Spheroid Formation Research Articles

Exploiting Cancer Dormancy Signaling Mechanisms in Epithelial Ovarian Cancer Through Spheroid and Organoid Analysis

Epithelial ovarian cancer (EOC) exhibits a unique mode of metastasis, involving spheroid formation in the peritoneum. Our research on EOC spheroid cell biology has provided valuable insights into the signaling plasticity associated with metastasis. We speculate that EOC cells modify their biology between tumour and spheroid states during cancer dormancy, although the specific mechanisms underlying this transition remain unknown. Here, we present novel findings from direct comparisons between cultured EOC spheroids and organoids. Our results indicated that AMP-activated protein kinase (AMPK) activity was significantly upregulated and protein kinase B (Akt) was downregulated in EOC spheroids compared to organoids, suggesting a clear differential phenotype. Through RNA sequencing analysis, we further supported these phenotypic differences and highlighted the significance of cell cycle regulation in organoids. By inhibiting the G2/M checkpoint via kinase inhibitors, we confirmed that this pathway is essential for organoids. Interestingly, our results suggest that specifically targeting aurora kinase A (AURKA) may represent a promising therapeutic strategy since our cells were equally sensitive to Alisertib treatment as both spheroids and organoids. Our findings emphasize the importance of studying cellular adaptations of EOC cells, as there may be different therapeutic targets depending on the step of EOC disease progression.

Dynamic Culture of Bioprinted Liver Tumor Spheroids in a Pillar/Perfusion Plate for Predictive Screening of Anticancer Drugs.

Recent advancements in three-dimensional (3D) cell culture technologies, such as cell spheroids, organoids, and 3D bioprinted tissue constructs, have significantly improved the physiological relevance of in vitro models. These models better mimic tissue structure and function, closely emulating in vivo characteristics and enhancing phenotypic analysis, critical for basic research and drug screening in personalized cancer therapy. Despite their potential, current 3D cell culture platforms face technical challenges, which include user-unfriendliness in long-term dynamic cell culture, incompatibility with rapid cell encapsulation in biomimetic hydrogels, and low throughput for compound screening. To address these issues, we developed a 144-pillar plate with sidewalls and slits (144PillarPlate) and a complementary 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for dynamic 3D cell culture and predictive compound screening. To accelerate biomimetic tissue formation, small Hep3B liver tumor spheroids suspended in alginate were printed and encapsulated on the 144PillarPlate rapidly by using microsolenoid valve-driven 3D bioprinting technology. The microarray bioprinting technology enabled precise and rapid loading of small spheroids in alginate on the pillar plate, facilitating reproducible and scalable formation of large tumor spheroids with minimal manual intervention. The bioprinted Hep3B spheroids on the 144PillarPlate were dynamically cultured in the 144PerfusionPlate and tested with anticancer drugs to measure drug effectiveness and determine the concentration required to inhibit 50% of the cell viability (IC50 value). The perfusion plate enabled the convenient dynamic culture of tumor spheroids and facilitated the dynamic testing of anticancer drugs with increased sensitivity. It is envisioned that the integration of microarray bioprinting of tumor spheroids onto the pillar plate, along with dynamic 3D cell culture in the perfusion plate, could more accurately replicate tumor microenvironments. This advancement has the potential to enhance the predictive drug screening process in personalized cancer therapy significantly.

A nuclear spiral in a dusty star-forming galaxy at z=2.78

The nuclear structure of dusty star-forming galaxies is largely unexplored but harbours critical information about their structural evolution. Here, we present long-baseline Atacama Large (sub-)Millimetre Array (ALMA) continuum observations of a gravitationally lensed dusty star-forming galaxy at z=2.78. We use a pixellated lens modelling analysis to reconstruct the rest-frame 230 dust emission with a mean resolution of ≈55 pc and demonstrate that the inferred source properties are robust to changes in lens modelling methodology. The central 1 kpc is characterised by an exponential profile, a dual spiral arm morphology and an apparent super-Eddington compact central starburst. We find tentative evidence for a nuclear bar in the central 300 pc. These features may indicate that secular dynamical processes play a role in accumulating a high concentration of cold gas that fuels the rapid formation of a compact stellar spheroid and black hole accretion. We propose that the high spatial resolution provided by long-baseline ALMA observations and strong gravitational lensing will give key insights into the formation mechanisms of massive galaxies.

Urokinase Plasminogen Activation System Modulation in Transformed Cell Lines

The role of the plasminogen activation system is to regulate the activity of the extracellular protease plasmin. It comprises the urokinase plasminogen activator (uPA), a specific extracellular protease which activates plasminogen, its inhibitor PAI1, and the urokinase plasminogen activator receptor, uPAR, which localizes the urokinase activity. The plasminogen activation system is involved in tissue remodeling through extracellular matrix degradation, and therefore participates in numerous physiological and pathological processes, which make it a potential biomarker. To investigate the role of these molecules in the cellular processes, we cloned human uPA, PAI1, and uPAR and overexpressed them in two cell lines, the glioblastoma line A1235 and the transformed human embryonal kidney cells HEK 293. We analyzed the urokinase activity and the expression of plasminogen activation system elements on the protein and RNA level by Western blot analysis and RTqPCR. Cell proliferation was followed up by cell counting, cell migration and invasion by wound-healing and the transwell assays, respectively, and cell adhesion and dispersal by spheroid formation. The cells transfected with urokinase sequence had increased urokinase activity and uPA expression, while the PAI1-transfected cells decreased urokinase activity, increased PAI1 expression, and decreased cell migration. HEK 293 cells expressing PAI formed only small spheroids. The effects of the uPA system molecules depended on their interactions with each other and with other molecules in the microenvironment, as well as on the cell-type-specific signaling.

ZNFX1 functions as a master regulator of epigenetically induced pathogen mimicry and inflammasome signaling in cancer.

DNA methyltransferase and poly (ADP-ribose) polymerase inhibitors (DNMTis, PARPis) induce a stimulator of interferon genes (STING)-dependent pathogen mimicry response (PMR) in ovarian and other cancers. Here, we showed that combining DNMTis and PARPis upregulates expression of the nucleic-acid sensor NFX1-type zinc finger-containing 1 protein (ZNFX1). ZNFX1 mediated induction of PMR in mitochondria, serving as a gateway for STING-dependent interferon/inflammasome signaling. Loss of ZNFX1 in ovarian cancer cells promoted proliferation and spheroid formation in vitro and tumor growth in vivo. In patient ovarian cancer databases, expression of ZNFX1 was elevated in advanced stage disease, and ZNFX1 expression alone significantly correlated with an increase in overall survival in a phase 3 trial for therapy-resistant ovarian cancer patients receiving bevacizumab in combination with chemotherapy. RNA-sequencing revealed an association between inflammasome signaling through ZNFX1 and abnormal vasculogenesis. Together, this study identified that ZNFX1 as a tumor suppressor that controls PMR signaling through mitochondria and may serve as a biomarker to facilitate personalized therapy in ovarian cancer patients.

Leveraging the nanotopography of filamentous fungal chitin-glucan nano/microfibrous spheres (FNS) coated with collagen (type I) for scaffolded fibroblast spheroids in regenerative medicine.

Numerous naturally occurring biological structures have inspired the development of innovative biomaterials for a wide range of applications. Notably, the nanotopographical architectures found in natural materials have been leveraged in biomaterial design to enhance cell adhesion and proliferation and improve tissue regeneration for biomedical applications. In this study, we fabricated three-dimensional (3D) chitin-glucan micro/nanofibrous fungal-based spheres coated with collagen (type I) to mimic the native extracellular matrix (ECM) microenvironment. These collagen-coated fungal nano/microfibrous spheres (C-FNS) were utilized to construct 3D scaffolded spheroids of human fibroblasts through suspension culture for tissue engineering and regenerative medicine. The particle sizes of C-FNS ranged from 1.4 to 3.25 µm (average: 2.27 ± 0.38 µm), with a porosity of 81.17 %. Field emission-scanning electron microscopy (FE-SEM) revealed that C-FNS comprised continuous chitin-glucan fibers with an average diameter of 363 ± 61 nm (range: 203-512 nm), exhibiting a highly interconnected structure. The reduced arithmetic average roughness (Ra) and root mean square roughness (Rq) values of C-FNS compared to uncoated FNS suggested that collagen coating reduced surface roughness, resulting in a smoother surface that enhanced hydrophilicity, crucial for mammalian cell adhesion and spheroid formation. Moreover, the in vitro cytocompatibility of C-FNS with fibroblasts was evaluated using a resazurin-based PrestoBlue assay, which demonstrated a time-dependent increase in the metabolic activity of C-FNS/fibroblast spheroids during suspension culture for up to 14 days. FE-SEM images of C-FNS/fibroblast spheroids further revealed enhanced adhesion and proliferation of fibroblasts on the nano/microfibrous mycelial architecture, accompanied by the secretion of ECM components and formation of multilayered cell sheets over the 14-day culture period. Similarly, an assessment of the hemocompatibility of C-FNS with erythrocytes revealed the non-hemolytic properties of the biomaterial. Overall, the interaction between collagen-coated fungal chitin-glucan nano/microfibrous structures and mammalian cells holds significant potential for the development of novel, sustainable biomaterials with tailored properties for a myriad of biomedical applications, including tissue engineering, regenerative medicine, drug screening, and wound healing.

Targeting BMP-1 enhances anti-tumoral effects of doxorubicin in metastatic mammary cancer: common and distinct features of TGF-β inhibition.

Mammary carcinoma is comprised heterogeneous groups of cells with different metastatic potential. 4T1 mammary carcinoma cells metastasized to heart (4THM), liver (4TLM) and brain (4TBM) and demonstrate cancer-stem cell phenotype. Using these cancer cells we found thatTGF-β is the top upstream regulator of metastatic process. In addition, secretion of bone morphogenetic protein 1 (BMP-1), which is crucial for the proteolytic release of TGF-β, was markedly high in metastatic mammary cancer cells compared to non-metastatic cells. Although TGF-β inhibitors are in clinical trials, systemic inhibition of TGF-β may produce heavy side effects. We here hypothesize that inhibition of BMP-1 proteolytic activity inhibits TGF-β activity and induces anti-tumoral effects. Effects of specific BMP-1 inhibitor on liver and brain metastatic murine mammary cancer cells (4TLM and 4TBM), as well as on human mammary cancer MDA-MB-231 and MCF-7 cells, were examined and compared with the results of TGF-β inhibition. Inhibition of BMP-1 activity markedly suppressed proliferation of cancer cells and enhanced anti-tumoral effects of doxorubicin. Inhibition of BMP-1 activity but not of TGF-β activity decreased colony and spheroid formation. Differential effects of BMP-1 and TGF-β inhibitors on TGF-β secretion was also observed. These results demonstrated for the first time that the inhibition of BMP-1 activity has therapeutic potential for treatment of metastatic mammary cancer and enhances the anti-tumoral effects of doxorubicin.

N6-methyladenosine-modification of USP15 regulates chemotherapy resistance by inhibiting LGALS3 ubiquitin-mediated degradation via AKT/mTOR signaling activation pathway in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is among the most malignant tumors and seriously threatens human health worldwide, and its incidence rate is increasing annually. USP15 is a member of the ubiquitination-specific protease (USP) family, which can regulate protein ubiquitination, thereby affecting their stability, and is dysregulated in many cancers, but its expression and regulatory mechanism in HCC are unclear. The aims of this study were to explore the role and mechanism of USP15 in regulating HCC cell stemness, proliferation, and lenvatinib resistance. Immunohistochemistry and high-throughput sequencing analyses of tumor and adjacent normal tissue samples from 52 patients with HCC were conducted. Functional analyses of immortalized human liver and HCC cell lines were conducted, including quantitative real-time PCR; western blot; plasmid, lentivirus, and siRNA transfection; co-immunoprecipitation; mass spectrometry; MeRIP-qPCR; and ubiquitination, cell growth, colony formation, and spheroid formation assays. HCC tumor growth was also assessed using cell transplantation in nude mice. We found that USP15 is upregulated in HCC and affects patient prognosis. Our results demonstrated that USP15 can increase LGALS3 stability in HCC through deubiquitination modification, and affect the stemness, proliferation, and lenvatinib resistance of HCC cells by activating the AKT/mTOR pathway. USP15 expression levels were positively correlated with HCC cell stemness, proliferation, and lenvatinib resistance. In addition, methyltransferase-like protein 3 (Mettl3) N6-methyladenosine (m6A) modified USP15 to upregulate its levels by increasing its mRNA stability. These findings provide a theoretical basis for the potential discovery of new HCC oncogenes, as well as the identification of effective targets and development of novel anti-HCC drugs and clinical applications.

Molecular Characterization of Cancer Preventive and Therapeutic Potential of Three Antistress Compounds, Triethylene Glycol, Withanone, and Withaferin A

The molecular link between stress and carcinogenesis and the positive outcomes of stress intervention in cancer therapy have recently been well documented. Cancer stem cells (CSCs) facilitate cancer malignancy, drug resistance, and relapse and, hence, have emerged as a new therapeutic target. Here, we aimed to investigate the effect of three previously described antistress compounds (triethylene glycol, TEG; Withanone, Wi-N, and Withaferin A, Wi-A) on the stemness and differentiation characteristics of cancer cells. Breast carcinoma, glioblastoma, and neuroblastoma cells were treated with a non-toxic concentration of TEG (0.1%), Wi-N (5 µM), and Wi-A (0.1 µM) in 2D and 3D cultures. The results demonstrated that TEG, Wi-N, and Wi-A suppressed the stemness properties, which was linked with their inhibition of epithelial–mesenchymal transition (EMT) signaling. In particular, Wi-N and TEG caused a stronger reduction in the self-renewal capability of CSCs than Wi-A, as evidenced by a tumor spheroid formation assay and analyses of stemness-related genes (ALDH1, CD44, NANOG, CD133, SOX2). Furthermore, TEG and Wi-N caused the differentiation of cancer cells. Each of these was supported by (i) the upregulation of KRT18, KRT19, E-cadherin, and downregulation of vimentin in breast carcinoma; (ii) increased levels of GFAP, MAP2, and PSD-95 in astrocytoma; and (iii) increased NeuN, GAP-43, and NF200 levels in neuroblastoma. Furthermore, a reduction in cancer progression-related proteins (PI3K, N-myc) was recorded in treated cells. Our results suggest that TEG and Wi-N may be recruited to target cancer cell stemness and differentiation therapy.

The design of the spheroids-based in vitro tumor model determines its biomimetic properties.

Cancer, one of the world's deadliest diseases, is expected to claim an estimated 16 million lives by 2040. Three-dimensional (3D) models of cancer have become invaluable tools for the study of tumor biology and the development of new therapies. The tumor microenvironment (TME) is a determinant of tumor progression and has implications for clinical therapies. Cancer-associated fibroblasts (CAFs) are one of the most important components of the TME. Modeling the interactions between cancer cells and CAFs in vitro can help to create biomimetic tumor equivalents for elucidating the causes of cancer growth and assessing the effectiveness of therapies. Here, we are investigated the effect of the mutual arrangement of tumor cells and fibroblasts on the formation of tumor models and their biomimetic properties. Pancreatic tumor models of three different designs were formed by the bioprinting method. Gelatin-alginate hydrogels with and without PANC-1 (pancreatic cancer) and NIH/3T3 (mouse fibroblasts) cells, as well as their homo- and heterospheroids, were used as bioink. To enable bioprinting, we have chosen the most suitable compositions of alginate and gelatin that provide both good printability and cell proliferation activity. We also have investigated the kinetics of spheroid formation to identify the optimal cultivation parameters for achieving spheroid sizes suitable for bioprinting. All tumor models remained viable for 3-4weeks. At the same time, the patterns of model development in the cultivation process and the biomimetic properties of the final tissue-engineered structures depended on the model design.

Production and Cryopreservation of 3d Cultures

Three-dimensional (3D) culture systems, which include spheroids (SPs), provide a unique platform for studying complex biological processes in vivo and for enhancing the capabilities of in vitro test systems. Their uniqueness lies in the 3D organization of cells and in the reproduction of complex intercellular interactions, similar to those in native tissues and organs. These "mini-organs" can be used for fundamental research, tissue-engineering constructs, development of preclinical models for testing pharmacological drugs, etc. Important and current issues regarding SPs involve improving methods for their production and cryopreservation. Solving these issues will expand the range and effectiveness of their use in tissue engineering. Here, we describe the authors' research and experience on factors influencing the formation of SPs, which can enhance the understanding of their correct application and standardization. A crucial aspect of this review is the information on applying theoretical approaches based on physico-mathematical calculations to improve the quality of existing cryopreservation protocols for SPs.

Current perspectives on the dynamic culture of mesenchymal stromal/stem cell spheroids.

Mesenchymal stromal/stem cells (MSCs) are promising candidates for regenerative medicine owing to their self-renewal properties, multilineage differentiation, immunomodulatory effects, and angiogenic potential. MSC spheroids fabricated by 3Dculture have recently shown enhanced therapeutic potential. MSC spheroids create a specialized niche with tight cell-cell and cell-extracellular matrix interactions, optimizing their cellular function by mimicking the in vivo environment. Methods for 3D cultivation of MSCs can be classified into 2 main forms: static suspension culture and dynamic suspension culture. Numerous studies have reported the beneficial influence of these methods on MSCs, which is displayed by increased differentiation, angiogenic, immunomodulatory, and anti-apoptotic effects, and stemness of MSC spheroids. Particularly, recent studies highlighted the benefits of dynamic suspension cultures of the MSC spheroids in terms of faster and more compact spheroid formation and the long-term maintenance of stemness properties. However, only a few studies have compared the behavior of MSC spheroids formed using static and dynamic suspension cultures, considering the significant differences between their culture conditions. This review summarizes the differences between static and dynamic suspension culture methods and discusses the biological outcomes of MSC spheroids reported in the literature. In particular, we highlight the advantages of the dynamic suspension culture of MSC spheroids and contemplate its future applications for various diseases.

Injectable and 3D-Printable Semi-Interpenetrating Polymer Networks Based on Modified Sodium Alginate for Cell Spheroid Formation.

We report on 3D-printable polymer networks based on the combination of modified alginate-based polymer blends; two alginate polymers were prepared, namely, a thermoresponsive polymer grafted with P(NIPAM86-co-NtBAM14)-NH2 copolymer chains and a second polymer modified with diol/pH-sensitive 3-aminophenylboronic acid. The gelation properties were determined by the hydrophobic association of the thermosensitive chains and the formation of boronate esters. At a mixing ratio of 70/30 wt % of the thermo/diol-responsive polymers, the semi-interpenetrating network exhibited an optimum storage modulus ranging from ca. 150 Pa at 20 °C up to ca. 480 Pa at 37 °C due to the stimulated cross-linking synergism. The resulting bioink blends could promote the rapid formation of cell spheroids with an average diameter of 62.5 μm within 24 h. The network could easily be dissociated by the addition of free glucose, acting as an antagonistic disruptor of the cross-links. The proposed material was found to be nontoxic, with adequate injectability and 3D printability.

Synergistic Approach of High-Precision 3D Printing and Low Cell Adhesion for Enhanced Self-Assembled Spheroid Formation

Spheroids, as three-dimensional (3D) cell aggregates, can be prepared using various methods, including hanging drops, microwells, microfluidics, magnetic manipulation, and bioreactors. However, current spheroid manufacturing techniques face challenges such as complex workflows, the need for specialized personnel, and poor batch reproducibility. In this study, we designed a support-free, 3D-printed microwell chip and developed a compatible low-cell-adhesion process. Through simulation and experimental validation, we rapidly optimized microwell size and the coating process. We successfully formed three types of spheroids—human immortalized epidermal cells (HaCaTs), umbilical cord mesenchymal stem cells (UC-MSCs), and human osteosarcoma cells (MG63s)—on the chip. Fluorescent viability staining confirmed the biocompatibility and reliability of the chip. Finally, drug response experiments were conducted using the chip. Compared to traditional methods, our proposed strategy enables high-throughput production of size-controlled spheroids with excellent shape retention, while enhanced gas exchange during culture improves differentiation marker expression. This platform provides an efficient and cost-effective solution for biosensing applications, such as drug screening, disease modeling, and personalized therapy monitoring. Furthermore, the chip shows significant potential for real-time in vitro monitoring of cellular viability, reaction kinetics, and drug sensitivity, offering valuable advancements in biosensor technology for life sciences and medical applications.

Development of Pyramidal Microwells for Enhanced Cell Spheroid Formation in a Cell-on-Chip Microfluidic System for Cardiac Differentiation of Mouse Embryonic Stem Cells.

Three-dimensional (3D) tissue culture models provide in vivo-like conditions for studying cell physiology. This study aimed to examine the efficiency of pyramidal microwell geometries in microfluidic devices on spheroid formation, cell growth, viability, and differentiation in mouse embryonic stem cells (mESCs). The static culture using the hanging drop (HD) method served as a control. The microfluidic chips were fabricated to have varying pyramidal tip angles, including 66°, 90°, and 106°. From flow simulations, when the tip angle increased, streamline distortion decreased, resulting in more uniform flow and a lower velocity gradient near the spheroids. These findings demonstrate the significant influence of microwell geometry on fluid dynamics. The 90° microwells provide optimal conditions, including uniform flow and reduced shear stress, while maintaining the ability for waste removal, resulting in superior spheroid growth compared to the HD method and other microwell designs. From the experiments, by Day 3, spheroids in the 90° microwells reached approximately 400 µm in diameter which was significantly larger than those in the 66° microwells, 106° microwells, and HD cultures. Brachyury gene expression in the 90° microwells was four times higher than the HD method, indicating enhanced mesodermal differentiation essential for cardiac differentiation. Immunofluorescence staining confirmed cardiomyocyte differentiation. In conclusion, microwell geometry significantly influences 3D cell culture outcomes. The pyramidal microwells with a 90° tip angle proved most effective in promoting spheroid growth and cardiac differentiation of mESC differentiation, providing insights for optimizing microfluidic systems in tissue engineering and regenerative medicine.

USP21-EGFR signaling axis is functionally implicated in metastatic colorectal cancer

The emerging role of ubiquitin-specific peptidase 21 (USP21) in stabilizing Fra-1 (FOSL1) highlights its involvement in promoting colorectal cancer (CRC) metastasis. Additionally, a reciprocal link between EGFR signaling and Fra-1 activation has been identified, mediated through matrix metalloproteinases (MMPs). However, the functional implications of the USP21-EGFR signaling axis in metastatic CRC (mCRC) are not fully understood. To investigate the clinical correlation between USP21 and EGFR expression, RNA-Seq data from tumor tissues (n = 27) and matched normal tissues (n = 27) of 27 mCRC patients were analyzed. Functional studies were performed, including the use of CRISPR/Cas9 to generate USP21-knockout (USP21-KO) CRC cells, in vitro assays for cancer progression and tumor formation, in vivo xenograft assays in NSG mice. Additionally, the therapeutic effect of the USP21 inhibitor, BAY-805, was evaluated. We found that elevated levels of USP21 and EGFR expression in mCRC patients were associated with poorer survival outcomes. Mechanistically, USP21 was found to enhance EGFR stability by deubiquitinating EGFR, leading to reduced EGFR degradation. USP21-KO colon cancer cells exhibited significantly reduced proliferation, migration, colony formation, and 3D tumor spheroid formation in response to EGF. Furthermore, the tumorigenic activity in vivo was markedly diminished in NSG mice xenografted with USP21-KO colon cancer cells. Importantly, BAY-805 demonstrated a notable inhibitory effect on the formation of 3D tumor spheroids in colorectal cancer cells stimulated with EGF. These findings suggest that USP21 could be a valuable therapeutic target and predictive biomarker for managing mCRC driven by EGF.

TFDP1 transcriptionally activates KIF22 to enhance aggressiveness and stemness in endometrial cancer: implications for prognosis and targeted therapy.

This study aims to elucidate the role of Kinesin Family Member 22 (KIF22) as a critical regulator of aggressive behavior in endometrial cancer (uterine corpus endometrial carcinoma, UCEC) and to uncover its underlying mechanisms, thereby providing a molecular rationale for future targeted treatment. Bioinformatics analyses were employed to assess KIF22 and TFDP1 expression in UCEC, examining their prognostic value and associations with disease progression. Expression levels were validated in UCEC tissues using qRT-PCR and western blotting. Potential TFDP1 binding sites on the KIF22 promoter were predicted using the JASPAR database and confirmed via dual-luciferase reporter assays. Functional assays, including CCK-8, transwell, and spheroid formation assays, were conducted to evaluate the effects of KIF22 knockdown on UCEC cell behavior. A mouse xenograft model was utilized to investigate the in vivo impact of KIF22 suppression on tumor growth and stemness. KIF22 expression was significantly elevated in UCEC tissues, correlating with reduced overall survival in patients with high KIF22 levels. Overexpression of KIF22 enhanced the proliferation, migration, and sphere formation of UCEC cells. Similarly, high TFDP1 expression was associated with poorer patient outcomes. KIF22 was found to be positively regulated by the TFDP1 transcription factor, which bound to the KIF22 promoter and activated its expression in UCEC cells. In vivo, KIF22 knockdown markedly impeded the tumor formation of cells and reduced stemness marker expression. KIF22, upregulated by TFDP1, enhances UCEC cell aggressiveness and is linked to poor prognosis, highlighting its potential as a target for therapeutic intervention in endometrial cancer.

Evaluation of the Cancer-Preventive Effect of Resveratrol-Loaded Nanoparticles on the Formation and Growth of In Vitro Lung Tumor Spheroids.

Resveratrol (RSV) is a natural polyphenol that offers antioxidant, anti-inflammatory, and chemopreventive benefits. This project determined the ability of RSV-loaded nanoparticles (NP) to inhibit the growth of lung tumor spheroids in vitro. RSV was encapsulated in NP comprised of the biodegradable polymer, acetalated dextran. A549 lung cancer cells in two-dimensional and three-dimensional cell culture models were exposed to free RSV and RSV NP to evaluate their effect on cell proliferation and spheroid formation and growth. For prevention studies, spheroids were exposed to free RSV and RSV NP on day 0, and for treatment studies, spheroids were dosed with the same formulations on day 5 after the spheroids had fully formed. The resulting RSV NP were 200 nm in diameter with neutral surface charge and exhibited the ability to control the release of RSV in vitro based on environmental pH. In comparison to free RSV, the RSV NP exerted a greater inhibitory effect on the proliferation and growth of cancer cells and spheroids. RSV NP have the potential to be used as a chemopreventive agent for lung cancer.

Plasma-Activated Medium Inhibited the Proliferation and Migration of Non-Small Cell Lung Cancer A549 Cells in 3D Culture.

Lung cancer is the most common type of malignant tumor worldwide. Plasma-activated medium (PAM) is an innovative cancer treatment method that has received considerable scientific attention. The objective of this study is to evaluate the effects of PAM on the anti-tumor characteristics of non-small cell lung cancer (NSCLC) cells in two-dimensional (2D) and three-dimensional (3D) cultures. The effects of PAM treatment on the proliferative and migratory capabilities of A549 cells in 2D and 3D cultures were assessed using MTT, migration, invasion assays, and cell cycle, respectively. The study also investigated the impact of PAM treatment on the changes in the content of intracellular and extracellular reactive species and analyzed protein expression using the Western Blot method. PAM treatment inhibited the viability, migration, and invasion abilities of A549 cells in both 2D and 3D cultures, suppressed the epithelial-mesenchymal transition (EMT) process, and downregulated the expression of the RAS/ERK signaling pathway, which effectively inhibited tumor spheroid formation. Additionally, the effect of PAM on A549 cells was mediated through ROS-induced oxidative reactions, and PAM treatment exhibited greater cytotoxicity in 2D culture compared to 3D culture. As compared to 2D, the 3D cell culture model provides a viable in vitro cell model for studying the mechanisms of PAM treatment in lung cancer. PAM represents an effective new treatment for NSCLC.

Establishment and characterization of NCC-GCTB10-C1: a novel cell line derived from a patient with recurrent giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is a rare osteolytic tumor composed of mononuclear stromal cells, macrophages, and osteoclast-like giant cells. While generally benign, GCTB has a high risk of local recurrence and can occasionally undergo malignant transformation or metastasis, posing significant clinical challenges. The primary treatment is complete surgical resection; however, effective management strategies for recurrent or advanced GCTB remain elusive, underscoring the need for further preclinical research. This study reports the establishment of a novel cell line, NCC-GCTB10-C1, derived from a recurrent GCTB lesion. NCC-GCTB10-C1 retains the characteristic H3-3A G34W mutation, which is central to the tumor's pathogenesis, and demonstrates significant growth potential, spheroid formation capability, and invasive properties. Extensive drug screening of NCC-GCTB10-C1, along with nine previously established GCTB cell lines, revealed a distinct drug response profile, with the cell line showing resistance to many previously effective agents. However, doxorubicin, foretinib, and ceritinib were identified as promising therapeutic candidates due to their low IC50 values in NCC-GCTB10-C1. The establishment of NCC-GCTB10-C1 offers a critical resource for further research into GCTB, especially in the context of recurrent disease, and holds potential for the development of more effective treatment strategies.