Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 15 de 15
Filtrar
Mais filtros








Base de dados
Intervalo de ano de publicação
1.
Methods Cell Biol ; 174: 1-16, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36710044

RESUMO

The immunomodulatory properties of local hypofractionated radiotherapy are known to promote the generation of anti-tumor immune responses. Such responses are largely due to the infiltration of cytotoxic lymphocytes (TILs) into the tumors that are able to destroy malignant lesions. In this context, characterizing the tumor immune microenvironment following radiotherapy is crucial for the study of its mechanism of action. Flow cytometry-based analyses are frequently used to elucidate changes in the tumor immune microenvironment. The use of a fluorochrome-conjugated antibody panel is currently a standard technique to assess the number and phenotype of immune cell populations infiltrating the tumors. Here, we describe a method to isolate and quantify TILs based on flow-cytometry in mammary carcinoma-bearing mice that undergo a local hypofractionated radiotherapy regimen consisting of 3 consecutive doses of 8 Gy. With some adaptations, this protocol can be successfully applied to a diverse range of transplantable and inducible solid mouse tumors of different origins.


Assuntos
Neoplasias , Camundongos , Animais , Citometria de Fluxo , Neoplasias/patologia , Linfócitos do Interstício Tumoral/patologia , Microambiente Tumoral
2.
Methods Cell Biol ; 174: 127-136, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36710046

RESUMO

The clonogenic assay is an in vitro method based on the ability of a single cell to proliferate indefinitely into a colony. This assay is the gold standard method to analyze cell viability and quantify reproductive cell survival fraction after treatment with ionizing radiation and other cytotoxic agents in vitro. After the cytotoxic effect, only some cells retain their ability to grow from one cell and form colonies. The colony is defined to consist of at least 50 cells. The radiosensitivity of each cell line may vary. Thus, characterizing cell sensitivity following radiation is crucial to choose the optimum radiotherapy dose. Here, we describe a method to test the in vitro capability of cell lines to form colonies following radiation treatment. This assay allows to analyze the efficacy of specific treatments on the cell reproductivity of cell lines With some adaptations, this protocol can be essentially applied to analyze the cell proliferation rate after different doses of irradiation on many different cell lines.


Assuntos
Neoplasias , Tolerância a Radiação , Linhagem Celular , Sobrevivência Celular , Técnicas In Vitro
3.
Methods Cell Biol ; 172: 179-189, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36064224

RESUMO

The rapid proliferation of cancer cells and the aberrant vasculature present in most solid tumors frequently result in the lack of oxygen generating a hypoxic tumor microenvironment. Low levels of oxygen not only affect the tumor cell biology and tumorigenesis, but also the other components of the tumor microenvironment such as the tumor stroma and the immune infiltrate, promoting a more suppressive environment. In addition, tumor hypoxia has been associated with reduced sensitivity to chemotherapy (CH) and radiotherapy (RT), leading to poor outcomes in cancer patients. Therefore, the evaluation of tumor oxygen status has become clinically relevant. Tumor hypoxia can be assessed by different methods that include the analysis of the oxygen concentration or the expression of endogenous markers directly related to hypoxia. In this paper, we focus on the use of the hypoxia-specific marker pimonidazole as a straightforward way to measure tumor hypoxia following radiotherapy in a preclinical melanoma model.


Assuntos
Hipóxia , Neoplasias , Biomarcadores/metabolismo , Hipóxia Celular , Humanos , Hipóxia/metabolismo , Neoplasias/radioterapia , Nitroimidazóis , Oxigênio/metabolismo , Coloração e Rotulagem , Microambiente Tumoral
4.
Eur J Immunol ; 51(9): 2274-2280, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33963542

RESUMO

In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.


Assuntos
Quimiotaxia/imunologia , Armadilhas Extracelulares/imunologia , Interleucina-8/imunologia , Neutrófilos/imunologia , Acetilcisteína/metabolismo , Humanos , Espécies Reativas de Oxigênio/metabolismo , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/antagonistas & inibidores , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais/imunologia
5.
Oncoimmunology ; 9(1): 1760676, 2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32934876

RESUMO

Checkpoint inhibitors have improved the survival of patients with advanced tumors and show a manageable toxicity profile. However, auto-immune colitis remains a relevant side effect, and combinations of anti-PD1/PDL1 and anti-CTLA-4 increase its incidence and severity. Here, we report the case of a 50-year-old patient diagnosed with stage IV cervical cancer that relapsed following radical surgery, external radiation/brachytherapy and standard chemotherapy. She was subsequently treated with Nivolumab and Ipilimumab combination and developed grade 2 colitis presenting a dissociation between endoscopic and pathological findings. At cycle 10 the patient reported grade 3 diarrhea and abdominal discomfort, without blood or mucus in the stools. Immunotherapy was withheld and a colonoscopy was performed, showing normal mucosa in the entire colon. Puzzlingly, histologic evaluation of randomly sampled mucosal biopsy of the distal colon showed an intense intraepithelial lymphocyte infiltration with crypt loss and some regenerating crypts with a few apoptotic bodies set in a chronically inflamed lamina propria, consistent with the microscopic diagnosis of colitis. Treatment with methylprednisolone 2 mg/kg was initiated which led to a decrease in the number of stools to grade 1. Additional investigations to exclude other causes of diarrhea rendered negative results. The patient experienced a major partial response and, following the resolution of diarrhea, she was re-challenged again with immunotherapy, with the reappearance of grade 2 diarrhea, leading to permanent immunotherapy interruption. We conclude and propose that performing random colonic biopsies should be considered in cases of immune checkpoint-associated unexplained diarrhea, even when colonoscopy shows macroscopically normal colonic mucosa inflammatory lesions.


Assuntos
Colite , Inibidores de Checkpoint Imunológico , Colite/induzido quimicamente , Transtornos Dissociativos , Feminino , Humanos , Ipilimumab/efeitos adversos , Pessoa de Meia-Idade , Nivolumabe
6.
Methods Enzymol ; 635: 111-125, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32122540

RESUMO

Focal radiation therapy has the potential to generate systemic tumor-targeting immune responses so potent as to eradicate anatomically distant, non-irradiated malignant lesions, a phenomenon commonly referred to as "the abscopal response." In cancer patients, bona fide abscopal responses are rare, although the recent introduction of immune checkpoint blockers into the clinical practice has significantly increased their incidence. In rodents, abscopal responses can be conveniently modeled by establishing two, slightly asynchronous and anatomically distant subcutaneous tumors in syngeneic immunocompetent hosts, provided that the therapeutic partners of radiation potentially included in the regimen of choice do not mediate systemic anticancer effects per se. Here, we describe such method to monitor abscopal responses based on mammary carcinoma TSA cells implanted in syngeneic immunocompetent BALB/c mice. With minor variations, the same technique can be conveniently applied to a variety of transplantable mouse tumors.


Assuntos
Camundongos Endogâmicos BALB C , Animais , Linhagem Celular Tumoral , Humanos , Camundongos
7.
Nat Immunol ; 21(2): 120-134, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31873291

RESUMO

Therapeutic irradiation of the tumor microenvironment causes differential activation of pro-survival and pro-death pathways in malignant, stromal, endothelial and immune cells, hence causing a profound cellular and biological reconfiguration via multiple, non-redundant mechanisms. Such mechanisms include the selective elimination of particularly radiosensitive cell types and consequent loss of specific cellular functions, the local release of cytokines and danger signals by dying radiosensitive cells, and altered cytokine secretion by surviving radioresistant cells. Altogether, these processes create chemotactic and immunomodulatory cues for incoming and resident immune cells. Here we discuss how cytoprotective and cytotoxic signaling modules activated by radiation in specific cell populations reshape the immunological tumor microenvironment.


Assuntos
Morte Celular/efeitos da radiação , Transdução de Sinais/imunologia , Transdução de Sinais/efeitos da radiação , Microambiente Tumoral/imunologia , Microambiente Tumoral/efeitos da radiação , Animais , Humanos
8.
Oncoimmunology ; 8(11): e1655364, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31646103

RESUMO

Caspases are known for their ability to precipitate apoptosis. Our findings indicate that accelerating the terminal inactivation of cells dying in response to radiation therapy limit their immunogenicity as a consequence of reduced type I interferon secretion. Thus, caspase inhibitors stand out as promising combinatorial partners to improve the immunogenicity of radiation therapy in the clinic.

9.
Oncoimmunology ; 8(11): e1655964, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31646105

RESUMO

Caspase 3 (CASP3) has a key role in the execution of apoptosis, and many cancer cells are believed to disable CASP3 as a mechanism of resistance to cytotoxic therapeutics. Alongside, CASP3 regulates stress-responsive immunomodulatory pathways, including secretion of type I interferon (IFN). Here, we report that mouse mammary carcinoma TSA cells lacking Casp3 or subjected to chemical caspase inhibition were as sensitive to the cytostatic and cytotoxic effects of radiation therapy (RT) in vitro as their control counterparts, yet secreted increased levels of type I IFN. This effect originated from the accrued accumulation of irradiated cells with cytosolic DNA, likely reflecting the delayed breakdown of cells experiencing mitochondrial permeabilization in the absence of CASP3. Casp3-/- TSA cells growing in immunocompetent syngeneic mice were more sensitive to RT than their CASP3-proficient counterparts, and superior at generating bona fide abscopal responses in the presence of an immune checkpoint blocker. Finally, multiple genetic signatures of apoptotic proficiency were unexpectedly found to have robust negative (rather than positive) prognostic significance in a public cohort of breast cancer patients. However, these latter findings were not consistent with genetic signatures of defective type I IFN signaling, which were rather associated with improved prognosis. Differential gene expression analysis on patient subgroups with divergent prognosis (as stratified by independent signatures of apoptotic proficiency) identified SLC7A2 as a new biomarker with independent prognostic value in breast cancer patients. With the caveats associated with the retrospective investigation of heterogeneous, public databases, our data suggest that apoptotic caspases may influence the survival of breast cancer patients (or at least some subsets thereof) via mechanisms not necessarily related to type I IFN signaling as they identify a novel independent prognostic biomarker that awaits prospective validation.

10.
Nature ; 569(7756): 428-432, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31043740

RESUMO

Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer1-3. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients4. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2-/-Il2rg-/- mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno CTLA-4/antagonistas & inibidores , Imunoterapia/efeitos adversos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Inibidores do Fator de Necrose Tumoral/farmacologia , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Antígeno CTLA-4/imunologia , Colite/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/imunologia , Sulfato de Dextrana/farmacologia , Feminino , Doença Enxerto-Hospedeiro , Hepatite/tratamento farmacológico , Humanos , Ipilimumab/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nivolumabe/efeitos adversos , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Cancer Treat Rev ; 53: 79-97, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28088073

RESUMO

The discovery of reliable biomarkers to predict efficacy and toxicity of anticancer drugs remains one of the key challenges in cancer research. Despite its relevance, no efficient study designs to identify promising candidate biomarkers have been established. This has led to the proliferation of a myriad of exploratory studies using dissimilar strategies, most of which fail to identify any promising targets and are seldom validated. The lack of a proper methodology also determines that many anti-cancer drugs are developed below their potential, due to failure to identify predictive biomarkers. While some drugs will be systematically administered to many patients who will not benefit from them, leading to unnecessary toxicities and costs, others will never reach registration due to our inability to identify the specific patient population in which they are active. Despite these drawbacks, a limited number of outstanding predictive biomarkers have been successfully identified and validated, and have changed the standard practice of oncology. In this manuscript, a multidisciplinary panel reviews how those key biomarkers were identified and, based on those experiences, proposes a methodological framework-the DESIGN guidelines-to standardize the clinical design of biomarker identification studies and to develop future research in this pivotal field.


Assuntos
Biomarcadores Tumorais/análise , Pesquisa Biomédica/métodos , Quinase do Linfoma Anaplásico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Pesquisa Biomédica/normas , Estudos Clínicos como Assunto , Ensaios Clínicos como Assunto , Receptores ErbB/genética , Humanos , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-kit/genética , Receptores Proteína Tirosina Quinases/genética , Receptor ErbB-2/análise , Proteínas ras/genética
13.
Cancer Res ; 76(10): 2863-7, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-27308833

RESUMO

The recent approval by the FDA of the combination of anti-CTLA4 and anti-PD-1 mAbs for the treatment of BRAF-unmutated unresectable or metastatic melanoma is a landmark for the development of cancer immunotherapy. On October 18 to 22, 2015, a symposium was held in Pamplona (Spain) to present and discuss the basic and clinical discoveries that have brought us to this milestone and to explore other targets and immunotherapy strategies aimed at attaining more efficacious oncology practice in the short term. Cancer Res; 76(10); 2863-7. ©2016 AACR.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno CTLA-4/imunologia , Imunomodulação/efeitos dos fármacos , Imunoterapia , Melanoma/terapia , Receptor de Morte Celular Programada 1/imunologia , Animais , Antígeno CTLA-4/antagonistas & inibidores , Humanos , Melanoma/imunologia , Melanoma/secundário , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Relatório de Pesquisa
14.
Radiat Oncol ; 10: 25, 2015 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-25612575

RESUMO

BACKGROUND: Stereotactic ablative body radiation (SABR) is a novel and sophisticated radiation modality that involves the irradiation of extracranial tumors through precise and very high doses in patients with oligometastatic lung disease and primary lung tumors. CASE PRESENTATION: A 52-year-old female with subclinical idiopathic interstitial lung disease (ILD) and oligometastatic lung disease from squamous urethral cancer who was treated with SABR for a metastatic lesion located in the right lower pulmonary lobe. The patient received a hypo-fractionated course of SABR. A 3D-conformal multifield technique was used with six coplanar and one non-coplanar statics beams. A 48 Gy total dose in three fractions over six days was prescribed to the 95% of the PTV. The presence of idiopathic ILD and other identifiable underlying lung conditions were not taken into account as a constraint to prescribe a different than standard total dose or fractionation schedule. Six months after the SABR treatment, a CT-scan showed the presence of a pneumomediastinum with air outside the bronchial tree and within the subcutaneous tissue without co-existing pneumothorax. To our knowledge, this is the first case of pneumomediastinum appearing 6 months after SABR treatment for a lung metastasis located in the perihiliar/central tumors region as defined by the RTOG protocols as the proximal bronchial tree. CONCLUSION: Radiation oncologist should be aware of the potential risk of severe lung toxicity caused by SABR in patients with ILD, especially when chemotherapy-induced pulmonary toxicity is administered in a short time interval.


Assuntos
Neoplasias Pulmonares/radioterapia , Enfisema Mediastínico/etiologia , Radiocirurgia/efeitos adversos , Radioterapia Conformacional/efeitos adversos , Feminino , Humanos , Neoplasias Pulmonares/secundário , Pessoa de Meia-Idade , Neoplasias Uretrais/patologia
15.
Radiat Oncol ; 7: 50, 2012 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-22455311

RESUMO

BACKGROUND: Stereotactic body radiation therapy (SBRT) is a radiation technique used in patients with oligometastatic lung disease. Lung and chest wall toxicities have been described in the patients but pathological vertebral fracture is an adverse effect no reported in patients treated with SBRT for lung metastases. CASE PRESENTATION: A 68-year-old woman with the diagnosis of a recurrence of a single lung metastatic nodule of urothelial carcinoma after third line of chemotherapy. The patient received a hypo-fractionated course of SBRT.A 3D-conformal multifield technique was used with six coplanar and one non-coplanar statics beams. A total dose of 48 Gy in three fractions over six days was prescribed to the 95% of the CTV. Ten months after the SBRT procedure, a CT scan showed complete response of the metastatic disease without signs of radiation pneumonitis. However, rib and vertebral bone toxicities were observed with the fracture-collapse of the 7th and 8th vertebral bodies and a fracture of the 7th and 8th left ribs. We report a unique case of pathological vertebral fracture appearing ten months after SBRT for an asymptomatic growing lung metastases of urothelial carcinoma. CONCLUSION: Though SBRT allows for minimization of normal tissue exposure to high radiation doses SBRT tolerance for vertebral bone tissue has been poorly evaluated in patients with lung tumors. Oncologists should be alert to the potential risk of fatal bone toxicity caused by this novel treatment. We recommend BMD testing in all woman over 65 years old with clinical risk factors that could contribute to low BMD. If low BMD is demonstrated, we should carefully restrict the maximum radiation dose in the vertebral body in order to avoid intermediate or low radiation dose to the whole vertebral body.


Assuntos
Carcinoma de Células de Transição/secundário , Fraturas Espontâneas/etiologia , Neoplasias Pulmonares/secundário , Radiocirurgia/efeitos adversos , Radioterapia Conformacional/efeitos adversos , Fraturas das Costelas/etiologia , Costelas/efeitos da radiação , Fraturas da Coluna Vertebral/etiologia , Vértebras Torácicas/efeitos da radiação , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Densidade Óssea , Carboplatina/administração & dosagem , Carcinoma de Células de Transição/tratamento farmacológico , Carcinoma de Células de Transição/cirurgia , Vértebras Cervicais/efeitos da radiação , Cisplatino/administração & dosagem , Terapia Combinada , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Feminino , Glutamatos/administração & dosagem , Guanina/administração & dosagem , Guanina/análogos & derivados , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/cirurgia , Recidiva Local de Neoplasia/radioterapia , Osteoporose Pós-Menopausa/complicações , Pemetrexede , Neoplasias da Bexiga Urinária/tratamento farmacológico , Gencitabina
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA