Integrating tumor and healthy epithelium in a micro-physiology multi-compartment approach to study renal cell carcinoma pathophysiology.

The advent of micro-physiological systems (MPS) in biomedical research has enabled the introduction of more complex and relevant physiological into in vitro models. The recreation of complex morphological features in three-dimensional environments can recapitulate otherwise absent dynamic interactions in conventional models. In this study we developed an advanced in vitro Renal Cell Carcinoma (RCC) that mimics the interplay between healthy and malignant renal tissue. Based on the TissUse Humimic platform our model combines healthy renal proximal tubule epithelial cells (RPTEC) and RCC. Co-culturing reconstructed RPTEC tubules with RCC spheroids in a closed micro-perfused circuit resulted in significant phenotypical changes to the tubules. Expression of immune factors revealed that interleukin-8 (IL-8) and tumor necrosis factor-alfa (TNF-α) were upregulated in the non-malignant cells while neutrophil gelatinase-associated lipocalin (NGAL) was downregulated in both RCC and RPTEC. Metabolic analysis showed that RCC prompted a shift in the energy production of RPTEC tubules, inducing glycolysis, in a metabolic adaptation that likely supports RCC growth and immunogenicity. In contrast, RCC maintained stable metabolic activity, emphasizing their resilience to external factors. RNA-seq and biological process analysis of primary RTPTEC tubules demonstrated that the 3D tubular architecture and MPS conditions reverted cells to a predominant oxidative phosphorylate state, a departure from the glycolytic metabolism observed in 2D culture. This dynamic RCC co-culture model, approximates the physiology of healthy renal tubules to that of RCC, providing new insights into tumor-host interactions. Our approach can show that an RCC-MPS can expand the complexity and scope of pathophysiology and biomarker studies in kidney cancer research.

A novel approach to determine the critical survival threshold of cellular oxygen within spheroids via integrating live/dead cell imaging with oxygen modeling.

Defining the oxygen level that induces cell death within 3-D tissues is vital for understanding tissue hypoxia; however, obtaining accurate measurements has been technically challenging. In this study, we introduce a noninvasive, high-throughput methodology to quantify critical survival partial oxygen pressure (pO2) with high spatial resolution within spheroids by using a combination of controlled hypoxic conditions, semiautomated live/dead cell imaging, and computational oxygen modeling. The oxygen-permeable, micropyramid patterned culture plates created a precisely controlled oxygen condition around the individual spheroid. Live/dead cell imaging provided the geometric information of the live/dead boundary within spheroids. Finally, computational oxygen modeling calculated the pO2 at the live/dead boundary within spheroids. As proof of concept, we determined the critical survival pO2 in two types of spheroids: isolated primary pancreatic islets and tumor-derived pseudoislets (2.43 ± 0.08 vs. 0.84 ± 0.04 mmHg), indicating higher hypoxia tolerance in pseudoislets due to their tumorigenic origin. We also applied this method for evaluating graft survival in cell transplantations for diabetes therapy, where hypoxia is a critical barrier to successful transplantation outcomes; thus, designing oxygenation strategies is required. Based on the elucidated critical survival pO2, 100% viability could be maintained in a typically sized primary islet under the tissue pO2 above 14.5 mmHg. This work presents a valuable tool that is potentially instrumental for fundamental hypoxia research. It offers insights into physiological responses to hypoxia among different cell types and may refine translational research in cell therapies.NEW & NOTEWORTHY Our study introduces an innovative combinatory approach for noninvasively determining the critical survival oxygen level of cells within small cell spheroids, which replicates a 3-D tissue environment, by seamlessly integrating three pivotal techniques: cell death induction under controlled oxygen conditions, semiautomated imaging that precisely identifies live/dead cells, and computational modeling of oxygen distribution. Notably, our method ensures high-throughput analysis applicable to various cell types, offering a versatile solution for researchers in diverse fields.

Characterization of Single-Spheroid Oxygen Consumption Using a Microfluidic Platform and Fluorescence Lifetime Imaging Microscopy.

Oxygen consumption has been used to evaluate various cellular activities. In addition, three-dimensional (3D) spheroids have been broadly exploited as advanced in vitro cell models for various biomedical studies due to their capability of mimicking 3D in vivo microenvironments and cell arrangements. However, monitoring the oxygen consumption of live 3D spheroids poses challenges because existing invasive methods cause structural and cell damage. In contrast, optical methods using fluorescence labeling and microscopy are non-invasive, but they suffer from technical limitations like high cost, tedious procedures, and poor signal-to-noise ratios. To address these challenges, we developed a microfluidic platform for uniform-sized spheroid formation, handling, and culture. The platform is further integrated with widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) to efficiently characterize the lifetime of an oxygen-sensitive dye filling the platform for oxygen consumption characterization. In the experiments, osteosarcoma (MG-63) cells are exploited as the spheroid model and for the oxygen consumption analysis. The results demonstrate the functionality of the developed approach and show the accurate characterization of the oxygen consumption of the spheroids in response to drug treatments. The developed approach possesses great potential to advance spheroid metabolism studies with single-spheroid resolution and high sensitivity.

Real-Time Cell Cycle Imaging in a 3D Cell Culture Model of Melanoma, Quantitative Analysis, Optical Clearing, and Mathematical Modeling.

Aberrant cell cycle progression is a hallmark of solid tumors. Therefore, cell cycle analysis is an invaluable technique to study cancer cell biology. However, cell cycle progression has been most commonly assessed by methods that are limited to temporal snapshots or that lack spatial information. In this chapter, we describe a technique that allows spatiotemporal real-time tracking of cell cycle progression of individual cells in a multicellular context. The power of this system lies in the use of 3D melanoma spheroids generated from melanoma cells engineered with the fluorescent ubiquitination-based cell cycle indicator (FUCCI). This technique, combined with mathematical modeling, allows us to gain further and more detailed insight into several relevant aspects of solid cancer cell biology, such as tumor growth, proliferation, invasion, and drug sensitivity.

Spheroid architecture strongly enhances miR-221/222 expression and promotes oxidative phosphorylation in an ovarian cancer cell line through a mechanism that includes restriction of miR-9 expression.

BACKGROUND: Tumor cell spheroids are organized multicellular structures that form during the expansive growth of carcinoma cells. Spheroids formation is thought to contribute to metastasis by supporting growth and survival of mobile tumor cell populations. METHODS AND RESULTS: We investigated how spheroid architecture affects OXPHOS activity, microRNA expression, and intraperitoneal survival of an ovarian carcinoma cell line using high resolution respirometry, quantitative RT-PCR, and a rodent intraperitoneal growth model. Rates of oxidative phosphorylation/respiration per cell of cells growing as spheroids were nearly double those of a variant of the same cell type growing in suspension as loosely aggregated cells. Further, inhibition of spheroid formation by treatment with CDH2 (N-cadherin) siRNA reduced the rate of OXPHOS to that of the non-spheroid forming variant. Cells growing as spheroids showed greatly enhanced expression of miR-221/222, an oncomiR that targets multiple tumor suppressor genes and promotes invasion, and reduced expression of miR-9, which targets mitochondrial tRNA-modification enzymes and inhibits OXPHOS. Consistent with greater efficiency of ATP generation, tumor cells growing as spheroids injected into the nutrient-poor murine peritoneum survived longer than cells growing in suspension as loosely associated aggregates. CONCLUSIONS: The data indicate that growth in spheroid form enhances the OXPHOS activity of constituent tumor cells. In addition, spheroid architecture affects expression of microRNA genes involved in growth control and mitochondrial function. During the mobile phase of metastasis, when ovarian tumor cells disperse through nutrient-poor environments such as the peritoneum, enhanced OXPHOS activity afforded by spheroid architecture would enhance survival and metastatic potential.

Interrogating the Impact of Protease Activity on Tumor Progression Using 3D Spheroid Models.

Cancers have a complex relationship with the surrounding environment that regulates everything from progression to response to treatment. Cell-cell and cell-matrix interactions are heavily influenced by protease biology. Studies on the tumor microenvironment have revealed a new complexity for proteases, describing novel substrates for classic proteases, and protease-independent roles for these enzymes. The rapid expansion of 3D in vitro model systems provides excellent tools to study the intricate influence of proteases on the tumor microenvironment. Here we describe a spheroid invasion assay, providing a platform to interrogate key protease-matrix interactions in the context of early-stage breast cancer. Incorporation of pharmacological inhibition and RNAi techniques enables the elucidation of key protease-dependent pathways and can be complemented with immunofluorescence analysis to visualize matrix cleavage events and visualize cell behavior during collective cell invasion.

Therapeutic targeting of tumor spheroids in a 3D microphysiological renal cell carcinoma-on-a-chip system.

Metastatic renal cell carcinoma (RCC) remains an incurable disease for most patients highlighting an urgent need for new treatments. However, the preclinical investigation of new therapies is limited by traditional two-dimensional (2D) cultures which do not recapitulate the properties of tumor cells within a collagen extracellular matrix (ECM), while human tumor xenografts are time-consuming, expensive and lack adaptive immune cells. We report a rapid and economical human microphysiological system ("RCC-on-a-chip") to investigate therapies targeting RCC spheroids in a 3D collagen ECM. We first demonstrate that culture of RCC cell lines A498 and RCC4 in a 3D collagen ECM more faithfully reproduces the gene expression program of primary RCC tumors compared to 2D culture. We next used bortezomib as a cytotoxin to develop automated quantification of dose-dependent tumor spheroid killing. We observed that viable RCC spheroids exhibited collective migration within the ECM and demonstrated that our 3D system can be used to identify compounds that inhibit spheroid collective migration without inducing cell death. Finally, we demonstrate the RCC-on-a-chip as a platform to model the trafficking of tumor-reactive T cells into the ECM and observed antigen-specific A498 spheroid killing by engineered human CD8+ T cells expressing an ROR1-specific chimeric antigen receptor. In summary, the phenotypic differences between the 3D versus 2D environments, rapid imaging-based readout, and the ability to carefully study the impact of individual variables with quantitative rigor will encourage adoption of the RCC-on-a-chip system for testing a wide range of emerging therapies for RCC.

Proteomic and Phosphoproteomic Reprogramming in Epithelial Ovarian Cancer Metastasis.

Epithelial ovarian cancer (EOC) is a high-risk cancer presenting with heterogeneous tumors. The high incidence of EOC metastasis from primary tumors to nearby tissues and organs is a major driver of EOC lethality. We used cellular models of spheroid formation and readherence to investigate cellular signaling dynamics in each step toward EOC metastasis. In our system, adherent cells model primary tumors, spheroid formation represents the initiation of metastatic spread, and readherent spheroid cells represent secondary tumors. Proteomic and phosphoproteomic analyses show that spheroid cells are hypoxic and show markers for cell cycle arrest. Aurora kinase B abundance and downstream substrate phosphorylation are significantly reduced in spheroids and readherent cells, explaining their cell cycle arrest phenotype. The proteome of readherent cells is most similar to spheroids, yet greater changes in the phosphoproteome show that spheroid cells stimulate Rho-associated kinase 1 (ROCK1)-mediated signaling, which controls cytoskeletal organization. In spheroids, we found significant phosphorylation of ROCK1 substrates that were reduced in both adherent and readherent cells. Application of the ROCK1-specific inhibitor Y-27632 to spheroids increased the rate of readherence and altered spheroid density. The data suggest ROCK1 inhibition increases EOC metastatic potential. We identified novel pathways controlled by Aurora kinase B and ROCK1 as major drivers of metastatic behavior in EOC cells. Our data show that phosphoproteomic reprogramming precedes proteomic changes that characterize spheroid readherence in EOC metastasis.

3D Volumetric Mechanosensation of MCF7 Breast Cancer Spheroids in a Linear Stiffness Gradient GelAGE.

The tumor microenvironment presents spatiotemporal shifts in biomechanical properties with cancer progression. Hydrogel biomaterials like GelAGE offer the stiffness tuneability to recapitulate dynamic changes in tumor tissues by altering photo-energy exposures. Here, a tuneable hydrogel with spatiotemporal control of stiffness and mesh-network is developed. The volume of MCF7 spheroids encapsulated in a linear stiffness gradient demonstrates an inverse relationship with stiffness (p < 0.0001). As spheroids are exposed to increased crosslinking (stiffer) and greater mechanical confinement, spheroid stiffness increases. Protein expression (TRPV4, ß1 integrin, E-cadherin, and F-actin) decreases with increasing stiffness while showing strong correlations to spheroid volume (r2  > 0.9). To further investigate the role of volume, MCF7 spheroids are grown in a soft matrix for 5 days prior to a second polymerisation which presents a stiffness gradient to equally expanded spheroids. Despite being exposed to variable stiffness, these spheroids show even protein expression, confirming volume as a key regulator. Overall, this work showcases the versatility of GelAGE and demonstrates volume expansion as a key regulator of 3D mechanosensation in MCF7 breast cancer spheroids. This platform has the potential to further investigation into the role of stiffness and dimensionality in 3D spheroid culture for other types of cancers and diseases.

Evaluating variations in bilirubin glucuronidation activity by protease inhibitors in canine and human primary hepatocytes cultured in a 3D culture system.

Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin. The culture supernatant was collected for bilirubin glucuronidation assessment and the cells were used to evaluate viability. On day 8 of culture, both canine and human hepatocyte spheroids showed high albumin secretion and distinct spheroid formation, and their bilirubin glucuronidation activities were evaluated considering cell viability. Treatment with atazanavir and ritonavir remarkably inhibited bilirubin glucuronide formation, wherein atazanavir showed the highest inhibition, particularly in human hepatocyte spheroids. These results may reflect the effects on cellular uptake of bilirubin and its intracellular metabolic function. Thus, primary hepatocytes cultured in a 3D culture system may be a useful in vitro system for the comprehensive evaluation of bilirubin metabolic function and risk assessment in bilirubin metabolic disorders for drug development.

Multicellular spheroids containing synthetic mineral particles: an advanced 3D tumor model system to investigate breast precancer malignancy potential according to the mineral type.

Mineral particles that form in soft tissues in association with disease conditions are heterogeneous in their composition and physiochemical properties. Hence, it is challenging to study the effect of mineral type on disease progression in a high-throughput and realistic manner. For example, most early breast precancer lesions, termed ductal carcinoma in situ (DCIS), contain microcalcifications (MCs), calcium-containing pathological minerals. The most common type of MCs is calcium phosphate crystals, mainly carbonated apatite; it is associated with either benign or malignant lesions. In vitro studies indicate that the crystal properties of apatite MCs can affect breast cancer progression. A less common type of MCs is calcium oxalate dihydrate (COD), which is almost always found in benign lesions. We developed a 3D tumor model of multicellular spheroids of human precancer cells containing synthetic MC analogs that link the crystal properties of MCs with the progression of breast precancer to invasive cancer. Using this 3D model, we show that apatite crystals induce Her2 overexpression in DCIS cells. This tumor-triggering effect is increased when the carbonate fraction in the MCs decreases. COD crystals, in contrast, decrease Her2 expression in the spheroids, even compared with a control group with no added MC analogs. Furthermore, COD decreases cell proliferation and migration in DCIS monolayers compared to untreated cells and cells incubated with apatite crystals. This finding suggests that COD is not randomly located only in benign lesions-it may actively contribute to suppressing precancer progression in its surroundings. Our model provides an easy-to-manipulate platform to better understand the interactions between mineral particles and their biological microenvironment. A better understanding of the effect of the crystal properties of MCs on precancer progression will potentially provide new directions for better precancer prognosis and treatment.

Establishment and Characterization of Free-Floating 3D Macrophage Programming Model in the Presence of Cancer Cell Spheroids.

Reprogramming of tumor-associated macrophages (TAMs) is a promising strategy for cancer immunotherapy. Several studies have shown that cancer cells induce/support the formation of immunosuppressive TAMs phenotypes. However, the specific factors that orchestrate this immunosuppressive process are unknown or poorly studied. In vivo studies are expensive, complex, and ethically constrained. Therefore, 3D cell interaction models could become a unique framework for the identification of important TAMs programming factors. In this study, we have established and characterized a new in vitro 3D model for macrophage programming in the presence of cancer cell spheroids. First, it was demonstrated that the profile of cytokines, chemokines, and surface markers of 3D-cultured macrophages did not differ conceptually from monolayer-cultured M1 and M2-programmed macrophages. Second, the possibility of reprogramming macrophages in 3D conditions was investigated. In total, the dynamic changes in 6 surface markers, 11 cytokines, and 22 chemokines were analyzed upon macrophage programming (M1 and M2) and reprogramming (M1→M2 and M2→M1). According to the findings, the reprogramming resulted in a mixed macrophage phenotype that expressed both immunosuppressive and anti-cancer immunostimulatory features. Third, cancer cell spheroids were shown to stimulate the production of immunosuppressive M2 markers as well as pro-tumor cytokines and chemokines. In summary, the newly developed 3D model of cancer cell spheroid/macrophage co-culture under free-floating conditions can be used for studies on macrophage plasticity and for the development of targeted cancer immunotherapy.

Mechanistic study for drug induced cholestasis using batch-fabricated 3D spheroids developed by agarose-stamping method.

Cell spheroid culture can recapitulate the tissue microstructure and cellular responses in vivo. While there is a strong need to understand the modes of toxic action using the spheroid culture method, existing preparation techniques suffer from low efficiency and high cost. Herein, we developed a metal stamp containing hundreds of protrusions for batch bulk preparation of cell spheroids in each well of the culture plates. The agarose matrix imprinted by the stamp can form an array of hemispherical pits, which facilitated the fabrication of hundreds of uniformly sized rat hepatocyte spheroids in each well. Chlorpromazine (CPZ) was used as a model drug to investigate the mechanism for drug induced cholestasis (DIC) by agarose-stamping method. Hepatocyte spheroids showed a more sensitive detection of hepatotoxicity compared to 2D and Matrigel-based culture systems. Cell spheroids were also collected for staining of cholestatic protein and showed a CPZ-concentration-dependent decrease of bile acid efflux related proteins (BSEP and MRP2) and tight junction (ZO-1). In addition, the stamping system successfully delineated the DIC mechanism by CPZ that may be associated with the phosphorylation of MYPT1 and MLC2, two central proteins in the Rho-associated protein kinase pathway (ROCK), which were significantly attenuated by ROCK inhibitors. Our results demonstrated a large-scale fabrication of cell spheroids by the agarose-stamping method, with promising benefits for exploring the mechanisms for drug hepatotoxic responses.

Eye-Mimicked Neural Network Composed of Photosensitive Neural Spheroids with Human Opsin Proteins.

An in vitro model, composed of the short-wavelength human opsins and rhodopsins, is created. Two types of photosensitive neural spheroids are transfected for selective reaction under bluish-purple and green lights. These are employed to two devices with intact neuron and neural-spheroid to study the interaction. By photostimulation, the photosensitive spheroid initiated photoactivation, and the signal generated from its body is transmitted to adjacent neural networks. Specifically, the signal traveled through the axon bundle in narrow gap from photosensitive spheroid to intact spheroid as an eye-to-brain model including optic nerve. The whole process with photosensitive spheroid is monitored by calcium ion detecting fluorescence images. The results of this study can be applied to examine vision restoration and novel photosensitive biological systems with spectral sensitivity.

In Vitro 3D Spheroid Model Preserves Tumor Microenvironment of Hot and Cold Breast Cancer Subtypes.

Dynamic interaction of cancer, immune, and stromal cells with extracellular matrix components modulates and resists the response of standard care therapies. To mimic this, an in vitro 3D spheroid model is designed using liquid overlay method to simulate hot (MDA-MB-231) and cold (MCF-7) breast tumor microenvironment (TME). This study shows increased mesenchymal phenotype, stemness, and suppressive microenvironment in MDA-MB-231-spheroids upon exposure to doxorubicin. Intriguingly, the presence of human dermal fibroblasts enhances cancer-associated fibroblast phenotype in MDA-MB-231-spheroids through increased expression of CXCL12 and FSP-1, leading to higher infiltration of immune cells (THP-1 monocytes). However, a suppressive TME is observed in both subtypes, as seen by upregulation of M2-macrophage-specific CD68 and CD206 markers. Specifically, increased PDL-1 expressing tumor-associated macrophages along with FoxP3 expressing T regulatory cells are found in MDA-MB-231-spheroids when cultured with peripheral blood mononuclear cells. Further, it is found that the addition of 1-methyl-tryptophan, a potent indoleamine-2,3-dioxygenase-1 inhibitor, subsides the suppressive phenotype by decreasing the M2 polarization via downregulation of tryptophan metabolism and IL10 expression, particularly in MCF-7 triculture spheroids. Thus, the in vitro 3D spheroid model of TME can be utilized in therapeutics to validate immunomodulatory drugs for various breast cancer subtypes.

Controlling Cell Organization in 3D Coculture Spheroids Using DNA Interactions.

The role of stromal and immune cells in transforming the tumor microenvironment is a key consideration in understanding tumor cell behavior and anticancer drug development. To better model these systems in vitro, 3D coculture tumor spheroids have been engineered using a variety of techniques including centrifugation to microwells, hanging drop, low adhesion cultures, and culture of cells in a microfluidic platform. Aside from using bioprinting, however, it has remained more challenging to direct the spatial organization of heterotypic cells in standalone 3D spheroids. To address this, we present an in vitro 3D coculture tumor model where we modulated the interactions between cancer cells and fibroblasts through DNA hybridization. When native heterotypic cells are simply mixed, the cell aggregates typically show cell sorting behavior to form phase separated structures composed of single cell types. In this work, we demonstrate that when MDA-MB-468 breast cancer and NIH/3T3 fibroblasts are directed to associate via complementary DNA, a uniform distribution of the two cell types within a single spheroid was observed. In contrast, in the absence of specific DNA interactions between the cancer cells and fibroblasts, individual clusters of the NIH/3T3 cells formed in each spheroid due to cell sorting. To better understand the effect of heterotypic cell organization on either cell-cell contacts or matrix protein production, the spheroids were further stained with anti-E-cadherin and antifibronectin antibodies. While the amounts of E-cadherin appeared to be similar between the spheroids, a significantly higher amount of fibronectin secretion was observed in the coculture spheroids with uniform mixing of two cell types. This result showed that different heterotypic cell distributions within 3D architecture can influence the ECM protein production that can again alter the properties of the tumor or tumor microenvironment. The present study thus describes the use of DNA templating to direct the organization of cells in coculture spheroids, which can provide mechanistic biological insight into how heterotypic distribution in tumor spheroids can influence tumor progression, metastasis, and drug resistance.

3D melanoma spheroid model for the development of positronium biomarkers.

It was recently demonstrated that newly invented positronium imaging may be used for improving cancer diagnostics by providing additional information about tissue pathology with respect to the standardized uptake value currently available in positron emission tomography (PET). Positronium imaging utilizes the properties of positronium atoms, which are built from the electrons and positrons produced in the body during PET examinations. We hypothesized that positronium imaging would be sensitive to the in vitro discrimination of tumor-like three-dimensional structures (spheroids) built of melanoma cell lines with different cancer activities and biological properties. The lifetime of ortho-positronium (o-Ps) was evaluated in melanoma spheroids from two cell lines (WM266-4 and WM115) differing in the stage of malignancy. Additionally, we considered parameters such as the cell number, spheroid size and melanoma malignancy to evaluate their relationship with the o-Ps lifetime. We demonstrate pilot results for o-Ps lifetime measurement in extracellular matrix-free spheroids. With the statistical significance of two standard deviations, we demonstrated that the higher the degree of malignancy and the rate of proliferation of neoplastic cells, the shorter the lifetime of ortho-positronium. In particular, we observed the following indications encouraging further research: (i) WM266-4 spheroids characterized by a higher proliferation rate and malignancy showed a shorter o-Ps lifetime than WM115 spheroids characterized by a lower growth rate. (ii) Both cell lines showed a decrease in the lifetime of o-Ps after spheroid generation on day 8 compared to day 4 in culture, and the mean o-Ps lifetime was longer for spheroids formed from WM115 cells than for those formed from WM266-4 cells, regardless of spheroid age. The results of this study revealed that positronium is a promising biomarker that may be applied in PET diagnostics for the assessment of the degree of cancer malignancy.

A Method for Culturing 3D Tumoroids of Lung Adenocarcinoma Cells at the Air-Liquid Interface.

Three-dimensional (3D) tumor spheroids and tumoroids are among the most exploited cell culture methods in the lung cancer field, finding applications in the investigation of tumor growth and proliferation, invasion, and drug screening. However, 3D tumor spheroids and tumoroids cannot fully mimic the architecture of the human lung adenocarcinoma tissue and, in particular, the direct contact of the lung adenocarcinoma cells with the air, as they lack polarity. Our method allows to overcome this limitation by enabling to grow tumoroids of lung adenocarcinoma cells and healthy lung fibroblasts at the Air-Liquid Interface (ALI). This ensures straightforward access to both the apical and basal surface of the cancer cell culture, with several advantages in drug screening applications.

Collagen-Laponite Nanoclay Hydrogels for Tumor Spheroid Growth.

The extracellular matrix (ECM) plays an important regulatory role in the development and progression of tumoral tissue. Its functions and properties are crucial in determining tumor cell behavior such as invasion, migration, and malignancy development. Our study explores the role of collagen type I in cancer development and spread using engineered tumor models like multicellular spheroids grown in collagen-based hydrogels to simulate early tumor formation. We employ microfluidic techniques to test the hypothesis that (i) adding Laponite nanoclay to collagen hydrogels modifies mechanical and rheological properties and (ii) changing the stiffness of the collagen microenvironment affects tumor spheroid growth. Our findings support our theories and suggest the use of ECM components and engineered tumor models in cancer research, offering a biocompatible and biomimetic method to tailor the mechanical properties of conventional collagen hydrogels.

A mechanobiological model for tumor spheroid evolution with application to glioblastoma: A continuum multiphysics approach.

BACKGROUND: Spheroids are in vitro quasi-spherical structures of cell aggregates, eventually cultured within a hydrogel matrix, that are used, among other applications, as a technological platform to investigate tumor formation and evolution. Several interesting features can be replicated using this methodology, such as cell communication mechanisms, the effect of gradients of nutrients, or the creation of realistic 3D biological structures. The main objective of this work is to link the spheroid evolution with the mechanical activity of cells, coupled with nutrient consumption and the subsequent cell dynamics. METHOD: We propose a continuum mechanobiological model which accounts for the most relevant phenomena that take place in tumor spheroid evolution under in vitro suspension, namely, nutrient diffusion in the spheroid, kinetics of cellular growth and death, and mechanical interactions among the cells. The model is qualitatively validated, after calibration of the model parameters, versus in vitro experiments of spheroids of different glioblastoma cell lines. RESULTS: Our model is able to explain in a novel way quite different setups, such as spheroid growth (up to six times the initial configuration for U-87 MG cell line) or shrinking (almost half of the initial configuration for U-251 MG cell line); as the result of the mechanical interplay of cells driven by cellular evolution. CONCLUSIONS: Glioblastoma tumor spheroid evolution is driven by mechanical interactions of the cell aggregate and the dynamical evolution of the cell population. All this information can be used to further investigate mechanistic effects in the evolution of tumors and their role in cancer disease.