Three-dimensional Tumor Cell Culture Research Articles

An effective way for targeting EGFR-mediated carcinogenesis: an in vitro study

Introduction: EGFR-activating overexpression or somatic mutations are common in different human cancers. In this regard, the search for promising ways to control the carcinogenic transformation of tumor cells and the progression of malignant tumors expressing EGFR seems to be one of the most promising and developing areas of modern molecular pathology and pharmacology. Material and Methods: An antitumor activity of a novel compound, a pyridine carboxylic acid derivative LHT-17-19, was studied. The molecule was developed and synthesized at the Department of Chemistry, Drug Design and Technology of All-Russian Research Center for Biological Active Compounds Safety (Russia). The study was carried out in cell cultures of stomach cancer (Hs746T, AGS and MKN1) and patient-derived organoid (PDO) model of breast cancer (BC) expressing wild-type EGFR. Results: It was shown that LHT-17-19 induced concentration-dependent cytotoxicity of EGFR-expressing gastric cancer cells of all the aforementioned cultures. Pathomorphological, immunohistochemical and molecular validation of BC organoids derived from ductal breast carcinoma cells of a 68-year-old patient was done. PDOs were established as ER-negative, PR-negative, Her2/neu-negative, EGFR-positive with 35% of the Ki-67 expression index. In addition, the tumor cells translocation was resulted in a loss of ER expression and PDOs molecular pattern conversion towards a more aggressive triple negative type. PDOs incubation with 0.5-60.0 µM LHT-17-19 was accompanied not only by inhibition of their growth and proliferation, but also by significant cytoreduction. Conclusion: Thus, in two-dimensional and three-dimensional tumor cell cultures, the possibility of controlling the oncogenic expression of EGFR with the acridone compound 9-ammonium-3,3-dimethyl-3,4-dihydroacridine-1(2H)-OH L-2-hydroxybutanedivacate (LHT-17-19) was shown.

THREE-DIMENSIONAL IN VITRO MODELS FOR STUDYING THE CHEMOSENSITIVITY OF BREAST CANCER CELLS

Scientists agree that three-dimensional tumor cell cultures reflect the in vitro biological features of malignant tumors to a greater extent than it is the case with the conventional monolayer cultures. In particular, the results of assessing the effectiveness of anticancer drugs obtained in three-dimensional cell models are considered as more accurate and consistent with the tumor response in vivo, where sensitivity is usually lower than that found in the in vitro experiments. Some of the most developed cellular models are structures obtained using three-dimensional bioprinting, which include not only malignant cells, but also biogels to imitate the chemical and mechanical properties of the extracellular matrix of the tumor.

A novel xenograft model of human hepatocellular carcinoma in immunocompetent mice based on the microcarrier-6

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors that threaten human health; thus, the establishment of an animal model with clinical features similar to human hepatocellular carcinoma is of important practical significance. MethodsTaking advantage of the novel microcarrier-6, human HCC cells were injected into immunocompetent mice to establish a novel human HCC patient-derived xenograft (PDX) model. Primary HCC cells were isolated from fresh hepatocellular carcinoma tissues, which were subsequently co-cultured with microcarrier-6 to construct a three-dimensional tumor cell culture model in vitro. The HCC-microcarrier complex was implanted into mice by subcutaneous inoculation, and the tumor formation time, tumor formation rate, and pathological manifestation were recorded. Changes of immune parameters in mice were detected by flow cytometry. ResultsThe success rate was 60% (6/10) in the establishment of hepatocellular carcinoma PDX mouse model, and the total tumor formation rate of the tumor-forming model is 90–100%. H&E staining and immunohistochemical experiments indicate that the model well retained the characteristics of the primary tumor. Interestingly, M2 macrophages in tumor-bearing mice increased significantly, and the levels of CD4+ T cells were significantly reduced. ConclusionsThrough the application of the microcarrier-6 in immunocompetent mice, we successfully established a novel human HCC PDX model, which can be used to better study and further elucidate the occurrence and pathogenic mechanism of HCC, improve the predictability of toxicity and drug sensitivity in HCC.

Construction of a new patient-derived xenograft model of human liver cancer in mice with normal immunity

Objective To establish a new patient-derived xenograft (PDX) model of human liver cancer by inoculating the complex of human primary liver cancer cells and a novel microcarrier (microcarrier 6) into mice with normal immune function. Methods Primary liver cancer cells were isolated and extracted from the fresh human liver cancer tissue of five patients and were then co-cultured with microcarrier 6 to construct a three-dimensional tumor cell culture model in vitro. According to the type of graft, 75 male C57BL/6 mice were divided into cell control group, microcarrier control group, and experimental group (each sample corresponded to three groups, with 15 groups in total and 5 mice in each group). The liver cancer cell-microcarrier complex was implanted into the mice by subcutaneous inoculation, and tumor formation time, tumor formation rate, and histopathological manifestations were observed. The Fisher's exact test was used for comparison of categorical data between two groups. Results As for the liver cancer cells from the five patients, tumor formation was observed in the mice corresponding to three patients. In these three experiments, tumor formation was not observed in the control groups and was only observed in the experimental groups, and 12 of the 15 mice in the experimental groups had successful tumor formation, with a tumor formation rate as high as 80%, which was significantly different from that in the cell control groups and the microcarrier control groups (all P Conclusion This experiment successfully establishes a new PDX model of human liver cancer based on the complex of microcarrier 6 and human primary liver cancer cells in mice with normal immunity. This model can be used to better elucidate the mechanism of the development and progression of liver cancer in the body with normal immunity, and besides, it also provides a new animal model with higher value for the precise treatment of liver cancer.

Three-Dimensional Tumor Cell Cultures and the Role of Tissue Biorepositories in Personalized Medicine

Three-Dimensional Tumor Cell Cultures and the Role of Tissue Biorepositories in Personalized Medicine

Three-dimensional tumor cell cultures employed in virotherapy research.

Oncolytic virotherapy constitutes an upcoming alternative treatment option for a broad spectrum of cancer entities. However, despite great research efforts, there is still only a single US Food and Drug Administration/European Medicines Agency-approved oncolytic virus available for clinical use. One reason for that is the gap between promising preclinical data and limited clinical success. Since oncolytic viruses are biological agents, they might require more realistic in vitro tumor models than common monolayer tumor cell cultures to provide meaningful predictive preclinical evaluation results. For more realistic invitro tumor models, three-dimensional tumor cell-culture systems can be employed in preclinical virotherapy research. This review provides an overview of spheroid and hydrogel tumor cell cultures, organotypic tumor-tissue slices, organotypic raft cultures, and tumor organoids utilized in the context of oncolytic virotherapy. Furthermore, we also discuss advantages, disadvantages, techniques, and difficulties of these three-dimensional tumor cell-culture systems when applied specifically in virotherapy research.

Comparative proteome analysis of monolayer and spheroid culture of canine osteosarcoma cells

Osteosarcoma is an aggressive bone tumor with high metastasis rate in the lungs and affects both humans and dogs in a similar way. Three-dimensional tumor cell cultures mimic the in vivo situation of micro-tumors and metastases and are therefore better experimental in vitro models than the often applied two-dimensional monolayer cultures. The aim of the present study was to perform comparative proteomics of standard monolayer cultures of canine osteosarcoma cells (D17) and three-dimensional spheroid cultures, to better characterize the 3D model before starting with experiments like migration assays. Using DIGE in combination with MALDI-TOF/TOF we found 27 unique canine proteins differently represented between these two culture systems, most of them being part of a functional network including mainly chaperones, structural proteins, stress-related proteins, proteins of the glycolysis/gluconeogenesis pathway and oxidoreductases. In monolayer cells, a noticeable shift to more acidic pI values was noticed for several proteins of medium to high abundance; two proteins (protein disulfide isomerase A3, stress-induced-phosphoprotein 1) showed an increase of phosphorylated protein species. Protein distribution within the cells, as detected by immunohistochemistry, displayed a switch of stress-induced-phosphoprotein 1 from the cytoplasm (in monolayer cultures) to the nucleus (in spheroid cultures). Additionally, Western blot testing revealed upregulated concentrations of metastasin (S100A4), triosephosphate isomerase 1 and septin 2 in spheroid cultures, in contrast to decreased concentrations of CCT2, a subunit of the T-complex. Results indicate regulation of stress proteins in the process of three-dimensional organization characterized by a hypoxic and nutrient-deficient environment comparable to tumor micro-metastases. SignificanceOsteosarcoma is an aggressive bone tumor that early spreads to the lungs. Three-dimensional tumor cell cultures represent the avascular stage of micro-tumors and metastases, and should therefore represent a better experimental in vitro model compared to two-dimensional monolayer cultures. Significant differences have been reported in response to drug and radiation treatment between these two culture systems. A gel-based proteomic investigation was performed to compare protein patterns of a canine osteosarcoma cell line cultivated under those two conditions, to learn more about altered cell composition and its impact on cell behaviour. Due to the fact that the canine osteosarcoma is an accepted model for the human disease, results will be relevant for the human species as well.

Facile preparation of a novel mulberry silk fibroin scaffold for three-dimensional tumor cell culture

Three-dimensional (3D) culture methods, building a bridge between the traditional two-dimensional (2D) monolayer cell culture and the in-vivo conditions, launch a new era in cell culture. A reliable and stable method for 3D cell culture is required for further cytology research or chemosensitivity tests, considering its high similarity with conditions in vivo. Here we present a novel 3D tumor cell culture model based on mulberry silk fibroin derived from Bombyx mori, which is easy to access in China. This is a biocompatible 3D silk fibroin scaffold, without adding any toxic organic solvents, surfactants or other toxic agents during the preparation process. We investigated its microstructure and confirmed that the gastric cancer cell lines were successfully seeded and grown on the scaffold. In conclusion, the silk fibroin scaffold, as a promising 3D cell culture model, has the potential for more accurate cytology research and clinical chemosensitivity tests in the future.

Three-Dimensional Tumor Cell Cultures and the Role of Tissue Biorepositories in Personalized Medicine

Tissue biorepositories have long been critically important for medical research by providing human tissues for scholarly investigations. In addition to this traditional research role, tissue banks are rapidly assuming new roles in the clinical side of medicine. In the modern era of personalized medicine, tissue biorepositories are increasingly expected to establish close collaborations with clinical and laboratory units and provide banked tissues for a variety of tests that in turn will guide clinical decision making tailored to the patient [1-4]. This new role of biorepositories requires the development of novel complex standard operating procedures (SOPs) for tissue collection, processing and storage that can provide tissues ideal for a variety of novel molecular and other tests [1,3,4].

Abstract 4942: Can three-dimensional cell cultures be used to predict in vivo drug response and synergistic combinations

Abstract The discovery of synergistic combinations of standard-of-care drugs or new chemical or biological entities shows much promise in the treatment of cancer, and there is much interest in this strategy. However, the increasing number of possible combinations makes the task of selecting the best regimens particularly difficult. As a response to this problem, we developed a computerized PK-PD model of a growing tumor, called the Virtual Tumor™. We demonstrated that this platform can successfully simulate the outcome of various drug combination schedules in xenografts, as well as predict optimal drug schedules and combinations. Although xenografts represent a convenient and relatively inexpensive approach to assessing the likely efficacy of proposed dosing regimens in vivo, the number of permutations that can be tested is still limited by practical considerations. We have therefore explored the use of three-dimensional tumor cell cultures (microtissues) as a more cost-effective alternative to xenografts for validating Virtual Tumor™ predictions. Using the Virtual Tumor™, we previously predicted that the efficacy of a gemcitabine-docetaxel combination could vary greatly depending on the scheduling of the drug administration, and verified these findings in vivo in MX-1 xenograft mouse model. We have now conducted a comparable study using MX-1 microtissues, in which the cultures were treated with these same two drugs in isolation or in combination, according to various regimens. Here we show how the microtissue Virtual Tumor™ model can be employed to simulate microtissue growth and response to drug treatment, and the capability of this model to predict drug synergy in vivo in xenografts. Furthermore, we discuss whether microtissues can be used as a surrogate for xenografts, in conjunction with the Virtual Tumor™, for designing new drug regimens, testing possible schedules for combinations of different drugs and prioritizing the most effective drug combinations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4942. doi:1538-7445.AM2012-4942

Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation

BackgroundThere is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed.ResultsTo address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)γ inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease.ConclusionsWe have established and validated a suite of highly reproducible tumor microplate three-dimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds.

Abstract A191: The application of three-dimensional cell cultures in combination with the Virtual Tumor for designing optimal drug dosing schedules.

Abstract There is currently a great deal of interest in determining synergistic drug combinations, however, it is not easy to determine which schedules should be tested, since the number of different possible schedules increases combinatorially when more than one drug is considered. We have therefore developed a predictive PK-PD “Virtual Tumor” model that allows rational design of schedules for drug combinations. We previously built a Virtual Tumor that was capable of successfully simulating the outcome of various drug combination schedules in xenografts; using this model we were also able to propose new optimal administration schemas. Although xenografts represent a convenient and relatively inexpensive approach to assessing the likely efficacy of proposed dosing regimens in vivo, the number of permutations that can be tested is still limited by practical considerations. We have therefore explored the use of three-dimensional tumor cell cultures (microtissues) as a more cost-effective alternative to xenografts for validating Virtual Tumor predictions. Here we present a microtissue Virtual Tumor that is analogous to our xenograft model, and a comparison of the utility of each in simulating and optimising drug dosing schedules. The application of microtissues in combination with the Virtual Tumor technology can be used to design new regimens with proprietary compounds as well as standards of care, small molecules or biotherapeutical agents; help test possible schedules for combinations of different drugs that would be effectively impossible to investigate experimentally; and allow prioritisation of the most effective drug combinations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A191.

Magnetic levitating polymeric nano/microparticular substrates for three-dimensional tumor cell culture

Herein, we describe magnetic cell levitation models using conventional polymeric microparticles or nanoparticles as a substrate for the three-dimensional tumor cell culture. When the magnetic force originating from the ring-shaped magnets overcame the gravitational force, the magnetic field-levitated KB tumor cells adhered to the surface area of magnetic iron oxide (Fe(3)O(4))-encapsulated nano/microparticles and concentrated clusters of levitated cells, ultimately developing tumor cells to tumor spheroids. These simple cell culture models may prove useful for the screening of anticancer drugs and their formulations.