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1.
Methods Mol Biol ; 2269: 37-47, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687670

RESUMO

Ionizing radiation is a critical component of glioblastoma (GBM) therapy. Recent data have implicated glioblastoma stem-like cells (GSCs) as determinants of GBM development, maintenance, and treatment response. Understanding the response of GSCs to radiation should thus provide insight into the development of improved GBM treatment strategies. Towards this end, in vitro techniques for the analysis of GSC radiosensitivity are an essential starting point. One such method, the clonogenic survival assay has been adapted to assessing the intrinsic radiosensitivity of GSCs and is described here. As an alternative method, the limiting dilution assay is presented for defining the radiosensitivity of GSC lines that do not form colonies or only grow as neurospheres. In addition to these cellular strategies, we describe γH2AX foci analysis, which provides a surrogate marker for radiosensitivity at the molecular level. Taken together, the in vitro methods presented here provide tools for defining intrinsic radiosensitivity of GSCs and for testing agents that may enhance GBM radioresponse.


Assuntos
Biomarcadores Tumorais , Loci Gênicos , Glioblastoma , Histonas , Proteínas de Neoplasias , Células-Tronco Neoplásicas , Tolerância a Radiação , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioblastoma/radioterapia , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
2.
Methods Mol Biol ; 2269: 49-61, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33687671

RESUMO

In solid tumors, mesenchymal stem cells (MSCs) are recognized to establish complex intercommunication networks with cancer cells and to significantly influence their invasion and metastasis potential. Such bidirectional interplay occurs between both tissue resident/tumor-associated MSCs (TA-MSCs) and also tumor infiltrating MSCs (TM-MSCs) that migrate from distant sites such as the bone marrow. Interestingly, malignant cells interactions with MSCs in the tumor microenvironment extends beyond conventional exchanges of signaling factors and extracellular vesicles, including unconventional direct exchanges of intracellular components, or cancer cells cannibalism of MSCs. In the context of 3D in vitro tumor models, cell tracking assays making use of cell-labeling probes such as membrane penetrating dyes, can be leveraged to shed light on these events, and allow researchers to analyze overtime cell-to-cell spatial distribution, fusion, internal organization, and changes in co-cultured populations ratios. Herein, we describe a high-throughput compatible method through which MSCs positioning and permanence within in vitro 3D multicellular tumor spheroid models (3D-MCTS) can be tracked overtime. Although we have focused on the interactions of human bone marrow-derived MSCs (hBM-MSCs) within heterotypic lung cancer A549 3D-MCTS, these procedures can be implemented for other 3D tumor spheroid models and types of cells, taking into consideration that optimization steps are undertaken.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Modelos Biológicos , Neoplasias/metabolismo , Esferoides Celulares/metabolismo , Microambiente Tumoral , Humanos , Células-Tronco Mesenquimais/patologia , Neoplasias/patologia , Esferoides Celulares/patologia
3.
Int J Mol Sci ; 22(4)2021 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-33670029

RESUMO

Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.


Assuntos
Derme/citologia , Queratinócitos/citologia , Microgéis/química , Polietilenoglicóis/farmacologia , Adesão Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Linhagem Celular , Técnicas de Cocultura , Proteínas de Fluorescência Verde/metabolismo , /efeitos dos fármacos , Humanos , Hidrogéis/farmacologia , Proteínas Luminescentes/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Proteínas Wnt/metabolismo
4.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671198

RESUMO

Near-infrared (NIR) fluorescence-guided surgery is an innovative technique for the real-time visualization of resection margins. The aim of this study was to develop a head and neck multicellular tumor spheroid model and to explore the possibilities offered by it for the evaluation of cameras for NIR fluorescence-guided surgery protocols. FaDu spheroids were incubated with indocyanine green (ICG) and then included in a tissue-like phantom. To assess the capability of Fluobeam® NIR camera to detect ICG in tissues, FaDu spheroids exposed to ICG were embedded in 2, 5 or 8 mm of tissue-like phantom. The fluorescence signal was significantly higher between 2, 5 and 8 mm of depth for spheroids treated with more than 5 µg/mL ICG (p < 0.05). The fluorescence intensity positively correlated with the size of spheroids (p < 0.01), while the correlation with depth in the tissue-like phantom was strongly negative (p < 0.001). This multicellular spheroid model embedded in a tissue-like phantom seems to be a simple and reproducible in vitro tumor model, allowing a comparison of NIR cameras. The ideal configuration seems to be 450 µm FaDu spheroids incubated for 24 hours with 0.05 mg/ml of ICG, ensuring the best stability, toxicity, incorporation and signal intensity.


Assuntos
Cabeça/diagnóstico por imagem , Imageamento Tridimensional , Modelos Biológicos , Pescoço/diagnóstico por imagem , Neoplasias/cirurgia , Fotografação/instrumentação , Espectroscopia de Luz Próxima ao Infravermelho , Esferoides Celulares/citologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Fluorescência , Humanos , Verde de Indocianina/toxicidade , Cinética , Imagens de Fantasmas
5.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671292

RESUMO

Anticancer drugs, such as fluorouracil (5-FU), oxaliplatin, and doxorubicin (Dox) are commonly used to treat colorectal cancer (CRC); however, owing to their low response rate and adverse effects, the development of efficient drug delivery systems (DDSs) is required. The cellular prion protein PrPC, which is a cell surface glycoprotein, has been demonstrated to be overexpressed in CRC, however, there has been no research on the development of PrPC-targeting DDSs for targeted drug delivery to CRC. In this study, PrPC aptamer (Apt)-conjugated gold nanoparticles (AuNPs) were synthesized for targeted delivery of Dox to CRC. Thiol-terminated PrPC-Apt was conjugated to AuNPs, followed by hybridization of its complementary DNA for drug loading. Finally, Dox was loaded onto the AuNPs to synthesize PrPC-Apt-functionalized doxorubicin-oligomer-AuNPs (PrPC-Apt DOA). The PrPC-Apt DOA were spherical nanoparticles with an average diameter of 20 nm. Treatment of CRC cells with PrPC-Apt DOA induced reactive oxygen species generation by decreasing catalase and superoxide dismutase activities. In addition, treatment with PrPC-Apt DOA inhibited mitochondrial functions by decreasing the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, complex 4 activity, and oxygen consumption rates. Compared to free Dox, PrPC-Apt DOA decreased proliferation and increased apoptosis of CRC cells to a greater degree. In this study, we demonstrated that PrPC-Apt DOA targeting could effectively deliver Dox to CRC cells. PrPC-Apt DOA can be used as a treatment for CRC, and have the potential to replace existing anticancer drugs, such as 5-FU, oxaliplatin, and Dox.


Assuntos
Aptâmeros de Nucleotídeos/química , Neoplasias Colorretais/tratamento farmacológico , Doxorrubicina/administração & dosagem , Doxorrubicina/uso terapêutico , Sistemas de Liberação de Medicamentos , Ouro/química , Nanopartículas Metálicas/química , Proteínas Priônicas/química , Apoptose/efeitos dos fármacos , Catalase/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Superóxido Dismutase/metabolismo
6.
Methods Mol Biol ; 2265: 141-154, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704712

RESUMO

Three-dimensional (3D) cell culture has allowed a deeper understanding of complex pathological and physiological processes, overcoming some of the limitations of 2D cell culture on plastic and avoiding the costs and ethical issues related to experiments involving animals. Here we describe a protocol to embed single melanoma cells alone or together with primary human lymphatic endothelial cells in a 3D cross-linked matrix, to investigate the invasion and molecular crosstalk between these two cell types, respectively. After fixation and staining with antibodies and fluorescent conjugates, phenotypic changes in both cell types can be specifically analyzed by confocal microscopy.


Assuntos
Técnicas de Cocultura/métodos , Células Endoteliais/metabolismo , Melanoma/metabolismo , Esferoides Celulares/metabolismo , Linhagem Celular Tumoral , Células Endoteliais/citologia , Imunofluorescência/métodos , Humanos , Melanoma/patologia , Microscopia Confocal , Invasividade Neoplásica
7.
Methods Mol Biol ; 2265: 155-171, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704713

RESUMO

Researchers often aim to incorporate microenvironmental variables such as the dimensionality and composition of the extracellular matrix into their cell-based assays. A technical challenge created by introduction of these variables is quantification of single-cell measurements and control of environmental reproducibility. Here, we detail a methodology to quantify viability and proliferation of melanoma cells in 3D collagen-based culture platforms by automated microscopy and 3D image analysis to yield robust, high-throughput results of single-cell responses to drug treatment.


Assuntos
Antineoplásicos/farmacologia , Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Processamento de Imagem Assistida por Computador/métodos , Melanoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Análise de Célula Única/métodos , Sobrevivência Celular/efeitos dos fármacos , Colágeno , Imidazóis/farmacologia , Melanoma/patologia , Oximas/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Esferoides Celulares
8.
Methods Mol Biol ; 2265: 173-183, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704714

RESUMO

Most currently available three-dimensional melanoma models have either focused on simplicity or were optimized for physiological relevance. Accordingly, these paradigms have been either composed of malignant cells only or they were sophisticated human skin equivalents featuring multiple cell types and skin-like organization. Here, an intermediate spheroid-based assay system is presented, which uses tri-cultures of human CCD-1137Sk fibroblasts, HaCaT keratinocytes, and SK-MEL-28 melanoma cells. Being made of cell lines, these spheroids can be reliably reproduced without any special equipment using standard culture procedures, and they feature different aspects of skin and early stage melanoma. Therefore, this kind of model can be useful for lead-compound testing or addressing fundamental principles of early melanoma formation.


Assuntos
Antineoplásicos/farmacologia , Técnicas de Cocultura/métodos , Fibroblastos/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Esferoides Celulares/metabolismo , Linhagem Celular Tumoral , Docetaxel/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
9.
Methods Mol Biol ; 2265: 185-199, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704715

RESUMO

Sphere assays are widely used in vitro techniques to enrich and evaluate the stem-like cell behavior of both normal and cancer cells. Utilizing three-dimensional in vitro sphere culture conditions provide a better representation of tumor growth in vivo than the more common monolayer cultures. We describe how to perform primary and secondary sphere assays, used for the enrichment and self-renewability studies of melanoma/melanocyte stem-like cells. Spheres are generated by growing melanoma cells at low density in nonadherent conditions with stem cell media. We provide protocols for preparing inexpensive and versatile polyHEMA-coated plates, setting up primary and secondary sphere assays in almost any tissue culture format and quantification methods using standard inverted microscopy. Our protocol is easily adaptable to laboratories with basic cell culture capabilities, without the need for expensive fluidic instruments.


Assuntos
Técnicas de Cultura de Células/métodos , Melanoma/patologia , Células-Tronco Neoplásicas/citologia , Esferoides Celulares/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Methods Mol Biol ; 2279: 59-73, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33683686

RESUMO

In an era of precision medicine important treatment decisions are dictated by expression of clinically informative tumor protein biomarkers. These biomarkers can be detected by immunohistochemistry (IHC) performed in tumor tissue sections obtained from biopsies or resections. Like all experimental procedures, IHC needs optimization for several of its steps. However, the investigator must avoid optimizing the IHC procedure using valuable human biopsy samples which may be difficult to obtain. Ideally, valuable biopsy samples should only be subjected to IHC once the IHC protocol has been optimized. In this chapter, we describe a procedure for IHC optimization using tri-dimensional (3D) cellular spheroids created from cultured cells. In this approach, cultured cells are pelleted into 3D spheroids, which are then processed just like a tissue sample, namely, fixed, embedded, sectioned, mounted on slides, and stained with IHC just like a human tissue sample. These 3D cellular spheroids have a tissue-like architecture and cellularity resembling a tumor section, and both cellular and antigen structure are preserved. This method is therefore acceptable for IHC optimization before proceeding to the IHC staining of human tumor samples.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Imuno-Histoquímica , Neoplasias Pulmonares , Inclusão em Parafina , Esferoides Celulares , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Formaldeído/química , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia
11.
J Vis Exp ; (168)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33720119

RESUMO

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.


Assuntos
Neoplasias Encefálicas/patologia , Comunicação Celular , Movimento Celular , Técnicas de Cocultura/métodos , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Neurônios/patologia , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Rastreamento de Células , Glioma/patologia , Humanos , Processamento de Imagem Assistida por Computador , Laminina/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia
12.
J Vis Exp ; (168)2021 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-33720122

RESUMO

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles' integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.


Assuntos
/química , Magnetometria , Células-Tronco Mesenquimais/metabolismo , Endocitose , Humanos , Células-Tronco Mesenquimais/ultraestrutura , Micro-Ondas , Soluções , Esferoides Celulares/metabolismo , Esferoides Celulares/ultraestrutura
13.
Molecules ; 26(4)2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670440

RESUMO

Cancer stem cells (CSCs) represent a small subpopulation within a tumour. These cells possess stem cell-like properties but also initiate resistance to cytotoxic agents, which contributes to cancer relapse. Natural compounds such as curcumin that contain high amounts of polyphenols can have a chemosensitivity effect that sensitises CSCs to cytotoxic agents such as cisplatin. This study was designed to investigate the efficacy of curcumin as a chemo-sensitiser in CSCs subpopulation of non-small cell lung cancer (NSCLC) using the lung cancer adenocarcinoma human alveolar basal epithelial cells A549 and H2170. The ability of curcumin to sensitise lung CSCs to cisplatin was determined by evaluating stemness characteristics, including proliferation activity, colony formation, and spheroid formation of cells treated with curcumin alone, cisplatin alone, or the combination of both at 24, 48, and 72 h. The mRNA level of genes involved in stemness was analysed using quantitative real-time polymerase chain reaction. Liquid chromatography-mass spectrometry was used to evaluate the effect of curcumin on the CSC niche. A combined treatment of A549 subpopulations with curcumin reduced cellular proliferation activity at all time points. Curcumin significantly (p < 0.001) suppressed colonies formation by 50% and shrank the spheroids in CSC subpopulations, indicating inhibition of their self-renewal capability. This effect also was manifested by the down-regulation of SOX2, NANOG, and KLF4. Curcumin also regulated the niche of CSCs by inhibiting chemoresistance proteins, aldehyde dehydrogenase, metastasis, angiogenesis, and proliferation of cancer-related proteins. These results show the potential of using curcumin as a therapeutic approach for targeting CSC subpopulations in non-small cell lung cancer.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/farmacologia , Curcumina/farmacologia , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Cisplatino/uso terapêutico , Curcumina/uso terapêutico , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo
14.
Anticancer Res ; 41(3): 1183-1195, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33788709

RESUMO

BACKGROUND/AIM: Resistance to glioblastoma (GB) therapy is attributed to the presence of glioblastoma stem cells (GSC). Here, we defined the behavior of GSC as it pertains to proliferation, migration, and angiogenesis. MATERIALS AND METHODS: Human-derived GSC were isolated and cultured from GB patient tumors. Xenograft GSC were extracted from the xenograft tumors, and spheroids were created and compared with human GSC spheroids by flow cytometry, migration, proliferation, and angiogenesis assays. Oct3/4 and Sox2, GFAP, and Ku80 expression was assessed by immunoanalysis. RESULTS: The xenograft model showed the formation of two different tumors with distinct characteristics. Tumors formed at 2 weeks were less aggressive with well-defined margins, whereas tumors formed in 5 months were diffuse and aggressive. Expression of Oct3/4 and Sox2 was positive in both human and xenograft GSC. Positive Ku80 expression in xenograft GSC confirmed their human origin. Human and xenograft GSC migrated vigorously in collagen and Matrigel, respectively. Xenograft GSC displayed a higher rate of migration and invasion than human GSC. CONCLUSION: Human GSC were more aggressive in growth and proliferation than xenograft GSC, while xenograft GSC had increased invasion and migration compared to human GSC. A simple in vitro spheroid system for GSC provides a superior platform for the development of precision medicine in the treatment of GB.


Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Esferoides Celulares/fisiologia , Antígeno AC133/análise , Animais , Neoplasias Encefálicas/irrigação sanguínea , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Glioblastoma/irrigação sanguínea , Humanos , Masculino , Camundongos , Células-Tronco Neoplásicas/fisiologia , Neovascularização Patológica/etiologia
15.
Adv Exp Med Biol ; 1295: 223-242, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33543462

RESUMO

Nanotechnology is a rapid-growing field with an extreme potential to revolutionize cancer treatments. However, despite the rapid advances, the clinical translation is still scarce. One of the main hurdles contributing for this setback is the lack of reliable in vitro models for preclinical testing capable of predicting the outcomes in an in vivo setting. In fact, the use of 2D monolayers, considered the gold-standard in vitro technique, leads to the creation of misleading data that might not be completely observed in in vivo or clinical setting. Thus, there is the need to use more complex models capable of better mimicking the tumor microenvironment. For that purpose, the development and use of multicellular tumor spheroids, three-dimensional (3D) cell cultures which recapitulate numerous aspects of the tumors, represents an advantageous approach to test the developed anticancer therapies. In this chapter, we identify and discuss the advantages of the use of these 3D cellular models compared to the 2D models and how they can be utilized to study nanoparticle-cancer cell interaction in a more reliable way to predict the treatment outcome in vivo.


Assuntos
Nanopartículas , Neoplasias , Comunicação Celular , Linhagem Celular Tumoral , Humanos , Neoplasias/tratamento farmacológico , Esferoides Celulares , Microambiente Tumoral
16.
Adv Exp Med Biol ; 1295: 243-270, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33543463

RESUMO

The anticancer activity of compounds and nanoparticles is most often determined in the cell monolayer. However, three-dimensional (3D) systems, such as tumor spheroids, are more representing the natural tumor microenvironment. They have been shown to have higher invasiveness and resistance to cytotoxic agents and radiotherapy compared to cells growing in 2D monolayer. Furthermore, to improve the prediction of clinical efficacy of drugs, in the past decades, even more sophisticated systems, such as multicellular 3D cultures, closely representing natural tumor microenvironment have been developed. Those cultures are formed from either cell lines or patient-derived tumor cells. Such models are very attractive and could improve the selection of tested materials for clinical trials avoiding unnecessary expensive tests in vivo. The microenvironment in tumor spheroids is different, and those differences or the interaction between several cell populations may contribute to different tumor response to the treatment. Also, different types of nanoparticles may have different behavior in 3D models, depending on their nature, physicochemical properties, the presence of targeting ligands on the surface, etc. Therefore, it is very important to understand in which cases which type of tumor spheroid is more suitable for testing specific types of nanoparticles, which conditions should be used, and which analytical method should be applied.


Assuntos
Nanopartículas , Esferoides Celulares , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Humanos , Microambiente Tumoral
17.
Adv Exp Med Biol ; 1295: 271-299, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33543464

RESUMO

Multiple studies about tumor biology have revealed the determinant role of the tumor microenvironment in cancer progression, resulting from the dynamic interactions between tumor cells and surrounding stromal cells within the extracellular matrix. This malignant microenvironment highly impacts the efficacy of anticancer nanoparticles by displaying drug resistance mechanisms, as well as intrinsic physical and biochemical barriers, which hamper their intratumoral accumulation and biological activity.Currently, two-dimensional cell cultures are used as the initial screening method in vitro for testing cytotoxic nanocarriers. However, this fails to mimic the tumor heterogeneity, as well as the three-dimensional tumor architecture and pathophysiological barriers, leading to an inaccurate pharmacological evaluation.Biomimetic 3D in vitro tumor models, on the other hand, are emerging as promising tools for more accurately assessing nanoparticle activity, owing to their ability to recapitulate certain features of the tumor microenvironment and thus provide mechanistic insights into nanocarrier intratumoral penetration and diffusion rates.Notwithstanding, in vivo validation of nanomedicines remains irreplaceable at the preclinical stage, and a vast variety of more advanced in vivo tumor models is currently available. Such complex animal models (e.g., genetically engineered mice and patient-derived xenografts) are capable of better predicting nanocarrier clinical efficiency, as they closely resemble the heterogeneity of the human tumor microenvironment.Herein, the development of physiologically more relevant in vitro and in vivo tumor models for the preclinical evaluation of anticancer nanoparticles will be discussed, as well as the current limitations and future challenges in clinical translation.


Assuntos
Antineoplásicos , Nanopartículas , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Nanomedicina , Esferoides Celulares , Microambiente Tumoral
18.
J Vis Exp ; (167)2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33522508

RESUMO

Cancer cachexia (CC) presents itself as a syndrome with multiple manifestations, causing a marked multi-organ metabolic imbalance. Recently, cachectic wasting has been proposed to be stimulated by several inflammatory mediators, which may disrupt the integrative physiology of adipose tissues and other tissues such as the brain and muscle. In this scenario, the tumor can survive at the host's expense. In recent clinical research, the intensity of depletion of the different fat deposits has been negatively correlated with the patient's survival outcome. Studies have also shown that various metabolic disorders can alter white adipose tissue (WAT) remodeling, especially in the early stages of cachexia development. WAT dysfunction resulting from tissue remodeling is a contributor to overall cachexia, with the main modifications in WAT consisting of morpho-functional changes, increased adipocyte lipolysis, accumulation of immune cells, reduction of adipogenesis, changes in progenitor cell population, and the increase of "niches" containing beige/brite cells. To study the various facets of cachexia-induced WAT remodeling, particularly the changes progenitor cells and beige remodeling, two-dimensional (2D) culture has been the first option for in vitro studies. However, this approach does not adequately summarize WAT complexity. Improved assays for the reconstruction of functional AT ex vivo help the comprehension of physiological interactions between the distinct cell populations. This protocol describes an efficient three-dimensional (3D) printing tissue culture system based on magnetic nanoparticles. The protocol is optimized for investigating WAT remodeling induced by cachexia induced factors (CIFs). The results show that a 3D culture is an appropriate tool for studying WAT modeling ex vivo and may be useful for functional screens to identify bioactive molecules for individual adipose cell populations applications and aid the discovery of WAT-based cell anticachectic therapy.


Assuntos
Adipócitos/patologia , Tecido Adiposo Branco/patologia , Caquexia/patologia , Técnicas de Cultura de Células/métodos , Modelos Biológicos , Adipócitos/metabolismo , Animais , Carcinoma Pulmonar de Lewis/patologia , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Humanos , Camundongos Endogâmicos C57BL , Nanopartículas/química , Perilipina-1/metabolismo , Esferoides Celulares/patologia , Células Estromais/patologia , Proteína Desacopladora 1/metabolismo
19.
J Vis Exp ; (167)2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33554962

RESUMO

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.


Assuntos
Neoplasias do Colo/patologia , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/metabolismo , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Enterócitos/efeitos dos fármacos , Enterócitos/patologia , Imunofluorescência , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Coloração e Rotulagem
20.
Int J Nanomedicine ; 16: 789-802, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33568906

RESUMO

Purpose: The aims of this study were to test the feasibility, targeting specificity and anticancer therapeutic efficacy of CendR motif tLyP-1 functionalized at the N-terminal of ferritin for paclitaxel (PTX) delivery. Methods: A tumor homing and penetrating peptide tLyP-1 was fused to the N-terminal of human H chain ferritin (HFtn) to generate a dual-targeting nanoparticle delivery system. PTX molecules were encapsulated into the HFtn nanocage using the disassembly/assembly method by adjusting pHs. Cellular uptake was examined by confocal laser scanning microscopy (CLSM) and flow cytometry. The MTT assay was used to test the cytotoxicity of various PTX-loaded NPs against MDA-MB-231 and SMMC-7721 tumor cells. The wound healing and cell migration assays were conducted to assess the inhibitory effect on cell motility and metastasis. The inhibition effect on the SMMC-7721 tumor spheroids was studied and penetration ability was evaluated by CLSM. The antitumor efficacy of PTX-loaded NPs was assessed in MDA-MB-231 breast cancer xenografted in female BALB/c nude mice. Results: Compared with HFtn-PTX, in vitro studies demonstrated that the tLyP-1-HFtn-PTX displayed enhanced intracellular delivery and better cytotoxicity and anti-invasion ability against both SMMC-7721 and MDA-MB-231 cells. The better penetrability and growth inhibitory effect on SMMC-7721 tumor spheroids were also testified. In vivo distribution and imaging demonstrated that the tLyP-1-HFtn-PTX NPs were selectively accumulated and penetrated at the tumor regions. Verified by the breast cancer cells model in BABL/c nude mice, tLyP-1-HFtn-PTX displayed higher in vivo therapeutic efficacy with lower systemic toxicity. Conclusion: Ferritin decorated with tumor-homing penetration peptide tLyP-1 at the N terminal could deliver PTX specifically inside the cell via receptor-mediated endocytosis with better efficacy. The peptide tLyP-1 which is supposed to work only at the C terminus showed enhanced tumor tissue penetration and antitumor efficacy, demonstrating that it also worked at the N-terminal of HFtn.


Assuntos
Apoferritinas/química , Sistemas de Liberação de Medicamentos , Paclitaxel/administração & dosagem , Peptídeos Cíclicos/química , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Liberação Controlada de Fármacos , Endocitose/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Nanopartículas/ultraestrutura , Paclitaxel/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia , Cicatrização/efeitos dos fármacos
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