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The dysregulation of pH has been linked to the onset of chronic conditions, such as cancer and neurological diseases. Consequently, the development of a highly sensitive tool for intracellular pH sensing is imperative to investigate the interplay between pH and the biochemical changes accompanying disease pathogenesis. Here, we present the development of a ratiometric fluorescent nanoprobe, NpRhoDot, designed for precisely measuring pH levels. We demonstrate its efficacy in sensitively reporting intracellular pH in monolayer A549 lung cancer cells, primary fibroblast cells, and 3D tumor spheroids derived from the DLD-1 colorectal adenocarcinoma cell line. NpRhoDot leverages a novel design, where stable carbon dots are functionalized with a pH-responsive ratiometric fluorescent probe comprising a naphthalimide-rhodamine moiety, NpRho1. This design confers NpRhoDot with the high pH sensitivity characteristics of organic fluorescent probes, along with excellent photostability up to 1 h and biocompatibility of carbon dots. Through one-photon and two-photon fluorescence microscopy, we validate the reliability of NpRhoDot for biosensing intracellular pH in monolayer and three-dimensional tumor models from pH 4 to 7.
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Carbono , Colorantes Fluorescentes , Puntos Cuánticos , Humanos , Concentración de Iones de Hidrógeno , Carbono/química , Colorantes Fluorescentes/química , Puntos Cuánticos/química , Línea Celular Tumoral , Esferoides Celulares/patología , Células A549 , Rodaminas/químicaRESUMEN
Systemic delivery of oncolytic and immunomodulatory adenoviruses may be required for optimal effects on human malignancies. Mesenchymal stromal cells (MSCs) can serve as delivery systems for cancer therapeutics due to their ability to transport and shield these agents while homing to tumors. We now use MSCs to deliver a clinically validated binary oncolytic and helper-dependent adenovirus combination (CAdVEC) to tumor cells. We show successful oncolysis and helper-dependent virus function in tumor cells even in the presence of plasma from adenovirus-seropositive donors. In both two- and three-dimensional cultures, CAdVEC function is eliminated even at high dilutions of seropositive plasma but is well sustained when CAdVEC is delivered by MSCs. These results provide a robust in vitro model to measure oncolytic and helper-dependent virus spread and demonstrate a beneficial role of using MSCs for systemic delivery of CAdVEC even in the presence of a neutralizing humoral response.
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Three-dimensional (3D) tumor spheroids are regarded as promising models for utilization as preclinical assessments of chemo-sensitivity. However, the creation of these tumor spheroids presents challenges, given that not all tumor cell lines are able to form consistent and regular spheroids. In this context, we have developed a novel layer-by-layer coating of cellulose nanofibril-polyelectrolyte bilayers for the generation of spheroids. This technique builds bilayers of cellulose nanofibrils and polyelectrolytes and is used here to coat two distinct 96-well plate types: nontreated/non-sterilized and Nunclon Delta. In this work, we optimized the protocol aimed at generating and characterizing spheroids on difficult-to-grow pancreatic tumor cell lines. Here, diverse parameters were explored, encompassing the bilayer count (five and ten) and multiple cell-seeding concentrations (10, 100, 200, 500, and 1000 cells per well), using four pancreatic tumor cell lines-KPCT, PANC-1, MiaPaCa-2, and CFPAC-I. The evaluation includes the quantification (number of spheroids, size, and morphology) and proliferation of the produced spheroids, as well as an assessment of their viability. Notably, our findings reveal a significant influence from both the number of bilayers and the plate type used on the successful formation of spheroids. The novel and simple layer-by-layer-based coating method has the potential to offer the large-scale production of spheroids across a spectrum of tumor cell lines.
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Drug combination therapy is a highly effective approach for enhancing the therapeutic efficacy of anti-cancer drugs and overcoming drug resistance. However, the innumerable possible drug combinations make it impractical to screen all synergistic drug pairs. Moreover, biological insights into synergistic drug pairs are still lacking. To address this challenge, we systematically analyzed drug combination datasets curated from multiple databases to identify drug pairs more likely to show synergy. We classified drug pairs based on their MoA and discovered that 110 MoA pairs were significantly enriched in synergy in at least one type of cancer. To improve the accuracy of predicting synergistic effects of drug pairs, we developed a suite of machine learning models that achieve better predictive performance. Unlike most previous methods that were rarely validated by wet-lab experiments, our models were validated using two-dimensional cell lines and three-dimensional tumor slice culture (3D-TSC) models, implying their practical utility. Our prediction and validation results indicated that the combination of the RTK inhibitors Lapatinib and Pazopanib exhibited a strong therapeutic effect in breast cancer by blocking the downstream PI3K/AKT/mTOR signaling pathway. Furthermore, we incorporated molecular features to identify potential biomarkers for synergistic drug pairs, and almost all potential biomarkers found connections between drug targets and corresponding molecular features using protein-protein interaction network. Overall, this study provides valuable insights to complement and guide rational efforts to develop drug combination treatments.
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Antineoplásicos , Neoplasias de la Mama , Humanos , Femenino , Fosfatidilinositol 3-Quinasas , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Transducción de Señal , Neoplasias de la Mama/tratamiento farmacológico , Sistemas de Liberación de MedicamentosRESUMEN
Patient-derived xenografts represent the gold standard in pre-clinical research models. The chicken embryo chorioallantoic membrane (CAM) is used in functional studies for studying biological processes such as blood vessel development and embryogenesis, biocompatible material testing, and more recently three-dimensional patient-derived xenograft (PDX) tumor modeling. We describe here a detailed method used to readily engraft established mouse PDX and primary patient tumor specimens on the CAM with as little as 25 mg of tissue per embryonated egg.
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Pollos , Membrana Corioalantoides , Animales , Embrión de Pollo , Modelos Animales de Enfermedad , Embrión de Mamíferos , Xenoinjertos , Humanos , RatonesRESUMEN
The tumor microenvironment (TME) of oral carcinomas has highly complex contents and a dynamic nature which is difficult to study using oversimplified two-dimensional (2D) cell culture systems. By contrast, three dimensional (3D) in vitro models such as spheroids, organoids, and scaffold-based constructs have been able to replicate tumors three-dimensionality and have allowed a better understanding of the role of various microenvironmental cues in the initiation and progression of cancer. However, the heterogeneity of TME cannot be fully reproduced by these traditional tissue engineering strategies since they are unable to control the organization of multiple cell types in a complex architecture. 3D bioprinting is an emerging field that can be leveraged to produce biomimetic and complex tissue structures. Bioprinting allows for controllable and precise placement of multicomponent bioinks composed of multiple biomaterials, different types of cells, and soluble factors according to the natural compartments of the target tissue, aiming to reproduce the equivalent of the complex tissue. As such, 3D bioprinting provides a unique opportunity to fabricate in vitro tumor models with a complexity similar to that of the in vivo oral carcinoma. This will facilitate a thorough investigation of cellular physiology, cancer progression, and anti-cancer drug screening with unprecedented control and reproducibility. In this review, we discuss the role of 3D bioprinting in reconstituting oral cancer, the prospects of application to fill the literature gap, and the challenges that need to be addressed in order to exploit this emerging technology for future work in oral cancer research.
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BACKGROUND: Cancer has been responsible for a large number of human deaths in the 21st century. Establishing a controllable, biomimetic, and large-scale analytical platform to investigate the tumor-associated pathophysiological and preclinical events, such as oncogenesis and chemotherapy, is necessary. METHODS AND RESULTS: This study presents antitumor investigation in a parallel, large-scale, and tissue-mimicking manner based on well-constructed chemical gradients and heterotypic three-dimensional (3D) tumor cocultures using a multifunction-integrated device. The integrated microfluidic device was engineered to produce a controllable and steady chemical gradient by manipulative optimization. Array-like and size-homogeneous production of heterotypic 3D tumor cocultures with in vivo-like features, including similar tumor-stromal composition and functional phenotypic gradients of metabolic activity and viability, was successfully established. Furthermore, temporal, parallel, and high-throughput analyses of tumor behaviors in different antitumor stimulations were performed in a device based on the integrated operations involving gradient generation and coculture. CONCLUSION: This achievement holds great potential for applications in the establishment of multifunctional tumor platforms to perform tissue-biomimetic neoplastic research and therapy assessment in the fields of oncology, bioengineering, and drug discovery.
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Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas , Biomimética , Línea Celular Tumoral , Técnicas de Cocultivo , HumanosRESUMEN
Osteosarcoma (OS) is a highly aggressive primary bone tumor. The mainstay for its treatment is multiagent chemotherapy and surgical resection, with a 50-70% 5-year survival rate. Despite the huge effort made by clinicians and researchers in the past 30 years, limited progress has been made to improve patient outcomes. As novel therapeutic approaches for OS become available, such as monoclonal antibodies, small molecules, and immunotherapies, the need for OS preclinical model development becomes equally pressing. Three-dimensional (3D) OS models represent an alternative system to study this tumor: In contrast to two-dimensional monolayers, 3D matrices can recapitulate key elements of the tumor microenvironment (TME), such as the cellular interaction with the bone mineralized matrix. The advancement of tissue engineering and biofabrication techniques enables the incorporation of specific TME aspects into 3D models, to investigate the contribution of individual components to tumor progression and enhance understanding of basic OS biology. The use of biomaterials that mimic the extracellular matrix could also facilitate the testing of drugs targeting the TME itself, allowing a larger range of therapeutics to be tested, while averting the ethical implications and high cost associated with in vivo preclinical models. This review aims at serving as a practical guide by delineating the OS TME ("what it is like") and, in turn, propose various biofabrication strategies to create a 3D model ("how to recreate it"), to improve the in vitro representation of the OS tumor and ultimately generate more accurate drug response profiles.
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Neoplasias Óseas , Osteosarcoma , Neoplasias Óseas/terapia , Comunicación Celular , Humanos , Osteosarcoma/terapia , Ingeniería de Tejidos , Microambiente TumoralRESUMEN
Treating systemic metastases at the micrometastatic stage is a potential strategy to inhibit cancer metastasis. This study aims to establish an apoptosis sensor-based platform for rapid, effective, and noninvasive identification of drugs that can inhibit the proliferation of micrometastatic cancer cells. We stably transfected the plasmid DNA encoding the fluorescence resonance energy transfer-based caspase-3 sensor into highly metastatic melanoma B16F10 cells. The resulting B16F10-C3 cells were applied for screening of antiproliferative and proapoptotic drugs in two-dimensional (2D) monolayer, three-dimensional (3D) spheroids, and zebrafish xenotransplantation tumors. All studies were conducted in 96-well plates in a high throughput manner. Fourteen compounds including six chemotherapeutic drugs and eight kinase inhibitors were tested. Thirteen compounds failed the tests due to: Drug resistance, low efficacy, poor pharmacokinetic profile, and/or high side effects to zebrafish. The only compound that passed all tests was pan-phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, which inhibited the proliferation of B16F10-C3 cells in both 2D and 3D cultures. More important, it significantly reduced the xenograft tumor size in zebrafish by decreasing the viability of metastatic cancer cells. Our study suggests that the PI3K/AKT pathway is a potential therapeutic target for the reactivation of tumor dormancy and proliferation of micrometastases. Moreover, this integrated approach is effective for rapid identification of systemic antimetastases drugs.
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Antineoplásicos/aislamiento & purificación , Caspasa 3/análisis , Cromonas/aislamiento & purificación , Evaluación Preclínica de Medicamentos/métodos , Morfolinas/aislamiento & purificación , Metástasis de la Neoplasia/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Apoptosis , Técnicas Biosensibles/métodos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromonas/administración & dosificación , Cromonas/farmacología , Modelos Animales de Enfermedad , Transferencia Resonante de Energía de Fluorescencia , Humanos , Melanoma/tratamiento farmacológico , Melanoma/secundario , Modelos Teóricos , Morfolinas/administración & dosificación , Morfolinas/farmacología , Esferoides Celulares , Factores de Tiempo , Trasplante Heterólogo , Células Tumorales Cultivadas , Pez CebraRESUMEN
Detachment of non-malignant epithelial cells from the extracellullar matrix (ECM) triggers their growth arrest and apoptosis. Conversely, carcinoma cells can grow without adhesion to the ECM. This capacity for anchorage-independent growth is thought to be critical for tumor progression. ErbB2/Her2 oncoprotein is overproduced by a significant fraction of breast cancers and promotes anchorage-independent tumor cell growth by poorly understood mechanisms. In an effort to understand them we found that in order to produce ErbB2, detached breast cancer cells require the activity of an ErbB2 effector protein kinase Mek and that Mek-driven ErbB2 expression is neccesary for anchorage-independent growth of such cells. We observed that Mek inhibition does not alter ErbB2 mRNA levels in detached cancer cells and that ErbB2 protein loss induced by this inhibition can be blocked by a lysosomal inhibitor. We also noticed that an increase of the density of cancer cells detached from the ECM downregulates a Mek effector protein kinase Erk and causes ErbB2 loss. Those cells that survive after ErbB2 loss display resistance to trastuzumab, an anti-ErbB2 antibody used for ErbB2-positive breast cancer treatment. Thus, Mek-induced ErbB2 stabilization in detached breast cancer cells is critical for their ability to grow anchorage-independently and their trastuzumab sensitivity.
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In cancer biology, the architectural concept "form follows function" is reflected by cell morphology, migration, and epithelial-mesenchymal transition protein pattern. In vivo, features of epithelial-mesenchymal transition have been associated with tumor budding, which correlates significantly with patient outcome. Hereby, the majority of tumor buds are not truly detached but still connected to a major tumor mass. For detailed insights into the different tumor bud types and the process of tumor budding, we quantified tumor cells according to histomorphological and immunohistological epithelial-mesenchymal transition characteristics. Three-dimensional reconstruction from adenocarcinomas (pancreatic, colorectal, lung, and ductal breast cancers) was performed as published. Tumor cell morphology and epithelial-mesenchymal transition characteristics (represented by zinc finger E-box-binding homeobox 1 and E-Cadherin) were analyzed qualitatively and quantitatively in a three-dimensional context. Tumor buds were classified into main tumor mass, connected tumor bud, and isolated tumor bud. Cell morphology and epithelial-mesenchymal transition marker expression were assessed for each tumor cell. Epithelial-mesenchymal transition characteristics between isolated tumor bud and connected tumor bud demonstrated no significant differences or trends. Tumor cell count correlated significantly with epithelial-mesenchymal transition and histomorphological characteristics. Regression curve analysis revealed initially a loss of membranous E-Cadherin, followed by expression of cytoplasmic E-Cadherin and subsequent expression of nuclear zinc finger E-box-binding homeobox 1. Morphologic changes followed later in this sequence. Our data demonstrate that connected and isolated tumor buds are equal concerning immunohistochemical epithelial-mesenchymal transition characteristics and histomorphology. Our data also give an insight in the process of tumor budding. While there is a notion that the epithelial-mesenchymal transition zinc finger E-box-binding homeobox 1-E-Cadherin cascade is initiated by zinc finger E-box-binding homeobox 1, our results are contrary and outline other possible pathways influencing the regulation of E-Cadherin.
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Adenocarcinoma/genética , Cadherinas/biosíntesis , Transición Epitelial-Mesenquimal/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/biosíntesis , Adenocarcinoma/patología , Cadherinas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Análisis de Regresión , Transducción de Señal/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genéticaRESUMEN
Cancer is a serious death causing disease having 8.2 million deaths in 2012. In the last decade, only about 10% of chemotherapeutic compounds showed productivity in drug screening. Two-dimensional culture assays are the most common in vitro drug screening models, which do not precisely model the in vivo condition for reliable preclinical drug screening. Three-dimensional scaffold-based cell cultures perhaps mimic tumor microenvironment and recapitulate physiologically more relevant tumor. This study was carried out to develop bi-functional oxidized dextran-based cell instructive hydrogel that provides three-dimensional environment to cancer cells for inducing microtumor. Oxidized dextran was blended with thiolated chitosan to fabricate an in situ self-gelable hydrogel (modified dextran-chitosan) in a one-step process. The hydrogels characterization revealed cross-linked network structure with highly porous structure and water absorption. The modified dextran-chitosan hydrogel showed reduced hydrophobicity and has reduced protein absorption, which resulted in changing the A549 cell adhesiveness, and encouraged them to form microtumor. The cells were proliferated in clusters having spherical morphology with randomly oriented stress fiber and large nucleus. Further microtumors were studied for hypoxia where reactive oxygen species generation demonstrated 15-fold increase as compared to monolayer culture. Drug-sensitivity results showed that microtumors generated on modified dextran-chitosan hydrogel showed resistance to doxorubicin with having 33%-58% increased growth than two-dimensional monolayer model at concentrations of 25-100 µM. In summary, the modified dextran-chitosan scaffold can provide surface chemistry that induces three-dimensional microtumors with physiologically relevant properties to in vivo tumor including growth, morphology, extracellular matrix production, hypoxic phenotype, and drug response. This model can be potentially utilized for drug toxicity studies and cancer disease modeling to understand tumor phenotype and progression.
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We investigated the effect of anticancer drug-loaded functional polymeric nanoparticles on drug resistance of three-dimensional (3D) breast tumor spheroids. 3D tumor models were built using concave microwells with different diameters (300-700µm) and nanoparticles were prepared using thermo-responsive poly(N-isopropylacrylamide) (PNIPAM)-co-acrylic acid (AA). Upon culturing with doxorubicin-loaded PNIPAM-co-AA nanoparticles for 96hours, the smallest tumor spheroids were extensively disrupted, resulting in a reduction in spheroid diameter. In contrast, the sizes of the largest tumor spheroids were not changed. Scanning electron microscopy revealed that the circular shape of 3D spheroids treated with doxorubicin-loaded PNIPAM-co-AA nanoparticles had collapsed severely. Cell viability assays also demonstrated that the largest tumor spheroids cultured with doxorubicin-loaded PNIPAM-co-AA nanoparticles were highly resistant to the anticancer drug. We confirmed that tight cell-cell contacts within largest tumor spheroids significantly improved the anticancer drug resistance. Therefore, this uniform-sized 3D breast tumor model could be a potentially powerful tool for anticancer drug screening applications. FROM THE CLINICAL EDITOR: The battle against cancer is a big challenge. With new anti-cancer drugs being developed under the nanotechnology platform, there is a need to have a consistent and reliable testing system that mimics the in-vivo tumor scenario. The authors successfully designed a 3D tumor model using concave microwells to produce different tumor diameters. This will be of value for future drug screening.
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Acrilatos/química , Resinas Acrílicas/química , Antibióticos Antineoplásicos/administración & dosificación , Técnicas de Cultivo de Célula/métodos , Doxorrubicina/administración & dosificación , Ensayos de Selección de Medicamentos Antitumorales/métodos , Nanopartículas/química , Antibióticos Antineoplásicos/farmacología , Antineoplásicos , Mama/efectos de los fármacos , Mama/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Técnicas de Cultivo de Célula/instrumentación , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales/instrumentación , Diseño de Equipo , Femenino , Humanos , Células MCF-7 , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
Physiologically relevant in vitro models can serve as biological analytical platforms for testing novel treatments and drug delivery systems. We describe the first steps in the development of a 3D human brain tumour co-culture model that includes the interplay between normal and tumour tissue along with nutrient gradients, cell-cell and cell-matrix interactions. The human medulloblastoma cell line UW228-3 and human foetal brain tissue were marked with two supravital fluorescent dyes (CDCFDASE, Celltrace Violet) and cultured together in ultra-low attachment 96-well plates to form reproducible single co-culture spheroids (d = 600 µm, CV% = 10%). Spheroids were treated with model cytotoxic drug etoposide (0.3-100 µM) and the viability of normal and tumour tissue quantified separately using flow cytometry and multiphoton microscopy. Etoposide levels of 10 µM were found to maximise toxicity to tumours (6.5% viability) while stem cells maintained a surviving fraction of 40%. The flexible cell marking procedure and high-throughput compatible protocol make this platform highly transferable to other cell types, primary tissues and personalised screening programs. The model's key anticipated use is for screening and assessment of drug delivery strategies to target brain tumours, and is ready for further developments, e.g. differentiation of stem cells to a range of cell types and more extensive biological validation.