In situ crosslinkable multi-functional and cell-responsive alginate 3D matrix via thiol-maleimide click chemistry.

In vivo, cells interact with the extracellular matrix (ECM), which provides a multitude of biophysical and biochemical signals that modulate cellular behavior. Inspired by this, we explored a new methodology to develop a more physiomimetic polysaccharide-based matrix for 3D cell culture. Maleimide-modified alginate (AlgM) derivatives were successfully synthesized using DMTMM to activate carboxylic groups. Thiol-terminated cell-adhesion peptides were tethered to the hydrogel network to promote integrin binding. Rapid and efficient in situ hydrogel formation was promoted by thiol-Michael addition "click" chemistry via maleimide reaction with thiol-flanked protease-sensitive peptides. Alginate derivatives were further ionically crosslinked by divalent ions present in the medium, which led to greater stability and allowed longer cell culture periods. By tailoring alginate's biofunctionality we improved cell-cell and cell-matrix interactions, providing an ECM-like 3D microenvironment. We were able to systematically and independently vary biochemical and biophysical parameters to elicit specific cell responses, creating custom-made 3D matrices. DMTMM-mediated maleimide incorporation is a promising approach to synthesizing AlgM derivatives that can be leveraged to produce ECM-like matrices for a broad range of applications, from in vitro tissue modeling to tissue regeneration.

A Pump-Free Strategy for the Controllable Generation of Alginate Microgels as Cellular Microcarriers.

Microgels are advanced scaffolds for tissue engineering due to their proper biodegradability, good biocompatibility, and high specific surface area for effective oxygen and nutrient transfer. However, most of the current monodispersed microgel fabrication systems rely heavily on various precision pumps, which highly increase the cost and complexity of their downstream application. In this work, we developed a simple and facile system for the controllable generation of uniform alginate microgels by integrating a gas-shearing strategy into a glass microfluidic device. Importantly, the cell-laden microgels can be rapidly prepared in a pump-free manner under an all-aqueous environment. The three-dimensional cultured green fluorescent protein-human A549 cells in alginate microgels exhibited enhanced stemness and drug resistance compared to those under two-dimensional conditions. The pancreatic cancer organoids in alginate microgels exhibited some of the key features of pancreatic cancer. The proposed microgels showed decent monodispersity, biocompatibility, and versatility, providing great opportunities in various biomedical applications such as microcarrier fabricating, organoid engineering, and high-throughput drug screening.

Cell-induced collagen alignment in a 3D in vitro culture during extracellular matrix production.

The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized with carbonated hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we used a 3D cell culture system in which osteogenic cells deposit and orient the collagen matrix that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenically induced cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration through the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenic differentiation and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.

Advances in the application of extracellular vesicles derived from three-dimensional culture of stem cells.

Stem cells (SCs) have been used therapeutically for decades, yet their applications are limited by factors such as the risk of immune rejection and potential tumorigenicity. Extracellular vesicles (EVs), a key paracrine component of stem cell potency, overcome the drawbacks of stem cell applications as a cell-free therapeutic agent and play an important role in treating various diseases. However, EVs derived from two-dimensional (2D) planar culture of SCs have low yield and face challenges in large-scale production, which hinders the clinical translation of EVs. Three-dimensional (3D) culture, given its ability to more realistically simulate the in vivo environment, can not only expand SCs in large quantities, but also improve the yield and activity of EVs, changing the content of EVs and improving their therapeutic effects. In this review, we briefly describe the advantages of EVs and EV-related clinical applications, provide an overview of 3D cell culture, and finally focus on specific applications and future perspectives of EVs derived from 3D culture of different SCs.

Arabinoxylan-based psyllium seed hydrocolloid: Single-step aqueous extraction and use in tissue engineering.

Biomacromolecules derived from natural sources offer superior biocompatibility, biodegradability, and water-holding capacity, which make them promising scaffolds for tissue engineering. Psyllium seed has gained attention in biomedical applications recently due to its gel-forming ability, which is provided by its polysaccharide-rich content consisting mostly of arabinoxylan. This study focuses on the extraction and gelation of Psyllium seed hydrocolloid (PSH) in a single-step water-based protocol, and scaffold fabrication using freeze-drying method. After characterization of the scaffold, including morphological, mechanical, swelling, and protein adsorption analyses, 3D cell culture studies were done using NIH-3 T3 fibroblast cells on PSH scaffold, and cell viability was assessed using Live/Dead and Alamar Blue assays. Starting from day 1, high cell viability was obtained, and it reached 90 % at the end of 15-day culture period. Cellular morphology on PSH scaffold was monitored via SEM analysis; cellular aggregates then spheroid formation were observed throughout the study. Collagen Type-I and F-actin expressions were followed by immunostaining revealing a 9- and 10-fold increase during long-term culture. Overall, a single-step and non-toxic protocol was developed for extraction and gelation of PSH. Obtained results unveiled that PSH scaffold provided a favorable 3D microenvironment for cells, holding promise for further tissue engineering applications.

Microfluidic Chip Fabrication for Tumor Cell 3D Culture Based on Microwell Arrays.

Compared with traditional 2D cell culture, 3D cell culture more closely resembles the original state of cells in vivo and enables the establishment of in vivo-like microenvironments and cell-cell interactions, thereby providing valuable cellular materials for numerous studies. The direct establishment of in vitro patient tumor models can enhance drug testing, cancer research, and individualized precision therapy. In this study, we propose a microfluidic chip based on microwell arrays for 3D tumor cell culture. This chip combines nanoscale channels and microwell arrays to precisely control cell distribution and nutrient diffusion, thus closely mimicking the tumor microenvironment. The incorporation of microwell arrays allows for simple and rapid high-throughput preparation of tumor spheroids, while promoting the formation of cell-cell and cell-matrix interactions, ultimately enhancing cell viability and function. Preliminary experiments using tumor cell lines validate the ability of the chip to support 3D tumor growth with enhanced physiological relevance. The microfluidic chip serves as a reliable and scalable platform for studying tumor biology and evaluating therapeutic efficacy and is anticipated to expedite cancer research and drug discovery.

A Pillar/Perfusion Plate Enhances Cell Growth, Reproducibility, Throughput, and User Friendliness in Dynamic 3D Cell Culture.

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from a necrotic core due to limited nutrient and oxygen diffusion and waste removal and have a limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids were loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow velocity was maintained within perfusion wells and the pillar plate was separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in a dynamic 3D cell culture.

Polyphenolic dendrimers as carriers of anticancer siRNA.

Dendrimers have emerged as an important group of nanoparticles to transport drugs, DNA, or RNA into target cells in cancer and other diseases. Various functional modifications can be imposed on dendrimers to increase the efficacy and specificity in delivering their cargo to the target cells and decrease their toxicity. In the present work, we evaluated the potential of carbosilane polyphenolic dendrimers modified with caffeic acid (CA) and polyethylene glycol (PEG) to deliver proapoptotic Mcl-1 and Bcl-2 siRNAs to A549 cancer cells. Dendrimers formed stable complexes with siRNAs as assessed by transmission electron microscopy and gel electrophoresis. Modification of dendrimers with PEG reduced the size and the zeta potential of dendrimer/siRNA complexes. The presence of PEG caused a red shift of the CD spectrum, and this effect was the more pronounced, the higher the dendrimer/siRNA ratio was. The nanocomplexes were internalized by A549. All studied dendrimer/siRNA formulations inhibited tumor cell migration and adhesion and caused an increase in the population of early apoptotic cells. Among four tested dendrimers, the polyphenolic compound containing two caffeic acid moieties complexed with siRNA demonstrated the lowest polydispersity index and showed an excellent transfection profile. In conclusion, this dendrimer are a promising candidate for the delivery of siRNA into cancer cells in further in vivo studies.

Gradually Self-Strengthen DNA Supramolecular Hydrogels.

The dynamic mechanical strength of the extracellular matrix (ECM) has been demonstrated to play important role in determining the cell behavior. Growing evidences suggest that the gradual stiffening process of the matrix is particularly decisive during tissue development and wound healing. Herein, a novel strategy to prepare hydrogels with gradually enhanced mechanical strength is provided. Such hydrogels could maintain the dynamic properties at their initial states, such as self-healing and shear-thinning properties. With subsequent slow covalent crosslinking, the stability and mechanical properties would be gradually improved. This method is useful for sequence programmability and oxidation strategies, which has provided an alternated tool to study cell behavior during dynamic increase in mechanical strength of ECM.

Deep learning-assisted concentration gradient generation for the study of 3D cell cultures in hydrogel beads of varying stiffness.

The study of dose-response relationships underpins analytical biosciences. Droplet microfluidics platforms can automate the generation of microreactors encapsulating varying concentrations of an assay component, providing datasets across a large chemical space in a single experiment. A classical method consists in varying the flow rate of multiple solutions co-flowing into a single microchannel (producing different volume fractions) before encapsulating the contents into water-in-oil droplets. This process can be automated through controlling the pumping elements but lacks the ability to adapt to unpredictable experimental scenarios, often requiring constant human supervision. In this paper, we introduce an image-based, closed-loop control system for assessing and adjusting volume fractions, thereby generating unsupervised, uniform concentration gradients. We trained a shallow convolutional neural network to assess the position of the laminar flow interface between two co-flowing fluids and used this model to adjust flow rates in real-time. We apply the method to generate alginate microbeads in which HEK293FT cells could grow in three dimensions. The stiffnesses ranged from 50 Pa to close to 1 kPa in Young modulus and were encoded with a fluorescent marker. We trained deep learning models based on the YOLOv4 object detector to efficiently detect both microbeads and multicellular spheroids from high-content screening images. This allowed us to map relationships between hydrogel stiffness and multicellular spheroid growth.

Research progress on optimization of in vitro isolation, cultivation and preservation methods of dental pulp stem cells for clinical application.

Due to high proliferative capacity, multipotent differentiation, immunomodulatory abilities, and lack of ethical concerns, dental pulp stem cells (DPSCs) are promising candidates for clinical application. Currently, clinical research on DPSCs is in its early stages. The reason for the failure to obtain clinically effective results may be problems with the production process of DPSCs. Due to the different preparation methods and reagent formulations of DPSCs, cell characteristics may be affected and lead to inconsistent experimental results. Preparation of clinical-grade DPSCs is far from ready. To achieve clinical application, it is essential to transit the manufacturing of stem cells from laboratory grade to clinical grade. This review compares and analyzes experimental data on optimizing the preparation methods of DPSCs from extraction to resuscitation, including research articles, invention patents and clinical trials. The advantages and disadvantages of various methods and potential clinical applications are discussed, and factors that could improve the quality of DPSCs for clinical application are proposed. The aim is to summarize the current manufacture of DPSCs in the establishment of a standardized, reliable, safe, and economic method for future preparation of clinical-grade cell products.

Fabrication of a cell culture scaffold that mimics the composition and structure of bone marrow extracellular matrix.

Cell culture models that mimic tissue environments are useful for cell and extracellular matrix (ECM) function analysis. Decellularized tissues with tissue-specific ECM are expected to be applied as cell culture scaffolds, however, it is often difficult for seeded cells to permeate their structures. In this study, we evaluated the adhesion and proliferation of mouse fibroblasts seeded onto decellularized bone marrow scaffolds that we fabricated from adult and fetal porcine. Decellularized fetal bone marrow displays more cell attachment and faster cell proliferation than decellularized adult bone marrow. Our findings suggest that decellularized fetal bone marrow is useful as a cell culture scaffold with bone marrow ECM and structure.

Nanofunctionalized Microparticles for Glucose Delivery in Three-Dimensional Cell Assemblies.

Three-dimensional (3D) cell assemblies, such as multicellular spheroids, can be powerful biological tools to closely mimic the complexity of cell-cell and cell-matrix interactions in a native-like microenvironment. However, potential applications of large spheroids are limited by the insufficient diffusion of oxygen and nutrients through the spheroids and, thus, result in the formation of a necrotic core. To overcome this drawback, we present a new strategy based on nanoparticle-coated microparticles. In this study, microparticles function as synthetic centers to regulate the diffusion of small molecules, such as oxygen and nutrients, within human mesenchymal stem cell (hMSC) spheroids. The nanoparticle coating on the microparticle surface acts as a nutrient reservoir to release glucose locally within the spheroids. We first coated the surface of the poly(lactic-co-glycolic acid) (PLGA) microparticles with mesoporous silica nanoparticles (MSNs) based on electrostatic interactions and then formed cell-nanofunctionalized microparticle spheroids. Next, we investigated the stability of the MSN coating on the microparticles' surface during 14 days of incubation in cell culture medium at 37 °C. Then, we evaluated the influence of MSN-coated PLGA microparticles on spheroid aggregation and cell viability. Our results showed the formation of homogeneous spheroids with good cell viability. As a proof of concept, fluorescently labeled glucose (2-NBD glucose) was loaded into the MSNs at different concentrations, and the release behavior was monitored. For cell culture studies, glucose was loaded into the MSNs coated onto the PLGA microparticles to sustain local nutrient release within the hMSC spheroids. In vitro results demonstrated that the local delivery of glucose from MSNs enhanced the cell viability in spheroids during a short-term hypoxic culture. Taken together, the newly developed nanofunctionalized microparticle-based delivery system may offer a versatile platform for local delivery of small molecules within 3D cellular assemblies and, thus, improve cell viability in spheroids.

Development of a 3D Brain Model to Study Sex-Specific Neuroinflammation After Hemorrhagic Stroke.

Subarachnoid hemorrhage (SAH) accounts for 5% of stroke, with women having a decreased inflammatory response compared to men; however, this mechanism has yet to be identified. One hurdle in SAH research is the lack of human brain models. Studies in murine models are helpful, but human models should be used in conjunction for improved translatability. These observations lead us to develop a 3D system to study the sex-specific microglial and neuroglial function in a novel in vitro human SAH model and compare it to our validated in vivo SAH model. Our lab has developed a 3D, membrane-based in vitro cell culture system with human astrocytes, microglia, and neurons from both sexes. The 3D cultures were incubated with male and female cerebrospinal fluid from SAH patients in the Neuro-ICU. Furthermore, microglial morphology, erythrophagocytosis, microglial inflammatory cytokine production, and neuronal apoptosis were studied and compared with our murine SAH models. The human 3D system demonstrated intercellular interactions and proportions of the three cell types similar to the adult human brain. In vitro and in vivo models of SAH showed concordance in male microglia being more inflammatory than females via morphology and flow cytometry. On the contrary, both in vitro and in vivo models revealed that female microglia were more phagocytic and less prone to damaging neurons than males. One possible explanation for the increased phagocytic ability of female microglia was the increased expression of CD206 and MerTK. Our in vitro, human, 3D cell culture SAH model showed similar results to our in vivo murine SAH model with respect to microglial morphology, inflammation, and phagocytosis when comparing the sexes. A human 3D brain model of SAH may be a useful adjunct to murine models to improve translation to SAH patients.

Electrophysiological properties of dorsal root ganglion neurons cultured on 3D silicon micro-pillar substrates.

BACKGROUND: Silicon-based micro-pillar substrates (MPS), as three-dimensional cell culture platforms with vertically aligned micro-patterned scaffolding structures, are known to facilitate high-quality growth and morphology of dorsal root ganglion (DRG) sensory neurons, promote neurite outgrowth and enhance neurite alignment. However, the electrophysiological aspects of DRG neurons cultured on silicon MPSs have not been thoroughly investigated, which is of greatest importance to ensure that such substrates do not disrupt neuronal homeostasis and function before their widespread adoption in diverse biomedical applications. NEW METHOD: We conducted whole-cell patch-clamp recordings to explore the electrophysiological properties of DRG neurons cultured on MPS arrays, utilizing a custom-made upright patch-clamp setup. RESULTS: Our findings revealed that DRG neurons exhibited similar electrophysiological responses on patterned MPS samples when compared to the control planar glass surfaces. Notably, there were no significant differences observed in the action potential parameters or firing patterns of action potentials between neurons grown on either substrate. COMPARISON WITH EXISTING METHODS: In the current study we for the first time confirmed that successful electrophysiological recordings can be obtained from the cells grown on MPS. CONCLUSION: Our results imply that, despite the potential alterations caused by the cumulative trauma of tissue harvest and cell dissociation, essential functional cell properties of DRG neurons appear to be relatively maintained on MPS surfaces. Therefore, vertically aligned silicon MPSs could be considered as a potentially effective three-dimensional system for supporting a controlled cellular environment in culture.

Monte Carlo simulation-guided design for size-tuned tumor spheroid formation in 3D printed microwells.

Tumor spheroid models have garnered significant attention in recent years as they can efficiently mimic in vivo models, and in addition, they offer a more controlled and reproducible environment for evaluating the efficacy of cancer drugs. In this study, we present the design and fabrication of a micromold template to form multicellular spheroids in a high-throughput and controlled-sized fashion. Briefly, polydimethylsiloxane-based micromolds at varying sizes and geometry were fabricated via soft lithography using 3D-printed molds as negative templates. The efficiency of spheroid formation was assessed using GFP-expressing human embryonic kidney 293 cells (HEK-293). After 7 days of culturing, circularity and cell viability of spheroids were >0.8 and 90%, respectively. At 1500 cells/microwell of cell seeding concentration, the spheroids were 454 ± 15 µm, 459 ± 7 µm, and 451 ± 18 µm when cultured in microwells with the diameters of 0.4, 0.6, and 0.8 µm, respectively. Moreover, the distance between each microwell and surfactant treatment before cell seeding notably impacted the uniform spheroid formation. The centrifugation was the key step to collect cells on the bottom of the microwells. Our findings were further verified using a commercial microplate. Furthermore, Monte Carlo simulation confirmed the seeding conditions where the spheroids could be formed. This study showed prominent steps in investigating spheroid formation, thereby leveraging the current know-how on the mechanism of tumor growth.

Organoid models: the future companions of personalized drug development.

High failure rates of the current drug development process are driving exemplary changes toward methodologies centered on human disease in-vitro modeling. Organoids are self-organized tissue sub-units resembling their organ of origin and are widely acknowledged for their unique potential in recapitulating human physio-pathological mechanisms. They are transformative for human health by becoming the platform of choice to probe disease mechanisms and advance new therapies. Furthermore, the compounds' validation as therapeutics represents another point of the drug development pipeline where organoids may provide key understandings and help pharma organizations replace or reduce animal research. In this review, we focus on gastrointestinal organoid models, which are currently the most advanced organoid models in drug development. We focus on experimental validations of their value, and we propose avenues to enhance their use in drug discovery and development, as well as precision medicine and diagnostics.

Advances in the application of iron oxide nanoparticles (IONs and SPIONs) in three-dimensional cell culture systems.

BACKGROUND: The field of tissue engineering has remarkably progressed through the integration of nanotechnology and the widespread use of magnetic nanoparticles. These nanoparticles have resulted in innovative methods for three-dimensional (3D) cell culture platforms, including the generation of spheroids, organoids, and tissue-mimetic cultures, where they play a pivotal role. Notably, iron oxide nanoparticles and superparamagnetic iron oxide nanoparticles have emerged as indispensable tools for non-contact manipulation of cells within these 3D environments. The variety and modification of the physical and chemical properties of magnetic nanoparticles have profound impacts on cellular mechanisms, metabolic processes, and overall biological function. This review article focuses on the applications of magnetic nanoparticles, elucidating their advantages and potential pitfalls when integrated into 3D cell culture systems. This review aims to shed light on the transformative potential of magnetic nanoparticles in terms of tissue engineering and their capacity to improve the cultivation and manipulation of cells in 3D environments.

Comparative analysis of traction forces in normal and glaucomatous trabecular meshwork cells within a 3D, active fluid-structure interaction culture environment.

This study presents a 3D in vitro cell culture model, meticulously 3D printed to replicate the conventional aqueous outflow pathway anatomical structure, facilitating the study of trabecular meshwork (TM) cellular responses under glaucomatous conditions. Glaucoma affects TM cell functionality, leading to extracellular matrix (ECM) stiffening, enhanced cell-ECM adhesion, and obstructed aqueous humor outflow. Our model, reconstructed from polyacrylamide gel with elastic moduli of 1.5 and 21.7 kPa, is based on serial block-face scanning electron microscopy images of the outflow pathway. It allows for quantifying 3D, depth-dependent, dynamic traction forces exerted by both normal and glaucomatous TM cells within an active fluid-structure interaction (FSI) environment. In our experimental design, we designed two scenarios: a control group with TM cells observed over 20 hours without flow (static setting), focusing on intrinsic cellular contractile forces, and a second scenario incorporating active FSI to evaluate its impact on traction forces (dynamic setting). Our observations revealed that active FSI results in higher traction forces (normal: 1.83-fold and glaucoma: 2.24-fold) and shear strains (normal: 1.81-fold and glaucoma: 2.41-fold), with stiffer substrates amplifying this effect. Glaucomatous cells consistently exhibited larger forces than normal cells. Increasing gel stiffness led to enhanced stress fiber formation in TM cells, particularly in glaucomatous cells. Exposure to active FSI dramatically altered actin organization in both normal and glaucomatous TM cells, particularly affecting cortical actin stress fiber arrangement. This model while preliminary offers a new method in understanding TM cell biomechanics and ECM stiffening in glaucoma, highlighting the importance of FSI in these processes. STATEMENT OF SIGNIFICANCE: This pioneering project presents an advanced 3D in vitro model, meticulously replicating the human trabecular meshwork's anatomy for glaucoma research. It enables precise quantification of cellular forces in a dynamic fluid-structure interaction, a leap forward from existing 2D models. This advancement promises significant insights into trabecular meshwork cell biomechanics and the stiffening of the extracellular matrix in glaucoma, offering potential pathways for innovative treatments. This research is positioned at the forefront of ocular disease study, with implications that extend to broader biomedical applications.

Matrix confinement modulates 3D spheroid sorting and burst-like collective migration.

While it is known that cells with differential adhesion tend to segregate and preferentially sort, the physical forces governing sorting and invasion in heterogeneous tumors remain poorly understood. To investigate this, we tune matrix confinement, mimicking changes in the stiffness and confinement of the tumor microenvironment, to explore how physical confinement influences individual and collective cell migration in 3D spheroids. High levels of confinement lead to cell sorting while reducing matrix confinement triggers the collective fluidization of cell motion. Cell sorting, which depends on cell-cell adhesion, is crucial to this phenomenon. Burst-like migration does not occur for spheroids that have not undergone sorting, regardless of the degree of matrix confinement. Using computational Self-Propelled Voronoi modeling, we show that spheroid sorting and invasion into the matrix depend on the balance between cell-generated forces and matrix resistance. The findings support a model where matrix confinement modulates 3D spheroid sorting and unjamming in an adhesion-dependent manner, providing insights into the mechanisms of cell sorting and migration in the primary tumor and toward distant metastatic sites. STATEMENT OF SIGNIFICANCE: The mechanical properties of the tumor microenvironment significantly influence cancer cell migration within the primary tumor, yet how these properties affect intercellular interactions in heterogeneous tumors is not well understood. By utilizing calcium and calcium chelators, we dynamically alter collagen-alginate hydrogel stiffness and investigate tumor cell behavior within co-culture spheroids in response to varying degrees of matrix confinement. High confinement is found to trigger cell sorting while reducing confinement for sorted spheroids facilitates collective cell invasion. Notably, without prior sorting, spheroids do not exhibit burst-like migration, regardless of confinement levels. This work establishes that matrix confinement and intercellular adhesion regulate 3D spheroid dynamics, offering insights into cellular organization and migration within the primary tumor.