Deep learning unlocks label-free viability assessment of cancer spheroids in microfluidics.

Despite recent advances in cancer treatment, refining therapeutic agents remains a critical task for oncologists. Precise evaluation of drug effectiveness necessitates the use of 3D cell culture instead of traditional 2D monolayers. Microfluidic platforms have enabled high-throughput drug screening with 3D models, but current viability assays for 3D cancer spheroids have limitations in reliability and cytotoxicity. This study introduces a deep learning model for non-destructive, label-free viability estimation based on phase-contrast images, providing a cost-effective, high-throughput solution for continuous spheroid monitoring in microfluidics. Microfluidic technology facilitated the creation of a high-throughput cancer spheroid platform with approximately 12 000 spheroids per chip for drug screening. Validation involved tests with eight conventional chemotherapeutic drugs, revealing a strong correlation between viability assessed via LIVE/DEAD staining and phase-contrast morphology. Extending the model's application to novel compounds and cell lines not in the training dataset yielded promising results, implying the potential for a universal viability estimation model. Experiments with an alternative microscopy setup supported the model's transferability across different laboratories. Using this method, we also tracked the dynamic changes in spheroid viability during the course of drug administration. In summary, this research integrates a robust platform with high-throughput microfluidic cancer spheroid assays and deep learning-based viability estimation, with broad applicability to various cell lines, compounds, and research settings.

A cost-effective three-dimensional culture platform functionally mimics the adipose tissue microenvironment surrounding the kidney.

There is an increasing interest in studying the crosstalk between tumor-associated adipose tissue and tumor progression. In proximity to the primary site of kidney tumors, perinephric adipose tissue has direct contact with cancer cells when kidney cancer becomes invasive. To mimic the perinephric adipose tissue microenvironment, we applied the liquid overlay-based technique, which cost-effectively generated functional adipocyte spheroids using mesenchymal stem cells isolated from human perinephric adipose tissue. Thereafter, we co-cultured adipocyte spheroids with unpolarized macrophages and discovered an M2 phenotype skew in macrophages. Moreover, we discovered that, in the presence of adipocyte spheroids, M2 macrophages exhibited stronger invasive capacity than M1 macrophages. We further showed that the perinephric adipose tissue sampled from metastatic kidney cancer exhibited high expression of M2 macrophages. In conclusion, the liquid overlay-based technique can generate a novel three-dimensional platform enabling investigation of the interactions of adipocytes and other types of cells in a tumor microenvironment.

Agent-based model of multicellular tumor spheroid evolution including cell metabolism.

Computational models aiming at the spatio-temporal description of cancer evolution are a suitable framework for testing biological hypotheses from experimental data, and generating new ones. Building on our recent work (J. Theor. Biol. 389, 146 (2016)) we develop a 3D agent-based model, capable of tracking hundreds of thousands of interacting cells, over time scales ranging from seconds to years. Cell dynamics is driven by a Monte Carlo solver, incorporating partial differential equations to describe chemical pathways and the activation/repression of "genes", leading to the up- or down-regulation of specific cell markers. Each cell-agent of different kind (stem, cancer, stromal etc.) runs through its cycle, undergoes division, can exit to a dormant, senescent, necrotic state, or apoptosis, according to the inputs from its systemic network. The basic network at this stage describes glucose/oxygen/ATP cycling, and can be readily extended to cancer-cell specific markers. Eventual accumulation of chemical/radiation damage to each cell's DNA is described by a Markov chain of internal states, and by a damage-repair network, whose evolution is linked to the cell systemic network. Aimed at a direct comparison with experiments of tumorsphere growth from stem cells, the present model will allow to quantitatively study the role of transcription factors involved in the reprogramming and variable radio-resistance of simulated cancer-stem cells, evolving in a realistic computer simulation of a growing multicellular tumorsphere.

Tumor spheroid-on-a-chip: a standardized microfluidic culture platform for investigating tumor angiogenesis.

The field of microfluidics-based three-dimensional (3D) cell culture system is rapidly progressing from academic proof-of-concept studies to valid solutions to real-world problems. Polydimethylsiloxane (PDMS)-based platform has been widely adopted as in vitro platforms for mimicking tumor microenvironment. However, PDMS has not been welcomed as a standardized commercial application for preclinical screening due to inherent material limitations that make it difficult to scale-up production. Here, we present an injection-molded plastic array 3D spheroid culture platform (Sphero-IMPACT). The platform is made of polystyrene (PS) in a standardized 96-well plate format with a user-friendly interface. This interface describes a simpler design that incorporates a tapered hole in the center of the rail to pattern a large spheroid with 3D extracellular matrix and various cell types. This hole is designed to accommodate standard pipette tip for automated system. The platform that mediate open microfluidics allows implement spontaneous fluid patterning with high repeatability from the end user. To demonstrate versatile use of the platform, we developed 3D perfusable blood vessel network and tumor spheroid assays. In addition, we established a tumor spheroid induced angiogenesis model that can be applicable for drug screening. Sphero-IMPACT has the potential to provide a robust and reproducible in vitro assay related to vascularized cancer research. This easy-to-use, ready-to-use platform can be translated into an enhanced preclinical model that faithfully reflects the complex tumor microenvironment.

Organotypic 3D HepaRG Liver Model for Assessment of Drug-Induced Cholestasis.

Cholestasis remains a major challenge in drug-induced liver injury, and therefore warrants identification of chemical entities that may lead to cholestasis. Recent advances in cell culture methods enable 3D spheroid models to remain viable for much longer periods of time than conventional sandwich cultures of primary human hepatocytes while maintaining native tissue-like functionality, such as drug metabolism activity, receptor signaling functionality, and physiological relevance. These spheroid models enable us to study repeated exposure effects associated with chemicals and their metabolites that may ultimately progress to cholestasis and liver injury. HepaRG cells cultured as spheroids are viable for more than 4 weeks with cytochrome P450 enzymatic activities comparable to ranges observed in freshly isolated/cryopreserved suspensions of primary human hepatocytes. HepaRG spheroids form bile canalicular structures with potential application as a model to study biliary excretion processes and intrahepatic obstruction of bile flow, leading to hepatocellular damage and death. In this chapter, we describe methods to culture 3D spheroids of HepaRG cells with extensive bile canalicular structures/networks, image transport of bile acid (cholyl-lysyl-fluorescein) to the bile canaliculi, and measure cholestatic drug-induced cytotoxicity.

Possible repurposing of pyrvinium pamoate for the treatment of mesothelioma: A pre-clinical assessment.

Malignant mesothelioma (MM) is a very aggressive asbestos-related cancer, whose incidence is increasing worldwide. Unfortunately, no effective therapies are currently available and the prognosis is extremely poor. Recently, the anti-helminthic drug pyrvinium pamoate has attracted a strong interest for its anti-cancer activity, which has been demonstrated in many cancer models. Considering the previously established inhibitory effect of pyrvinium pamoate on the Wnt/ß-catenin pathway and given the important role of this pathway in MM, we investigated the potential anti-tumor activity of this drug in MM cell lines. We observed that pyrvinium pamoate significantly impairs MM cell proliferation, cloning efficiency, migration, and tumor spheroid formation. At the molecular level, our data show that pyrvinium pamoate down-regulates the expression of ß-catenin and Wnt-regulates genes. Overall, our study suggests that the repurposing of pyrvinium pamoate for MM treatment could represent a new promising therapeutic approach.

3D pulmospheres serve as a personalized and predictive multicellular model for assessment of antifibrotic drugs.

Idiopathic pulmonary fibrosis (IPF) is a fatal progressive fibrotic lung disease characterized by the presence of invasive myofibroblasts in the lung. Currently, there are only two FDA-approved drugs (pirfenidone and nintedanib) for the treatment of IPF. There are no defined criteria to guide specific drug therapy. New methodologies are needed not only to predict personalized drug therapy, but also to screen novel molecules that are on the horizon for treatment of IPF. We have developed a model system that exploits the invasive phenotype of IPF lung tissue. This ex vivo 3D model uses lung tissue from patients to develop pulmospheres. Pulmospheres are 3D spheroids composed of cells derived exclusively from primary lung biopsies and inclusive of lung cell types reflective of those in situ, in the patient. We tested the pulmospheres of 20 subjects with IPF and 9 control subjects to evaluate the responsiveness of individual patients to antifibrotic drugs. Clinical parameters and outcomes were also followed in the same patients. Our results suggest that pulmospheres simulate the microenvironment in the lung and serve as a personalized and predictive model for assessing responsiveness to antifibrotic drugs in patients with IPF.

Spheroid Formation of Hepatocarcinoma Cells in Microwells: Experiments and Monte Carlo Simulations.

The formation of spherical aggregates during the growth of cell population has long been observed under various conditions. We observed the formation of such aggregates during proliferation of Huh-7.5 cells, a human hepatocarcinoma cell line, in a microfabricated low-adhesion microwell system (SpheroFilm; formed of mass-producible silicone elastomer) on the length scales up to 500 µm. The cell proliferation was also tracked with immunofluorescence staining of F-actin and cell proliferation marker Ki-67. Meanwhile, our complementary 3D Monte Carlo simulations, taking cell diffusion and division, cell-cell and cell-scaffold adhesion, and gravity into account, illustrate the role of these factors in the formation of spheroids. Taken together, our experimental and simulation results provide an integrative view of the process of spheroid formation for Huh-7.5 cells.

Spheroid-Formation (Colonosphere) Assay for in Vitro Assessment and Expansion of Stem Cells in Colon Cancer.

Colorectal cancers (CRCs) form a disorganized hierarchy of heterogeneous cell populations on which current chemotherapy regimens fail to exert their distinctive cytotoxicity. A small sub-population of poorly differentiated cancer stem-like cells (CSCs), also known as cancer initiating cells, may exhibit embryonic and/or adult stem-cell gene expression signatures. Self-renewal and survival signals are also dominant over differentiation in CSCs. However, inducers of differentiation exclusive to CSC may affect cellular pathways required for the formation and progression of a tumor, which are not utilized in normal adult stem-cells. Nevertheless, assays for targeting CSCs have been hindered by expanding and maintaining rare CSCs in vitro. However, CRC-CSCs are able to form floating spheroids (known as colonospheres) 3-dimentinionally (3D) in a serum-free defined medium. Therefore, great efforts have been paid to improve colonosphere forming assay as a preclinical model to study tumor biology and to conduct drug screening in cancer research. The 3D-colonosphere culture model may also represent in vivo conditions for the spontaneous aggregation of cancer cells in spheroids. This protocol describes the development of an enrichment/culture assay using CRC-CSCs to facilitate colorectal cancer research through immunofluorescence staining of colonospheres. We have developed colonospheres from HCT116 CRC cell line to compare and link CRC-CSC markers to the NANOG expression level using an immunofluorescence assay. Our data also show that the immunostaining assay of colonosphere is a useful method to explore the role and dynamics of CRC-CSCs division between self-renewal and cell lineage differentiation of cancer cells. In principle, this method is applicable to a variety of primary cells and cell lines of epithelial origin. Furthermore, this protocol may also allow screening of libraries of compounds to identify bona fide CRC-CSC differentiation inducers.

A simple, low-cost staining method for rapid-throughput analysis of tumor spheroids.

Tumor spheroids are becoming an important tool for the investigation of cancer stem cell (CSC) function in tumors; thus, low-cost and high-throughput methods for drug screening of tumor spheroids are needed. Using neurospheres as non-adherent three-dimensional (3-D) cultures, we developed a simple, low-cost acridine orange (AO)-based method that allows for rapid analysis of live neurospheres by fluorescence microscopy in a 96-well format. This assay measures the cross-section area of a spheroid, which corresponds to cell viability. Our novel method allows rapid screening of a panel of anti-proliferative drugs to assess inhibitory effects on the growth of cancer stem cells in 3-D cultures.

Biomimicry 3D gastrointestinal spheroid platform for the assessment of toxicity and inflammatory effects of zinc oxide nanoparticles.

Our current mechanistic understanding on the effects of engineered nanoparticles (NPs) on cellular physiology is derived mainly from 2D cell culture studies. However, conventional monolayer cell culture may not accurately model the mass transfer gradient that is expected in 3D tissue physiology and thus may lead to artifactual experimental conclusions. Herein, using a micropatterned agarose hydrogel platform, the effects of ZnO NPs (25 nm) on 3D colon cell spheroids of well-defined sizes are examined. The findings show that cell dimensionality plays a critical role in governing the spatiotemporal cellular outcomes like inflammatory response and cytotoxicity in response to ZnO NPs treatment. More importantly, ZnO NPs can induce different modes of cell death in 2D and 3D cell culture systems. Interestingly, the outer few layers of cells in 3D model could only protect the inner core of cells for a limited time and periodically slough off from the spheroids surface. These findings suggest that toxicological conclusions made from 2D cell models might overestimate the toxicity of ZnO NPs. This 3D cell spheroid model can serve as a reproducible platform to better reflect the actual cell response to NPs and to study a more realistic mechanism of nanoparticle-induced toxicity.

A bioengineered 3D ovarian cancer model for the assessment of peptidase-mediated enhancement of spheroid growth and intraperitoneal spread.

Cancer-associated proteases promote peritoneal dissemination and chemoresistance in malignant progression. In this study, kallikrein-related peptidases 4, 5, 6, and 7 (KLK4-7)-cotransfected OV-MZ-6 ovarian cancer cells were embedded in a bioengineered three-dimensional (3D) microenvironment that contains RGD motifs for integrin engagement to analyze their spheroid growth and survival after chemotreatment. KLK4-7-cotransfected cells formed larger spheroids and proliferated more than controls in 3D, particularly within RGD-functionalized matrices, which was reduced upon integrin inhibition. In contrast, KLK4-7-expressing cell monolayers proliferated less than controls, emphasizing the relevance of the 3D microenvironment and integrin engagement. In a spheroid-based animal model, KLK4-7-overexpression induced tumor growth after 4 weeks and intraperitoneal spread after 8 weeks. Upon paclitaxel administration, KLK4-7-expressing tumors declined in size by 91% (controls: 87%) and showed 90% less metastatic outgrowth (controls: 33%, P < 0.001). KLK4-7-expressing spheroids showed 53% survival upon paclitaxel treatment (controls: 51%), accompanied by enhanced chemoresistance-related factors, and their survival was further reduced by combination treatment of paclitaxel with KLK4/5/7 (22%, P = 0.007) or MAPK (6%, P = 0.006) inhibition. The concomitant presence of KLK4-7 in ovarian cancer cells together with integrin activation drives spheroid formation and proliferation. Combinatorial approaches of paclitaxel and KLK/MAPK inhibition may be more efficient for late-stage disease than chemotherapeutics alone as these inhibitory regimens reduced cancer spheroid growth to a greater extent than paclitaxel alone.

Integrating intracellular dynamics using CompuCell3D and Bionetsolver: applications to multiscale modelling of cancer cell growth and invasion.

In this paper we present a multiscale, individual-based simulation environment that integrates CompuCell3D for lattice-based modelling on the cellular level and Bionetsolver for intracellular modelling. CompuCell3D or CC3D provides an implementation of the lattice-based Cellular Potts Model or CPM (also known as the Glazier-Graner-Hogeweg or GGH model) and a Monte Carlo method based on the metropolis algorithm for system evolution. The integration of CC3D for cellular systems with Bionetsolver for subcellular systems enables us to develop a multiscale mathematical model and to study the evolution of cell behaviour due to the dynamics inside of the cells, capturing aspects of cell behaviour and interaction that is not possible using continuum approaches. We then apply this multiscale modelling technique to a model of cancer growth and invasion, based on a previously published model of Ramis-Conde et al. (2008) where individual cell behaviour is driven by a molecular network describing the dynamics of E-cadherin and ß-catenin. In this model, which we refer to as the centre-based model, an alternative individual-based modelling technique was used, namely, a lattice-free approach. In many respects, the GGH or CPM methodology and the approach of the centre-based model have the same overall goal, that is to mimic behaviours and interactions of biological cells. Although the mathematical foundations and computational implementations of the two approaches are very different, the results of the presented simulations are compatible with each other, suggesting that by using individual-based approaches we can formulate a natural way of describing complex multi-cell, multiscale models. The ability to easily reproduce results of one modelling approach using an alternative approach is also essential from a model cross-validation standpoint and also helps to identify any modelling artefacts specific to a given computational approach.

Assessment of drug induced developmental toxicity using human embryonic stem cells.

Embryonic stem (ES) cells are unique as they have the potential to be generated in large numbers and the ability to differentiate into the three germ layers via embryoid body (EB) formation. This property could be utilized as an index to study initial mammalian development. We have investigated the utility of a comprehensively characterized human ES (hES) cell line (ReliCellhES1) for testing the embryotoxic effects of compounds using cytotoxicity assays. Further, we performed real time gene expression analysis to check the alterations in germ layer markers expression upon drug treatment. The results show that assays using hES cells could serve as a reliable, sensitive and robust method to assess embryotoxic potential of compounds. They also provide a proof of concept that hES cells can be used as an in vitro model to demonstrate developmental toxicity, and to examine the germ layer-specific effects on differentiating EBs.

Dynamic assessment of angiogenesis in renal cell carcinoma spheroids by intravital microscopy.

PURPOSE: Renal cell cancer represents a suitable tumor model for in vivo observation of neo-angiogenesis. We used intravital microscopy and the well established dorsal skin fold chamber model to characterize neo-angiogenesis in freely implanted renal cell cancer spheroids. MATERIAL AND METHODS: Tumor spheroids were implanted into dorsal skin fold chambers of 8 nude mice. At days 3, 6, 10 and 14 after implantation the newly vascularized spheroid area, density of perfused microvessels in the spheroid versus the periphery, capillary center erythrocyte velocity and capillary diameter were recorded by intravital microscopy. Video images were analyzed by a computer assisted image analysis device. After the experiments the chambers were analyzed morphologically. RESULTS: The model enabled quantitative analysis of microcirculation and angiogenesis in the renal cell cancer spheroids during 14 days of observation. Mean spheroid center perfused microvessel density +/- SEM increased from 3 +/- 2 to 269 +/- 21 cm.-1 on days 3 to 10 and subsequently decreased to 189 +/- 38 cm.-1 on day 14. Spheroid periphery perfused microvessel density was significantly higher throughout the experiments, attaining a mean maximum of 522 +/- 34 cm. on day 14. Mean capillary diameter decreased continuously from 14.2 +/- 0.9 to 8.4 +/- 0.4 microm. on days 3 to 14. In contrast, mean capillary center erythrocyte velocity significantly increased during 14 days of observation from 0.09 + 0.02 mm. per second on day 3 to 0.24 +/- 0.08 mm. per second on day 14. Histological analysis after 14 days revealed the spheroids as cell clusters in the upper layers of the dorsal skin fold chamber. CONCLUSIONS: The model is suitable for the analysis of renal cell cancer angiogenesis. Although it is heterotopic, angiogenesis in renal cell cancer spheroids mimics important characteristics of human renal cell cancer.

Modeling tumor growth and irradiation response in vitro--a combination of high-performance computing and web-based technologies including VRML visualization.

A simplified three-dimensional Monte Carlo simulation model of in vitro tumor growth and response to fractionated radiotherapeutic schemes is presented in this paper. The paper aims at both the optimization of radiotherapy and the provision of insight into the biological mechanisms involved in tumor development. The basics of the modeling philosophy of Duechting have been adopted and substantially extended. The main processes taken into account by the model are the transitions between the cell cycle phases, the diffusion of oxygen and glucose, and the cell survival probabilities following irradiation. Specific algorithms satisfactorily describing tumor expansion and shrinkage have been applied, whereas a novel approach to the modeling of the tumor response to irradiation has been proposed and implemented. High-performance computing systems in conjunction with Web technologies have coped with the particularly high computer memory and processing demands. A visualization system based on the MATLAB software package and the virtual-reality modeling language has been employed. Its utilization has led to a spectacular representation of both the external surface and the internal structure of the developing tumor. The simulation model has been applied to the special case of small cell lung carcinoma in vitro irradiated according to both the standard and accelerated fractionation schemes. A good qualitative agreement with laboratory experience has been observed in all cases. Accordingly, the hypothesis that advanced simulation models for the in silico testing of tumor irradiation schemes could substantially enhance the radiotherapy optimization process is further strengthened. Currently, our group is investigating extensions of the presented algorithms so that efficient descriptions of the corresponding clinical (in vivo) cases are achieved.