Cell Spheroid Formation Research Articles

CALML3-AS1 enhances malignancies and stemness of small cell lung cancer cells through interacting with DAXX protein and promoting GLUT4-mediated aerobic glycolysis

The lncRNA CALML3 antisense RNA 1 (CALML3-AS1) is a biomarker for various cancers, including non-small cell lung cancer (NSCLC). However, the role of CALM3-AS1 in small cell lung cancer (SCLC) is still unclear. Here, we found that the CALML3-AS1 was upregulated in SCLC tissues and cells. SCLC cells (NCI-H69 and NCI-H466 cells) were transfected with small interfering RNA of CALML-AS1 (si-CALML3-AS1) and Death domain-associated protein (DAXX) (si-DAXX) or an overexpression vector of CALML-AS1 (dCas9-CALML3-AS1) and DAXX (dCas9-DAXX). The results showed that silencing CALML3-AS1 inhibited SCLC cell proliferation, colony formation, migration, invasion, and spheroid formation, and reduced the expression of stemness marker proteins (Nanog. Oct4, and Lin28). Moreover, silencing CALML3-AS1 reduced glycolysis rate, glucose utilization, and lactate production, and decreased the levels of key glycolytic regulatory proteins (GLUT1, GLUT4, HK2, and PKM2) in SCLC cells, while overexpression of CALML3-AS1 promoted malignant growth and stemness and enhanced glucose transporters type 4 (GLUT4)-mediated aerobic glycolysis by interacting with DAXX in NCI-H69 and NCI-H466 cells. Silencing DAXX or GLUT4, or treatment with 2-Deoxy-d-glucose (2-DG, a glycolysis inhibitor) reversed the effects of CALML3-AS1 overexpression on aerobic glycolysis, malignant growth, and stemness of SCLC cells. Finally, NCI-H69 cells transfected with CALML3-AS1, sh-CALML3-AS1, and sh-DAXX lentiviral vectors were subcutaneously injected into nude mice to construct xenograft models. Knockdown of CALML3-AS1 or DAXX inhibited tumor growth in SCLC in vivo. In conclusion, CALML3-AS1, an oncogene, promotes the malignancy and stemness of SCLC cells by interacting with DAXX to enhance GLUT4-mediated aerobic glycolysis, thereby promoting SCLC progression.

Doubly self-assembled dermatan sulfate/chitosan nanoparticles for targeted siRNA delivery in cancer therapy

RNA interference, a naturally occurring regulatory mechanism in which small interfering RNA (siRNA) molecules are responsible for the sequence-specific suppression of gene expression, emerged as one of the most promising gene therapies in cancer. In this work, we investigate a microfluidics double self-assembly method based on micellization and polyelectrolyte complex formation for the encapsulation of siRNA targeting the BIRC5 gene, a member of the inhibitor of apoptosis gene family, that codes for survivin a protein of theinhibitorof apoptosis protein family that is involved in triple-negative breast cancer (TNBC) proliferation and metastasis within nanoparticles of an amphiphilic chitosan-graft-poly(methyl methacrylate) copolymer and low-molecular weight dermatan sulfate, a polysaccharide targeting the CD44 receptor overexpressed in this tumor. Nanoparticles are spherical and display a hydrodynamic diameter of ∼ 200 nm, as measured by dynamic light scattering and scanning electron microscopy. In addition, these colloidal systems exhibit a strongly negative zeta-potential that confers them excellent physical stability for at least four months owing to electrostatic repulsion and evidences the exposure of the polyanionic dermatan sulfate on the surface. The key role of dermatan sulfate in the active targeting and intracellular delivery of the cargo in the murine breast cancer cell line 4T1, a model of TNBC, is confirmed by confocal laser scanning microscopy and imaging flow cytometry. Finally, the silencing efficiency is demonstrated in 4T1 cell viability, migration, proliferation and spheroid formation assays in vitro. Overall results highlight the promise of this simple, reproducible and scalable method for the nanoencapsulation of siRNA and other therapeutic nucleic acids.

Design of a non-oxidative adhesive dopamine-grafted hyaluronic acid/NOCC hydrogel for enhanced cell spheroid formation and soft tissue regeneration

Tissue adhesive hydrogels from dopamine represent a compelling material with diverse applications across engineering and medical fields. Although numerous designed adhesive hydrogels have been developed, significant limitations have arisen from the reliance on hydrogen peroxide as an activating agent for crosslinking hydrogels. To address this, our study investigates an innovative approach to create an adhesive and biocompatible hydrogel from dopamine conjugated-hyaluronic acid (Da-HA) and N, O-Carboxymethyl Chitosan (NOCC) without the need for oxidative agent treatment for use in biomedical engineering application. The reactions were performed under mild alkaline conditions, vigorous stirring, and curing, resulting in DHC hydrogels. Proper modification and reactions in the hydrogel matrix were confirmed by NMR spectroscopy and Fourier transform infrared spectroscopy (FTIR). The gelling and tissue adhesive properties of the produced DHC hydrogel are proportional to the Da-HA content in the hydrogel. Scanning electron microscopy revealed changes in pore walls among samples and pore diameters ranging from 100 to 300 nm. Rheological analysis demonstrated a high visco-elastic material and self-healing properties. Importantly, the growth of L929 cell spheroids was noted upon their cultivation atop the designed hydrogel. The material holds significant promise for 3D printing and cell encapsulation therapies, as well as in situ tissue repair, particularly for mechanically dynamic tissues like the dermis.

Abstract A023: RNA secondary structures mediate circular RNA stability and function

Abstract Our group recently discovered that the circular RNA (circRNA), CDR1as, promotes lung cancer metastasis in part through the stabilization of the coding gene, CDR1. The purpose of this study was to identify intramolecular RNA secondary structures of CDR1as and determine whether there is a functional relationship between the CDR1as secondary structure and non-small cell lung cancer (NSCLC) metastasis. We employed the chemical probing approach selective 2’- hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to experimentally inform structure predictions of CDR1as. The SHAPE-MaP profiling experiments revealed that CDR1as is remarkably unstructured overall, with an exception of a highly probable and consistent stem-loop structure embedding the CDR1as backsplice junction (BSJ). Additionally, BSJ-embedding secondary structures were identified in all other mammalian circRNAs studied in a similar chemical probing experiment. To evaluate the role of the BSJ structure on CDR1as expression, we sterically disrupted the BSJ structure using a chemically modified, inert antisense oligonucleotide (BSJ-ASO) lacking a gapmer in order to block RNAse H activity. After transfection of the structure disrupting BSJ-ASO into several cell lines, we observed a 90-95% reduction of CDR1as expression by the BSJ-ASO by 24 hours. Furthermore, the BSJ-ASO significantly inhibited cancer cell proliferation and spheroid formation in several NSCLC models. Strikingly, using RNA stability assays we observed that the BSJ-ASO caused dramatic reductions in CDR1as levels within 5 minutes of transfection, suggesting a possibly hydrolytic process. A functional screen of silencing curated CDR1as RNA-binding proteins failed to rescue the loss of CDR1as expression following BSJ-ASO treatment. We next tested whether an RNA-catalyzed mechanism of action is possible. By incubating total RNA with the BSJ-ASO in buffers of varying magnesium chloride concentrations in vitro, we observed a dramatic decrease in CDR1as levels similar to that of the BSJ-ASO cellular transfection experiments, suggestive of ribozyme-like activity. Interestingly, use of a primer mapping approach indicated two putative cut sites within CDR1as. Further chemical probing following treatment with the BSJ-ASO revealed a new, highly probable secondary structure upstream of the BSJ, which appears similar to a hairpin ribozyme when modeled two-dimensionally. Taken together, our work reveals that intramolecular RNA secondary structures can have critical roles in maintaining circRNA cellular stability and function, and strategic steric disruption of these structures may induce circRNA self-cleavage. Citation Format: Aaron C Chack, Emily B Harrison, Caroline J Aufgebauer, Mark A Boerneke, Patrick S Irving, Edgar M Faison, Jingyu Zhao, Qi Zhang, Kevin M Weeks, Chad V Pecot. RNA secondary structures mediate circular RNA stability and function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A023.

CSIG-14. ACROLEIN INDUCES THE MIR-30A-5P/NCAM AXIS THROUGH THE AKT/GSK3Β/Β-CATENIN PATHWAY, PROMOTING GLIOMA PROGRESSION

Abstract Glioblastoma (GBM), the most aggressive brain tumor, exhibits resistance to treatment and thrives in low-oxygen (hypoxic) environments, leading to the accumulation of reactive oxygen species (ROS). Hypoxia activates the hypoxia-inducible factor (HIF)-1α pathway, enhancing tumor malignancy. Acrolein, a reactive aldehyde produced during lipid peroxidation in hypoxia, induces toxicity through DNA damage, inflammation, mitochondrial dysfunction, and oxidative stress. Our previous study found elevated acrolein levels in hypoxic glioma cells and linked acrolein-induced DNA damage (Acr-dG) with poor GBM prognosis. However, the role of acrolein in GBM progression remains unclear. This study aims to elucidate the impact of acrolein on GBM. In vitro experiments demonstrated acrolein production under hypoxia, which increased glioma cell migration and spheroid formation. RNA sequencing identified five affected pathways: cell adhesion molecules, PI3K-AKT signaling, ECM-receptor interaction, MAPK signaling, and neuroactive ligand-receptor interaction. Acrolein downregulated NCAM (neural cell adhesion molecule) via the miR-30a-5p-AKT-GSK3β/β-catenin axis in GBM cell lines and patient-derived cells. Overexpressing NCAM reduced cell migration and spheroid formation. Lower NCAM levels in GBM tissues correlated with poor patient survival. This study suggests the potential of NCAM as a therapeutic target for GBM and provides insights into early detection and treatment strategies for this malignancy.

CSIG-15. ACROLEIN INDUCES THE MIR-30A-5P/NCAM AXIS VIA THE AKT/GSK3Β/Β-CATENIN PATHWAY, PROMOTING GLIOMA PROGRESSION

Abstract Glioblastoma (GBM), the most aggressive brain tumor, exhibits resistance to treatment and thrives in low-oxygen (hypoxic) environments, leading to the accumulation of reactive oxygen species (ROS). Hypoxia activates the hypoxia-inducible factor (HIF)-1α pathway, enhancing tumor malignancy. Acrolein, a reactive aldehyde produced during lipid peroxidation in hypoxia, induces toxicity through DNA damage, inflammation, mitochondrial dysfunction, and oxidative stress. Our previous study found elevated acrolein levels in hypoxic glioma cells and linked acrolein-induced DNA damage (Acr-dG) with poor GBM prognosis. However, the role of acrolein in GBM progression remains unclear. This study aims to elucidate the impact of acrolein on GBM. In vitro experiments demonstrated acrolein production under hypoxia, which increased glioma cell migration and spheroid formation. RNA sequencing identified five affected pathways: cell adhesion molecules, PI3K-AKT signaling, ECM-receptor interaction, MAPK signaling, and neuroactive ligand-receptor interaction. Acrolein downregulated NCAM (neural cell adhesion molecule) via the miR-30a-5p-AKT-GSK3β/β-catenin axis in GBM cell lines and patient-derived cells. Overexpressing NCAM reduced cell migration and spheroid formation. Lower NCAM levels in GBM tissues correlated with poor patient survival. This study suggests the potential of NCAM as a therapeutic target for GBM and provides insights into early detection and treatment strategies for this malignancy.

TMIC-23. INVESTIGATING THE FUNCTIONAL ROLE OF SECRETED FERRITIN FROM GLIOBLASTOMA CELL TYPES

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor in adults with a survival rate ~5%. Our laboratory previously showed the inhibitory effect of iron and ferritin-bound iron on migration and spheroid formation in GBM cells. Furthermore, studies have shown the release of extracellular vesicles (EVs) and their iron-related cargoes are regulated by cellular iron concentrations. Building on these findings, this study aims to investigate the impact of EV-associated ferritin on migration in GBM. To achieve this, we examined the influence of iron on EV release from four GBM cell lines, comprising two cancer stem cells (CSCs) and two non-CSCs: T387, T3691, LN229, and T98G. Using Nanoparticle tracking analysis (NTA), we demonstrated that 100 μM of ferric ammonium citrate (FAC) significantly increases EV release in the CSC line after 24 hours (p-value <0.01). Conversely, there was no significant impact on the non-CSC line under the same conditions. Based on these results, the ability of iron to regulate EV release is dependent on CSC versus non-CSC status. Given the functional role of ferritin on migration potential in GBM, we used immunoblotting to examine ferritin release from cells following iron treatment. We observed a reduction in ferritin release from CSCs following FAC treatment compared to the control, while non-CSCs, in contrast, released more ferritin after treatment. Interestingly, ferritin released from CSCs was predominantly within EVs, whereas ferritin released from non-CSCs was not associated with EVs. To assess the functional impact of ferritin release on migration, we utilized a Transwell migration system, and observed that EVs from iron-treated cells reduced migration in the recipients. Together, these findings expand our understanding of differential responses of ferritin in CSC and non-CSC EVs to iron treatment and will allow us to identify new targets and strategies for managing GBM progression.

NSDHL contributes to breast cancer stem-like cell maintenance and tumor-initiating capacity through TGF-β/Smad signaling pathway in MCF-7 tumor spheroid

BackgroundNAD(P)-dependent steroid dehydrogenase-like protein (NSDHL), which is involved in breast tumor growth and metastasis, has been implicated in the maintenance of cancer stem cells. However, its role in regulating breast cancer stem-like cells (BCSCs) remains unclear. We have previously reported the clinical significance of NSDHL in patients with estrogen receptor-positive (ER +) breast cancer. This study aimed to elucidate the molecular mechanisms by which NSDHL regulates the capacity of BCSCs in the ER + human breast cancer cell line, MCF-7.MethodsNSDHL knockdown suppressed tumor spheroid formation in MCF-7 human breast cancer cells grown on ultralow-attachment plates. RNA sequencing revealed that NSDHL knockdown induced widespread transcriptional changes in the MCF-7 spheroids. TGF-β signaling pathway was the most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (fold change ≥ 2, P ≤ 0.05) identified in NSDHL-knockdown MCF-7 spheroids compared with the control. In orthotopic tumor models injected with NSDHL-knockdown MCF-7 spheroids, tumor initiation and growth were strongly suppressed compared with those in the control.ResultsBCSC populations with CD44+/CD24- and CD49f+/EpCAM + phenotypes and high ALDH activity were decreased in NSDHL-knockdown MCF-7 spheroids and xenograft tumors relative to controls, along with decreased secretion of TGF-β1 and 3, phosphorylation of Smad2/3, and expression of SOX2. In RNA-sequencing data from The (TCGA) database, a positive correlation between the expression of NSDHL and SOX2 was found in luminal-type breast cancer specimens (n = 998). Our findings revealed that NSDHL plays an important role in maintaining the BCSC population and tumor-initiating capacity of ER-positive MCF-7 spheroids, suggesting that NSDHL is an attractive therapeutic target for eliminating BCSCs, thus preventing breast cancer initiation and progression.ConclusionsOur findings suggest that NSDHL regulates the BCSC/tumor-initiating cell population in MCF-7 spheroids and xenograft tumors.

Islet cell spheroids produced by a thermally sensitive scaffold: a new diabetes treatment

The primary issues in treating type 1 diabetes mellitus (T1DM) through the transplantation of healthy islets or islet β-cells are graft rejection and a lack of available donors. Currently, the majority of approaches use cell encapsulation technology and transplant replacement cells that can release insulin to address transplant rejection and donor shortages. However, existing encapsulation materials merely serve as carriers for islet cell growth. A new treatment approach for T1DM could be developed by creating a smart responsive material that encourages the formation of islet cell spheroids to replicate their 3D connections in vivo and controls the release of insulin aggregates. In this study, we used microfluidics to create thermally sensitive porous scaffolds made of poly(N-isopropyl acrylamide)/graphene oxide (PNIPAM/GO). The material was carefully shrunk under near-infrared light, enriched with mouse insulinoma pancreatic β cells (β-TC-6 cells), encapsulated, and cultivated to form 3D cell spheroids. The controlled contraction of the thermally responsive porous scaffold regulated insulin release from the spheroids, demonstrated using the glucose-stimulated insulin release assay (GSIS), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assay. Eventually, implantation of the spheroids into C57BL/6 N diabetic mice enhanced the therapeutic effect, potentially offering a novel approach to the management of T1DM.Graphical

Composition Tuning of Semi-Open Cell Carriers via Phase Freeze-Shrink Self-Molding.

Extracellular matrix (ECM)-mimicking microsized cell carriers featuring a semi-isolated chamber facilitate the study of cellular heterogeneity as well as intercellular communication. However, the semiopen shaping of the designated gel mixture remains unattainable with current methods. We report an oil-phase freeze-shrink self-molding mechanism for generating size- and composition-tunable cradle-shaped microgels (microcradles) from water-in-oil droplets. The universality of this shape transition principle is demonstrated with six types of polysaccharides dispersed in a poly(ethylene glycol) diacrylate (PEGDA) or methacrylate gelatin (GelMA) matrix. By doping the microcradles with the major ECM component, hyaluronic acid sodium, we demonstrate a label-free selective culture of CD44 receptor-rich cells and the formation of cell spheroids within 3 days. This cryo-induced cradle-shaping strategy enables the functionalization of microcarriers for selective cell culture, thereby allowing them to be used for intercellular communication, drug delivery, and the construction of structural units for osteogenesis and 3D printing.

Enhanced Anticancer Activity of 7MeERT over Ertredin: A Comparative Study on Cancer Cell Proliferation and NDUFA12 Binding.

We have previously identified Ertredin (3-(2-amino-5-bromophenyl) quinoxalin-2(1H)-one) as a compound that suppresses 3D spheroid formation and tumorigenesis in NIH3T3 cells induced by Epidermal Growth Factor Receptor variant III (EGFRvIII) transduction. One of its targets has been shown to be NDUFA12 (NADH Dehydrogenase (Ubiquinone) 1 Alpha Subcomplex Subunit 12), a component protein of oxidative phosphorylation complex I. In this report, we compared the growth inhibitory activity of Ertredin with its methylated analogue 7MeERT (3-(2-amino-5-bromophenyl)-7-methylquinoxalin-2(1H)-one) on human cancer cells. 7MeERT induced the inhibition of the proliferation of various cancer cells similarly to Ertredin and showed higher activity in glioblastoma cells, A431 cells overexpressing EGFR (wild type), and multiple myeloma cells. Molecular docking analysis and a Cellular Thermal Shift Assay (CETSA) suggested that 7MeERT binds to NDUFA12 similarly to Ertredin. The binding of 7MeERT and Ertredin to NDUFA12 in glioblastoma was further supported by the inhibition of the oxygen consumption rate. These results suggest that 7MeERT also binds to NDUFA12, inhibits oxidative phosphorylation, and has a higher anti-cancer cell growth inhibitory activity than Ertredin.

Platelet and epithelial cell interations can be modeled in cell culture, and are not affected by dihomo-gamma-linolenic acid.

Increasing evidence is implicating roles for platelets in the development and progression of ovarian cancer, a highly lethal disease that can arise from the fallopian tubes, and has no current method of early detection or prevention. Thrombosis is a major cause of mortality of ovarian cancer patients suggesting that the cancer alters platelet behavior. The objective of this study was to develop a cell culture model of the pathological interactions of human platelets and ovarian cancer cells, using normal FT epithelial cells as a healthy control, and to test effects of the anti-platelet dihomo-gamma-linolenic acid (DGLA) in the model. Both healthy and cancer cells caused platelet aggregation, however platelets only affected spheroid formation by cancer cells and had no effect on healthy cell spheroid formation. When naturally-formed spheroids of epithelial cells were exposed to platelets in transwell inserts that did not allow direct interactions of the two cell types, platelets caused increased size of the spheroids formed by cancer cells, but not healthy cells. When cancer cell spheroids formed using magnetic nanoshuttle technology were put in direct physical contact with platelets, the platelets caused spheroid condensation. In ovarian cancer cells, DGLA promoted epithelial-to-mesenchymal (EMT) transition at doses as low as 100 μM, and inhibited metabolic viability and induced apoptosis at doses ≥150 μM. DGLA doses ≤150 μM used to avoid direct DGLA effects on cancer cells, had no effect on the pathological interactions of platelets and ovarian cancer cells in our models. These results demonstrate that the pathological interactions of platelets with ovarian cancer cells can be modeled in cell culture, and that DGLA has no effect on these interactions, suggesting that targeting platelets is a rational approach for reducing cancer aggressiveness and thrombosis risk in ovarian cancer patients, however DGLA is not an appropriate candidate for this strategy.

Jiedu Xiaozheng Yin extract targets cancer stem cells by Wnt signaling pathway in colorectal cancer

Ethnopharmacological relevanceThe clinical application of the traditional Chinese medicinal formula Jiedu Xiaozheng Yin (JXY) for gastrointestinal tumors, particularly colorectal cancer (CRC), is well-established, yet the precise biological mechanism underlying its efficacy in CRC treatment remains elusive. Aims of the studyThis study endeavors to unravel the intricate mechanism through which JXY modulates colorectal cancer stem cells, thus elucidating the pathways by which it exerts its potent anti-tumor effects. Materials and methodsIn this study, the regulatory impact of JXY on the signaling pathway and function of CRC cells was analyzed through Network pharmacology. The ethyl acetate extract of JXY was detected the major compounds using HPLC and then treated the HCT-116 cells for RNA-Sequencing (RNA-Seq). Protein expression and stemness of HCT-15 and HCT-116 cells following JXY extract treatment were assessed using Western blot analysis and matrigel spheroid assays. Additionally, the β-catenin transcriptional activity was evaluated using a TOPflash reporter assay with or without Lithium chloride (LiCl) stimulation. Patient-derived organoids of CRC (CRC PDOs) were cultured using a stemness maintenance medium, and their viability was measured using ATP assays after treatment of JXY extract. Furthermore, the anti-tumor efficacy of JXY extract was assessed using a xenograft mice model derived from HCT-15 cells. ResultsNetwork pharmacology emphasized the influence of JXY on cancer stem cells and the Wnt signaling pathway. HPLC analysis confirmed that the JXY extract contained the three most prevalent pharmaceutical compounds among the four herbs documented in the Chinese Pharmacopoeia (rosmarinic acid, quercetin, and kaempferol). RNA-Seq results further elucidated the effect of JXY extract, particularly targeting cancer stem cells and the Wnt signaling pathway. Furthermore, JXY extract inhibited spheroid formation in CRC cells and downregulated CRC CSC markers (CD133, DCLK1, and C-MYC). Additionally, JXY extract suppressed the β-catenin expression and transcriptional activity as well as the Wnt pathway target proteins, including C-MYC and Cyclin D1. Consistent with findings from cell lines, JXY extract suppressed the growth of CRC PDOs exhibiting stemness characteristics. And JXY extract demonstrated a significant inhibitory effect on tumor growth, C-MYC, and β-catenin protein levels in xenograft tumors. ConclusionsThese results highlight the novel function of JXY extract in targeting CRC CSCs by regulating Wnt signaling pathway, underscoring its potential as a therapeutic agent for treating CRC.

High-Quality Three-Dimensionally Cultured Cells Using Interfaces of Diblock Copolymers Containing Different Ratios of Zwitterionic N-Oxides.

To control three-dimensional (3D) cell spheroid formation, it is well-known the surface physicochemical and mechanical properties of cell culture materials are important; however, the formation and function of 3D cells are still unclear. This study demonstrated the precise control of the formation of 3D cells and 3D cell functions using diblock copolymers containing different ratios of a zwitterionic trimethylamine N-oxide group. The diblock copolymers were composed of poly(n-butyl methacrylate) (PBMA) as the hydrophobic unit for surface coating on a cell culture dish and stabilization in water, and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) as the precursor of N-oxide. The zwitterionic N-oxide converted from 0 to 100% using PDMAEMA. The wettability and surface zeta potential varied with different ratios of N-oxide diblock copolymer-coated surfaces, and the amount of protein adsorbed in the cell culture medium decreased monotonically with increasing N-oxide ratio. 3D cell spheroid formations were observed by seeding human umbilical cord mesenchymal stem cells (hUC-MSCs) in diblock copolymer-coated flat-bottom well plates, and the N-oxide ratio was over 40%. The cells proliferated in two-dimensions (2D) and did not form spheroids when the N-oxide ratio was less than 20%. Interestingly, the expression of undifferentiated markers of hUC-MSCs was higher on surfaces that adsorbed proteins to some extent and formed 50-150 μm spheroids in the range of 40-70% of N-oxide ratio. We revealed that a moderately protein-adsorbed surface allows precise control of spheroid formation and undifferentiated 3D cells and has potential applications for high-quality spheroids in regenerative medicine and drug screening.

Formation and culture of cell spheroids by using magnetic nanostructures resembling a crown of thorns

In contrast to traditional two-dimensional cell-culture conditions, three-dimensional (3D) cell-culture models closely mimic complexin vivoconditions. However, constructing 3D cell culture models still faces challenges. In this paper, by using micro/nano fabrication method, including lithography, deposition, etching, and lift-off, we designed magnetic nanostructures resembling a crown of thorns. This magnetic crown of thorns (MCT) nanostructure enables the isolation of cells that have endocytosed magnetic particles. To assess the utility of this nanostructure, we used high-flux acquisition of Jurkat cells, an acute-leukemia cell line exhibiting the native phenotype, as an example. The novel structure enabled Jurkat cells to form spheroids within just 30 min by leveraging mild magnetic forces to bring together endocytosed magnetic particles. The size, volume, and arrangement of these spheroids were precisely regulated by the dimensions of the MCT nanostructure and the array configuration. The resulting magnetic cell clusters were uniform in size and reached saturation after 1400 s. Notably, these cell clusters could be easily separated from the MCT nanostructure through enzymatic digestion while maintaining their integrity. These clusters displayed a strong proliferation rate and survival capabilities, lasting for an impressive 96 h. Compared with existing 3D cell-culture models, the approach presented in this study offers the advantage of rapid formation of uniform spheroids that can mimicin vivomicroenvironments. These findings underscore the high potential of the MCT in cell-culture models and magnetic tissue enginerring.

Patterned PVA Hydrogels with 3D Petri Dish® Micro-Molds of Varying Topography for Spheroid Formation of HeLa Cancer Cells: In Vitro Assessment.

Cell spheroids are an important three-dimensional (3D) model for in vitro testing and are gaining interest for their use in clinical applications. More natural 3D cell culture environments that support cell-cell interactions have been created for cancer drug discovery and therapy applications, such as the scaffold-free 3D Petri Dish® technology. This technology uses reusable and autoclavable silicone micro-molds with different topographies, and it conventionally uses gelled agarose for hydrogel formation to preserve the topography of the selected micro-mold. The present study investigated the feasibility of using a patterned Poly(vinyl alcohol) hydrogel using the circular topography 12-81 (9 × 9 wells) micro-mold to form HeLa cancer cell spheroids and compare them with the formed spheroids using agarose hydrogels. PVA hydrogels showed a slightly softer, springier, and stickier texture than agarose hydrogels. After preparation, Fourier transform infrared (FTIR) spectra showed chemical interactions through hydrogen bonding in the PVA and agarose hydrogels. Both types of hydrogels favor the formation of large HeLa spheroids with an average diameter of around 700-800 µm after 72 h. However, the PVA spheroids are more compact than those from agarose, suggesting a potential influence of micro-mold surface chemistry on cell behavior and spheroid formation. This was additionally confirmed by evaluating the spheroid size, morphology, integrity, as well as E-cadherin and Ki67 expression. The results suggest that PVA promotes stronger cell-to-cell interactions in the spheroids. Even the integrity of PVA spheroids was maintained after exposure to the drug cisplatin. In conclusion, the patterned PVA hydrogels were successfully prepared using the 3D Petri Dish® micro-molds, and they could be used as suitable platforms for studying cell-cell interactions in cancer drug therapy.

Formation of cell spheroids using Standing Surface Acoustic Wave (SSAW).

3D bioprinting becomes one of the popular approaches in the tissue engineering. In this emerging application, bioink is crucial for fabrication and functionality of constructed tissue. The use of cell spheroids as bioink can enhance the cell-cell interaction and subsequently the growth and differentiation of cells in the 3D printed construct with the minimum amount of other biomaterials. However, the conventional methods of preparing the cell spheroids have several limitations, such as long culture time, low-throughput, and medium modification. In this study, the formation of cell spheroids by SSAW was evaluated both numerically and experimentally in order to overcome the aforementioned limitations. The effects of excitation frequencies on the cell accumulation time, diameter of the formed cell spheroids, and subsequently, the growth and viability of cell spheroids in the culture medium over time were studied. Using the high-frequency (23.8 MHz) excitation, cell accumulation time to the pressure nodes could be reduced in comparison to that of the low-frequency (10.4 MHz) excitation, but in a smaller spheroid size. SSAW excitation at both frequencies does not affect the cell viability up to 7 days, > 90% with no statistical difference compared with the control group. In summary, SSAW can effectively prepare the cell spheroids as bioink for the future 3D bioprinting and various biotechnology applications (e.g., pharmaceutical drug screening and tissue engineering).

Evaluation of allylated gelatin as a bioink supporting spontaneous spheroid formation of HepG2 cells

The spheroid culture system has gained significant attention as an effective in vitro model to mimic the in vivo microenvironment. Even though numerous studies were focused on developing spheroids, the structural organization of encapsulated cells within hydrogels remains a challenge. Allylated gelatin or GelAGE is used as a bioink due to its excellent physicochemical properties. In this study, GelAGE was evaluated for its capacity to induce spontaneous spheroid formation in encapsulated HepG2 cells. GelAGE was synthesized and characterized using 1HNMR spectroscopy and ninhydrin assay. Then the physicochemical and biological attributes of GelAGE hydrogel was examined. The results demonstrate that GelAGE has remarkable ability to induce the encapsulated cells to self-organize into spheroids.

The demethylase ALKBH5 mediates ZKSCAN3 expression through the m6A modification to activate VEGFA transcription and thus participates in MNNG-induced gastric cancer progression

N-Nitroso compounds (NOCs) are recognized as important factors that promote gastric cancer development, but the specific effects and potential mechanisms by which NOC exposure promotes gastric cancer are still poorly understood. In this study, we explored the effects and potential molecular mechanisms of NOCs on the promotion of gastric cancer using methylnitronitrosoguanidine (MNNG), a classical direct carcinogen of NOC. The results of in vivo and in vitro experiments showed that chronic and low-concentration MNNG exposure significantly promoted the malignant progression of tumors, including cell migration, cell invasion, vasculogenic mimicry (VM) formation, cell spheroid formation, stem cell-like marker expression, and gastric cancer growth and metastasis. Mechanistically, we revealed that demethylase ALKBH5 regulated the level of the N6‑methyladenosine (m6A) modification in the 3′UTR and CDS region of the ZKSCAN3 mRNA to promote ZKSCAN3 expression, mediated the binding of ZKSCAN3 to the VEGFA promoter region to regulate VEGFA transcription, and participated in MNNG-induced gastric cancer cell migration, invasion, VM formation, cell spheroid formation, stem cell-like marker expression and ultimately gastric cancer progression. In addition, our study revealed that ALKBH5-ZKSCAN3-VEGFA signaling was significantly activated during MNNG-induced gastric carcinogenesis, and further studies in gastric cancer patients showed that ALKBH5, ZKSCAN3, and VEGFA expression were upregulated in cancers compared with paired gastric mucosal tissues, that ALKBH5, ZKSCAN3, and VEGFA could serve as important biomarkers for determining patient prognosis, and that the molecular combination showed greater prognostic value. These findings provide a theoretical basis for developing gastric cancer interventions for NOCs and for determining gastric cancer progression.

Rationally designed β-cyclodextrin-crosslinked polyacrylamide hydrogels for cell spheroid formation and 3D tumor model construction

In vitro tumor models are essential for understanding tumor behavior and evaluating tumor biological properties. Hydrogels that can mimic the tumor extracellular matrix have become popular for creating 3D in vitro tumor models. However, designing biocompatible hydrogels with appropriate chemical and physical properties for constructing tumor models is still a challenge. In this study, we synthesized a series of β-cyclodextrin (β-CD)-crosslinked polyacrylamide hydrogels with different β-CD densities and mechanical properties and evaluated their potential for use in 3D in vitro tumor model construction, including cell capture and spheroid formation. By utilizing a combination of β-CD-methacrylate (CD-MA) and a small amount of N,N′-methylene bisacrylamide (BIS) as hydrogel crosslinkers and optimizing the CD-MA/BIS ratio, the hydrogels performed excellently for tumor cell 3D culture and spheroid formation. Notably, when we co-cultured L929 fibroblasts with HeLa tumor cells on the hydrogel surface, co-cultured spheroids were formed, showing that the hydrogel can mimic the complexity of the tumor extracellular matrix. This comprehensive investigation of the relationship between hydrogel mechanical properties and biocompatibility provides important insights for hydrogel-based in vitro tumor modeling and advances our understanding of the mechanisms underlying tumor growth and progression.