Loss of LKB1-NUAK1 signalling enhances NF-κB activity in a spheroid model of high-grade serous ovarian cancer.

High-grade serous ovarian cancer (HGSOC) is an aggressive malignancy often diagnosed at an advanced stage. Although most HGSOC patients respond initially to debulking surgery combined with cytotoxic chemotherapy, many ultimately relapse with platinum-resistant disease. Thus, improving outcomes requires new ways of limiting metastasis and eradicating residual disease. We identified previously that Liver kinase B1 (LKB1) and its substrate NUAK1 are implicated in EOC spheroid cell viability and are required for efficient metastasis in orthotopic mouse models. Here, we sought to identify additional signalling pathways altered in EOC cells due to LKB1 or NUAK1 loss-of-function. Transcriptome analysis revealed that inflammatory signalling mediated by NF-κB transcription factors is hyperactive due to LKB1-NUAK1 loss in HGSOC cells and spheroids. Upregulated NF-κB signalling due to NUAK1 loss suppresses reactive oxygen species (ROS) production and sustains cell survival in spheroids. NF-κB signalling is also activated in HGSOC precursor fallopian tube secretory epithelial cell spheroids, and is further enhanced by NUAK1 loss. Finally, immunohistochemical analysis of OVCAR8 xenograft tumors lacking NUAK1 displayed increased RelB expression and nuclear staining. Our results support the idea that NUAK1 and NF-κB signalling pathways together regulate ROS and inflammatory signalling, supporting cell survival during each step of HGSOC pathogenesis. We propose that their combined inhibition may be efficacious as a novel therapeutic strategy for advanced HGSOC.

Designing and interpreting 4D tumour spheroid experiments.

Tumour spheroid experiments are routinely used to study cancer progression and treatment. Various and inconsistent experimental designs are used, leading to challenges in interpretation and reproducibility. Using multiple experimental designs, live-dead cell staining, and real-time cell cycle imaging, we measure necrotic and proliferation-inhibited regions in over 1000 4D tumour spheroids (3D space plus cell cycle status). By intentionally varying the initial spheroid size and temporal sampling frequencies across multiple cell lines, we collect an abundance of measurements of internal spheroid structure. These data are difficult to compare and interpret. However, using an objective mathematical modelling framework and statistical identifiability analysis we quantitatively compare experimental designs and identify design choices that produce reliable biological insight. Measurements of internal spheroid structure provide the most insight, whereas varying initial spheroid size and temporal measurement frequency is less important. Our general framework applies to spheroids grown in different conditions and with different cell types.

A machine learning pipeline revealing heterogeneous responses to drug perturbations on vascular smooth muscle cell spheroid morphology and formation.

Machine learning approaches have shown great promise in biology and medicine discovering hidden information to further understand complex biological and pathological processes. In this study, we developed a deep learning-based machine learning algorithm to meaningfully process image data and facilitate studies in vascular biology and pathology. Vascular injury and atherosclerosis are characterized by neointima formation caused by the aberrant accumulation and proliferation of vascular smooth muscle cells (VSMCs) within the vessel wall. Understanding how to control VSMC behaviors would promote the development of therapeutic targets to treat vascular diseases. However, the response to drug treatments among VSMCs with the same diseased vascular condition is often heterogeneous. Here, to identify the heterogeneous responses of drug treatments, we created an in vitro experimental model system using VSMC spheroids and developed a machine learning-based computational method called HETEROID (heterogeneous spheroid). First, we established a VSMC spheroid model that mimics neointima-like formation and the structure of arteries. Then, to identify the morphological subpopulations of drug-treated VSMC spheroids, we used a machine learning framework that combines deep learning-based spheroid segmentation and morphological clustering analysis. Our machine learning approach successfully showed that FAK, Rac, Rho, and Cdc42 inhibitors differentially affect spheroid morphology, suggesting that multiple drug responses of VSMC spheroid formation exist. Overall, our HETEROID pipeline enables detailed quantitative drug characterization of morphological changes in neointima formation, that occurs in vivo, by single-spheroid analysis.

GlomSpheres as a 3D co-culture spheroid model of the kidney glomerulus for rapid drug-screening.

The glomerulus is the filtration unit of the kidney. Injury to any component of this specialised structure leads to impaired filtration and eventually fibrosis and chronic kidney disease. Current two and three dimensional (2D and 3D) models that attempt to recreate structure and interplay between glomerular cells are imperfect. Most 2D models are simplistic and unrepresentative, and 3D organoid approaches are currently difficult to reproduce at scale and do not fit well with current industrial drug-screening approaches. Here we report a rapidly generated and highly reproducible 3D co-culture spheroid model (GlomSpheres), better demonstrating the specialised physical and molecular structure of a glomerulus. Co-cultured using a magnetic spheroid formation approach, conditionally immortalised (CI) human podocytes and glomerular endothelial cells (GEnCs) deposited mature, organized isoforms of collagen IV and Laminin. We demonstrate a dramatic upregulation of key podocyte (podocin, nephrin and podocalyxin) and GEnC (pecam-1) markers. Electron microscopy revealed podocyte foot process interdigitation and endothelial vessel formation. Incubation with pro-fibrotic agents (TGF-ß1, Adriamycin) induced extracellular matrix (ECM) dysregulation and podocyte loss, which were attenuated by the anti-fibrotic agent Nintedanib. Incubation with plasma from patients with kidney disease induced acute podocyte loss and ECM dysregulation relative to patient matched remission plasma, and Nintedanib reduced podocyte loss. Finally, we developed a rapid imaging approach to demonstrate the model's usefulness in higher throughput pharmaceutical screening. GlomSpheres therefore represent a robust, scalable, replacement for 2D in vitro glomerular disease models.

Stress generation, relaxation and size control in confined tumor growth.

Experiments on tumor spheroids have shown that compressive stress from their environment can reversibly decrease tumor expansion rates and final sizes. Stress release experiments show that nonuniform anisotropic elastic stresses can be distributed throughout. The elastic stresses are maintained by structural proteins and adhesive molecules, and can be actively relaxed by a variety of biophysical processes. In this paper, we present a new continuum model to investigate how the growth-induced elastic stresses and active stress relaxation, in conjunction with cell size control feedback machinery, regulate the cell density and stress distributions within growing tumors as well as the tumor sizes in the presence of external physical confinement and gradients of growth-promoting chemical fields. We introduce an adaptive reference map that relates the current position with the reference position but adapts to the current position in the Eulerian frame (lab coordinates) via relaxation. This type of stress relaxation is similar to but simpler than the classical Maxwell model of viscoelasticity in its formulation. By fitting the model to experimental data from two independent studies of tumor spheroid growth and their cell density distributions, treating the tumors as incompressible, neo-Hookean elastic materials, we find that the rates of stress relaxation of tumor tissues can be comparable to volumetric growth rates. Our study provides insight on how the biophysical properties of the tumor and host microenvironment, mechanical feedback control and diffusion-limited differential growth act in concert to regulate spatial patterns of stress and growth. When the tumor is stiffer than the host, our model predicts tumors are more able to change their size and mechanical state autonomously, which may help to explain why increased tumor stiffness is an established hallmark of malignant tumors.

Autotaxin May Have Lysophosphatidic Acid-Unrelated Effects on Three-Dimension (3D) Cultured Human Trabecular Meshwork (HTM) Cells.

PURPOSE: The objective of the current study was to evaluate the effects of the autotaxin (ATX)-lysophosphatidic acid (LPA) signaling axis on the human trabecular meshwork (HTM) in two-dimensional (2D) and three-dimensional (3D) cultures of HTM cells. METHODS: The effects were characterized by transendothelial electrical resistance (TEER) and FITC-dextran permeability (2D), measurements of size and stiffness (3D), and the expression of several genes, including extracellular matrix (ECM) molecules, their modulators, and endoplasmic reticulum (ER) stress-related factors. RESULTS: A one-day exposure to 200 nM LPA induced significant down-sizing effects of the 3D HTM spheroids, and these effects were enhanced slightly on longer exposure. The TEER and FITC-dextran permeability data indicate that LPA induced an increase in the barrier function of the 2D HTM monolayers. A one-day exposure to a 2 mg/L solution of ATX also resulted in a significant decrease in the sizes of the 3D HTM spheroids, and an increase in stiffness was also observed. The gene expression of several ECMs, their regulators and ER-stress related factors by the 3D HTM spheroids were altered by both ATX and LPA, but in different manners. CONCLUSIONS: The findings presented herein suggest that ATX may have additional roles in the human TM, in addition to the ATX-LPA signaling axis.

Quantitative analysis of tumour spheroid structure.

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Spheroids through Improved Oxygen Supply.

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to generate O2 for 3 d. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. When human hepatocellular carcinoma (HepG2) spheroids were prepared using the conventional microwells, the O2 concentration in the culture medium reduced to approx. 67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high cell viability.

The major evolutionary transitions and codes of life.

Major evolutionary transitions as well as the evolution of codes of life are key elements in macroevolution which are characterized by increase in complexity Major evolutionary transitions ensues by a transition in individuality and by the evolution of a novel mode of using, transmitting or storing information. Here is where codes of life enter the picture: they are arbitrary mappings between different (mostly) molecular species. This flexibility allows information to be employed in a variety of ways, which can fuel evolutionary innovation. The collation of the list of major evolutionary transitions and the list of codes of life show a clear pattern: codes evolved prior to a major evolutionary transition and then played roles in the transition and/or in the transformation of the new individual. The evolution of a new code of life is in itself not a major evolutionary transition but allow major evolutionary transitions to happen. This could help us to identify new organic codes.

DOC-2/DAB2 interactive protein destabilizes c-Myc to impair the growth and self-renewal of colon tumor-repopulating cells.

Colorectal carcinoma (CRC) remains a huge challenge in clinical treatment due to tumor metastasis and recurrence. Stem cell-like colon tumor-repopulating cells (TRCs) are a subpopulation of cancer cells with highly tumorigenic and chemotherapy resistant properties. The core transcription factor c-Myc is essential for maintaining cancer stem-like cell phenotypes, yet its roles and regulatory mechanisms remain unclear in colon TRCs. We report that elevated c-Myc protein supported formation and growth of TRC spheroids. The tumor suppressor DOC-2/DAB2 interactive protein (DAB2IP) suppressed c-Myc expression to inhibit TRC expansion and self-renewal. Particularly, DAB2IP disrupted c-Myc stability through glycogen synthase kinase 3ß/protein phosphatase 2A-B56α-mediated phosphorylation and dephosphorylation cascade on c-Myc protein, leading to its eventual degradation through the ubiquitin-proteasome pathway. The expression of DAB2IP was negatively correlated with c-Myc in CRC specimens. Overall, our results improved mechanistic insight into how DAB2IP suppressed TRC growth and self-renewal.

3D tumor spheroid microarray for high-throughput, high-content natural killer cell-mediated cytotoxicity.

Immunotherapy has emerged as a promising approach to treating several forms of cancer. Use of immune cells, such as natural killer (NK) cells, along with small molecule drugs and antibodies through antibody dependent cell-mediated cytotoxicity (ADCC) has been investigated as a potential combination therapy for some difficult to treat solid tumors. Nevertheless, there remains a need to develop tools that support co-culture of target cancer cells and effector immune cells in a contextually relevant three-dimensional (3D) environment to provide a rapid means to screen for and optimize ADCC-drug combinations. To that end, here we have developed a high throughput 330 micropillar-microwell sandwich platform that enables 3D co-culture of NK92-CD16 cells with pancreatic (MiaPaCa-2) and breast cancer cell lines (MCF-7 and MDA-MB-231). The platform successfully mimicked hypoxic conditions found in a tumor microenvironment and was used to demonstrate NK-cell mediated cell cytotoxicity in combination with two monoclonal antibodies; Trastuzumab and Atezolizumab. The platform was also used to show dose response behavior of target cancer cells with reduced EC50 values for paclitaxel (an anti-cancer chemotherapeutic) when treated with both NK cells and antibody. Such a platform may be used to develop more personalized cancer therapies using patient-derived cancer cells.

Patterning of interconnected human brain spheroids.

Brain spheroids are emerging as valuable in vitro models that are accelerating the pace of research in various diseases. For Alzheimer's disease (AD) research, these models are enhanced using genetically engineered human neural progenitor cells and novel cell culture methods. However, despite these advances, it remains challenging to study the progression of AD in vitro as well as the propagation of pathogenic amyloid-ß (Aß) and tau tangles between diseased and healthy neurons using the brain spheroids model. To address this need, we designed a microfluidic system of connected microwells for arranging two types of brain spheroids in complex patterns and enabling the formation of thick bundles of neurites between the brain spheroids and the accumulation of pathogenic Aß within the spheroids.

Mechanical activities of self-beating cardiomyocyte aggregates under mechanical compression.

Since the discovery of synchronous pulsations in cardiomyocytes (CMs), electrical communication between CMs has been emphasized; however, recent studies suggest the possibility of mechanical communication. Here, we demonstrate that spherical self-beating CM aggregates, termed cardiac spheroids (CSs), produce enhanced mechanical energy under mechanical compression and work cooperatively via mechanical communication. For single CSs between parallel plates, compression increased both beating frequency and beating energy. Contact mechanics revealed a scaling law on the beating energy, indicating that the most intensively stressed cells in the compressed CSs predominantly contributed to the performance of mechanical work against mechanical compression. For pairs of CSs between parallel plates, compression immediately caused synchronous beating with mechanical coupling. Compression tended to strengthen and stabilize the synchronous beating, although some irregularity and temporary arrest were observed. These results suggest that mechanical compression is an indispensable control parameter when evaluating the activities of CMs and their aggregates.

The hypertrophic cartilage induction influences the building-block capacity of human adipose stem/stromal cell spheroids for biofabrication.

As an alternative to the classical tissue engineering approach, bottom-up tissue engineering emerges using building blocks in bioassembly technologies. Spheroids can be used as building blocks to reach a highly complex ordered tissue by their fusion (bioassembly), representing the foundation of biofabrication. In this study, we analyzed the biomechanical properties and the fusion capacity of human adipose stem/stromal cell (ASC) we spheroids during an in vitro model of hypertrophic cartilage established by our research group. Hypertrophic induced-ASC spheroids showed a statistically significant higher Young's modulus at weeks 2 (P < .001) and 3 (P < .0005) compared with non-induced. After fusion, non-induced and induced-ASC spheroids increased the contact area and decreased their pairs' total length. At weeks 3 and 5, induced-ASC spheroids did not fuse completely, and the cells migrate preferentially in the fusion contact region. Alizarin red O staining showed the highest intensity of staining in the fused induced-ASC spheroids at week 5, together with intense staining for collagen type I and osteocalcin. Transmission electron microscopy and element content analysis (X-ray Energy Dispersive Spectroscopy) revealed in the fused quartet at week 3 a crystal-like structure. Hypertrophic induction interferes with the intrinsic capacity of spheroids to fuse. The measurements of contact between spheroids during the fusion process, together with the change in viscoelastic profile to the plastic, will impact the establishment of bioassembly protocols using hypertrophic induced-ASC spheroids as building blocks in biofabrication.

CB1 antagonism increases excitatory synaptogenesis in a cortical spheroid model of fetal brain development.

The endocannabinoid system (ECS) plays a complex role in the development of neural circuitry during fetal brain development. The cannabinoid receptor type 1 (CB1) controls synaptic strength at both excitatory and inhibitory synapses and thus contributes to the balance of excitatory and inhibitory signaling. Imbalances in the ratio of excitatory to inhibitory synapses have been implicated in various neuropsychiatric disorders associated with dysregulated central nervous system development including autism spectrum disorder, epilepsy, and schizophrenia. The role of CB1 in human brain development has been difficult to study but advances in induced pluripotent stem cell technology have allowed us to model the fetal brain environment. Cortical spheroids resemble the cortex of the dorsal telencephalon during mid-fetal gestation and possess functional synapses, spontaneous activity, an astrocyte population, and pseudo-laminar organization. We first characterized the ECS using STORM microscopy and observed synaptic localization of components similar to that which is observed in the fetal brain. Next, using the CB1-selective antagonist SR141716A, we observed an increase in excitatory, and to a lesser extent, inhibitory synaptogenesis as measured by confocal image analysis. Further, CB1 antagonism increased the variability of spontaneous activity within developing neural networks, as measured by microelectrode array. Overall, we have established that cortical spheroids express ECS components and are thus a useful model for exploring endocannabinoid mediation of childhood neuropsychiatric disease.

Flow cytometric quantification of apoptotic and proliferating cells applying an improved method for dissociation of spheroids.

Spheroids are a promising tool for many cell culture applications, but their microscopic analysis is limited. Flow cytometry on a single cell basis, which requires a gentle but also efficient dissociation of spheroids, could be an alternative analysis. Mono-culture and coculture spheroids consisting of human fibroblasts and human endothelial cells were generated by the liquid overlay technique and were dissociated using AccuMax as a dissociation agent combined with gentle mechanical forces. This study aimed to quantify the number of apoptotic and proliferative cells. We were able to dissociate spheroids of differing size, age, and cellular composition in a single-step dissociation protocol within 10 min. The number of single cells was higher than 95% and in most cases, the viability of the cells after dissociation was higher than 85%. Coculture spheroids exhibited a higher sensitivity as shown by lower viability, higher amount of cellular debris, and a higher amount of apoptotic cells. Considerable expression of the proliferation marker Ki67 could only be seen in 1-day-old spheroids but was already downregulated on Day 3. In summary, our dissociation protocol enabled a fast and gentle dissociation of spheroids for the subsequent flow cytometric analysis. The chosen cell type had a strong influence on cell viability and apoptosis. Initially high rates of proliferative cells decreased rapidly and reached values of healthy tissue 3 days after generation of the spheroids. In conclusion, the flow cytometry of dissociated spheroids could be a promising analytical tool, which could be ideally combined with microscopic techniques.

Neurospheres: a potential in vitro model for the study of central nervous system disorders.

Neurogenesis was believed to end after the period of embryonic development. However, the possibility of obtaining an expressive number of cells with functional neuronal characteristics implied a great advance in experimental research. New techniques have emerged to demonstrate that the birth of new neurons continues to occur in the adult brain. Two main rich sources of these cells are the subventricular zone (SVZ) and the subgranular zone of the hippocampal dentate gyrus (SGZ) where adult neural stem cells (aNSCs) have the ability to proliferate and differentiate into mature cell lines. The cultivation of neurospheres is a method to isolate, maintain and expand neural stem cells (NSCs) and has been used extensively by several research groups to analyze the biological properties of NSCs and their potential use in injured brains from animal models. Throughout this review, we highlight the areas where this type of cell culture has been applied and the advantages and limitations of using this model in experimental studies for the neurological clinical scenario.

Core-shell hydrogel microcapsules enable formation of human pluripotent stem cell spheroids and their cultivation in a stirred bioreactor.

Cellular therapies based on human pluripotent stem cells (hPSCs) offer considerable promise for treating numerous diseases including diabetes and end stage liver failure. Stem cell spheroids may be cultured in stirred bioreactors to scale up cell production to cell numbers relevant for use in humans. Despite significant progress in bioreactor culture of stem cells, areas for improvement remain. In this study, we demonstrate that microfluidic encapsulation of hPSCs and formation of spheroids. A co-axial droplet microfluidic device was used to fabricate 400 µm diameter capsules with a poly(ethylene glycol) hydrogel shell and an aqueous core. Spheroid formation was demonstrated for three hPSC lines to highlight broad utility of this encapsulation technology. In-capsule differentiation of stem cell spheroids into pancreatic ß-cells in suspension culture was also demonstrated.

Understanding the Biological Basis of Glioblastoma Patient-derived Spheroids.

BACKGROUND/AIM: Resistance to glioblastoma (GB) therapy is attributed to the presence of glioblastoma stem cells (GSC). Here, we defined the behavior of GSC as it pertains to proliferation, migration, and angiogenesis. MATERIALS AND METHODS: Human-derived GSC were isolated and cultured from GB patient tumors. Xenograft GSC were extracted from the xenograft tumors, and spheroids were created and compared with human GSC spheroids by flow cytometry, migration, proliferation, and angiogenesis assays. Oct3/4 and Sox2, GFAP, and Ku80 expression was assessed by immunoanalysis. RESULTS: The xenograft model showed the formation of two different tumors with distinct characteristics. Tumors formed at 2 weeks were less aggressive with well-defined margins, whereas tumors formed in 5 months were diffuse and aggressive. Expression of Oct3/4 and Sox2 was positive in both human and xenograft GSC. Positive Ku80 expression in xenograft GSC confirmed their human origin. Human and xenograft GSC migrated vigorously in collagen and Matrigel, respectively. Xenograft GSC displayed a higher rate of migration and invasion than human GSC. CONCLUSION: Human GSC were more aggressive in growth and proliferation than xenograft GSC, while xenograft GSC had increased invasion and migration compared to human GSC. A simple in vitro spheroid system for GSC provides a superior platform for the development of precision medicine in the treatment of GB.

Understanding the influence of substrate when growing tumorspheres.

BACKGROUND: Cancer stem cells are important for the development of many solid tumors. These cells receive promoting and inhibitory signals that depend on the nature of their environment (their niche) and determine cell dynamics. Mechanical stresses are crucial to the initiation and interpretation of these signals. METHODS: A two-population mathematical model of tumorsphere growth is used to interpret the results of a series of experiments recently carried out in Tianjin, China, and extract information about the intraspecific and interspecific interactions between cancer stem cell and differentiated cancer cell populations. RESULTS: The model allows us to reconstruct the time evolution of the cancer stem cell fraction, which was not directly measured. We find that, in the presence of stem cell growth factors, the interspecific cooperation between cancer stem cells and differentiated cancer cells induces a positive feedback loop that determines growth, independently of substrate hardness. In a frustrated attempt to reconstitute the stem cell niche, the number of cancer stem cells increases continuously with a reproduction rate that is enhanced by a hard substrate. For growth on soft agar, intraspecific interactions are always inhibitory, but on hard agar the interactions between stem cells are collaborative while those between differentiated cells are strongly inhibitory. Evidence also suggests that a hard substrate brings about a large fraction of asymmetric stem cell divisions. In the absence of stem cell growth factors, the barrier to differentiation is broken and overall growth is faster, even if the stem cell number is conserved. CONCLUSIONS: Our interpretation of the experimental results validates the centrality of the concept of stem cell niche when tumor growth is fueled by cancer stem cells. Niche memory is found to be responsible for the characteristic population dynamics observed in tumorspheres. The model also shows why substratum stiffness has a deep influence on the behavior of cancer stem cells, stiffer substrates leading to a larger proportion of asymmetric doublings. A specific condition for the growth of the cancer stem cell number is also obtained.