Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system, a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

Simple droplet microfluidics platform for drug screening on cancer spheroids.

3D in vitro biological systems are progressively replacing 2D systems to increase the physiological relevance of cellular studies. Microfluidics-based approaches can be powerful tools towards such biomimetic systems, but often require high-end complicated and expensive processes and equipment for microfabrication. Herein, a drug screening platform is proposed, minimizing technicality and manufacturing steps. It provides an alternate way of spheroid generation in droplets in tubes. Droplet microfluidics then elicit multiple droplets merging events at programmable times, to submit sequentially the spheroids to chemotherapy and to reagents for cytotoxicity screening. After a comprehensive study of tumorogenesis within the droplets, the system is validated for drug screening (IC50) with chemotherapies in cancer cell lines as well as cells from a patient-derived-xenografts (PDX). As compared to microtiter plates methods, our system reduces the initial number of cells up to 10 times and opens new avenues towards primary tumors drug screening approaches.

A tumour-spheroid manufacturing and cryopreservation process that yields a highly reproducible product ready for direct use in drug screening assays.

If it were possible to purchase tumour-spheroids as a standardised product, ready for direct use in assays, this may contribute to greater research reproducibility, potentially reducing costs and accelerating outcomes. Herein, we describe a workflow where uniformly sized cancer tumour-spheroids are mass-produced using microwell culture, cryopreserved with high viability, and then cultured in neutral buoyancy media for drug testing. C4-2B prostate cancer or MCF-7 breast cancer cells amalgamated into uniform tumour-spheroids after 48 h of culture. Tumour-spheroids formed from 100 cells each tolerated the cryopreservation process marginally better than tumour-spheroids formed from 200 or 400 cells. Post-thaw, tumour-spheroid metabolic activity was significantly reduced, suggesting mitochondrial damage. Metabolic function was rescued by thawing the tumour-spheroids into medium supplemented with 10 µM N-Acetyl-l-cysteine (NAC). Following thaw, the neutral buoyancy media, Happy Cell ASM, was used to maintain tumour-spheroids as discrete tissues during drug testing. Fresh and cryopreserved C4-2B or MCF-7 tumour-spheroids responded similarly to titrations of Docetaxel. This protocol will contribute to a future where tumour-spheroids may be available for purchase as reliable and reproducible products, allowing laboratories to efficiently replicate and build on published research, in many cases, making tumour-spheroids simply another cell culture reagent.

Polyglycerol-Based Biomedical Matrix for Immunomagnetic Circulating Tumor Cell Isolation and Their Expansion into Tumor Spheroids for Drug Screening.

Circulating tumor cells (CTCs) are established as distinct cancer biomarkers for diagnosis, as preclinical models, and therapeutic targets. Their use as preclinical models is limited owing to low purity after isolation and the lack of effective techniques to create 3D cultures that accurately mimic in vivo conditions. Herein, a two-component system for detecting, isolating, and expanding CTCs to generate multicellular tumor spheroids that mimic the physiology and microenvironment of the diseased organ is proposed. First, an antifouling biointerface on magnetic beads is fabricated by adding a bioinert polymer layer and conjugation of biospecific ligands to isolate cancer cells, dramatically enhancing the selectivity and purity of the isolated cancer cells. Next, the isolated cells are encapsulated into self-degradable hydrogels synthesized using a thiol-click approach. The hydrogels are mechanochemically tuned to enable tumor spheroid growth to a size greater than 300 µm and to further release the grown spheroids while retaining their tumor-like characteristics. In addition, drug treatment highlights the need for 3D culture environments rather than conventional 2D culture. The designed biomedical matrix shows potential as a universal method to ensure mimicry of in vivo tumor characteristics in individual patients and to improve the predictability of preclinical screening of personalized therapeutics.

Spheroid on-demand printing and drug screening of endothelialized hepatocellular carcinoma model at different stages.

Hepatocellular carcinoma (HCC) poses a significant threat to human health and medical care. Its dynamic microenvironment and stages of development will influence the treatment strategies in clinics. Reconstructing tumor-microvascular interactions in different stages of the microenvironment is an urgent need forin vitrotumor pathology research and drug screening. However, the absence of tumor aggregates with paracancerous microvascular and staged tumor-endothelium interactions leads to bias in the antitumor drug responses. Herein, a spheroid-on-demand manipulation strategy was developed to construct staged endothelialized HCC models for drug screening. Pre-assembled HepG2 spheroids were directly printed by alternating viscous and inertial force jetting with high cell viability and integrity. A semi-open microfluidic chip was also designed to form a microvascular connections with high density, narrow diameter, and curved morphologies. According to the single or multiple lesions in stages Ⅰ or Ⅰ HCC, endothelialized HCC models from micrometer to millimeter scale with dense tumor cell aggregation and paracancerous endothelial distribution were successively constructed. A migrating stage Ⅰ HCC model was further constructed under TGF-ßtreatment, where the spheroids exhibited a more mesenchymal phenotype with a loose cell connection and spheroid dispersion. Finally, the stage ⅠHCC model showed stronger drug resistance compared to the stage Ⅰ model, while the stage III showed a more rapid response. The corresponding work provides a widely applicable method for the reproduction of tumor-microvascular interactions at different stages and holds great promise for the study of tumor migration, tumor-stromal cell interactions, and the development of anti-tumor therapeutic strategies.

Scaffold-based spheroid models of glioblastoma multiforme and its use in drug screening.

Among several types of brain cancers, glioblastoma multiforme (GBM) is a terminal and aggressive disease with a median survival of 15 months despite the most intensive surgery and chemotherapy. Preclinical models that accurately reproduce the tumor microenvironment are vital for developing new therapeutic alternatives. Understanding the complicated interactions between cells and their surroundings is essential to comprehend the tumor's microenvironment, however the monolayer cell culture approach falls short. Numerous approaches are used to develop GBM cells into tumor spheroids, while scaffold-based spheroids provides the opportunity to investigate the synergies between cells as well as cells and the matrix. This review summarizes the development of various scaffold-based GBM spheroid models and the prospective for their use as drug testing systems.

Multi-Inlet Spheroid Generator for High-Throughput Combinatorial Drug Screening Based on the Tumor Microenvironment.

Tumor spheroids are powerful tools for drug screening and understanding tumor physiology. Among spheroid formation methods, the hanging drop method is considered most suitable for high-throughput screening (HTS) of anticancer drugs because it does not require surface treatment. However, it still needs to increase the liquid-holding capacity because hanging drops often fall due to the increased pressure caused by the addition of drugs, cells, etc. Here, we report a multi-inlet spheroid generator (MSG) enabling the stable addition of liquid-containing drugs or cells into a spheroid through its side inlet. The MSG was able to load additional solutions through the side inlet without increasing the force applied to the hanging drop. The volume of the additional liquid was easily controlled by varying the diameter of the side inlet. Furthermore, the sequences of the solution injections were manipulated using multiple side inlets. The feasibility of the MSG in clinical application was demonstrated by testing the efficacy of drugs in patient-derived cancer (PDC) cells and controlling the stromal cell ratio in the tumor microenvironment (TME) containing spheroids. Our results suggest that the MSG is a versatile platform for HTS of anticancer drugs and recapitulating the TME.

Cell spheroids culture array with modifiable chemical gradients.

Cancer cell spheroids have been shown to mimic in vivo tumour microenvironment and are therefore suitable for in vitro drug screening. Microfluidic technology can provide conveniences for spheroid assays such as high-throughput, simplifying manual operation and saving reagent. Here, we propose a concentration gradient generator based on microfluidic technology for cell spheroid culture and assay. The chip consists of upper microchannels and lower microwells. After partitioning HepG2 suspension into the microwells with concave and non-adhesive bottoms, spheroids can spontaneously form. By controlling the fluid replacement and flow in microchannels, the doxorubicin solution is diluted automatically into a series of concentration gradients, which spanning more than one order of magnitude. And then the effect of doxorubicin on spheroids is measured in situ by fluorescent staining. This chip provides a very promising approach to achieve the high-throughput and standardized anti-cancer drug screening in future.

Multiplexed Viability Assays for High-Throughput Screening of Spheroids of Multiple Sizes.

High-throughput (HT) drug screening is in high demand for successful drug discovery and personalized medicine. Spheroids act as a promising preclinical model for HT drug screening, which may decrease drug failures in clinical trials. Numerous spheroid-forming technological platforms are currently under development, which include synchronous, jumbo-sized, hanging drop, rotary, and nonadherent surface spheroid growth. Initial cell seeding concentration and time of culture play a vital role for spheroids to mimic the extracellular microenvironment of natural tissue, especially for HT preclinical evaluation. Hence microfluidic platforms become a potential technology to provide a confined space for the oxygen and nutrient gradients within the tissues while controlling the cell count and spheroid size in an HT manner. We describe here a microfluidic platform capable of generating spheroids of multiple sizes in a controlled manner with a predefined cell concentration for HT drug screening. Ovarian cancer spheroids grown on this microfluidic platform were evaluated for viability using a confocal microscope and flow cytometer. In addition, screening of the HT chemotherapeutic drug carboplatin was carried out on-chip to evaluate the impact of spheroid size on drug toxicity. This chapter summarizes a detailed protocol on microfluidic platform fabrication for spheroid growth, on-chip multi-sized spheroid analysis, and chemotherapeutic drug screening.

The Variety of 3D Breast Cancer Models for the Study of Tumor Physiology and Drug Screening.

Breast cancer is the most common cancer in women and responsible for multiple deaths worldwide. 3D cancer models enable a better representation of tumor physiology than the conventional 2D cultures. This review summarizes the important components of physiologically relevant 3D models and describes the spectrum of 3D breast cancer models, e.g., spheroids, organoids, breast cancer on a chip and bioprinted tissues. The generation of spheroids is relatively standardized and easy to perform. Microfluidic systems allow control over the environment and the inclusion of sensors and can be combined with spheroids or bioprinted models. The strength of bioprinting relies on the spatial control of the cells and the modulation of the extracellular matrix. Except for the predominant use of breast cancer cell lines, the models differ in stromal cell composition, matrices and fluid flow. Organoids are most appropriate for personalized treatment, but all technologies can mimic most aspects of breast cancer physiology. Fetal bovine serum as a culture supplement and Matrigel as a scaffold limit the reproducibility and standardization of the listed 3D models. The integration of adipocytes is needed because they possess an important role in breast cancer.

Microwell-based flow culture increases viability and restores drug response in prostate cancer spheroids.

3D cancer spheroids represent a highly promising model for study of cancer progression and therapeutic development. Wide-scale adoption of cancer spheroids, however, remains a challenge due to the lack of control over hypoxic gradients that may cloud the assessment of cell morphology and drug response. Here, we present a Microwell Flow Device (MFD) that generates in-well laminar flow around 3D tissues via repetitive tissue sedimentation. Using a prostate cancer cell line, we demonstrate the spheroids in the MFD exhibit improved cell growth, reduced necrotic core formation, enhanced structural integrity, and downregulated expression of cell stress genes. The flow-cultured spheroids also exhibit an improved sensitivity to chemotherapy with greater transcriptional response. These results demonstrate how fluidic stimuli reveal the cellular phenotype previously masked by severe necrosis. Our platform advances 3D cellular models and enables study into hypoxia modulation, cancer metabolism, and drug screening within pathophysiological conditions.

Convenient tumor 3D spheroid arrays manufacturing via acoustic excited bubbles for in situ drug screening.

The quick and convenient fabrication of in vitro tumor spheroids models has been pursued for clinical drug discovery and personalized therapy. Here, uniform three-dimensional (3D) tumor spheroids are quickly constructed by acoustically excited bubble arrays in a microfluidic chip and performed drug response testing in situ. In detail, bubble oscillation excited by acoustic waves induces second radiation force, resulting in the cells rotating and aggregating into tumor spheroids, which obtain controllable sizes ranging from 30 to 300 µm. These spherical tumor models are located in microfluidic networks, where drug solutions with gradient concentrations are generated from 0 to 18 mg mL-1, so that the cell spheroids response to drugs can be monitored conveniently and efficiently. This one-step tumor spheroids manufacturing method significantly reduces the model construction time to less than 15 s and increases efficiency by eliminating additional transfer processes. These significant advantages of convenience and high-throughput manufacturing make the tumor models promising for use in tumor treatment and point-of-care diagnosis.

An Automated High-Throughput Screening (HTS) Spotter for 3D Tumor Spheroid Formation.

Three-dimensional (3D) culture platforms have been adopted in a high-throughput screening (HTS) system to mimic in vivo physiological microenvironments. The automated dispenser has been established commercially to enable spotting or distributing non-viscous or viscous biomaterials onto microplates. However, there are still challenges to the precise and accurate dispensation of cells embedded in hydrogels such as Alginate- and Matrigel-extracellular matrices. We developed and improved an automated contact-free dispensing machine, the ASFA SPOTTER (V5 and V6), which is compatible with 96- and 384-pillar/well plates and 330- and 532-micropillar/well chips for the support of 3D spheroid/organoid models using bioprinting techniques. This enables the distribution of non-viscous and viscous biosamples, including chemical drugs and cancer cells, for large-scale drug screening at high speed and small volumes (20 to 4000 nanoliters) with no damage to cells. The ASFA SPOTTER (V5 and V6) utilizes a contact-free method that minimizes cross-contamination for the dispensation of encapsulated tissue cells with highly viscous scaffolds (over 70%). In particular, the SPOTTER V6 does not require a washing process and offers the advantage of almost no dead volume (defined as additional required sample volume, including a pre-shot and flushing shot for dispensing). It can be successfully applied for the achievement of an organoid culture in automation, with rapid and easy operation, as well as miniaturization for high-throughput screening. In this study, we report the advantages of the ASFA SPOTTER, which distributes standard-sized cell spots with hydrogels onto a 384-pillar/well plate with a fast dispensing speed, small-scale volume, accuracy, and precision.

Gold nanostructure-integrated conductive microwell arrays for uniform cancer spheroid formation and electrochemical drug screening.

Cancer spheroids, which mimic distinct cell-to-cell and cell-extracellular matrix interactions of solid tumors in vitro, have emerged as a promising tumor model for drug screening. However, owing to the unique characteristics of spheroids composed of three-dimensionally densely-packed cells, the precise characterizations of cell viability and function with conventional colorimetric assays are challenging. Herein, we report gold nanostructure-integrated conductive microwell arrays (GONIMA) that enable both highly efficient uniform cancer spheroid formation and precise electrochemical detection of cell viability. A nanostructured gold on indium tin oxide (ITO) substrate facilitated the initial cell aggregation and further 3D cell growth, while the non-cytophilic polymer microwell arrays restricted the size and shape of the spheroids. As a result, approximately 150 human glioblastoma spheroids were formed on a chip area of 1.13 cm2 with an average diameter of 224 µm and a size variation of only 5% (±11.36 µm). The high uniformity of cancer spheroids contributed to the stability of electrical signals measuring cell viability. Using the fabricated GONIMA, the effects of a representative chemotherapeutic agent, hydroxyurea, on the glioblastoma spheroids were precisely monitored under conditions of varying drug concentrations (0-0.3 mg/mL) and incubation times (24-48 h). Therefore, we conclude that the newly developed platform is highly useful for rapid and precise in vitro drug screening, as well as for the pharmacokinetic analyses of specific drugs using 3D cellular cancer models.

Biofabrication of Modular Spheroids as Tumor-Scale Microenvironments for Drug Screening.

To streamline the drug discovery pipeline, there is a pressing need for preclinical models which replicate the complexity and scale of native tumors. While there have been advancements in the formation of microscale tumor units, these models are cell-line dependent, time-consuming and have not improved clinical trial success rates. In this study, two methods for generating 3D tumor microenvironments are compared, rapidly fabricated hydrogel microspheres and traditional cell-dense spheroids. These modules are then bioassembled into 3D printed thermoplastic scaffolds, using an automated biofabrication process, to form tumor-scale models. Modules are formed with SKOV3 and HFF cells as monocultures and cocultures, and the fabrication efficiency, cell architecture, and drug response profiles are characterized, both as single modules and as multimodular constructs. Cell-encapsulated Gel-MA microspheres are fabricated with high-reproducibility and dimensions necessary for automated tumor-scale bioassembly regardless of cell type, however, only cocultured spheroids form compact modules suitable for bioassembly. Chemosensitivity assays demonstrate the reduced potency of doxorubicin in coculture bioassembled constructs and a ≈five-fold increase in drug resistance of cocultured cells in 3D modules compared with 2D monolayers. This bioassembly system is efficient and tailorable so that a variety of relevant-sized tumor constructs could be developed to study tumorigenesis and modernize drug discovery.

Integrative In Situ Photodynamic Therapy-Induced Cell Death Measurement of 3D-Bioprinted MCF-7 Tumor Spheroids.

The development of new in vitro models that closely mimic the tumor microenvironment (TME) to evaluate the efficacy of anticancer drugs has received great attention. In this study, a three-dimensional (3D) bioprinted Michigan Cancer Foundation-7 (MCF-7) cancer spheroid-embedded hydrogel model was suggested for integrative in situ determination of the half-maximal inhibitory concentration (IC50) values of photosensitizers (PSs). The MCF-7 cell-laden alginate/gelatin hydrogel was printed for the fabrication of tumor spheroids. The hydrogel was used to mimic the extracellular matrix (ECM) surrounding the cancer cells in the TME. The fluorescence intensities corresponding to photodynamic therapy (PDT)-induced death of tumor spheroids probed by the laser showed a random distribution in the hydrogel, regardless of the focus of the laser and the vertical-axis direction in which the laser was passed. These results enabled integrative in situ measurement of all tumor spheroids probed by the laser without needing to separate the tumor spheroids in the hydrogel and measure them individually. When compared with two-dimensional (2D) monolayer cultures, very large IC50 values of the PSs, chlorin e6 (Ce6) and sulfonated tetraphenyl porphyrin (sTPP), were achieved in MCF-7 spheroid-embedded hydrogels mainly due to the drug resistance of the tumor spheroids. Additionally, the heterogenic PDT response of single MCF-7 cancer cells in a single tumor spheroid was observed through 3D imaging of irregular apoptosis in a single spheroid since single tumor spheroids showed a heterogenic PDT response. Furthermore, the laser-power-dependent IC50 values of PSs were obtained using the MCF-7 spheroid-embedded hydrogel model.

Integrated Acoustic Chip for Culturing 3D Cell Arrays.

Three-dimensional (3D) cell arrays provide an in vitro platform for clinical drug screening, but the bulky culture devices limit their application scenarios. Here, we demonstrate an integrated portable device that can realize contact-free construction of 3D cell spheroids. The interaction between the ultrasound generated by the portable device and the capillary results in periodic pressure nodes or anti-nodes, which lead to form a 3D cell array for cell culture. Such a 3D cell array pattern can be constructed in seconds and requires only 1 µL of cell samples. We further assessed the spheroids formed by the portable device and the impact of the acoustic field on spheroids and demonstrated the drug screening with assembled spheroids. More importantly, the integrated acoustic device can be further integrated with other components for more complex cell culture and all-round analysis. This portable and effective integrated device provides a new avenue for clinical biomedicine.

Flavone cirsimarin impairs cell proliferation, migration, and invasion in MCF-7 cells grown in 2D and 3D models.

The present study investigates the mechanisms underlying the in vitro antitumoral activity of cirsimarin (CIR 10 to 320 µM), a flavone extracted from the aerial parts of Scoparia dulcis L., on MCF-7 cells cultured in 2D and multicellular tumor spheroids (3D). CIR (from 40 µM) decreased cell viability in the resazurin assay and colony formation in the 2D model. In the same way, in the 3D model, CIR (from 40 µM) induced cell death (triple staining assay) and decreased spheroid integrity after 16 days with no induction of intracellular reactive species (CM-H2DCFDA). In 2D, CIR decreased the invasion (transwell) and horizontal migration (wound healing), while in 3D, CIR diminished cell migration (ECM® gel) and induced DNA damage (comet assay) possibly related to cell death. CIR mediated antitumoral effects in 3D spheroids by negative modulation of genes associated with cell proliferation (CCND1, CCNA2, CDK2, CDK4, and TNF) and death (BCL-XL, BAX, CASP9, and BIRC5). BIRC5 and CDKs inhibitors have been proposed as versatile anticancer drugs, which makes our results quite interesting. TNF negative modulation may also be related to the downregulation of MMP9 and MMP11 and anti-migration/invasion of MCF-7 cells cultured in 2D and 3D models. These are relevant properties for long-term strategies to avoid metastasis and improve the prognosis of breast cancer.

Advances in the use of 3D colorectal cancer models for novel drug discovery.

INTRODUCTION: Colorectal cancer (CRC) is one of the most common and deadly tumors worldwide. CRC in vitro and in vivo models that recapitulate key features of human disease are essential to the development of novel and effective therapeutics. However, two-dimensional (2D) in vitro culture systems are considered too simple and do not represent the complex nature of the human tumor. However, three-dimensional (3D) models have emerged in recent years as more advanced and complex cell culture systems, able to closely resemble key features of human cancer tissues. AREAS COVERED: The authors' review the currently established in vitro cell culture models and describe the advances in the development of 3D scaffold-free models to study CRC. The authors also discuss intestinal spheroids and organoids. As well as in vitro models for drug screening and metastatic CRC (mCRC). EXPERT OPINION: The ideal CRC in vitro model is not yet established. Spheroid-based 3D models represent one of the most used approaches to recapitulate the tumor environment, overcoming some limitations of 2D models. Mouse and patient-derived organoids are more advanced models that can mimic more closely the characteristics and properties of CRC, with the possibility of including cells derived from patients with metastatic CRC.

Recapitulating Tumor Microenvironment Using AXTEX-4DTM for Accelerating Cancer Research and Drug Screening.

OBJECTIVE: The formation of three-dimensional spheroid tumor model using the scaffold-based platforms has been demonstrated over many years now. 3D tumor models are generated mainly in non-scalable culture systems, using synthetic and biological scaffolds. Many of these models fail to reflect the complex tumor microenvironment and do not allow long-term monitoring of tumor progression. This has resulted in inconsistent data in drug testing assays during preclinical and clinical studies. METHODS: To overcome these limitations, we have developed 3D tissueoids model by using novel AXTEX-4D platform. RESULTS: Cancer 3D tissueoids demonstrated the basic features of 3D cell culture with rapid attachment, proliferation, and longevity with contiguous cytoskeleton and hypoxic core. This study also demonstrated greater drug resistance in 3D-MCF-7 tissueoids in comparison to 2D monolayer cell culture. CONCLUSION: In conclusion, 3D-tissueoids are more responsive than 2D-cultured cells in simulating important tumor characteristics, anti-apoptotic features, and their resulting drug resistance.