Multicellular Cancer Spheroids Research Articles

Modular Drug-Loaded Nanocapsules with Metal Dome Layers as a Platform for Obtaining Synergistic Therapeutic Biological Activities.

Multifunctional drug-loaded polymer-metal nanocapsules have attracted increasing attention in drug delivery due to their multifunctional potential endowed by drug activity and response to physicochemical stimuli. Current chemical synthesis methods of polymer/metal capsules require specific optimization of the different components to produce particles with precise properties, being particularly complex for Janus structures combining polymers and ferromagnetic and highly reactive metals. With the aim to generate tunable synergistic nanotherapeutic actuation with enhanced drug effects, here we demonstrate a versatile hybrid chemical/physical fabrication strategy to incorporate different functional metals with tailored magnetic, optical, or chemical properties on solid drug-loaded polymer nanoparticles. As archetypical examples, we present poly(lactic-co-glycolic acid) (PLGA) nanoparticles (diameters 100-150 nm) loaded with paclitaxel, indocyanine green, or erythromycin that are half-capped by either Fe, Au, or Cu layers, respectively, with application in three biomedical models. The Fe coating on paclitaxel-loaded nanocapsules permitted efficient magnetic enhancement of the cancer spheroid assembly, with 40% reduction of the cross-section area after 24 h, as well as a higher paclitaxel effect. In addition, the Fe-PLGA nanocapsules enabled external contactless manipulation of multicellular cancer spheroids with a speed of 150 μm/s. The Au-coated and indocyanine green-loaded nanocapsules demonstrated theranostic potential and enhanced anticancer activity in vitro and in vivo due to noninvasive fluorescence imaging with long penetration near-infrared (NIR) light and simultaneous photothermal-photodynamic actuation, showing a 3.5-fold reduction in the tumor volume growth with only 5 min of NIR illumination. Finally, the Cu-coated erythromycin-loaded nanocapsules exhibited enhanced antibacterial activity with a 2.5-fold reduction in the MIC50 concentration with respect to the free or encapsulated drug. Altogether, this technology can extend a nearly unlimited combination of metals, polymers, and drugs, thus enabling the integration of magnetic, optical, and electrochemical properties in drug-loaded nanoparticles to externally control and improve a wide range of biomedical applications.

Role of pelitinib in the regulation of migration and invasion of hepatocellular carcinoma cells via inhibition of Twist1

BackgroundOverexpression of Twist1, one of the epithelial-mesenchymal transition-transcription factors (EMT-TFs), is associated with hepatocellular carcinoma (HCC) metastasis. Pelitinib is known to be an irreversible epidermal growth factor receptor tyrosine kinase inhibitor that is used in clinical trials for colorectal and lung cancers, but the role of pelitinib in cancer metastasis has not been studied. This study aimed to investigate the anti-migration and anti-invasion activities of pelitinib in HCC cell lines.MethodsUsing three HCC cell lines (Huh7, Hep3B, and SNU449 cells), the effects of pelitinib on cell cytotoxicity, invasion, and migration were determined by cell viability, wound healing, transwell invasion, and spheroid invasion assays. The activities of MMP-2 and -9 were examined through gelatin zymography. Through immunoblotting analyses, the expression levels of EMT-TFs (Snail1, Twist1, and ZEB1) and EMT-related signaling pathways such as mitogen-activated protein kinases (MAPKs) and Akt signaling pathways were measured. The activity and expression levels of target genes were analyzed by reporter assay, RT-PCR, quantitative RT-PCR, and immunoblotting analysis. Statistical analysis was performed using one-way ANOVA with Dunnett's Multiple comparison tests in Prism 3.0 to assess differences between experimental conditions.ResultsIn this study, pelitinib treatment significantly inhibited wound closure in various HCC cell lines, including Huh7, Hep3B, and SNU449. Additionally, pelitinib was found to inhibit multicellular cancer spheroid invasion and metalloprotease activities in Huh7 cells. Further investigation revealed that pelitinib treatment inhibited the migration and invasion of Huh7 cells by inducing Twist1 degradation through the inhibition of MAPK and Akt signaling pathways. We also confirmed that the inhibition of cell motility by Twist1 siRNA was similar to that observed in pelitinib-treated group. Furthermore, pelitinib treatment regulated the expression of target genes associated with EMT, as demonstrated by the upregulation of E-cadherin and downregulation of N-cadherin.ConclusionBased on our novel finding of pelitinib from the perspective of EMT, pelitinib has the ability to inhibit EMT activity of HCC cells via inhibition of Twist1, and this may be the potential mechanism of pelitinib on the suppression of migration and invasion of HCC cells. Therefore, pelitinib could be developed as a potential anti-cancer drug for HCC.

Planar Waveguide NMR Gradients (B0 and B1) for spatiotemporal Investigations of Anticancer Drug Concentration in 3D Cancer Cell Cultures

Despite the progression in cancer therapeutic approaches, cancer is still the leading cause of death worldwide with a steadily increasing incidence rate. In Jordan, CRC is the most common type of cancer among men and the second most common among women, while being one of the most accruing cancers in Germany.
 The difficulties in cancer treatments arise from the poor correlation between in vivo and in in vitro anti-cancer drug testing results and cancer cells resistance to chemotherapy, which are attributed to the heterogeneity of cancer cells, and the interaction of cancer cells with non-cancerous cells in the tumor microenvironment (TME). In addition to, the several mechanisms, that governs DNA damages repair, epigenetics, epithelial to mesenchymal transition (EMT).
 Cell culture techniques have been developed to construct in vitro multicellular cancer spheroids (MTCS) that mimic cell-cells interaction and the cellular organization in the TME. They also illustrate the proliferation gradient of solid tumors, and the nutrition and gas supply to cancer cells at different depth in the spheroid. Therefore, they are considered an excellent in vitro cancer model to study the efficiency and to investigate the penetration depth of the anti-cancer materials.
 Nuclear Magnetic Resonance (NMR) spectroscopy is a reliable analytical method that gives comprehensive and rich chemical information, along with high speciation performance. It is one of the major noninvasive tools for metabolomics, therefore it has been utilized to study the metabolic profiling of freshly isolated- non-destructed tissues, and living in vitro cellular models.
 A micro-imaging technique for real-time NMR investigations is developed to study the penetration depth, rate, and effective diffusion coefficients of anti-cancer materials through cancer spheroids. This technique is based on our patented invention of planar waveguide micro slot NMR detector with on board thermal regulator integrated and a microfluidic device. Based on this innovative development, we are able to perform real-time magnetic resonance spectroscopic imaging (MRSI) on a living synthetic tissue constructed in micrometer scale to monitor drug penetration and diffusion, while providing spectroscopic information about the production and degradation of metabolites in picomoles concentrations.

Electron microscopy imaging and mechanical characterization of T47D multicellular tumor spheroids-Older spheroids reduce interstitial space and become stiffer.

Multicellular cancer spheroids are an in vitro tissue model that mimics the three-dimensional microenvironment. As spheroids grow, they develop the gradients of oxygen, nutrients, and catabolites, affecting crucial tumor characteristics such as proliferation and treatment responses. The measurement of spheroid stiffness provides a quantitative measure to evaluate such structural changes over time. In this report, we measured the stiffness of size-matched day 5 and day 20 tumor spheroids using a custom-built microscale force sensor and conducted transmission electron microscopy (TEM) imaging to compare the internal structures. We found that older spheroids reduce interstitial spaces in the core region and became significantly stiffer. The measured elastic moduli were 260±100 and 680±150 Pa, for day 5 and day 20 spheroids, respectively. The day 20 spheroids showed an optically dark region in the center. Analyzing the high-resolution TEM images of spheroid middle sections across the diameter showed that the cells in the inner region of the day 20 spheroids are significantly larger and more closely packed than those in the outer regions. On the other hand, the day 5 spheroids did not show a significant difference between the inner and outer regions. The observed reduction of the interstitial space may be one factor that contributes to stiffer older spheroids.

Autonomous Microlasers for Profiling Extracellular Vesicles from Cancer Spheroids

Self-propelled micro/nanomotors are emergent intelligent sensors for analyzing extracellular biomarkers in circulating biological fluids. Conventional luminescent motors are often masked by a highly dynamic and scattered environment, creating challenges to characterize biomarkers or subtle binding dynamics. Here we introduce a strategy to amplify subtle signals by coupling strong light-matter interactions on micromotors. A smart whispering-gallery-mode microlaser that can self-propel and analyze extracellular biomarkers is demonstrated through a liquid crystal microdroplet. Lasing spectral responses induced by cavity energy transfer were employed to reflect the abundance of protein biomarkers, generating exclusive molecular labels for cellular profiling of exosomes derived from 3D multicellular cancer spheroids. Finally, a microfluidic biosystem with different tumor-derived exosomes was employed to elaborate its sensing capability in complex environments. The proposed autonomous microlaser exhibits a promising method for both fundamental biological science and applications in drug screening, phenotyping, and organ-on-chip applications.

Core–Shell Spheroid‐Laden Microgels Crosslinked under Biocompatible Conditions for Probing Cancer‐Stromal Communication

Multicellular cancer spheroids (MCSs) have emerged as a promising in vitro model that recaptures many features of solid tumours in vivo. To generate cancer spheroids, cells are encapsulated in microgels with high throughput. While the biophysical properties of the cancer spheroid and biomaterial influence the function and behavior of the cells, characterization of these properties remains largely unexplored. In addition, many existing techniques lack control over cell positioning, resulting in the formation of spheroids with large variability. Herein, a versatile, microfluidic platform for generating biocompatible core–shell microgels with uniform cancer spheroids is reported. In addition, an in situ micromechanics measuring device to determine the stiffness of individual artificial cancer niches is used. The power of the microfluidics‐based method by making MCS‐laden microgels to model the interactions of stromal cancer cells is demonstrated. Together with in vivo data, it is shown that tumor cell proliferation is promoted by cocultured fibroblasts.

In Vitro Characterization of 177Lu-DOTA-M5A Anti-Carcinoembryonic Antigen Humanized Antibody and HSP90 Inhibition for Potentiated Radioimmunotherapy of Colorectal Cancer.

Carcinoembryonic antigen (CEA) is an antigen that is highly expressed in colorectal cancers and widely used as a tumor marker. 131I and 90Y-radiolabeled anti-CEA monoclonal antibodies (mAbs) have previously been assessed for radioimmunotherapy in early clinical trials with promising results. Moreover, the heat shock protein 90 inhibitor onalespib has previously demonstrated radiotherapy potentiation effects in vivo. In the present study, a 177Lu-radiolabeled anti-CEA hT84.66-M5A mAb (M5A) conjugate was developed and the potential therapeutic effects of 177Lu-DOTA-M5A and/or onalespib were investigated. The 177Lu radiolabeling of M5A was first optimized and characterized. Binding specificity and affinity of the conjugate were then evaluated in a panel of gastrointestinal cancer cell lines. The effects on spheroid growth and cell viability, as well as molecular effects from treatments, were then assessed in several three-dimensional (3D) multicellular colorectal cancer spheroid models. Stable and reproducible radiolabeling was obtained, with labeling yields above 92%, and stability was retained at least 48 h post-radiolabeling. Antigen-specific binding of the radiolabeled conjugate was demonstrated on all CEA-positive cell lines. Dose-dependent therapeutic effects of both 177Lu-DOTA-M5A and onalespib were demonstrated in the spheroid models. Moreover, effects were potentiated in several dose combinations, where spheroid sizes and viabilities were significantly decreased compared to the corresponding monotherapies. For example, the combination treatment with 350 nM onalespib and 20 kBq 177Lu-DOTA-M5A resulted in 2.5 and 2.3 times smaller spheroids at the experimental endpoint than the corresponding monotreatments in the SNU1544 spheroid model. Synergistic effects were demonstrated in several of the more effective combinations. Molecular assessments validated the therapy results and displayed increased apoptosis in several combination treatments. In conclusion, the combination therapy of anti-CEA 177Lu-DOTA-M5A and onalespib showed enhanced therapeutic effects over the individual monotherapies for the potential treatment of colorectal cancer. Further in vitro and in vivo studies are warranted to confirm the current study findings.

Evaluation of Anticancer Effects of Curcumin on Multicellular Breast Cancer Spheroids

Evaluation of Anticancer Effects of Curcumin on Multicellular Breast Cancer Spheroids

Biomedical Applications of Non-Small Cell Lung Cancer Spheroids.

Lung malignancies accounted for 11% of cancers worldwide in 2020 and remained the leading cause of cancer deaths. About 80% of lung cancers belong to non-small cell lung cancer (NSCLC), which is characterized by extremely high clonal and morphological heterogeneity of tumors and development of multidrug resistance. The improvement of current therapeutic strategies includes several directions. First, increasing knowledge in cancer biology results in better understanding of the mechanisms underlying malignant transformation, alterations in signal transduction, and crosstalk between cancer cells and the tumor microenvironment, including immune cells. In turn, it leads to the discovery of important molecular targets in cancer development, which might be affected pharmaceutically. The second direction focuses on the screening of novel drug candidates, synthetic or from natural sources. Finally, “personalization” of a therapeutic strategy enables maximal damage to the tumor of a patient. The personalization of treatment can be based on the drug screening performed using patient-derived tumor xenografts or in vitro patient-derived cell models. 3D multicellular cancer spheroids, generated from cancer cell lines or tumor-isolated cells, seem to be a helpful tool for the improvement of current NSCLC therapies. Spheroids are used as a tumor-mimicking in vitro model for screening of novel drugs, analysis of intercellular interactions, and oncogenic cell signaling. Moreover, several studies with tumor-derived spheroids suggest this model for the choice of “personalized” therapy. Here we aim to give an overview of the different applications of NSCLC spheroids and discuss the potential contribution of the spheroid model to the development of anticancer strategies.

Morphological Response in Cancer Spheroids for Screening Photodynamic Therapy Parameters.

Photodynamic therapy (PDT) is a treatment which uses light-activated compounds to produce reactive oxygen species, leading to membrane damage and cell death. Multicellular cancer spheroids are a preferable alternative for PDT evaluation in comparison to monolayer cell cultures due to their ability to better mimic in vivo avascular tumour characteristics such as hypoxia and cell-cell interactions, low cost, and ease of production. However, inconsistent growth kinetics and drug responsiveness causes poor experimental reproducibility and limits their usefulness. Herein, we used image analysis to establish a link between human melanoma C8161 spheroid morphology and drug responsiveness. Spheroids were pre-selected based on sphericity, area, and diameter, reducing variation in experimental groups before treatment. Spheroid morphology after PDT was analyzed using AnaSP and ReViSP, MATLAB-based open-source software, obtaining nine different parameters. Spheroids displayed a linear response between biological assays and morphology, with area (R2 = 0.7219) and volume (R2 = 0.6138) showing the best fit. Sphericity, convexity, and solidity were confirmed as poor standalone indicators of spheroid viability. Our results indicate spheroid morphometric parameters can be used to accurately screen inefficient treatment combinations of novel compounds.

Recent Advances in Multicellular Tumor Spheroid Generation for Drug Screening

Multicellular tumor spheroids (MCTs) have been employed in biomedical fields owing to their advantage in designing a three-dimensional (3D) solid tumor model. For controlling multicellular cancer spheroids, mimicking the tumor extracellular matrix (ECM) microenvironment is important to understand cell–cell and cell–matrix interactions. In drug cytotoxicity assessments, MCTs provide better mimicry of conventional solid tumors that can precisely represent anticancer drug candidates’ effects. To generate incubate multicellular spheroids, researchers have developed several 3D multicellular spheroid culture technologies to establish a research background and a platform using tumor modelingvia advanced materials science, and biosensing techniques for drug-screening. In application, drug screening was performed in both invasive and non-invasive manners, according to their impact on the spheroids. Here, we review the trend of 3D spheroid culture technology and culture platforms, and their combination with various biosensing techniques for drug screening in the biomedical field.

A Spheroid-Forming Hybrid Gold Nanostructure Platform That Electrochemically Detects Anticancer Effects of Curcumin in a Multicellular Brain Cancer Model.

In this study, a multifunctional platform that enables the highly efficient formation of 3D multicellular cancer spheroids and precise real-time assessments of the anticancer effects of curcumin in a brain tumor coculture model is reported. A highly conductive gold nanostructure (HCGN) is fabricated to facilitate cancer spheroid formation without using anti-cell adhesion molecules. A neuroblastoma (SH-SY5Y) and glioblastoma (U-87MG) coculture model is generated on HCGN with a specific cell-to-cell ratio (SH-SY5Y: U-87MG = 1:1), and their redox behaviors are successfully measured without destroying the distinct 3D structure of the multicellular spheroids. Using electrochemical signals as an indicator of spheroid viability, the effects of potential anticancer compounds on cocultured spheroids are further assessed. Remarkably, decreased cell viability in 3D spheroids caused by a low concentration of curcumin (30µM) is detectable using the electrochemical method (29.4%) but not with a conventional colorimetric assay (CCK-8). The detection is repeated more than ten times for both short- (63 h) and long-term cultivation (144 h) without damaging the spheroids, enabling real-time, non-destructive pharmacokinetic analysis of various drug candidates. Therefore, it can be concluded that the hybrid platform is a highly promising, precise, and high-throughput drug screening tool based on 3D cell cultivation.

Cytotoxicity of multicellular cancer spheroids, antibacterial, and antifungal of selected sulfonamide derivatives coupled with a salicylamide and/or anisamide scaffold

In an attempt to overcome the drawbacks of the cancer monolayers model (2D), the 3-dimensional (3D) multicellular cancer spheroids (MCS) have been developed. Nine of most active sulfonamide derivatives coupled with a salicylamide scaffold were screened for cytotoxicity on two human cancer cell line spheroids (MCF7 and HCT116) in addition to one human normal cell line spheroid (RPE-1). 5-Chloro-N-[(N-4-chlorophenyl) 4-sulfamoylbenzyl] salicylamide (9) was found to be the most active compound among all tested compounds. It showed 70% inhibition on HCT116 spheroids, almost double the activity of cisplatin, and higher activity than cisplatin on MCF7 spheroids. Also, 5-chloro-N-[(N-benzyl) 4-sulfamoylbenzyl] salicylamide (18) and 5-chloro-N-[(N-2-phenylethyl) 4-sulfamoylbenzyl] salicylamide (19) showed cytotoxicity against HCT116 slightly lower than that of cisplatin (32% and 31%, respectively) but with much lower cytotoxicity against the normal cell (4% and 10% vs. 39%, respectively). Based on in silico virtual screening against DHPS enzyme, some sulfonamide derivatives coupled with a salicylamide and/or anisamide scaffold were tested in vitro against four bacterial and fungal pathogens. 5-Chloro-N-[(N-2-nitro-4-methylphenyl) 4-sulfamoylbenzyl] salicylamide (17) and 5-chloro-N-[(N-2-nitro-4-methylphenyl) 4-sulfamoylbenzyl] anisamide (5) showed strong antifungal activity on the tested organism, while the first one (17) have the strongest antibacterial activity against the G +ve and G −ve bacterium. In vitro, dihydropterate synthase (DHPS) enzyme assay showed that, compounds 5 and 17 effectively inhibit dihydropterate synthase (DHPS) enzyme by 93.78% and 95.15%, respectively, while miconazole inhibit the enzyme with only 87.50%. In addition, their effect upon amylase, lipase and protease enzymes was reported. The most active compounds 5, 9, 17–19 could be subjected to in vivo investigation as new drugs.

Identification of a novel anticancer compound through screening of a drug library on multicellular spheroids

Background and objectives A multicellular cancer spheroid model has proven to mimic the in-vivo tumors more closely compared with the conventionally used monolayer model. Thus, the spheroid model estimates the in-vivo activity more accurately than its counterpart the monolayer model. Accordingly, a library of 320 chemically diverse compounds was screened for their cytotoxicity against MCF7 human breast carcinoma spheroids, aiming for identification of novel compounds active against this type of solid tumor. Materials and methods MCF7 spheroids were generated in 96-well plates by a centrifugation method. The spheroids took 5 days to reach ∼500-μm diameter and were ready for treatment. The initial screen was performed at 50 μM in triplicates. A dose–response study followed the initial screen. A counterscreen was carried out using RPE1 normal cell spheroids to identify the selectivity of active compounds. The acid phosphatase method was applied to measure the cytotoxicity of compounds. A clonogenic assay was used to investigate the viability of remaining cells after treatment with test compounds. Results and conclusion The compound (4,5-dibromo-6-oxo-1(6H)-pyridazinyl)methyl 3-chlorophenylcarbamate was identified in this study for the first time with reasonable toxicity on MCF7 cancer spheroids. This compound is suggested as a lead compound for the development of more active derivatives against solid tumors. Additionally, the multicellular spheroid model was proved as a useful and applicable platform for identification of novel compounds for the treatment of solid tumors.

SpheroidSim—Preliminary evaluation of a new computational tool to predict the influence of cell cycle time and phase fraction on spheroid growth

There is a relative paucity of research that integrates materials science and bioengineering with computational simulations to decipher the intricate processes promoting cancer progression. Therefore, a first-generation computational model, SpheroidSim, was developed that includes a biological data set derived from a bioengineered spheroid model to obtain a quantitative description of cell kinetics. SpheroidSim is a 3D agent-based model simulating the growth of multicellular cancer spheroids. Cell cycle time and phases mathematically motivated the population growth. SpheroidSim simulated the growth dynamics of multiple spheroids by individually defining a collection of specific phenotypic traits and characteristics for each cell. Experimental data derived from a hydrogel-based spheroid model were fit to the predictions providing insight into the influence of cell cycle time (CCT) and cell phase fraction (CPF) on the cell population. A comparison of the number of active cells predicted for each analysis showed that the value and method used to define CCT had a greater effect on the predicted cell population than CPF. The model predictions were similar to the experimental results for the number of cells, with the predicted total number of cells varying by 8% and 12%, respectively, compared to the experimental data. SpheroidSim is a first step in developing a biologically based predictive tool capable of revealing fundamental elements in cancer cell physiology. This computational model may be applied to study the effect of the microenvironment on spheroid growth and other cancer cell types that demonstrate a similar multicellular clustering behavior as the population develops. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1335-1343, 2018.

Abstract 5022: Precision medicine: High-throughput 3D bioprinting of embedded multicellular cancer spheroids

Abstract Three-dimensional (3D) cancer cell models, such as 3D multicellular spheroids, are superior models of in vivo systems than the more popular two-dimensional (2D) cell culture. 3D cancer spheroids provide a closer reflection of tumor gene expression and biology than 2D cultures. This is particularly relevant for drug development and precision medicine programs were cell culture conditions that better reflect the 3D tumor environments, including matrix stiffness, would be highly advantageous. A key challenge for the routine application of embedded 3D cancer spheroids in precision and drug programs is the lack of high throughput and uniform production of spheroids in a biocompatible matrix. To address this, we have developed a high-throughput method of producing 3D multicellular cancer spheroids embedded inside a tissue-like matrix using a custom-built drop-on-demand 3D bioprinter. The 3D bioprinter is capable of printing a cell-suspension ink with up to 300 million cells/mL and a cell viability greater than 95%. We have validated the 3D bioprinting using glioblastoma, neuroblastoma and lung cancer cells. The 3D bioprinted spheroids were confirmed to maintain all the biological characteristics typically found in a cancer spheroid. In particular, the 3D bioprinted cancer spheroids were shown to be viable and proliferating, preserved the apoptotic and cancer-stemness characteristics and using super-resolution lattice-light sheet microscopy, we showed the spheroids maintained the same structural integrity as manually formed spheroids. The potential application of the 3D bioprinted spheroids for high-throughput drug screening in 3D environments was demonstrated using doxorubicin as the model drug. 3D-bioprinting of patient-derived cancer cells for precision medicine applications is now underway. High-throughput embedded 3D bioprinting has enormous potential to accelerate precision medicine, drug discovery and cancer biology. Citation Format: Lakmali Atapattu, Robert Utama, Aiden O'Mahony, Christopher Fife, Jongho Baek, Théophile Allard, Kieran O'Mahony, Julio Ribeiro, Katharina Gaus, Justin Gooding, Maria Kavallaris. Precision medicine: High-throughput 3D bioprinting of embedded multicellular cancer spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5022.

Tissue Engineering Under Microgravity Conditions-Use of Stem Cells and Specialized Cells.

Experimental cell research studying three-dimensional (3D) tissues in space and on Earth using new techniques to simulate microgravity is currently a hot topic in Gravitational Biology and Biomedicine. This review will focus on the current knowledge of the use of stem cells and specialized cells for tissue engineering under simulated microgravity conditions. We will report on recent advancements in the ability to construct 3D aggregates from various cell types using devices originally created to prepare for spaceflights such as the random positioning machine (RPM), the clinostat, or the NASA-developed rotating wall vessel (RWV) bioreactor, to engineer various tissues such as preliminary vessels, eye tissue, bone, cartilage, multicellular cancer spheroids, and others from different cells. In addition, stem cells had been investigated under microgravity for the purpose to engineer adipose tissue, cartilage, or bone. Recent publications have discussed different changes of stem cells when exposed to microgravity and the relevant pathways involved in these biological processes. Tissue engineering in microgravity is a new technique to produce organoids, spheroids, or tissues with and without scaffolds. These 3D aggregates can be used for drug testing studies or for coculture models. Multicellular tumor spheroids may be interesting for radiation experiments in the future and to reduce the need for in vivo experiments. Current achievements using cells from patients engineered on the RWV or on the RPM represent an important step in the advancement of techniques that may be applied in translational Regenerative Medicine.

Patient-derived multicellular prostate cancer spheroids for in vitro cell culture and drug testing

Patient-derived multicellular prostate cancer spheroids for in vitro cell culture and drug testing

A novel ruthenium complex with xanthoxylin induces S-phase arrest and causes ERK1/2-mediated apoptosis in HepG2 cells through a p53-independent pathway

Ruthenium-based compounds have gained great interest due to their potent cytotoxicity in cancer cells; however, much of their potential applications remain unexplored. In this paper, we report the synthesis of a novel ruthenium complex with xanthoxylin (RCX) and the investigation of its cellular and molecular action in human hepatocellular carcinoma HepG2 cells. We found that RCX exhibited a potent cytotoxic effect in a panel of cancer cell lines in monolayer cultures and in a 3D model of multicellular cancer spheroids formed from HepG2 cells. This compound is detected at a high concentration in the cell nuclei, induces DNA intercalation and inhibits DNA synthesis, arresting the cell cycle in the S-phase, which is followed by the activation of the caspase-mediated apoptosis pathway in HepG2 cells. Gene expression analysis revealed changes in the expression of genes related to cell cycle control, apoptosis and the MAPK pathway. In addition, RCX induced the phosphorylation of ERK1/2, and pretreatment with U-0126, an MEK inhibitor known to inhibit the activation of ERK1/2, prevented RCX-induced apoptosis. In contrast, pretreatment with a p53 inhibitor (cyclic pifithrin-α) did not prevent RCX-induced apoptosis, indicating the activation of a p53-independent apoptosis pathway. RCX also presented a potent in vivo antitumor effect in C.B-17 SCID mice engrafted with HepG2 cells. Altogether, these results indicate that RCX is a novel anticancer drug candidate.

Molecular and functional assessment of multicellular cancer spheroids produced in double emulsions enabled by efficient airway resistance based selective surface treatment

Multicellular spheroids have garnered significant attention as an in vitro three-dimensional cancer model which can mimick the in vivo microenvironmental features. While microfluidics generated double emulsions have become a potential method to generate spheroids, challenges remain on the tedious procedures. Enabled by a novel ‘airway resistance’ based selective surface treatment, this study presents an easy and facile generation of double emulsions for the initiation and cultivation of multicellular spheroids in a scaffold-free format. Combining with our previously developed DNA nanosensors, intestinal spheroids produced in the double emulsions have shown an elevated activities of an essential DNA modifying enzyme, the topoisomerase I. The observed molecular and functional characteristics of spheroids produced in double emulsions are similar to the counterparts produced by the commercially available ultra-low attachment plates. However, the double emulsions excel for their improved uniformity, and the consistency of the results obtained by subsequent analysis of the spheroids. The presented technique is expected to ease the burden of producing spheroids and to promote the spheroids model for cancer or stem cell study.