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J Vis Exp ; (168)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33616115


Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

Commun Biol ; 4(1): 72, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33452400


Inflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of breast cancer with rapid onset and a strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC are largely unknown. Herein, we report that decorin (DCN), a small leucine-rich extracellular matrix proteoglycan, is downregulated in tumors from patients with IBC. Overexpression of DCN in IBC cells markedly decreased migration, invasion, and cancer stem cells in vitro and inhibited tumor growth and metastasis in IBC xenograft mouse models. Mechanistically, DCN functioned as a suppressor of invasion and tumor growth in IBC by destabilizing E-cadherin and inhibiting EGFR/ERK signaling. DCN physically binds E-cadherin in IBC cells and accelerates its degradation through an autophagy-linked lysosomal pathway. We established that DCN inhibits tumorigenesis and metastasis in IBC cells by negatively regulating the E-cadherin/EGFR/ERK axis. Our findings offer a potential therapeutic strategy for IBC, and provide a novel mechanism for IBC pathobiology.

J Cell Sci ; 129(24): 4622-4632, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27875278


Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orange-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods.

Ácidos/metabolismo , Laranja de Acridina/metabolismo , Autofagia , Técnicas Citológicas/métodos , Organelas/metabolismo , Animais , Tamanho Celular , Citometria de Fluxo , Fluorescência , Células HEK293 , Humanos , Microscopia Confocal , Ratos Wistar , Espectrometria de Fluorescência
Tumour Biol ; 37(10): 13743-13749, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27476170


Although cancer is a chronic disease, most of the in vitro experiments to assess the effectiveness of intervention are performed in hours or a few days. Moreover, none of the available methodologies to measure cell proliferation are adapted to provide information about the growth kinetic during and after treatment. Thus, the objective of this work is to provide a guide to assess long-term changes in cell population size to be used mainly in cancer research. Cumulative population doubling (CPD) graphs based on cell counting for in vitro or tumor volume for in vivo assays were used to calculate four parameters: relative end CPD (RendCPD), to quantify the end point analysis of proliferation; relative area under curve (rAUC), to describe the global chronic effect of a treatment; relative time to cross a threshold (RTCT), to indicate the delay in cell population recovery produced by a treatment; and relative proliferation rate (RPR), to describe the relative regrowth velocity of the cells that survived after treatment. These parameters describe not only the acute and chronic effects of a treatment but also the behavior of cells that are not eliminated by the treatment, providing crucial information about the growth kinetic of the surviving population. Moreover, the proposed analysis allowed the grouping of independent CPD experiments quantified at different time points and even the direct comparison of in vitro and in vivo experiments. Therefore, this new way to analyze long-term outcomes provides a global view of the effectiveness of an intervention, as an important tool for long-term studies.

Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Glioma/tratamento farmacológico , Glioma/patologia , Guias como Assunto , Animais , Área Sob a Curva , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Teóricos , Fatores de Tempo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
J Cell Biochem ; 109(5): 983-91, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-20069573


Gliomas have one of the worst prognosis among cancers. Their resistance to cell death induced by endogenous neurotoxic agents, such as extracellular ATP, seems to play an important role in their pathobiology since alterations in the degradation rate of extracellular ATP drastically affects glioma growth in rats. In the present work we characterized the mechanisms of cell death induced by extracellular ATP in a murine glioma cell line, GL261. ATP and BzATP, a P2X7 agonist, induced cell death at concentrations that are described to activate the P2X7 receptor in mouse. oATP, an antagonist of P2X7, blocked the ATP-induced cell death. Agonists of purinergic receptors expressed in GL261 such as adenosine, ADP, UTP did not cause any cell death, even at mM concentrations. A sub-population of cells more sensitive to ATP expressed more P2X7 when compared to a less sensitive subpopulation. Accordingly, RNA interference of the P2X7 receptor drastically reduced ATP-induced cell death, suggesting that this receptor is necessary for this effect. The mechanism of ATP-induced cell death is predominantly necrotic, since cells presented shrinkage accompanied by membrane permeabilization, but not apoptotic, since no phosphatidylserine externalization or caspase activity was observed. These data show the importance of P2X7 in ATP-induced cell death and shed light on the importance of ATP-induced cell death in glioma development.

Trifosfato de Adenosina/farmacologia , Glioma/patologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Glioma/genética , Camundongos , Necrose/patologia , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7