Multicellular tumor spheroids as a model for assessing delivery of oligonucleotides in three dimensions.
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ABSTRACT:
SUBMITTER: Carver K
PROVIDER: S-EPMC4027982 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Standard tissue culture has often been a poor model for predicting the efficacy of anti-cancer agents including oligonucleotides. In contrast to the simplicity of monolayer tissue cultures, a tumor mass includes tightly packed tumor cells, tortuous blood vessels, high levels of extracellular matrix, and stromal cells that support the tumor. These complexities pose a challenge for delivering therapeutic agents throughout the tumor, with many drugs limited to cells proximal to the vasculature. Multicellular tumor spheroids are superior to traditional monolayer cell culture for the assessment of cancer drug delivery, since they possess many of the characteristics of metastatic tumor foci. However, homogeneous spheroids comprised solely of tumor cells do not account for some of the key aspects of metastatic tumors, particularly the interaction with host cells such as fibroblasts. Further, homogeneous culture does not allow for the assessment of targeted delivery to tumor versus host cells. Here we have evaluated delivery of targeted and untargeted oligonucleotide nanoconjugates and of oligonucleotide polyplexes in both homogeneous and composite tumor spheroids. We find that inclusion of fibroblasts in the spheroids reduces delivery efficacy of the polyplexes. In contrast, targeted multivalent RGD-oligonucleotide nanoconjugates were able to effectively discriminate between melanoma cells and fibroblasts, thus providing tumor-selective uptake and pharmacological effects.
| S-EPMC4250944 | biostudies-literature
Project description:Tumor spheroids are important model systems due to the capability of capturing in vivo tumor complexity. In this work, the experimental design of metabolomics workflows using three-dimensional multicellular tumor spheroid (3D MTS) models is addressed. Non-scaffold based cultures of the HCT116 colon carcinoma cell line delivered highly reproducible MTSs with regard to size and other key parameters (such as protein content and fraction of viable cells) as a prerequisite. Carefully optimizing the multiple steps of sample preparation, the developed procedure enabled us to probe the metabolome of single MTSs (diameter range 790 ± 22 µm) in a highly repeatable manner at a considerable throughput. The final protocol consisted of rapid washing of the spheroids on the cultivation plate, followed by cold methanol extraction. 13C enriched internal standards, added upon extraction, were key to obtaining the excellent analytical figures of merit. Targeted metabolomics provided absolute concentrations with average biological repeatabilities of <20% probing MTSs individually. In a proof of principle study, MTSs were exposed to two metal-based anticancer drugs, oxaliplatin and the investigational anticancer drug KP1339 (sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]), which exhibit distinctly different modes of action. This difference could be recapitulated in individual metabolic shifts observed from replicate single MTSs. Therefore, biological variation among single spheroids can be assessed using the presented analytical strategy, applicable for in-depth anticancer drug metabolite profiling.
| S-EPMC6950217 | biostudies-literature
Project description:SignificanceThree-dimensional (3D) visualization of multicellular tumor spheroids (MCTS) in fluorescence microscopy can rapidly provide qualitative morphological information about the architecture of these cellular aggregates, which can recapitulate key aspects of their in vivo counterpart.AimThe present work is aimed at overcoming the shallow depth-of-field (DoF) limitation in fluorescence microscopy while achieving 3D visualization of thick biological samples under study.ApproachA custom-built fluorescence microscope with an electrically focus-tunable lens was developed to optically sweep in-depth the structure of MCTS. Acquired multifocus stacks were combined by means of postprocessing algorithms performed in the Fourier domain.ResultsImages with relevant characteristics as extended DoF, stereoscopic pairs as well as reconstructed viewpoints of MCTS were obtained without segmentation of the focused regions or estimation of the depth map. The reconstructed images allowed us to observe the 3D morphology of cell aggregates.ConclusionsComputational multifocus fluorescence microscopy can provide 3D visualization in MCTS. This tool is a promising development in assessing the morphological structure of different cellular aggregates while preserving a robust yet simple optical setup.
| S-EPMC9162503 | biostudies-literature
Project description:Solid tumors, such as neuroblastoma (NB), are associated with a heterogeneous cell environment. Multicellular tumor spheroid (MCTS) cultures have been shown to better mimic growth characteristics of in vivo solid tumors. Because tumor spheroid growth patterns may be quite different from standard two-dimensional culture systems, we sought to compare the protein expression profiles of two- and three-dimensional in vitro NB cultures, i.e., monolayers and MCTS.Human NB cells were grown as both monolayers and spheres. Nuclear and cytosolic proteins were analyzed for differentially secreted proteins by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and selected polypeptides were identified by mass spectrometry (LC-MS/MS).Several metabolic (transketolase, triosephosphate isomerase, pyruvate kinase M1/M2, alpha enolase, and phosphoglycerate mutase-1), cell stress response (heat shock proteins (HSP) 90, 70, and 60; antioxidant, thioredoxin), cell structure (septin 2, adenyl cyclase-associated protein-1), tubulin beta-2 chain, actin, translationally controlled tumor protein and cofilin), signal transduction (peptidyl prolyl cis/trans isomerase A), biosynthetic (phosphoserine aminotransferase) and transport (cellular retinoic acid binding protein 1) polypeptides were overexpressed in spheroids. Several protein groups were differentially expressed between NB monolayers and spheroids.The altered proteins among NB spheroids may represent an important link between monolayer cell cultures and in vivo experiments and thus a more ideal in vitro culture system for determining the precise three-dimensional microenvironment of NB.
| S-EPMC2804865 | biostudies-literature
Project description:Mathematical modeling provides the predictive ability to understand the metabolic reprogramming and complex pathways that mediate cancer cells' proliferation. We present a mathematical model using a multiscale, multicellular approach to simulate avascular tumor growth, applied to pancreatic cancer. The model spans three distinct spatial and temporal scales. At the extracellular level, reaction diffusion equations describe nutrient concentrations over a span of seconds. At the cellular level, a lattice-based energy driven stochastic approach describes cellular phenomena including adhesion, proliferation, viability and cell state transitions, occurring on the timescale of hours. At the sub-cellular level, we incorporate a detailed kinetic model of intracellular metabolite dynamics on the timescale of minutes, which enables the cells to uptake and excrete metabolites and use the metabolites to generate energy and building blocks for cell growth. This is a particularly novel aspect of the model. Certain defined criteria for the concentrations of intracellular metabolites lead to cancer cell growth, proliferation or death. Overall, we model the evolution of the tumor in both time and space. Starting with a cluster of tumor cells, the model produces an avascular tumor that quantitatively and qualitatively mimics experimental measurements of multicellular tumor spheroids. Through our model simulations, we can investigate the response of individual intracellular species under a metabolic perturbation and investigate how that response contributes to the response of the tumor as a whole. The predicted response of intracellular metabolites under various targeted strategies are difficult to resolve with experimental techniques. Thus, the model can give novel predictions as to the response of the tumor as a whole, identifies potential therapies to impede tumor growth, and predicts the effects of those therapeutic strategies. In particular, the model provides quantitative insight into the dynamic reprogramming of tumor cells at the intracellular level in response to specific metabolic perturbations. Overall, the model is a useful framework to study targeted metabolic strategies for inhibiting tumor growth.
| S-EPMC6588258 | biostudies-literature
Project description:Under hypoxic conditions, tumor cells undergo a series of adaptations that promote evolution of a more aggressive tumor phenotype including the activation of DNA damage repair proteins, altered metabolism, and decreased proliferation. Together these changes mitigate the negative impact of oxygen deprivation and allow preservation of genomic integrity and proliferative capacity, thus contributing to tumor growth and metastasis. As a result the presence of a hypoxic microenvironment is considered a negative clinical feature of many solid tumors. Hypoxic niches in tumors also represent a therapeutically privileged environment in which chemo- and radiation therapy is less effective. Although the negative impact of tumor hypoxia has been well established, the precise effect of oxygen deprivation on tumor cell behavior, and the molecular signals that allow a tumor cell to survive in vivo are poorly understood. Multicellular tumor spheroids (MCTS) have been used as an in vitro model for the avascular tumor niche, capable of more accurately recreating tumor genomic profiles and predicting therapeutic response. However, relatively few studies have used MCTS to study the molecular mechanisms driving tumor cell adaptations within the hypoxic tumor environment. Here we will review what is known about cell proliferation, DNA damage repair, and metabolic pathways as modeled in MCTS in comparison to observations made in solid tumors. A more precise definition of the cell populations present within 3D tumor models in vitro could better inform our understanding of the heterogeneity within tumors as well as provide a more representative platform for the testing of therapeutic strategies.
| S-EPMC5543535 | biostudies-other
Project description:During tumor progression, cancer cells rewire their metabolism to face their bioenergetic demands. In recent years, microRNAs (miRNAs) have emerged as regulatory elements that inhibit the translation and stability of crucial mRNAs, some of them causing direct metabolic alterations in cancer. In this study, we investigated the relationship between miRNAs and their targets mRNAs that control metabolism, and how this fine-tuned regulation is diversified depending on the tumor stage. To do so, we implemented a paired analysis of RNA-seq and small RNA-seq in a breast cancer cell line (MCF7). The cell line was cultured in multicellular tumor spheroid (MCTS) and monoculture conditions. For MCTS, we selected two-time points during their development to recapitulate a proliferative and quiescent stage and contrast their miRNA and mRNA expression patterns associated with metabolism. As a result, we identified a set of new direct putative regulatory interactions between miRNAs and metabolic mRNAs representative for proliferative and quiescent stages. Notably, our study allows us to suggest that miR-3143 regulates the carbon metabolism by targeting hexokinase-2. Also, we found that the overexpression of several miRNAs could directly overturn the expression of mRNAs that control glycerophospholipid and N-Glycan metabolism. While this set of miRNAs downregulates their expression in the quiescent stage, the same set is upregulated in proliferative stages. This last finding suggests an additional metabolic switch of the above mentioned metabolic pathways between the quiescent and proliferative stages. Our results contribute to a better understanding of how miRNAs modulate the metabolic landscape in breast cancer MCTS, which eventually will help to design new strategies to mitigate cancer phenotype.
| S-EPMC7793838 | biostudies-literature
Project description:Nanomaterials have been extensively investigated for cancer drug delivery and imaging applications. Nanoparticles that show promise in two-dimensional cell culture systems often fail in more complex environments, possibly due to the lack of penetration in dense, three-dimensional structures. Multicellular tumor spheroids are an emerging model system to investigate interactions of nanoparticles with 3D in vitro cell culture environments. Using the intrinsic near-infrared emission of semiconducting carbon nanotubes to optically reconstruct their localization within a three-dimensional volume, we resolved the relative permeability of two different multicellular tumor spheroids. Nanotube photoluminescence revealed that nanotubes rapidly internalized into MCF-7 breast cancer cell-derived spheroids, whereas they exhibited little penetration into spheroids derived from SK-136, a cell line that we developed from murine liver cancer. Characterization of the spheroids by electron microscopy and immunohistochemistry revealed large differences in the extracellular matrix and interstitial spacing, which correlated directly with nanotube penetration. This platform portends a new approach to characterize the permeability of living multicellular environments.
| S-EPMC4594636 | biostudies-literature
Project description:The multicellular spheroid model partly mimics tumor microenvironments in vivo and has been reported in plenty of studies regarding radiosensitivity. However, clear isolation of quiescent and proliferating cells in live conditions has been quite difficult owing to technical limitations; therefore, comprehensive characterization could not be done thus far. In this study, we succeeded in separately isolating different cell types using a fluorescent ubiquitination-based cell cycle indicator (Fucci) and determining their radiosensitivities. Unexpectedly, proliferating cells were more radioresistant than quiescent cells due to the contact effect when spheroids were disaggregated immediately after irradiation. However, the radiosensitivity of quiescent cells was not influenced by mild hypoxia (hypoxia-inducible factor-1?-positive but pimonidazole-negative), but their radioresistance became similar to that of proliferating cells due to potentially lethal damage repair when disaggregated 24 h after irradiation. The Fucci system further allowed long-term observation of cell kinetics inside of the spheroid following irradiation using real-time confocal fluorescence scanning. Repeated cycles of recruitment from the quiescent to the proliferating phase resulted in cell loss from the outside of the spheroid toward the inside, causing gradual shrinkage. Interestingly, the central region of the spheroid entered a dormant stage approximately 40 days after irradiation and survived for more than 2 months. Using the Fucci system, we were able to comprehensively characterize the radiosensitivity of spheroids for the first time, which highlights the importance of cell cycle kinetics after irradiation in determining the radiosensitivity under tumor microenvironments.
| S-EPMC5406603 | biostudies-literature
Project description:During tumor progression, cancer cells rewire their metabolism to face their bioenergetic demands. In recent years, microRNAs (miRNAs) have emerged as regulatory elements that inhibit the translation and stability of crucial mRNAs, some of them causing direct metabolic alterations in cancer. In this study, we investigated the relationship between miRNAs and their targets mRNAs that control metabolism, and how this fine-tuned regulation is diversified depending on the tumor stage. To do so, we implemented a paired analysis of RNA-seq and small RNA-seq in a breast cancer cell line (MCF7). The cell line was cultured in multicellular tumor spheroid (MCTS) and monoculture conditions. For MCTS, we selected two-time points during their development to recapitulate a proliferative and quiescent stage and contrast their miRNA and mRNA expression patterns associated with metabolism. As a result, we identified a set of new direct putative regulatory interactions between miRNAs and metabolic mRNAs representative for proliferative and quiescent stages. Notably, our study allows us to suggest that miR-3143 regulates the carbon metabolism by targeting hexokinase-2. Also, we found that the overexpression of several miRNAs could directly overturn the expression of mRNAs that control glycerophospholipid and N-Glycan metabolism. While this set of miRNAs downregulates their expression in the quiescent stage, the same set is upregulated in proliferative stages. This last finding suggests an additional metabolic switch of the above mentioned metabolic pathways between the quiescent and proliferative stages. Our results contribute to a better understanding of how miRNAs modulate the metabolic landscape in breast cancer MCTS, which eventually will help to design new strategies to mitigate cancer phenotype.
2020-11-11 | E-MTAB-9741 | biostudies-arrayexpress