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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.

Cells respond to physical stimuli, such as stiffness 1 , fluid shear stress 2 and hydraulic pressure 3 , 4 . Extracellular fluid viscosity is a key physical cue that varies under physiological and pathological conditions, such as cancer 5 . However, its influence on cancer biology and the mechanism by which cells sense and respond to changes in viscosity are unknown. Here we demonstrate that elevated viscosity counterintuitively increases the motility of various cell types on two-dimensional surfaces and in confinement, and increases cell dissemination from three-dimensional tumour spheroids. Increased mechanical loading imposed by elevated viscosity induces an actin-related protein 2/3 (ARP2/3)-complex-dependent dense actin network, which enhances Na + /H + exchanger 1 (NHE1) polarization through its actin-binding partner ezrin. NHE1 promotes cell swelling and increased membrane tension, which, in turn, activates transient receptor potential cation vanilloid 4 (TRPV4) and mediates calcium influx, leading to increased RHOA-dependent cell contractility. The coordinated action of actin remodelling/dynamics, NHE1-mediated swelling and RHOA-based contractility facilitates enhanced motility at elevated viscosities. Breast cancer cells pre-exposed to elevated viscosity acquire TRPV4-dependent mechanical memory through transcriptional control of the Hippo pathway, leading to increased migration in zebrafish, extravasation in chick embryos and lung colonization in mice. Cumulatively, extracellular viscosity is a physical cue that regulates both short- and long-term cellular processes with pathophysiological relevance to cancer biology. Elevated viscosity counterintuitively increases the motility of various cell types in vitro and imprints mechanical memory to tumour cells, which enables them to disseminate more efficiently in vivo.

Cancer genomics studies have identified thousands of putative cancer driver genes 1 . Development of high-throughput and accurate models to define the functions of these genes is a major challenge. Here we devised a scalable cancer-spheroid model and performed genome-wide CRISPR screens in 2D monolayers and 3D lung-cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulated those of in vivo tumours, and genes with differential sensitivities between 2D and 3D conditions were highly enriched for genes that are mutated in lung cancers. These analyses also revealed drivers that are essential for cancer growth in 3D and in vivo, but not in 2D. Notably, we found that carboxypeptidase D is responsible for removal of a C-terminal RKRR motif 2 from the α-chain of the insulin-like growth factor 1 receptor that is critical for receptor activity. Carboxypeptidase D expression correlates with patient outcomes in patients with lung cancer, and loss of carboxypeptidase D reduced tumour growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy for performing CRISPR screens in spheroids to reveal cancer vulnerabilities. CRISPR screens in a 3D spheroid cancer model system more accurately recapitulate cancer phenotypes than existing 2D models and were used to identify carboxypeptidase D, acting via the IGF1R, as a 3D-specific driver of cancer growth.

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.A knowledgebase developed for increased the transparency of reporting in spheroid research.

The potential of a spheroid tumor model composed of cells in different proliferative and metabolic states for the development of new anticancer strategies has been amply demonstrated. However, there is little or no information in the literature on the problems of reproducibility of data originating from experiments using 3D models. Our analyses, carried out using a novel open source software capable of performing an automatic image analysis of 3D tumor colonies, showed that a number of morphology parameters affect the response of large spheroids to treatment. In particular, we found that both spheroid volume and shape may be a source of variability. We also compared some commercially available viability assays specifically designed for 3D models. In conclusion, our data indicate the need for a pre-selection of tumor spheroids of homogeneous volume and shape to reduce data variability to a minimum before use in a cytotoxicity test. In addition, we identified and validated a cytotoxicity test capable of providing meaningful data on the damage induced in large tumor spheroids of up to diameter in 650 μm by different kinds of treatments.

Pancreatic ductal adenocarcinoma is characterised by a dense desmoplastic stroma composed of stromal cells and extracellular matrix (ECM). This barrier severely impairs drug delivery and penetration. Activated pancreatic stellate cells (PSCs) play a key role in establishing this unique pathological obstacle, but also offer a potential target for anti-tumour therapy. Here, we construct a tumour microenvironment-responsive nanosystem, based on PEGylated polyethylenimine-coated gold nanoparticles, and utilise it to co-deliver all- trans retinoic acid (ATRA, an inducer of PSC quiescence) and siRNA targeting heat shock protein 47 (HSP47, a collagen-specific molecular chaperone) to re-educate PSCs. The nanosystem simultaneously induces PSC quiescence and inhibits ECM hyperplasia, thereby promoting drug delivery to pancreatic tumours and significantly enhancing the anti-tumour efficacy of chemotherapeutics. Our combination strategy to restore homoeostatic stromal function by targeting activated PSCs represents a promising approach to improving the efficacy of chemotherapy and other therapeutic modalities in a wide range of stroma-rich tumours. Stromal-tumour interactions play an important role in pancreatic cancer progression. Here, they describe the development of a tumour microenvironment-responsive gold nanoparticle system incorporating all- trans retinoic acid (ATRA) and siRNA against heat shock protein 47 (HSP47), for use in pancreatic cancer treatment.

Multicellular 3D culture and interaction with stromal components are considered essential elements in establishing a 'more clinically relevant' tumor model. Matrix-embedded 3D cultures using a microfluidic chip platform can recapitulate the microscale interaction within tumor microenvironments. As a major component of tumor microenvironment, cancer-associated fibroblasts (CAFs) play a role in cancer progression and drug resistance. Here, we present a microfluidic chip-based tumor tissue culture model that integrates 3D tumor spheroids (TSs) with CAF in proximity within a hydrogel scaffold. HT-29 human colorectal carcinoma cells grew into 3D TSs and the growth was stimulated when co-cultured with fibroblasts as shown by 1.5-folds increase of % changes in diameter over 5 days. TS cultured for 6 days showed a reduced expression of Ki-67 along with increased expression of fibronectin when co-cultured with fibroblasts compared to mono-cultured TSs. Fibroblasts were activated under co-culture conditions, as demonstrated by increases in α-SMA expression and migratory activity. When exposed to paclitaxel, a survival advantage was observed in TSs co-cultured with activated fibroblasts. Overall, we demonstrated the reciprocal interaction between TSs and fibroblasts in our 7-channel microfluidic chip. The co-culture of 3D TS-CAF in a collagen matrix-incorporated microfluidic chip may be useful to study the tumor microenvironment and for evaluation of drug screening and evaluation.

To optimally penetrate biological hydrogels such as mucus and the tumor interstitial matrix, nanoparticles (NPs) require physicochemical properties that would typically preclude cellular uptake, resulting in inefficient drug delivery. Here, we demonstrate that (poly(lactic-co-glycolic acid) (PLGA) core)-(lipid shell) NPs with moderate rigidity display enhanced diffusivity through mucus compared with some synthetic mucus penetration particles (MPPs), achieving a mucosal and tumor penetrating capability superior to that of both their soft and hard counterparts. Orally administered semi-elastic NPs efficiently overcome multiple intestinal barriers, and result in increased bioavailability of doxorubicin (Dox) (up to 8 fold) compared to Dox solution. Molecular dynamics simulations and super-resolution microscopy reveal that the semi-elastic NPs deform into ellipsoids, which enables rotation-facilitated penetration. In contrast, rigid NPs cannot deform, and overly soft NPs are impeded by interactions with the hydrogel network. Modifying particle rigidity may improve the efficacy of NP-based drugs, and can be applicable to other barriers. Penetration of the mucus and tumor interstitial matrix is an important consideration for drug delivery devices. Here, the authors report on a study into the optimization of rigidity for the transport of nanoparticles through biological hydrogels using core-shell polymer-lipid nanoparticles.

Cancer-associated fibroblasts (CAFs) promote tumour invasion and metastasis. We show that CAFs exert a physical force on cancer cells that enables their collective invasion. Force transmission is mediated by a heterophilic adhesion involving N-cadherin at the CAF membrane and E-cadherin at the cancer cell membrane. This adhesion is mechanically active; when subjected to force it triggers β-catenin recruitment and adhesion reinforcement dependent on α-catenin/vinculin interaction. Impairment of E-cadherin/N-cadherin adhesion abrogates the ability of CAFs to guide collective cell migration and blocks cancer cell invasion. N-cadherin also mediates repolarization of the CAFs away from the cancer cells. In parallel, nectins and afadin are recruited to the cancer cell/CAF interface and CAF repolarization is afadin dependent. Heterotypic junctions between CAFs and cancer cells are observed in patient-derived material. Together, our findings show that a mechanically active heterophilic adhesion between CAFs and cancer cells enables cooperative tumour invasion. Cancer-associated fibroblasts (CAFs) promote metastasis by creating tracks for cancer cell migration. Labernadie  et al.  now show that heterotypic adhesions between E-cadherin on cancer cells and N-cadherin on CAFs transmit forces to drive invasion.

Patient‐derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high‐throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high‐throughput screening of 2427 drugs using the cancer tissue‐originated spheroid (CTOS) method. In this system, we apply the CTOS method in an ex vivo platform from xenograft tumors, using machines to handle CTOS and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. CTOS passages in xenograft tumors resulted in minimal changes of morphological and genomic status, and xenograft tumor generation efficiently expanded the number of CTOS to evaluate multiple drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease. We report an advanced system for the high‐throughput screening of 2427 drugs using organoids. In this system, we apply the cancer‐tissue originated spheroid (CTOS) method in an ex vivo platform from xenograft tumors, using machines to handle organoids and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease.