Spheroid Model Research Articles

A Demethylation-Switchable Aptamer Design Enables Lag-Free Monitoring of m6A Demethylase FTO with Energy Self-Sufficient and Structurally Integrated Features.

Cellular context profiling of modification effector proteins is critical for an in-depth understanding of their biological roles in RNA N6-methyladenosine (m6A) modification regulation and function. However, challenges still remain due to the high context complexities, which call for a versatile toolbox for accurate live-cell monitoring of effectors. Here, we propose a demethylation-switchable aptamer sensor engineered with a site-specific m6A (DSA-m6A) for lag-free monitoring of the m6A demethylase FTO activity in living cells. As a proof of concept, a DNA aptamer against adenosine triphosphate (ATP) is selected to construct the DSA-m6A model, as the "universal energy currency" role of ATP could guarantee the equally fast and spontaneous conformation change of DSA-m6A sensor upon demethylation and ATP binding in living organisms, thus enabling sensitive monitoring of FTO activity with neither time delay nor recourse to extra supply of substances. This ATP-driven DSA-m6A design facilitates biomedical research, including live-cell imaging, inhibitor screening, single-cell tracking of dynamic FTO nuclear translocation upon starvation stimuli, FTO characterization in a biomimetic heterotypic three-dimensional (3D) multicellular spheroid model, as well as the first report on the in vivo imaging of FTO activity. This strategy provides a simple yet versatile toolbox for clinical diagnosis, drug discovery, therapeutic evaluation, and biological study of RNA demethylation.

Spatiotemporally Resolved Approach for Profiling Ferroptosis-Associated Metabolic Vulnerabilities in Tumors Using Mass Spectrometry Imaging and Stable Isotope Tracing.

Ferroptosis, as an iron-dependent cell death mediated by lipid peroxidation, has sparked great interest in the tumor research community. Targeting ferroptosis has been proven to be a new therapeutic opportunity for inhibiting tumor growth. However, it is challenging to precisely characterize the metabolic pattern of ferroptosis in heterogeneous tumors and further identify ferroptosis-associated metabolic vulnerabilities for tumor treatment. In this work, we developed a spatiotemporally resolved method to image ferroptosis-associated metabolic alterations in 3D tumor spheroids by combining mass spectrometry imaging and stable isotope tracing techniques. The construction of a 3D tumor spheroid model allows for a more accurate simulation of ferroptosis, and the introduction of MALDI-MSI enables in situ screening of abnormal molecules in tumor tissues. Using this method, we showed that the expression proportion of nC═C = 4 polyunsaturated fatty acids, including arachidonic acid (FA-20:4) and adrenic acid (FA-22:4), were upregulated in RSL3-induced 3D tumor ferroptosis models. And the isotope tracing experiment revealed that the absorption of fatty acids and the biosynthesis of polyunsaturated fatty acids were significantly increased during ferroptosis. In addition, we discovered that arachidonic acid and adrenic acid supplementation render tumor cell sensitive to ferroptosis, thereby limiting the growth of tumor cells and the formation of 3D tumor spheroids. Such findings provide significant clues for understanding the metabolic signatures of tumor ferroptosis and raise the possibility to screen potential metabolic vulnerabilities for better tumor treatment in combination with ferroptosis.

Development and In-Vitro Evaluation of Doxorubicin-Loaded Methacrylate Gelatin (GelMa)-Acrylamide Hydrogels for the Treatment of Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is the most prevalent subtype of malignant mesothelioma that affects the pleural lining of the lungs. Conventionally, chemotherapy via systemic injections has shown limited efficacy due to off-target effects, and inefficacious deposition at the disease site. In our previous study, we reported the development and optimization of UV-initiated methacrylate gelatin (GelMa)-acrylamide based hydrogel formulation for local intracavitary administration of therapies. The current study utilizes a pre-established GelMa formulation for delivering a small molecule chemotherapeutic agent, Doxorubicin (Dox), against in-vitro MPM models. Dox-loaded hydrogel (DLH) precursor solution was prepared by dissolving Dox in the precursor solution. The gels were characterized for physical properties such as gelling time, swelling index, bio adhesion, and injectability and were compared to blank hydrogels. Dox-loaded hydrogels were also tested for therapeutic efficacy in MPM cells in various 2D and 3D cell culture models. It was revealed that Dox-loaded hydrogels retained similar physical properties, including gelling time (< 25s), swelling index (~ 1,200%), bio-adhesion (> 20g detachment force), and injectability (< 2N force for injecting precursor), compared to blank hydrogels. Moreover, the gel formulation effectively sustained the release of hydrophilic Dox HCl over a period of 12days by increasing the degree of crosslinking between GelMa and its crosslinkers. Further, the therapeutic efficacy of Dox was retained even after loading into hydrogels, indicating that no chemical interactions took place between gel excipients and the drug. Studies in MPM cell-based models revealed that DLH showed excellent potential in inhibiting 2D and 3D cell growth, with DLH being more effective than plain Dox in suppressing tumor growth in 3D spheroid models. Overall, the results of the present study suggest that Dox-loaded hydrogels (DLH) may be a good candidate for efficacy study in preclinical mesothelioma models, with strong potential for clinical translation.

Resveratrol Can Differentiate Human Melanoma Stem-like Cells from Spheroids Treated With All-trans Retinoic Acid.

Stem-like cancer cells are believed to be the leading cause of therapy resistance in malignant melanoma (MM). All-trans retinoic acid (ATRA) differentiation therapy is considered a promising approach to eradicate stem-like cancer cells, but some melanoma cells are resistant to ATRA. This study aimed to examine whether resveratrol (RS), a natural polyphenol compound, could improve the response of MM stem-like cells to ATRA and explore the possible underlying mechanisms. MM stem-like cells were established from a spheroid model of A375 human MM cell line. The response to RES alone and in combination with ATRA, was examined through analysis of cancer stemness, cell viability, and protein expression. The stem-like cells showed resistance to the anticancer drug docetaxel; however, the combination of RES and ATRA augmented the effects of docetaxel. Accordingly, these combinatorial effects were associated with significant inhibition of the expression levels of stemness markers CD133, OCT4, CD271, and ABCG2. The tested combinations also led to a significant increase in melanocyte differentiation marker SOX9, while efficiently suppressing the dedifferentiation marker SOX10. Notably, RES alone effectively up-regulated retinoic acid receptor beta (RARβ) expression and down-regulated crucial mediators like DNMT1, polycomb-group proteins EZH2, and BMI-1, which mechanistically explain how RES enhanced the differentiation-inducing effects of ATRA. The resistance of MM stem-like cells to ATRA can be attenuated by RES and combined applications of ATRA and RES provide a promising strategy for MM treatment.

ATR inhibition potentiates FOLFIRINOX cytotoxic effect in models of pancreatic ductal adenocarcinoma by remodelling the tumour microenvironment.

In pancreatic ductal adenocarcinoma (PDAC), the dense stroma rich in cancer-associated fibroblasts (CAFs) and the immunosuppressive microenvironment confer resistance to treatments. To overcome such resistance, we tested the combination of FOLFIRINOX (DNA damage-inducing chemotherapy drugs) with VE-822 (an ataxia-telangiectasia and RAD3-related inhibitor that targets DNA damage repair). PDAC spheroid models and organoids were used to assess the combination effects. Tumour growth and the immune and fibrotic microenvironment were evaluated by immunohistochemistry, single-cell analysis and spatial proteomics in patient-derived xenograft (PDX) and orthotopic immunocompetent KPC mouse models. The FOLFIRINOX and VE-822 combination had a strong synergistic effect in several PDAC cell lines, whatever their BRCA1, BRCA2 and ATM mutation status and resistance to standard chemotherapy agents. This was associated with high DNA damage and inhibition of DNA repair signalling pathways, leading to increased apoptosis. In immunocompetent and PDX mouse models of PDAC, the combination inhibited tumour growth more effectively than FOLFIRINOX alone. This was associated with tumour microenvironment remodelling, particularly decreased proportion of fibroblast activated protein-positive CAFs and increased anti-tumorigenic immune cell infiltration and interaction. The FOLFIRINOX and VE-822 combination is a promising strategy to improve FOLFIRINOX efficacy and overcome drug resistance in PDAC.

Reversible downregulation of MYC in a spheroid model of metastatic epithelial ovarian cancer.

Upon detachment from the primary tumour, epithelial ovarian cancer cells can form multicellular aggregates, also referred to as spheroids, that have the capacity to establish metastases at distant sites. These structures exhibit numerous adaptations that may facilitate metastatic transit and promote tumorigenic potential. One such adaptation is the acquisition of dormancy, characterized by decreased proliferation and molecular features of quiescence. One of the most frequently dysregulated genes in cancer is MYC, which encodes a transcription factor that promotes cell proliferation. In this study, we demonstrate that MYC protein abundance and associated gene expression is significantly decreased in EOC spheroids compared to adherent cells. This downregulation occurs rapidly upon cell detachment and is proteasome-dependent. Moreover, MYC protein abundance and associated gene expression is restored upon spheroid reattachment to an adherent culture surface. Overall, our findings suggest that suppression of MYC activity is a common feature of EOC spheroids and may contribute to the reversible acquisition of dormancy.

Synthesis and biological evaluation of a new class of azole urea compounds as Akt inhibitors with promising anticancer activity in pancreatic cancer models

The PI3K/Akt pathway is crucial in numerous cellular functions such as cell growth, survival proliferation and movement in both normal and cancer cells. It plays also a key role in epithelial-mesenchymal transitions and angiogenesis during the tumorigenesis processes. Since many transformative events in cancer are driven by increased PI3K/Akt pathway signaling, Akt is considered a valuable target for developing new therapies against various tumor types, including pancreatic cancer. This is because the PI3K/AKT/mTOR pathway is a key downstream effector of RAS, and RAS activation is the most prominent genetic alteration in pancreatic cancer. Herein we report the synthesis and the biological evaluation of a new series of azole urea compounds that exhibited promising antiproliferative and antimigratory activities against pancreatic cancer cells through an Akt inhibition mechanism. These effects were demonstrated using a variety of assays, including Sulforhodamine B, cell-cycle, wound-healing, and kinase activity, apotposis and ELISA assays. Additionally, the anticancer properties of the most active compound in the series were confirmed in the 3D spheroid model of PATU-T cells.

EXTH-61. TOWARDS A QUANTUM THERAPY FOR GLIOBLASTOMA: A NEW THERAPEUTIC PARADIGM IN MEDICINE

Abstract BACKGROUND A cell operates as an interconnected bioelectrical circuit, utilising electron transfer processes for intracellular communication, with cytochrome c (Cyt c) playing a pivotal role. The redox processes of Cyt c, occurring via electron tunnelling, are essential for its translocation into the cytosol and modulation of its conformation to bind apoptotic protease activating factor 1. This highlights the need for novel technologies capable of interacting with these processes at the atomic scale to control downstream effects and induce apoptosis in cancer cells. METHODS We demonstrate that ‘bio-nanoantennae’, when supplied with an electrical current, enable quantum biological tunnelling for electron transfer (QBET) and facilitate cellular apoptosis in patient-derived IDH wild-type glioblastoma from both the infiltrative tumour margin and proliferative core. The bio-nanoantennae were constructed from gold nanoparticles functionalised with reduced Cyt c and zinc porphyrin as a redox couple. RESULTS Electrical polarisation of these bio-nanoantennae via resonant alternating currents in preclinical glioblastoma cells led to decreased metabolic activity and reduced cell viability by oxidizing Cyt c, thus inducing cellular stress. No significant effect was observed in healthy human astrocyte counterparts. The cytosol localised bio-nanoantennae induced differential gene expression related to ion channels, apoptosis, cancer proliferation and tumour suppression upon activation, in tumour relative to astrocyte cell populations. The bio-nanoantennae were also tested in 3D glioblastoma spheroid models, showing similar effects, and in vivo studies demonstrated a significant reduction in glioblastoma xenograft tumour size. CONCLUSION We propose that bio-nanoantennae modulate the redox state of Cyt c under an electrical field through QBET. To validate this, we investigated the tunnel junction energy and plasmon resonance scattering, and developed a mathematical model to explain the system’s behaviour. This innovative wireless electrical–molecular nanodevice, capable of inducing cancer cell apoptosis, paves the way for further applications of quantum signalling as a new (non-pharmacological) therapeutic paradigm.

3D keloid spheroid model: Development and application for personalized drug response prediction

Research on keloid is limited by the lack of proper in vitro and animal model reflecting in vivo status. Based on heterogeneity of keloid and important role of endothelial cells in its pathogenesis, a novel 3D in vitro keloid spheroid prepared with keloid fibroblasts and endothelial cells was evaluated in this study. Commercial cell lines of keloid fibroblasts and endothelial cells were used at various cellular ratios to generate keloid spheroids to determine the optimal condition. Keloid spheroids from three keloid patients were also made and their usefulness as in vitro models, including their responses to drugs, were assessed. Spheroids with higher endothelial cell proportions exhibited increased viability and propagation ability. Patient-derived keloid spheroids showed heterogeneity which might reflect individual clinical conditions. The optimal ratio of fibroblasts to endothelial cells was determined to be 4:1 for keloid spheroids based on gene expression and viability analyses. Patient-derived keloid spheroid showed better keloidal changes in genetic expressions than 2D monolayer culture. Spheroids exhibited varied responses and resistance to each drug used for keloids, depending on the cell type used. 3D keloid spheroids might provide an effective in vitro model for investigating disease pathogenesis and appropriate treatment modalities for future precision medicine.

Recapitulating Glioma Stem Cell Niches Using 3D Spheroid Models for Glioblastoma Research.

Glioblastoma multiforme (GBM) is among the most aggressive brain cancers, and it contains glioma stem cells (GSCs) that drive tumor initiation, progression, and recurrence. These cells resist conventional therapies, contributing to high recurrence rates in GBM patients. Developing in vitro models that mimic the tumor microenvironment (TME), particularly the GSC niche, is crucial for understanding GBM growth and therapeutic resistance. Three-dimensional (3D) spheroid models provide a more physiologically relevant approach than traditional two-dimensional (2D) cultures, recapitulating key tumor features like hypoxia, cell heterogeneity, and drug resistance. This review examines scaffold-free and scaffold-based methods for generating 3D GBM spheroids, focusing on their applications in studying the cancer stem cell niche. The discussion encompasses methods such as the hanging drop, low-adhesion plates, and magnetic levitation, alongside advancements in embedding spheroids within extracellular matrix-based hydrogels and employing 3D bioprinting to fabricate more intricate tumor models. These 3D culture systems offer substantial potential for enhancing our understanding of GBM biology and devising more effective targeted therapies.

Improved visualisation of ACP-engineered osteoblastic spheroids: a comparative study of contrast-enhanced micro-CT and traditional imaging techniques

This study investigates osteoblastic cell spheroid cultivation methods, exploring flat-bottom, U-bottom, and rotary flask techniques with and without amorphous calcium phosphate (ACP) supplementation to replicate the 3D bone tissue microenvironment. ACP particles derived from eggshell waste exhibit enhanced osteogenic activity in 3D models. However, representative imaging of intricate 3D tissue-engineered constructs poses challenges in conventional imaging techniques due to notable scattering and absorption effects in light microscopy, and hence limited penetration depth. We investigated contrast-enhanced micro-CT as a methodological approach for comprehensive morphological 3D-analysis of thein-vitromodel and compared the technique with confocal laser scanning microscopy, scanning electron microscopy and classical histology. Phosphotungstic acid and iodine-based contrast agents were employed for micro-CT imaging in laboratory and synchrotron micro-CT imaging. Results revealed spheroid shape variations and structural integrity influenced by cultivation methods and ACP particles. The study underscores the advantage of 3D spheroid models over traditional 2D cultures in mimicking bone tissue architecture and cellular interactions, emphasising the growing demand for novel imaging techniques to visualise 3D tissue-engineered models. Contrast-enhanced micro-CT emerges as a promising non-invasive imaging method for tissue-engineered constructs containing ACP particles, offering insights into sample morphology, enabling virtual histology before further analysis.

Inhibiting ADAM17 enhances the efficacy of olaparib in ovarian cancer spheroids

Acquired or de novo resistance to poly (ADP-ribose) polymerase inhibitors (PARPi) is a major challenge to ovarian cancer treatment. Therefore, strategies to overcome PARPi resistance are critical to improve prognosis. The purpose of this study is to evaluate whether inhibition of ADAM17 sensitizes ovarian cancer to treatment with olaparib, a PARPi, thereby bypassing resistance mechanisms and improving treatment response. Thus, we analyzed the effect of olaparib in combination with the ADAM17 inhibitor GW280264X in ovarian cancer using a 2D monolayer and a 3D spheroid model followed by a multicontent readout (viability, caspase activation and cytotoxicity). To emphasize the translational aspect of our work, we performed corresponding experiments on primary cells derived from ovarian cancer patients initially screened for their mutation status of the breast cancer gene (BRCA 1/2). In 2D, we observed a significant reduction in cell viability and a subsequent increase in apoptosis of the combined treatment (olaparib + GW280264X) compared with olaparib mono-treatment. The combined treatment allows a substantial dose reduction of olaparib rendering a strong synergistic effect. Using a 3D spheroid model from primary cells, we confirmed the 2D monoculture results and demonstrated not only increased caspase activity under the combined treatment but also a substantial gain in cytotoxicity compared to the mono-treatment. Our study proposes ADAM17 inhibition sensitizing ovarian cancer to olaparib treatment and improving treatment response.

The role of mesenchymal cells in cholangiocarcinoma.

The tumour microenvironment (TME) significantly influences tumour formation and progression through dynamic interactions. Cholangiocarcinoma (CCA), a highly desmoplastic tumour, lacks early diagnostic biomarkers and has limited effective treatments due to an incomplete understanding of its molecular pathogenesis. Investigating the TME's role in CCA progression could lead to better therapies. RNA sequencing was performed on seven CCA PDXs and their corresponding patient samples. Differential expression analysis was conducted, and Qiagen Ingenuity Pathway Analysis (IPA) was used to predict dysregulated pathways and upstream regulators. PDX and cell line-derived spheroids, with and without immortalised mesenchymal stem cells, were grown and analysed for morphology, growth, and viability. Histological analysis confirmed biliary phenotypes. RNA sequencing indicated upregulation of ECM-receptor interaction and PI3K-Akt pathways in the presence of MSCs, with several genes linked to poor survival. MSCs restored the activity of inhibited cancer-associated kinases (ICAKs). This study shows that adding MSCs to CCA spheroid models restores key paracrine signalling pathways lost in PDXs, enhancing tumour growth and viability. These findings highlight the importance of including stromal components in cancer models to improve pre-clinical studies.

Development of Novel 3D Spheroids for Discrete Subaortic Stenosis.

In this study, we propose a new method for bioprinting 3D Spheroids to study complex congenital heart disease known as discrete subaortic stenosis (DSS). The bioprinter allows us to manipulate the extrusion pressure to change the size of the spheroids, and the alginate porosity increases in size over time. The spheroids are composed of human umbilical vein endothelial cells (HUVECs), and we demonstrated that pressure and time during the bioprinting process can modulate the diameter of the spheroids. In addition, we used Pluronic acid to maintain the shape and position of the spheroids. Characterization of HUVECs in the spheroids confirmed their uniform distribution and we demonstrated cell viability as a function of time. Compared to traditional 2D cell cultures, the 3D spheroids model provides more relevant physiological environments, making it valuable for drug testing and therapeutic applications.

Integrated Approach to Cyclopiazonic Acid Cytotoxicity Using In Vitro (2D and 3D Models) and In Silico Methods.

Cyclopiazonic acid (CPA) is an indole-tetramic acid neurotoxin produced by Aspergillus and Penicillium genera present mainly in fruit, cereals and nuts. This study compares the cytotoxicity produced by CPA after 24, 48 and 72 h of exposure using both monolayers and 3D spheroids in human neuroblastoma SH-SY5Y cells. Furthermore, CPA toxicokinetics was evaluated using in silico models. Cytotoxicity increased dose- and time-dependently, as shown by the MTT assay. The lowest CPA IC50 values were found in the monolayer study compared to the 3D spheroids at all exposure times (24 h: 864.01 vs. 1132; 48 h: 437 vs. 1069; 72 h: 392 vs. 567 nM). The CPA exposure on SH-SY5Y spheroid organization and morphology was also studied. Morphological changes, including spheroid disaggregation, were observed after mycotoxin exposure. The in silico methods, SwissADME and admetSAR, were used for short and full ADMEt profiles of CPA. The ADMEt predictive profile shows high gastrointestinal absorption and ability to penetrate the blood-brain barrier. Including in silico studies emphasizes the comprehensive approach to understanding mycotoxin toxicity and risk assessment. By combining in vitro 3D spheroid models with computational simulations, this study aims to provide a holistic perspective on the effects of CPA, enhancing the accuracy and relevance of our findings.

Amitraz mechanisms of cytotoxicity in a characterized SH-SY5Y cells spheroid model

In recent years, spheroids (tridimensional cell cultures) have emerged as a more physiologically relevant replacement for monolayer models. Their distinctive advantage is the formation of an extracellular matrix that facilitates enhanced cellular interaction and communication, approximating the conditions observed in vivo. Therefore, the potential for conducting intricate cellular and molecular techniques in these models could offer a more precise assessment of pivotal proteins within various cellular pathways of interest. Amitraz (AMZ), an acaricide classified as a formamidine chemical, has been detected in honey at concentrations exceeding legal limits. The objective of this study was to characterize a spheroid model of SH-SY5Y cells and determine the cytotoxic effect of AMZ and its mechanisms of action on this spheroid. The formation of mature spheroids was observed on the seventh day following seeding. The results obtained with SH-SY5Y spheroids were an IC50 of 238.8 ± 17 µM and 224.3 ± 19 µM, respectively, after 24 and 48 h of exposure by the MTT assay. The findings revealed that AMZ did not exhibit any indications of inflammatory over-expression markers in the spheroids. Nevertheless, at 238.8 µM of AMZ, an increase incidence of late apoptosis within spheroid cells and Bcl-2 protein expression in peripheral spheroid cells were observed through annexin V and propidium iodide probe and immunofluorescence analysis. In conclusion, the results demonstrated that spheroids could be useful for an accurate assessment of toxicity, representing a viable alternative method for determining the mechanisms of action of AMZ and related compounds.

Real-time modeling of transient crustal deformation through the quantification of uncertainty deduced from GNSS data

We propose a new method for real-time uncertainty monitoring of earthquake and volcano source models using data from the global navigation satellite system (GNSS) and explore its application concerning observation station placement. The Geospatial Information Authority of Japan operates two main types of GNSS earth observation network system (GEONET) coordinates for crustal deformation monitoring on different time scales: post-processing analysis values and real-time GEONET analysis system for rapid deformation monitoring (REGARD). REGARD uses the Markov Chain Monte Carlo (MCMC) method termed real-time automatic uncertainty estimation of a coseismic single rectangular model using GNSS data (RUNE) for single rectangular fault model estimation to handle uncertainty. Thus far, no GNSS monitoring system can automatically detect transient crustal deformation events, such as volcanic activity and earthquake swarms, on timescales of a day or less. We extended RUNE and developed a core program for a new monitoring system for earthquake and volcanic source models and their uncertainties. Our program achieved automatic and stable MCMC utilization for rectangular fault, dike, Mogi, and spheroid models by increasing the computational speed, improving search efficiency, and adjusting hyperparameters. The program automatically determines the standard deviation of the likelihood function assuming a normal distribution with weights for each observation station. The calculation time was within 15 s for 1 × 106 samples on a standard 1U server. We assessed the reliability of the developed method using synthetic and observed GNSS data from the 2015 Sakurajima volcanic event. The results were consistent with the assumed model and previous studies and indicated an advantage in automatically quantifying uncertainty in a short computation time. Based on MCMC samples, we developed a new visualization algorithm to indicate areas on a map in which the number of observation stations should be expanded. We assessed the reliability using data from the 2023 Noto Peninsula earthquake [Mj 6.5]. The results indicate that the algorithm is helpful in studying the placement of stations. The above model extensions and their application are essential to achieve a rapid quantitative understanding of disaster events near urban areas and for utilizing this information in emergency response activities.Graphical

Photodynamic Treatment of Glioblastoma/Endothelial Cell Spheroid Models: Accelerated Proliferation of Surviving Tumor Cells Due to iNOS/NO Upregulation

Photodynamic Treatment of Glioblastoma/Endothelial Cell Spheroid Models: Accelerated Proliferation of Surviving Tumor Cells Due to iNOS/NO Upregulation

Protein absorption alters the cellular targeting of glycopolymeric nanoparticles

Glycopolymeric nanoparticles are widely employed in biomedical applications due to their high targeting ability, biocompatibility, and biodegradability. The pendant saccharides serve as targeting ligands to be explicitly coordinated to receptors on cell membrane surfaces. However, proteins in the blood often absorb onto the nanoparticle surface, potentially impacting the exposure and effectiveness of the saccharide ligands for targeted delivery. To elucidate the protein absorption effects, we prepared micelles decorated with different percentages of fructose moieties and evaluated the bioactivity of nanoparticles using 2D cell culture, tumor spheroid, liver organoid, and coculture models. The 2D cell culture model did not show a significant difference in activity between protein-coated and non-coated micelles. However, a dramatic decrease in cellular uptake and penetration ability of micelles was observed in 3D tumor spheroids after protein absorption. Additionally, protein absorption notably increased micelle uptake in liver organoids. In the coculture model, protein absorption reduced micelle uptake in tumor spheroids while favoring uptake in liver organoids. Our work suggests that decreasing non-specific protein absorption of glycopolymeric nanoparticles could enhance their delivery efficiency. These findings underscore the importance of understanding protein interactions in nanoparticle applications for drug delivery.

Unraveling the mechanism of the anticancer potential of emodin using 2D and spheroid models of A549 cells

The increasing global cancer burden necessitates the development of new treatment options. Herbal medicine offers a viable alternative to conventional cancer treatments. Numerous studies have shown that 3-dimensional (3D) cell culture more accurately represents tumor characteristics in vivo. Therefore, this study utilized tumor spheroids to explore the therapeutic efficacy of emodin, a natural product-derived bioactive agent. We investigated differences in chemotherapeutic response between A549 cells cultured in 2D versus spheroids, assessing key factors influencing cancer progression, including apoptosis, cell proliferation, cell cycle, migration and invasion. The findings revealed that spheroid cells displayed increased resistance to emodin compared to cells cultured in 2D. Emodin exhibited a more pronounced cytostatic effect in 2D cells, while its cytotoxic effect was more prominent in spheroid cells. Moreover, emodin treatment diminished the migratory and invasive capabilities of the cells. Mechanistic investigations indicated that emodin triggered apoptosis in A549 cells via the mitochondrial apoptotic pathway. Emodin-treated cells exhibited a significant reduction in the phosphorylation of key cancer progression pathways, including JAK2, STAT3, FAK, and ERK, compared to untreated controls. Molecular docking analysis confirmed the interactions of emodin with JAK2 and FAK. These findings suggest that the JAK2/STAT3 and FAK/ERK signaling pathways may serve as critical drivers of the therapeutic effectiveness of emodin in A549 cells.