3D Cell Research Articles

Synthesis and characterization of self-healable supramolecular hydrogel based on carboxymethyl cellulose for biomedical applications

Hydrogels have been widely used in biomedical fields including tissue engineering, drug delivery and cell delivery and 3D cell delivery due to abundant water content in their hydrophilic three-dimensional networks and having soft tissue similar to the human body. In recent years, supramolecular hydrogels (SHG) formed by the inclusion complex between polyethylene glycol (PEG) and macrocycles such as cyclodextrin (CD) have attracted much interest due to their excellent biocompatibility and great potential in biomedical. In this research, a carboxymethyl cellulose (CMC)-based graft copolymer was prepared by using acrylic acid (AA) and maleic anhydride functionalized β-CD (β-CD-MA) as comonomers and ammonium persulfate (APS) as initiator. Then, a self-healable supramolecular hydrogel was synthesized by formation of a host-guest inclusion complex between CMC-g-poly (AA-co-β-CD-MA) as host molecule and cytosine- and guanine-modified PEG as guest molecules. The prepared hydrogel was characterized by Scanning Electron Microscope (SEM), X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Nuclear magnetic resonance spectroscopy (1H NMR). The thermal stability of hydrogel was also determined by thermal gravimetric (TGA) and differential scanning calorimetry (DSC) methods. In addition, the loading and release profiles of metformin hydrochloride (MH) drug as a model on hydrogel was investigated. The results indicated that the drug release from the hydrogel peaks around 360 min and aligns with the Ritger-Peppas model. The hydrogel's self-healing property was examined at ambient temperature and 37 °C. It showed 70 % healing in 1.5 h and completed recovery after 9 h.

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.

Polyvinyl alcohol assisted citrate based reduction of gold(III) ions: Theoretical design and experimental study on green synthesis of spherical and biocompatible gold nanoparticles

In this study, we demonstrate the synthesis of small gold nanoparticles (typically 8-10 nm) through a green synthesis approach. This method involves utilizing chloroauric acid as the gold precursor, trisodium citrate as a mild reducing and co-capping agent, and polyvinyl alcohol (PVA), as a co-capping and shape-directing agent. This novel synthesis method stands alone as a protocol that involves just mixing the reagents at room temperature and allowing the reaction to proceed at ambient temperature without any disturbance. In the absence of PVA, spherical particles are not formed and the reaction mixture slowly turns black followed by precipitation.The synthesis process was meticulously tracked using time-resolved monitoring of the growth of the localized surface plasmon resonance (LSPR) by UV-vis spectroscopy and transmission electron microscopy. The as-prepared colloidal gold solution was bright red, attributed to the small size (≤ 10 nm) and spherical geometry of nanoparticles. This colloidal solution could be stored indefinitely at room temperature without precipitation or a change in its absorption profile. The nanoparticles remained stable in adverse conditions such as 100 mM NaCl solution, 1×PBS and cell culture medium. Additionally, they could be easily loaded with drugs like doxorubicin by adsorption. The particles were thoroughly characterized using a range of techniques, including UV-vis spectroscopy, attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), high-resolution transmission electron microscopy (HRTEM), hyperspectral-enhanced dark field microscopy (HEDFM), dynamic light scattering (DLS), and X-ray photoelectron spectroscopy (XPS). Cell viability tests conducted on 2D cell lines and 3D spheroids revealed negligible toxicity even at high concentrations. Furthermore, we demonstrate that an advanced version of conventional diffusion-limited aggregation (DLA) schemes, which allows individual clusters to move freely within a domain to form larger aggregates, can effectively replicate the intricate interactions between chloroauric acid, trisodium citrate, and PVA, the agents involved in the synthesis of gold nanoparticles.

Antitumor Potential of Guttiferone E Combined With Carboplatin Against Osimertinib-resistant H1975 Lung Cancer Through Apoptosis.

Low selectivity and high frequency of side-effects are the major problems of currently used chemotherapeutics. Among natural compounds, the polyprenylated acylphloroglucinol, guttiferone E, isolated from Brazilian red propolis, has attracted attention due to its marked anticancer properties and was evaluated here for its role against osimertinib-resistant H1975 cells (with double mutations of epidermal growth factor receptor: EGFR L858R/T790M). Guttiferone E was obtained from red propolis using established extraction procedures. Guttiferone E was tested using the H1975 cell line in in vitro (2D and 3D) cell cultures and in vivo in BALB/c athymic nude mice. Live/dead assay was also performed to support the results. Tumor tissues obtained from in vivo studies were used for western blotting. Guttiferone E reduced H1975 cell viability in a concentration-dependent manner. The IC50 values in 2D and 3D cell lines were 2.56±0.12 μM and 11.25±0.34 μM. Furthermore, at 10 mg/kg intraperitoneally, guttiferone E significantly reduced the tumor volume in tumor xenografts when used alone and in combination with carboplatin. Guttiferone E and carboplatin displayed synergistic inhibition of H1975 cells and animal tumors. Co-treatment of guttiferone E with carboplatin induced more prominent apoptosis than treatment with either drug alone. Guttiferone E treatment induced cleavage of poly-ADP ribose polymerase and induced apoptosis by significantly reducing levels of mammalian target of rapamycin, sirtuin 1, sirtuin 7, superoxide dismutase, programmed death-ligand 1, and programmed cell death 1 in tumor tissues. Our results show guttiferone E to be a promising, novel and potent antitumor drug candidate for osimertinib-resistant lung cancer with EGFR L858R/T790M mutations.

Hyaluronan-Based Hydrogels for 3D Modeling of Tumor Tissues.

Although routine two-dimensional (2D) cell culture techniques have advanced basic cancer research owing to their simplicity, cost-effectiveness, and reproducibility, they have limitations that necessitate the development of advanced three-dimensional (3D) tumor models that better recapitulate the tumor microenvironment. Various biomaterials have been used to establish these 3D models, enabling the study of cancer cell behavior within different matrices. Hyaluronic acid (HA), a key component of the extracellular matrix (ECM) in tumor tissues, has been widely studied and employed in the development of multiple cancer models. This review first examines the role of HA in tumors, including its function as an ECM component and regulator of signaling pathways that affect tumor progression. It then explores HA-based models for various cancers, focusing on HA as a central component of the 3D matrix and its mobilization within the matrix for targeted studies of cell behavior and drug testing. The tumor models discussed included those for breast cancer, glioblastoma, fibrosarcoma, gastric cancer, hepatocellular carcinoma, and melanoma. The review concludes with a discussion of future prospects for developing more robust and high-throughput HA-based models to more accurately mimic the tumor microenvironment and improve drug testing. Impact Statement This review underscores the transformative potential of hyaluronic acid (HA)-based hydrogels in developing advanced tumor models. By exploring HA's dual role as a critical extracellular matrix component and a regulator of cancer cell dynamics, we highlight its unique contributions to replicating the tumor microenvironment. The recent advancements in HA-based models provide new opportunities for more accurate studies of cancer cell behavior and drug responses. Looking ahead, these innovations pave the way for high-throughput, biomimetic platforms that could revolutionize drug testing and accelerate the discovery of effective cancer therapies.

Hydrogel-Integrated Heart-on-a-Chip Platform for Assessment of Myocardial Ischemia Markers.

Organ-on-a-chip platform scans offer a controllable environment and a physiological similarity to mimic human pathophysiology. In this study, a single-channel PDMS microchip was fabricated, characterized, and optimized to obtain a heart-on-a-chip platform, which is integrated with a hydrogel scaffold suitable for cardiomyocyte growth inside its channel. Single-channel chips with a size of 20 × 12 mm and a channel height ranging from 60 to 100 μm were produced using photolithography and soft lithography techniques. A gelatin-embedded alginate network-based hydrogel was further augmented with 3% (v/v) collagen type I. Pore sizes were in the range of 74-153 μm for H9C2 implantation and biomimicry. The hydrogels are characterized both on PDMS surfaces and in capillaries. The primary feature distinguishing this study from previous microchip studies is that it mimics the cell microenvironment much better using different hydrogel formulations instead of creating a 2D cell culture by passing fluids, such as fibronectin, for cell adhesion. Instead of using complex microchip designs, the chip system we created intends to provide a physiologically relevant copy by using a 3D cell culture to its advantage and a simple, single-channel architecture. The microchip study was combined with cardiomyocytes to create the heart-on-a-chip system and tested under normoxic and hypoxic conditions to create a myocardial ischemia model inside this channel. As a result, this heart-on-a-chip platform was shown to be utilized for the detection of several small-size biomarkers such as adenosine, ADP, lactic acid, l-isoleucine, l-glutamic acid, and oxidized glutathione via LC-MS/MS from control conditions and a myocardial ischemia model. Cell-embedded and hydrogel matrix-supported versions of this heart-on-a-chip system were successfully prepared and shown to provide powerful outputs with myocardial ischemia markers. In light of this research, these outputs aim to develop simple and biologically effective organ-on-a-chip systems for future research.

Radical Protein Footprinting in Mammalian Whole Blood.

Hydroxyl Radical Protein Footprinting (HRPF) is a powerful method to probe the solvent-accessible surface area of proteins. It is mostly used to study the higher-order structure of proteins, as well as protein-protein and protein-carbohydrate interactions. Hydroxyl radicals are generated by the photolysis of hydrogen peroxide and these radicals modify the surface amino acids. Bottom-up proteomics is then applied and peptide oxidation is calculated and correlated with solvent accessibility. It is mainly performed in vitro ; however, it has been recently used in living systems, including live cells, live nematodes, and 3D cell cultures. Mammalian tissues are still out of reach as they absorb UV strongly, hindering radical generation. Here, we describe the first example of RPF in mammalian stabilized whole blood. Using photoactivation of persulfate with a commercially available FOX Photolysis System modified for sample handling and inline mixing, we demonstrate the first labeling of proteins in whole blood. We demonstrate that the RPF protocol does not alter the blood cell gross morphology outside of a moderate hypertonicity equivalent to sodium chloride exposure prior to labeling. We detail an improved quenching protocol to limit background labeling in persulfate RPF. We describe the labeling of the top ten most abundant proteins in the blood. We demonstrate the equivalence of ex vivo labeling in whole blood with labeling of the same structure in vitro using hemoglobin as a test system. Overall, these results now open the possibility of performing RPF-based structural proteomics in pre-clinical models and using readily available clinical samples.

The Use of Patient-Derived Organoids in the Study of Molecular Metabolic Adaptation in Breast Cancer

Around 13% of women will likely develop breast cancer during their lifetime. Advances in cancer metabolism research have identified a range of metabolic reprogramming events, such as altered glucose and amino acid uptake, increased reliance on glycolysis, and interactions with the tumor microenvironment (TME), all of which present new opportunities for targeted therapies. However, studying these metabolic networks is challenging in traditional 2D cell cultures, which often fail to replicate the three-dimensional architecture and dynamic interactions of real tumors. To address this, organoid models have emerged as powerful tools. Tumor organoids are 3D cultures, often derived from patient tissue, that more accurately mimic the structural and functional properties of actual tumor tissues in vivo, offering a more realistic model for investigating cancer metabolism. This review explores the unique metabolic adaptations of breast cancer and discusses how organoid models can provide deeper insights into these processes. We evaluate the most advanced tools for studying cancer metabolism in three-dimensional culture models, including optical metabolic imaging (OMI), matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), and recent advances in conventional techniques applied to 3D cultures. Finally, we explore the progress made in identifying and targeting potential therapeutic targets in breast cancer metabolism.

Photochemical Control of Network Topology in PEG Hydrogels.

Hydrogels are often synthesized through photoinitiated step-, chain-, and mixed-mode polymerizations, generating diverse network topologies and resultant material properties that depend on the underlying network connectivity. While many photocrosslinking reactions are available, few afford controllable connectivity of the hydrogel network. Herein, a versatile photochemical strategy is introduced for tuning the structure of poly(ethylene glycol) (PEG) hydrogels using macromolecular monomers functionalized with maleimide and styrene moieties. Hydrogels are prepared along a gradient of topologies by varying the ratio of step-growth (maleimide dimerization) to chain-growth (maleimide-styrene alternating copolymerization) network-forming reactions. The initial PEG content and final network physical properties (e.g., modulus, swelling, diffusivity) are tailored in an independent manner, highlighting configurable gel mechanics and reactivity. These photochemical reactions allow high-fidelity photopatterning and 3D printing and are compatible with 2D and 3D cell culture. Ultimately, this photopolymer chemistry allows facile control over network connectivity to achieve adjustable material properties for broad applications.

Shell-by-Shell functionalized nanoparticles as radiosensitizers and radioprotectors in radiation therapy of cancer cells and tumor spheroids

Shell-by-Shell (SbS)-functionalized NPs can be tailor-made by combining a metal oxide NP core of choice with any desired phosphonic acids and amphiphiles as 1st or 2nd ligand shell building blocks. The complementary composition of such highly hierarchical structures makes them interesting candidates for various biomedical applications, as certain active ingredients can be incorporated into the structure. Here, we used TiO2 and CoFe2O4 NPs as drug delivery tools and coated them with a hexadecylphosphonic acid and with hexadecyl ammonium phenolates (caffeate, p-coumarate, ferulate), that possess anticancer as well as antioxidant properties. These architectures were then incubated in 2D and 3D cell cultures of non-tumorigenic and tumorigenic breast cells and irradiated to study their anticancer effect. It was found that both, the functionalized TiO2 and CoFe2O4 NPs acted as strong protective agents in non-tumorigenic spheroids. In contrast, the functionalized CoFe2O4 NPs induce a higher damage in irradiated tumor spheroids compared to the functionalized TiO2 NPs. CoFe3O4 NPs act additionally as radiosensitizing agents to the tumor spheroids. The radio-enhancement of the CoFe2O4 NPs is due to the generation of highly toxic hydroxyl radicals during X-ray irradiation. The irradiation exposed the CoFe2O4 surface, releasing the anticancer drugs into the cytoplasm and making the surface Co2+ ions accessible. These surface ions catalyze the Fenton reaction. This combination of radiosensitizer and anticancer drug delivery proved to be a very effective nanotherapeutic in 2D and 3D cell cultures of breast cancer cells.

Evaluating ECM stiffness and liver cancer radiation response via shear-wave elasticity in 3D culture models

BackgroundThe stiffness of the tumor microenvironment (TME) directly influences cellular behaviors. Radiotherapy (RT) is a common treatment for solid tumors, but the TME can impact its efficacy. In the case of liver cancer, clinical observations have shown that tumors within a cirrhotic, stiffer background respond less to RT, suggesting that the extracellular matrix (ECM) stiffness plays a critical role in the development of radioresistance.MethodsThis study explored the effects of ECM stiffness and the inhibition of lysyl oxidase (LOX) isoenzymes on the radiation response of liver cancer in a millimeter-sized three-dimensional (3D) culture. We constructed a cube-shaped ECM-based millimeter-sized hydrogel containing Huh7 human liver cancer cells. By modulating the collagen concentration, we produced two groups of samples with different ECM stiffnesses to mimic the clinical scenarios of normal and cirrhotic livers. We used a single-transducer system for shear-wave-based elasticity measurement, to derive Young’s modulus of the 3D cell culture to investigate how the ECM stiffness affects radiosensitivity. This is the first demonstration of a workflow for assessing radiation-induced response in a millimeter-sized 3D culture.ResultsIncreased ECM stiffness was associated with a decreased radiation response. Moreover, sonoporation-assisted LOX inhibition with BAPN (β-aminopropionitrile monofumarate) significantly decreased the initial ECM stiffness and increased RT-induced cell death. Inhibition of LOX was particularly effective in reducing ECM stiffness in stiffer matrices. Combining LOX inhibition with RT markedly increased radiation-induced DNA damage in cirrhotic liver cancer cells, enhancing their response to radiation. Furthermore, LOX inhibition can be combined with sonoporation to overcome stiffness-related radioresistance, potentially leading to better treatment outcomes for patients with liver cancer.ConclusionsThe findings underscore the significant influence of ECM stiffness on liver cancer’s response to radiation. Sonoporation-aided LOX inhibition emerges as a promising strategy to mitigate stiffness-related resistance, offering potential improvements in liver cancer treatment outcomes.

Peptide-mediated targeting of Quantum Dots in a 3D model of head and neck cancer

BackgroundOral squamous cell carcinoma (OSCC) treatment mainly relies on surgery. The status of surgical margin is a major prognostic factor for patients as positive margins are associated with lower survival. However, the anatomical particularities of this area complicate margin establishment. Fluorescence guided surgery (FGS) could be employed as an intraoperative technique to improve tumor resection and margin investigation. Quantum dots (QDs) serve as ideal contrast agents in this technique due to their brightness and stability. Since αVβ6 integrin is overexpressed in OSCC, coupling QDs with A20FMDV2 peptide (QDs-A20) targeting the αVβ6 integrin constitute a real opportunity. This study investigates the accumulation of QDs-A20 in 2D and 3D tongue cancer models, as well as QDs coupled to a scrambled version of this peptide (QDs-Scr) or without peptide (QDs-SPP), for imaging purposes. MethodsCdSeCdS/ZnS quantum dots were coated with sulfobetaine polymers (QDs-SPP) and conjugated to A20FMDV2 peptide (QDs-A20) or its scrambled version (QDs-Scr). Two-dimensional (2D) and three-dimensional (3D) tongue cancer cells HSC-3 were employed to test the effectiveness of intracellular accumulation of all types of QDs. Targeting ability of each QDs was assessed by flow cytometry, while the depth of penetration into cancerous spheroids was assessed by fluorescence microscopy. ResultsQDs coating with sulfobetaines polymers (QDs-SPP) completely prevented their internalization by HSC-3 cells in 2D and 3D models, making QDs stealthy and preventing their non-specific accumulation. Conversely, peptides conjugated QDs (QDs-A20 & QDs-Scr) labeled HSC-3 monolayers and managed to label spheroid periphery up to 23 µm deep. However, no difference in accumulation was found between these two QDs whereas only A20 peptide could potentially target αVβ6 integrin. It appears that peptide conjugation increased QDs zeta potential, promoting their adsorption and subsequent endocytosis by cells, independently from αVβ6 integrin. ConclusionsThe present study highlighted the impact of peptide conjugation on QDs internalization in 2D and 3D tongue cancer cell models. QDs-SPP were stealthy and did not accumulate in cells. Peptides conjugated QDs could be used as contrast agents, but in a passive targeting approach. Modifications to surface chemistry are required to target αVβ6 integrin through active targeting. This study also highlights the need for controls such as scrambled peptides, the absence of which can lead to misinterpretation of results.

Enhancing T-cell recruitment in renal cell carcinoma with cytokine-armed adenoviruses

ABSTRACT Immunotherapy has emerged as a promising approach for cancer treatment, with oncolytic adenoviruses showing power as immunotherapeutic agents. In this study, we investigated the immunotherapeutic potential of an adenovirus construct expressing CXCL9, CXCL10, or IL-15 in clear cell renal cell carcinoma (ccRCC) tumor models. Our results demonstrated robust cytokine secretion upon viral treatment, suggesting effective transgene expression. Subsequent analysis using resistance-based transwell migration and microfluidic chip assays demonstrated increased T-cell migration in response to chemokine secretion by infected cells in both 2D and 3D cell models. Flow cytometry analysis revealed CXCR3 receptor expression across T-cell subsets, with the highest percentage found on CD8+ T-cells, underscoring their key role in immune cell migration. Alongside T-cells, we also detected NK-cells in the tumors of immunocompromised mice treated with cytokine-encoding adenoviruses. Furthermore, we identified potential immunogenic antigens that may enhance the efficacy and specificity of our armed oncolytic adenoviruses in ccRCC. Overall, our findings using ccRCC cell line, in vivo humanized mice, physiologically relevant PDCs in 2D and patient-derived organoids (PDOs) in 3D suggest that chemokine-armed adenoviruses hold promise for enhancing T-cell migration and improving immunotherapy outcomes in ccRCC. Our study contributes to the development of more effective ccRCC treatment strategies by elucidating immune cell infiltration and activation mechanisms within the tumor microenvironment (TME) and highlights the usefulness of PDOs for predicting clinical relevance and validating novel immunotherapeutic approaches. Overall, our research offers insights into the rational design and optimization of viral-based immunotherapies for ccRCC.

A light-addressable potentiometric sensor-based extracellular calcium dynamic monitoring and imaging platform for cellular calcium channel drug evaluation

Disruption and dysregulation of cellular calcium channel function can lead to diseases such as ischemic stroke, heart failure, and arrhythmias. Corresponding calcium channel drugs typically require preliminary efficacy evaluations using in vitro models such as cells and simulated tissues before clinical testing. However, traditional detection and evaluation methods often encounter challenges in long-term continuous monitoring and lack calcium specificity. In this study, a dynamic monitoring system based on ion-sensitive membranes for light-addressable potentiometric sensor (LAPS) was developed to meet the demand for monitoring changes in extracellular calcium ion (Ca2+) concentration in live cells. The effects of Ca2+ channel agonists and blockers on 2D and 3D HL-1 cells were investigated, with changes in extracellular Ca2+ concentration reflecting cellular calcium metabolism, facilitating drug evaluation. Additionally, calcium imaging technology with optical addressing capability complemented the LAPS system's ability to perceive 3D cell morphology, enhancing its drug evaluation capabilities. This work provides a novel, label-free, specific, and stable technique for monitoring cellular calcium metabolism. It achieves both continuous monitoring at single points and custom sensing area calcium imaging, holding significant implications for drug screening and disease treatment related to human calcium homeostasis.

Three-Dimensional-Bioprinted Non-Small Cell Lung Cancer Models in a Mouse Phantom for Radiotherapy Research.

Lung cancer continues to have one of the highest morbidity and mortality rates of any cancer. Although radiochemotherapy, in combination with immunotherapy, has significantly improved overall survival, new treatment options are urgently needed. However, preclinical radiotherapy testing is often performed in animal models, which has several drawbacks, including species-specific differences and ethical concerns. To replace animal models, this study used a micro-extrusion bioprinting approach to generate a three-dimensional (3D) human lung cancer model consisting of lung tumor cells embedded in human primary lung fibroblasts for radiotherapy research. The models were placed in a mouse phantom, i.e., a 3D-printed mouse model made of materials that mimic the X-ray radiation attenuation rates found in mice. In radiotherapy experiments, the model demonstrated a selective cytotoxic effect of X-rays on tumor cells, consistent with findings in 2D cells. Furthermore, the analysis of metabolic activity, cell death, apoptosis, and DNA damage-induced γH2AX foci formation revealed different results in the 3D model inside the phantom compared to those observed in irradiated models without phantom and 2D cells. The proposed setup of the bioprinted 3D lung model inside the mouse phantom provides a physiologically relevant model system to study radiation effects.

Alkaloid-rich extract of jujube seed regenerate the antiproliferative effect of paclitaxel on paclitaxel-resistant MDA-MB-231 breast cancer cell line in 2D and 3D cultures

IntroductionCancer is the second leading cause of death globally, and among all types of cancer, triple-negative breast cancer (TNBC) has one of the worst prognoses. The repeated use of chemotherapeutic drugs often leads to drug resistance, complicating treatment outcomes. This study aimed to investigate the effect of jujube alkaloid-rich extract on drug sensitivity and its antitumor effects on paclitaxel-resistant MDA-MB-231 cells in vitro. Materials and methodsThis study utilized the MDA-MB-231 breast cancer cell line and its paclitaxel-resistant variant. Cell viability was measured using the XTT assay, while apoptosis was detected through Annexin-PI assays. Various concentrations of jujube extract, alone and combined with paclitaxel, were assessed for synergistic effects in 2D and 3D culture models. ResultsThe presence of alkaloids in the jujube seed extract was confirmed via the Dragendorff test, with 0.395 g of alkaloids present in 5 g of jujube seed powder. A combination of paclitaxel and jujube extract exhibited dose-dependent cytotoxic effects on paclitaxel-resistant MDA-MB-231 cells. The combination of jujube extract and paclitaxel significantly decreased IC50 from 541.3 μg/ml to 124.3 μg/ml, and the IC20 decreased from 92.1 μg/ml to 23.9 μg/ml when combined with paclitaxel in 2D culture model. The cytotoxicity of the combination was reduced in the 3D culture model. Apoptosis rates were 0.75% in the control group, 13.89% in the alkaloid-rich extract group, 2.59% in the paclitaxel group, and 42.90% in the combination group. ConclusionCombining alkaloid-rich jujube seed extract with paclitaxel regenerates the cytotoxicity and apoptotic ability of paclitaxel in the paclitaxel-resistant MDA-MB-231 breast cancer cells in both 2D and 3D culture models. This suggests that such combinations might be a viable strategy to overcome drug resistance in TNBC treatments.

3D cell culture models in research: applications to lung cancer pharmacology.

Lung cancer remains one of the leading causes of cancer-related mortality worldwide, necessitating innovative research methodologies to improve treatment outcomes and develop novel strategies. The advent of three-dimensional (3D) cell cultures has marked a significant advancement in lung cancer research, offering a more physiologically relevant model compared to traditional two-dimensional (2D) cultures. This review elucidates the various types of 3D cell culture models currently used in lung cancer pharmacology, including spheroids, organoids and engineered tissue models, having pivotal roles in enhancing our understanding of lung cancer biology, facilitating drug development, and advancing precision medicine. 3D cell culture systems mimic the complex spatial architecture and microenvironment of lung tumours, providing critical insights into the cellular and molecular mechanisms of tumour progression, metastasis and drug responses. Spheroids, derived from commercialized cell lines, effectively model the tumour microenvironment (TME), including the formation of hypoxic and nutrient gradients, crucial for evaluating the penetration and efficacy of anti-cancer therapeutics. Organoids and tumouroids, derived from primary tissues, recapitulate the heterogeneity of lung cancers and are instrumental in personalized medicine approaches, supporting the simulation of in vivo pharmacological responses in a patient-specific context. Moreover, these models have been co-cultured with various cell types and biomimicry extracellular matrix (ECM) components to further recapitulate the heterotypic cell-cell and cell-ECM interactions present within the lung TME. 3D cultures have been significantly contributing to the identification of novel therapeutic targets and the understanding of resistance mechanisms against conventional therapies. Therefore, this review summarizes the latest findings in drug research involving lung cancer 3D models, together with the common laboratory-based assays used to study drug effects. Additionally, the integration of 3D cell cultures into lung cancer drug development workflows and precision medicine is discussed. This integration is pivotal in accelerating the translation of laboratory findings into clinical applications, thereby advancing the landscape of lung cancer treatment. By closely mirroring human lung tumours, these models not only enhance our understanding of the disease but also pave the way for the development of more effective and personalized therapeutic strategies.

SMoRe GloS: An efficient and flexible framework for inferring global sensitivity of agent-based model parameters.

Agent-based models (ABMs) have become essential tools for simulating complex biological, ecological, and social systems where emergent behaviors arise from the interactions among individual agents. Quantifying uncertainty through global sensitivity analysis is crucial for assessing the robustness and reliability of ABM predictions. However, most global sensitivity methods demand substantial computational resources, making them impractical for highly complex models. Here, we introduce SMoRe GloS (Surrogate Modeling for Recapitulating Global Sensitivity), a novel, computationally efficient method for performing global sensitivity analysis of ABMs. By leveraging explicitly formulated surrogate models, SMoRe GloS allows for comprehensive parameter space exploration and uncertainty quantification without sacrificing accuracy. We demonstrate our method's flexibility by applying it to two biological ABMs: a simple 2D cell proliferation assay and a complex 3D vascular tumor growth model. Our results show that SMoRe GloS is compatible with simpler methods like the Morris one-at-a-time method, and more computationally intensive variance-based methods like eFAST. SMoRe GloS accurately recovered global sensitivity indices in each case while achieving substantial speedups, completing analyses in minutes. In contrast, direct implementation of eFAST amounted to several days of CPU time for the complex ABM. Remarkably, our method also estimates sensitivities for ABM parameters representing processes not explicitly included in the surrogate model, further enhancing its utility. By making global sensitivity analysis feasible for computationally expensive models, SMoRe GloS opens up new opportunities for uncertainty quantification in complex systems, allowing for more in depth exploration of model behavior, thereby increasing confidence in model predictions.

Abstract C025: Enhanced efficacy of gemcitabine analogs and AZD 1775 combination therapy in patient-derived organoids

Abstract Pancreatic cancer (PCa), specifically Pancreatic Adenocarcinoma (PDAC), is projected to become the second leading cause of cancer related death by 2040. Currently, the 5-year survival rate for pancreatic cancer is between 7% - 12%. The standard treatment options for PDAC are chemotherapy and surgical resection. However, pancreatic cancer presents multiple challenges to successful treatment. First, PDAC is usually diagnosed in the advanced stages of the disease, even if diagnosed early only a small fraction of patients are eligible for surgical resection, about 13% - 15%. Second, PDAC is characterized by a stiff extracellular matrix. This stiffness can lead to poor vascularization, diminished immune responses and cell migration/metastasis. Finally, PDAC is notorious for being extremely resistant to chemotherapy regiments. In addition to these challenges, health disparities add to the dismal prognosis of pancreatic cancer. Examples of these disparities include unequal access to healthcare facilities and screenings, awareness of disease risk factors and symptoms and socioeconomic factors. Differences in genetics can influence the various outcomes seen in PDAC in terms of health disparities. Certain genetic mutations, some more prevalent in certain ethnic groups, can increase the risk for PDAC. Variation in tumor heterogeneity and genetic factors impacting drug metabolism can influence patient outcomes. To overcome some of these challenges, we have developed and tested novel gemcitabine analogs, conjugated with various fatty acid chains in collagen-based 3D gel, Patient Derived Organoid (PDO) models. The collagen-based gel allows tuning of the physical stiffness by varying the gelatin percentage, creating a more realistic tumor microenvironment with the intratumoral pressure can be upwards of 10 kPa. We have tested the gemcitabine analogs on patient derived cells, G43 and G46, from black and white patients respectively, in both soft and stiff matrices. Our results indicated that medium to long fatty acid chains (8, 10 and 18 carbon chains) have increased effectiveness, especially in softer matrices. Comparatively, G46 cells exhibited higher drug resistance across all environments than G43 cells. Recent studies have shown that combining Gemcitabine with AZD 1775, a WEE1 inhibitor may have synergistic effects. We tested the combinations of Gemcitabine analogs and AZD-1775 on both G43 and G46 patient derived cells in 2D and 3D environments. Our data suggests that short to medium length fatty acid chains (2-8 carbons) provide optimal combinational effectiveness. The combined treatment also demonstrated promising results in increasing treatment effectiveness in G46, which are typically chemoresistant. Further studies will examine the influence that tumor microenvironment may have on PDAC response to the combination therapy, as well as intrinsic difference between black patient-derived cells and white patient-derived cells and their sensitivity or resistance to treatment. Citation Format: Jonathan Barajas, Edward Agyare, Xueyou Zhu, Sherise Rogers, Ba Xuan Hoang, Bo Han. Enhanced efficacy of gemcitabine analogs and AZD 1775 combination therapy in patient-derived organoids [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C025.

The dynamics of capillary flow in an open-channel system featuring trigger valves.

Trigger valves are fundamental features in capillary-driven microfluidic systems that stop fluid at an abrupt geometric expansion and release fluid when there is flow in an orthogonal channel connected to the valve. The concept was originally demonstrated in closed-channel capillary circuits. We show here that trigger valves can be successfully implemented in open channels. We also show that a series of open-channel trigger valves can be placed alongside or opposite a main channel resulting in a layered capillary flow. We developed a closed form model for the dynamics of the flow at trigger valves based on the concept of average friction length and successfully validated the model against experiments. For the main channel, we discuss layered flow behavior in the light of the Taylor-Aris dispersion theory and in the channel turns by considering Dean theory of mixing. This work has potential applications in autonomous microfluidics systems for biosensing, at-home or point-of-care sample preparation devices, hydrogel patterning for 3D cell culture and organ-on-a-chip models.