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Recently, many types of in vitro 3‐D culture systems have been developed to recapitulate the in vivo growth conditions of cancer. The cancer 3‐D culture methods aim to preserve the biological characteristics of original tumors better than conventional 2‐D monolayer cultures, and include tumor‐derived organoids, tumor‐derived spheroids, organotypic multicellular spheroids, and multicellular tumor spheroids. The 3‐D culture methods differ in terms of cancer cell sources, protocols for cell handling, and the required time intervals. Tumor‐derived spheroids are unique because they are purposed for the enrichment of cancer stem cells (CSCs) or cells with stem cell‐related characteristics. These spheroids are grown as floating spheres and have been used as surrogate systems to evaluate the CSC‐related characteristics of solid tumors in vitro. Because eradication of CSCs is likely to be of clinical importance due to their association with the malignant nature of cancer cells, such as tumorigenicity or chemoresistance, the investigation of tumor‐derived spheroids may provide invaluable clues to fight against cancer. Spheroid cultures have been established from cancers including glioma, breast, colon, ovary, and prostate cancers, and their biological and biochemical characteristics have been investigated by many research groups. In addition to the investigation of CSCs, tumor‐derived spheroids may prove to be instrumental for a high‐throughput screening platform or for the cultivation of CSC‐related tumor cells found in the circulation or body fluids. Tumor‐derived spheroid culture is one of the representative 3D culture methods in which cancer cells with stem cell‐like features are expanded in vitro as floating spheres. In this review, we summarize the major discoveries from studies using tumor‐derived spheroids and future clinical applications.

Cancer is a multifactorial disease that is responsible for 10 million deaths per year. The intra- and inter-heterogeneity of malignant tumors make it difficult to develop single targeted approaches. Similarly, their diversity requires various models to investigate the mechanisms involved in cancer initiation, progression, drug resistance and recurrence. Of the in vitro cell-based models, monolayer adherent (also known as 2D culture) cell cultures have been used for the longest time. However, it appears that they are often less appropriate than the three-dimensional (3D) cell culture approach for mimicking the biological behavior of tumor cells, in particular the mechanisms leading to therapeutic escape and drug resistance. Multicellular tumor spheroids are widely used to study cancers in 3D, and can be generated by a multiplicity of techniques, such as liquid-based and scaffold-based 3D cultures, microfluidics and bioprinting. Organoids are more complex 3D models than multicellular tumor spheroids because they are generated from stem cells isolated from patients and are considered as powerful tools to reproduce the disease development in vitro. The present review provides an overview of the various 3D culture models that have been set up to study cancer development and drug response. The advantages of 3D models compared to 2D cell cultures, the limitations, and the fields of application of these models and their techniques of production are also discussed.

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.

Multicellular tumor spheroids and tumoroids are considered ideal in vitro models that reflect the features of the tumor microenvironment. Biomimetic components resembling the extracellular matrix form scaffolds to provide structure to 3-dimensional (3D) culture systems, supporting the growth of both spheroids and tumoroids. Although Matrigel has long been used to support 3D culture systems, batch variations, component complexity, and the use of components derived from tumors are complicating factors. To address these issues, we developed the ACD 3D culture system to provide better control and consistency. We evaluated spheroid and tumoroid formation using the ACD 3D culture system, including the assessment of cell viability and cancer marker expression. Under ACD 3D culture conditions, spheroids derived from cancer cell lines exhibited cancer stem cell characteristics, including a sphere-forming size and the expression of stem cell marker genes. The ACD 3D culture system was also able to support patient-derived primary cells and organoid cell cultures, displaying adequate cell growth, appropriate morphology, and resistance to oxaliplatin treatment. These spheroids could also be used for drug screening purposes. In conclusion, the ACD 3D culture system represents an efficient tool for basic cancer research and therapeutic development.

Tumor metastasis presents a formidable challenge in cancer treatment, necessitating effective tools for anti-cancer drug development. Conventional 2D cell culture methods, while considered the “gold standard” for invasive studies, exhibit limitations in representing cancer hallmarks and phenotypes. This study proposes an innovative approach that combines the advantages of 3D tumor spheroid culture with impedance-based biosensing technologies to establish a high-throughput 3D cell invasion assay for anti-metastasis drug screening through multicellular tumor spheroids. In addition, the xCELLigence device is employed to monitor the time-dependent kinetics of cell behavior, including attachment and invasion out of the 3D matrix. Moreover, an iron chelator (deferoxamine) is employed to monitor the inhibition of epithelial–mesenchymal transition in 3D spheroids across different tumor cell types. The above results indicate that our integrated 3D cell invasion assay with impedance-based sensing could be a promising tool for enhancing the quality of the drug development pipeline by providing a robust platform for predicting the efficacy and safety of anti-metastatic drugs before advancing into preclinical or clinical trials.

Glyco-quantum dots (glyco-QDs) have attracted significant interest in bioimaging applications, notably in cancer imaging, because they effectively combine the glycocluster effect with the exceptional optical properties of QDs. The key challenge now lies in how to eliminate the high heavy metal toxicity originating from traditional toxic Cd-based QDs for in vivo bioimaging. Herein, we report an eco-friendly pathway to prepare nontoxic Cd-free glyco-QDs in water by the “direct” reaction of thiol-ending monosaccharides with metal salts precursors. The formation of glyco-CuInS 2 QDs could be explained by a nucleation-growth mechanism following the LaMer model. As-prepared four glyco-CuInS 2 QDs were water-soluble, monodispersed, spherical in shape and exhibited size range of 3.0–4.0 nm. They exhibited well-separated dual emission in the visible region (500–590 nm) and near-infrared range (~ 827 nm), which may be attributable to visible excitonic emission and near-infrared surface defect emission. Meanwhile, the cell imaging displayed the reversibly distinct dual-color (green and red) fluorescence in tumor cells (HeLa, A549, MKN-45) and excellent membrane-targeting properties of glyco-CuInS 2 QDs based on their good biorecognition ability. Importantly, these QDs succeed in penetrating uniformly into the interior (the necrotic zone) of 3D multicellular tumor spheroids (MCTS) due to their high negative charge (zeta potential values ranging from − 23.9 to − 30.1 mV), which overcame the problem of poor penetration depth of existing QDs in in vitro spheroid models. So, confocal analysis confirmed their excellent ability to penetrate and label tumors. Thus, the successful application in in vivo bioimaging of these glyco-QDs verified that this design strategy is an effective, low cost and simple procedure for developing green nanoparticles as cheap and promising fluorescent bioprobes.

Invasive cancer cell migration is a key feature of metastatic human pancreatic ductal adenocarcinoma (PDAC), yet the underlying mechanisms remain poorly understood. Here, we investigated modes of cancer cell invasion using two pancreatic cancer cell lines with differential epithelial–mesenchymal status, PANC-1 and BxPC-3, under 3D culture conditions. Multicellular tumor spheroids (TSs) were grown in a collagen matrix co-cultured with pancreatic stellate cells (PSCs) using microchannel chips. PANC-1 cells showed individual migration from TSs via invadopodium formation. BxPC-3 cells showed plasticity between collective and individual migration in either mesenchymal mode, with filopodium-like protrusions, or blebby amoeboid mode. These two cell lines showed significantly different patterns of extracellular matrix (ECM) remodeling, with MMP-dependent degradation in a limited area of ECM around invadopodia for PANC-1 cells, or MMP-independent extensive deformation of ECM for BxPC-3 cells. Cancer cell migration out of the collagen channel significantly increased by PSCs and directional cancer cell migration was mediated by fibronectin deposited by PSCs. Our results highlight the phenotypic heterogeneity and plasticity of PDAC cell migration and ECM remodeling under 3D culture conditions. This 3D co-culture model of pancreatic cancer cells and PSCs offers a useful tool for studying cancer cell migration and ECM remodeling to identify and develop potential molecular targets and anti-cancer agents against human PDAC.

Anticancer drug development is a crucial step toward cancer treatment, that requires realistic predictions of malignant tissue development and sophisticated drug delivery. Tumors often acquire drug resistance and drug efficacy, hence cannot be accurately predicted in 2D tumor cell cultures. On the other hand, 3D cultures, including multicellular tumor spheroids (MCTSs), mimic the in vivo cellular arrangement and provide robust platforms for drug testing when grown in hydrogels with characteristics similar to the living body. Microparticles and liposomes are considered smart drug delivery vehicles, are able to target cancerous tissue, and can release entrapped drugs on demand. Microfluidics serve as a high‐throughput tool for reproducible, flexible, and automated production of droplet‐based microscale constructs, tailored to the desired final application. In this review, it is described how natural hydrogels in combination with droplet microfluidics can generate MCTSs, and the use of microfluidics to produce tumor targeting microparticles and liposomes. One of the highlights of the review documents the use of the bottom‐up construction methodologies of synthetic biology for the formation of artificial cellular assemblies, which may additionally incorporate both target cancer cells and prospective drug candidates, as an integrated “droplet incubator” drug assay platform. This review summarizes the recent progress of droplet technologies in anticancer drug development. Microfluidic‐formed droplets are utilized in both tumor spheroid formation and smart drug delivery. This work also highlights the current trend of using artificial cells materials for drug metabolism studies. Finally, a novel ‘droplet incubator’ concept as a prospective anticancer drug screening platform is introduced.

The prognosis, metastasis, and behavior of head and neck squamous cancer cells are influenced by numerous factors concerning the tumor microenvironment, intercellular communication, and epithelial-to-mesenchymal transition (EMT). The aim of this study was to examine the codependent interaction of the mesenchymal stroma with head and neck squamous cell carcinoma (HNSCC) in a 3D spheroid structure. To simulate stroma-rich and -poor 3D tumor microenvironments, cells of the established cell SCC-040 were cultured with human mesenchymal stromal cells (MSCs), forming 3D stroma-tumor spheroids (STSs). STSs were compared to uniform spheroids of SCC-040 and MSC, respectively. The expressions of CD24, β-catenin, SNAI2, and ZEB2 were analyzed via RT-qPCR. The immunohistochemical expressions of E-cadherin, connexin 43, vimentin, and emmprin were analyzed, and protein expression pathways as well as Akt signaling were assessed via protein analysis. A promotive effect on the expressions of EMT markers ZEB2 (p = 0.0099), SNAI2 (p = 0.0352), and β-catenin (p = 0.0031) was demonstrated in STSs, as was the expression of Akt pathway proteins mTOR (p = 0.007), Erk1/2 (p = 0.0045), and p70 S6 Kinase (p = 0.0016). Our study demonstrated a change in genetic expression patterns early on in tumor development, indicating a tumor turning point.

Au-Fe3O4 nanoheterodimers (NHD) were functionalized with the natural and synthetic anticancer drugs caffeic acid (CA), quercetin (Q) and 5-fluorocytidine (5FC). Their X-radiation dose-enhancing potential and chemotherapeutic efficacy for bimodal cancer therapy were investigated by designing multicellular tumor spheroids (MCTS) to in vitro avascular tumor models. MCTS were grown from the breast cancer cell lines MCF-7, MDA-MB-231, and MCF-10A. The MCF-7, MDA-MB-231 and MCF-10A MCTS were incubated with NHD-CA, NHD-Q, or NHD-5FC and then exposed to fractionated X-radiation comprising either a single 10 Gy dose, 2 daily single 5 Gy doses or 5 daily single 2 Gy doses. The NHD-CA, NHD-Q, and NHD-5FC affected the growth of X-ray irradiated and non-irradiated MCTS in a different manner. The impact of the NHDs on the glycolytic metabolism due to oxygen deprivation inside MCTS was assessed by measuring lactate secretion and glucose uptake by the MCTS. The NHD-CA and NHD-Q were found to act as X-radiation dose agents in MCF-7 MCTS and MDA-MB-231 MCTS and served as radioprotector in MCF-10A MCTS. X-ray triggered release of CA and Q inhibited lactate secretion and thereupon disturbed glycolytic reprogramming, whereas 5FC exerted their cytotoxic effects on both, healthy and tumor cells, after their release into the cytosol.