, function inhibition assays and immunoblotting. RESULTS: TBCP-1 cells naturally express high levels of HER2 but lack expression of hormone receptors. TBCP-1 tumours maintain a HER2-positive phenotype in vivo and give rise to a high incidence of spontaneous and experimental metastases in the brain and other organs. Cell proliferation/viability in vitro is inhibited by neratinib and by other HER2 inhibitors, but not by anti-oestrogens, indicating phenotypic and functional similarities to human HER2-positive breast cancer. Mechanistically, neratinib promotes a non-apoptotic form of cell death termed ferroptosis. Importantly, metastasis assays demonstrate that neratinib potently inhibits tumour growth and metastasis, including to the brain, and prolongs survival, particularly when used as a neoadjuvant therapy. CONCLUSIONS: The TBCP-1 model recapitulates the spontaneous spread of HER2-positive breast cancer to the brain seen in patients and provides a unique tool to identify novel therapeutics and biomarkers. Neratinib-induced ferroptosis provides new opportunities for therapeutic intervention. Further evaluation of neratinib neoadjuvant therapy is warranted.


Assuntos
Neoplasias Encefálicas/prevenção & controle , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ferroptose/efeitos dos fármacos , Quinolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Biologia Computacional/métodos , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Imuno-Histoquímica , Isoenxertos , Camundongos , Terapia de Alvo Molecular , Terapia Neoadjuvante , Quinolinas/uso terapêutico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo
19.
Sci Rep ; 9(1): 12286, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31439856

RESUMO

Pancreatic ductal adenocarcinoma (PDA) has a highly immunosuppressive microenvironment, which is contributed by the complex interaction between cancer cells and a heterogeneous population of stromal cells. Therefore, facile and trackable models are needed for integrative and dynamic interrogation of cancer-stroma interaction. Here, we tracked the immunoevolution of PDA in a genetically-defined transplantable model of mouse pancreatic tumour organoids that recapitulates the progression of the disease from early preinvasive lesions to metastatic carcinomas. We demonstrated that organoid-derived isografts (ODI) can be used as a biological source of biomarkers (NT5E, TGFB1, FN1, and ITGA5) of aggressive molecular subtypes of human PDA. In ODI, infiltration from leukocytes is an early event during progression of the disease as observed for autochthonous models. Neoplastic progression was associated to accumulation of Maf+ macrophages, which inversely correlated with CD8+ T cells infiltration. Consistently, levels of MAF were enriched in human PDA subtypes characterized by abundance of macrophage-related transcripts and indicated poor patients' survival. Density of MAF+ macrophages was higher in human PDA tissues compared to preinvasive lesions. Our results suggest that ODIs represent a suitable system for genotypic-immunophenotypic studies and support the hypothesis of MAF+ macrophages as a prominent immunosuppressive population in PDA.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/imunologia , Macrófagos/imunologia , Neoplasias Pancreáticas/imunologia , Microambiente Tumoral/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Feminino , Humanos , Isoenxertos , Macrófagos/patologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Pâncreas , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia
20.
J Immunol ; 203(4): 825-834, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31292216

RESUMO

Extracellular vesicles (EV) are candidates for cancer immunotherapy because of their capacity to stimulate tumor-specific activity in vivo. However, clinical trials using peptide-loaded autologous EVs have so far only showed moderate T cell responses, suggesting a need for optimization of EV-induced immunity in humans. We previously demonstrated that induction of Ag-specific CD8+ T cells and antitumor responses to whole Ag were independent of MHC class I on EVs and hypothesized that multiple injections of allogeneic EVs could potentiate Ag-specific responses. In this study, we show that the allogeneic EV from mouse bone marrow-derived dendritic cells enhances Ag-specific CD8+ T cell, follicular helper T cell, and Ag-specific Ab responses. EV-injected mice demonstrated Ag-specific memory after 4 mo, with the highest Ab avidity in mice receiving double allogeneic EV injections. Reduced B16mOVA melanoma tumor growth was shown in all EV-injected groups. Our findings support the application of allogeneic EVs for therapeutic use in clinical studies in which an adaptive immune response is desired.


Assuntos
Vesículas Extracelulares/transplante , Memória Imunológica/imunologia , Imunoterapia/métodos , Melanoma Experimental/imunologia , Aloenxertos , Animais , Células da Medula Óssea/imunologia , Células Dendríticas/imunologia , Vesículas Extracelulares/imunologia , Isoenxertos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL
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