Search

Apical-basal cell polarity and lumen formation are essential features of many epithelial tissues, which are disrupted in diseases like cancer. Here, we describe a proteomics-based screen to identify proteins involved in lumen formation in three-dimensional spheroid cultures. We established a suspension-based culture method suitable for generating polarized cysts in sufficient quantities for proteomic analysis. Using this approach, we identified several known and unknown proteins proximally associated with PAR6B, an apical protein involved in lumen formation. Functional analyses of candidates identified PARD3B (a homolog of PARD3), RALB, and HRNR as regulators of lumen formation. We also identified PTPN14 as a component of the Par-complex that is required for fidelity of apical-basal polarity. Cells transformed with KRASG12V exhibit lumen collapse/filling concomitant with disruption of the Par-complex and down-regulation of PTPN14. Enforced expression of PTPN14 maintained the lumen and restricted the transformed phenotype in KRASG12V-expressing cells. This represents an applicable approach to explore protein–protein interactions in three-dimensional culture and to identify proteins important for lumen maintenance in normal and oncogene-expressing cells.

The short survival time of transplanted adipose-derived mesenchymal stem cells (ASCs) is a problem for skin wound healing. Transplantation after the formation of cellular spheroids has been investigated as a promising method for prolonging cellular survival. However, there have been technical restrictions for transplantation of spheroids in clinical practice. Here, we show an effective method for transplantation of ASC spheroids onto skin wounds in order to efficiently cure refractory ulcers. To assist anchoring of spheroids onto skin wounds, we used a 120-nm-thick free-standing film (nanosheet) that has a highly adhesive property. Bioluminescence imaging showed that ASC spheroids carried by the nanosheet survived for 14 days, which is about two-times longer than that previously reported. Wounds treated with a nanosheet carrying ASC spheroids were 4-times smaller than untreated wounds on day 14. This method for transplantation of spheroids could be applied to cell therapy for various refractory skin wounds.

Studying medulloblastoma, the most common malignant paediatric brain tumour, requires simple yet realistic in vitro models. In this study, we optimised a robust, reliable, three-dimensional (3D) culture method for medulloblastoma able to recapitulate the spatial conformation, cell–cell and cell–matrix interactions that exist in vivo and in patient tumours. We show that, when grown under the same stem cell enriching conditions, SHH subgroup medulloblastoma cell lines established tight, highly reproducible 3D spheroids that could be maintained for weeks in culture and formed pathophysiological oxygen gradients. 3D spheroid culture also increased resistance to standard-of-care chemotherapeutic drugs compared to 2D monolayer culture. We exemplify how this model can enhance in vitro therapeutic screening approaches through dual-inhibitor studies and continual monitoring of drug response. Next, we investigated the initial stages of metastatic dissemination using brain-specific hyaluronan hydrogel matrices. RNA sequencing revealed downregulation of cell cycle genes and upregulation of cell movement genes and key fibronectin interactions in migrating cells. Analyses of these upregulated genes in patients showed that their expression correlated with early relapse and overall poor prognosis. Our 3D spheroid model is a significant improvement over current in vitro techniques, providing the medulloblastoma research community with a well-characterised and functionally relevant culture method.

Traction force microscopy (TFM) is an important family of techniques used to measure and study the role of cellular traction forces (CTFs) associated with many biological processes. However, current standard TFM methods rely on imaging techniques that do not provide the experimental capabilities necessary to study CTFs within 3D collective and dynamic systems embedded within optically scattering media. Traction force optical coherence microscopy (TF-OCM) was developed to address these needs, but has only been demonstrated for the study of isolated cells embedded within optically clear media. Here, we present computational 4D-OCM methods that enable the study of dynamic invasion behavior of large tumor spheroids embedded in collagen. Our multi-day, time-lapse imaging data provided detailed visualizations of evolving spheroid morphology, collagen degradation, and collagen deformation, all using label-free scattering contrast. These capabilities, which provided insights into how stromal cells affect cancer progression, significantly expand access to critical data about biophysical interactions of cells with their environment, and lay the foundation for future efforts toward volumetric, time-lapse reconstructions of collective CTFs with TF-OCM.

We present a quantitative study of the metabolic activity of a single spheroid culture of human cancer cells. NMR (nuclear magnetic resonance) spectroscopy is an ideal tool for observation of live systems due to its non-invasive nature. However, limited sensitivity has so far hindered its application in microfluidic culture systems. We have used an optimised micro-NMR platform to observe metabolic changes from a single spheroid. NMR spectra were obtained by directly inserting microfluidic devices containing spheroids ranging from 150 $$\upmu$$ μ m to 300 $$\upmu$$ μ m in diameter in 2.5 $$\upmu$$ μ L of culture medium into a dedicated NMR probe. Metabolite concentrations were found to change linearly with time, with rates approximately proportional to the number of cells in the spheroid. The results demonstrate that quantitative monitoring of a single spheroid with $$\le$$ ≤ 2500 cells is possible. A change in spheroid size by 600 cells leads to a clearly detectable change in the l-Lactic acid production rate ($$p=3.5\times 10^{-3}$$ p = 3.5 × 10 - 3 ). The consumption of d-Glucose and production of l-Lactic acid were approximately 2.5 times slower in spheroids compared to monolayer culture of the same number of cells. Moreover, while cells in monolayer culture were found to produce l-Alanine and l-Glutamine, spheroids showed slight consumption in both cases.

Organ and tissue development are highly coordinated processes; lens growth and functional integration into the eye (emmetropia) is a robust example. An epithelial monolayer covers the anterior hemisphere of the lens, and its organization is the key to lens formation and its optical properties throughout all life stages. To better understand how the epithelium supports lens function, we have developed a novel whole tissue imaging system using conventional confocal light microscopy and a specialized analysis software to produce three-dimensional maps for the epithelium of intact mouse lenses. The open source software package geometrically determines the anterior pole position, the equatorial diameter, and three-dimensional coordinates for each detected cell in the epithelium. The user-friendly cell maps, which retain global lens geometry, allow us to document age-dependent changes in the C57/BL6J mouse lens cell distribution characteristics. We evidence changes in epithelial cell density and distribution in C57/BL6J mice during the establishment of emmetropia between postnatal weeks 4–6. These epithelial changes accompany a previously unknown spheroid to lentoid shape transition of the lens as detected by our analyses. When combined with key findings from previous mouse genetic and cell biological studies, we suggest a cytoskeleton-based mechanism likely underpins these observations.

Osteochondral defects contain damage to both the articular cartilage and underlying subchon- dral bone, which remains a significant challenge in orthopedic surgery. Layered structure of bone, cartilage and the bone-cartilage interface must be taken into account in the case of biofabrication of the osteochondral (OC) interface. In this study, a dual layered OC interface was bioprinted using a newly developed aspiration-assisted bioprinting (AAB) technique, which has been the first time that scaffold-free bioprinting was applied to OC interface engineering. Tissue spheroids, made of human adipose-derived stem cells (ADSCs), were differentiated in three dimensions (3D) into chondrogenic and osteogenic spheroids, which were confirmed by immunostaining and histology qualitatively, and biochemistry assays and gene expression, quantitatively. Remarkably, the OC interface was bioprinted by accurate positioning of a layer of osteogenic spheroids onto a sacrificial alginate support followed by another layer of chondrogenic spheroids overlaid by the same support. Spheroids in individual zones fused and the maintenance of phenotypes in both zones confirmed the successful biofabrication of the histomorphologically-relevant OC interface. The biofabrication of OC tissue model without the use of polymeric scaffolds unveils great potential not only in regenerative medicine but also in drug testing and disease modeling for osteoarthritis.

Multicellular tumor spheroid (MCTS) systems provide an in vitro cell culture model system which mimics many of the complexities of an in vivo solid tumor and tumor microenvironment, and are often used to study cancer cell growth and drug efficacy. Here, we present a coupled experimental-computational framework to estimate phenotypic growth and biophysical tumor microenvironment properties. This novel framework utilizes standard microscopy imaging of MCTS systems to drive a biophysical mathematical model of MCTS growth and mechanical interactions. By extending our previous in vivo mechanically-coupled reaction-diffusion modeling framework we developed a microscopy image processing framework capable of mechanistic characterization of MCTS systems. Using MDA-MB-231 breast cancer MCTS, we estimated biophysical parameters of cellular diffusion, rate of cellular proliferation, and cellular tractions forces. We found significant differences in these model-based biophysical parameters throughout the treatment time course between untreated and treated MCTS systems, whereas traditional size-based morphometric parameters were inconclusive. The proposed experimental-computational framework estimates mechanistic MCTS growth and invasion parameters with significant potential to assist in better and more precise assessment of in vitro drug efficacy through the development of computational analysis methodologies for three-dimensional cell culture systems to improve the development and evaluation of antineoplastic drugs. National Institutes of Health - National Cancer InstituteUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [K25CA204599, P30CA012197]; Wake Forest Baptist Medical Center Comprehensive Cancer Center Signaling and Biotechnology Program Pilot Grant National Institutes of Health - National Cancer Institute K25CA204599 and P30CA012197. Wake Forest Baptist Medical Center Comprehensive Cancer Center Signaling and Biotechnology Program Pilot Grant.

Biological self-assembly is crucial in the processes of development, tissue regeneration, and maturation of bioprinted tissue-engineered constructions. The cell aggregates—spheroids—have become widely used model objects in the study of this phenomenon. Existing approaches describe the fusion of cell aggregates by analogy with the coalescence of liquid droplets and ignore the complex structural properties of spheroids. Here, we analyzed the fusion process in connection with structure and mechanical properties of the spheroids from human somatic cells of different phenotypes: mesenchymal stem cells from the limbal eye stroma and epithelial cells from retinal pigment epithelium. A nanoindentation protocol was applied for the mechanical measurements. We found a discrepancy with the liquid drop fusion model: the fusion was faster for spheroids from epithelial cells with lower apparent surface tension than for mesenchymal spheroids with higher surface tension. This discrepancy might be caused by biophysical processes such as extracellular matrix remodeling in the case of mesenchymal spheroids and different modes of cell migration. The obtained results will contribute to the development of more realistic models for spheroid fusion that would further provide a helpful tool for constructing cell aggregates with required properties both for fundamental studies and tissue reparation.

Tumor invasion within the interstitial space is critically regulated by the force balance between cell-extracellular matrix (ECM) and cell-cell interactions. Interstitial flows (IFs) are present in both healthy and diseased tissues. However, the roles of IFs in modulating cell force balance and subsequently tumor invasion are understudied. In this article, we develop a microfluidic model in which tumor spheroids are embedded within 3D collagen matrices with well-defined IFs. Using co-cultured tumor spheroids (1:1 mixture of metastatic and non-tumorigenic epithelial cells), we show that IFs downregulate the cell-cell adhesion molecule E-cadherin on non-tumorigenic cells and promote tumor invasion. Our microfluidic model advances current tumor invasion assays towards a more physiologically realistic model using tumor spheroids instead of single cells under perfusion. We identify a novel mechanism by which IFs can promote tumor invasion through an influence on cell-cell adhesion within the tumor and highlight the importance of biophysical parameters in regulating tumor invasion.

Pancreatic cancer is one of the most complex types of cancers to detect, diagnose, and treat. However, the field of nanomedicine has strong potential to address such challenges. When evaluating the diffusion and penetration of theranostic nanoparticles, the extracellular matrix (ECM) is of crucial importance because it acts as a barrier to the tumor microenvironment. In the present study, the penetration of functionalized, fluorescent gold nanorods into large (>500 μm) multicellular 3D tissue spheroids was studied using a multimodal imaging approach. The spheroids were generated by co-culturing pancreatic cancer cells and pancreatic stellate cells in multiple ratios to mimic variable tumor-stromal compositions and to investigate nanoparticle penetration. Fluorescence live imaging, photothermal, and photoacoustic analysis were utilized to examine nanoparticle behavior in the spheroids. Uniquely, the nanorods are intrinsically photoacoustic and photothermal, enabling multi-imaging detection even when fluorescence tracking is not possible or ideal.

This paper studies the position and stability of equilibrium points in the circular restricted three-body problem under the influence of small perturbations in the Coriolis and centrifugal forces when the primaries are radiating and heterogeneous oblate spheroids. It is seen that there exist five libration points as in the classical restricted three-body problem, three collinear $$L_{i} ,(i = 1,2,3)$$ L i , ( i = 1 , 2 , 3 ) and two triangular $$L_{i} ,(i = 4,5)$$ L i , ( i = 4 , 5 ) . It is also seen that the triangular points are no longer to form equilateral triangles with the primaries rather they form simple triangles with line joining the primaries. It is further observed that despite all perturbations the collinear points remain unstable while the triangular points are stable for $$ 0 < \mu < \mu _{c} $$ 0 < μ < μ c and unstable for $$ \mu _{c} \le \mu \le \frac{1}{2} $$ μ c ≤ μ ≤ 1 2 , where $$ \mu _{c} $$ μ c is the critical mass ratio depending upon aforementioned parameters. It is marked that small perturbation in the Coriolis force, radiation and heterogeneous oblateness of the both primaries have destabilizing tendencies. Their numerical examination is also performed.

Compared with traditional monolayer cell culture, the three-dimensional tumor spheroid has emerged as an essential in vitro model for cancer research due to the recapitulation of the architecture and physiology of solid human tumors. Herein, by implementing the rapid prototyping of a benchtop 3D printer, we developed a new strategy to generate and analyze tumor spheroids on a commonly used multi-well plate. In this method, the printed artifact can be directly mounted on a 96/384-well plate, enables hanging drop-based spheroid formation, avoiding the tedious fabrication process from micromechanical systems. Besides long-term spheroid culture (20 days), this method supports subsequent analysis of tumor spheroid by seamlessly dripping from the printed array, thereby eliminating the need for spheroids retrieval for downstream characterization. We demonstrated several tumor spheroid-based assays, including tumoroid drug testing, metastasis on or inside extracellular matrix gel, and tumor transendothelial (TEM) assay. Based on quantitative phenotypical and molecular analysis without any precarious retrieval and transfer, we found that the malignant breast cancer (MDA-MB-231) cell aggregate presents a more metastatic morphological phenotype than the non-malignant breast cancer (MCF-7) and colonial cancer (HCT-116) cell spheroid, and shows an up-regulation of epithelial-mesenchymal transition (EMT) relevant genes (fold change > 2). Finally, we validated this tumor malignancy by the TEM assay, which could be easily performed using our approach. This methodology could provide a useful workflow for expediting tumoroid modeled in vitro assay, allowing the “Lab-on-a-Cloud” scenario for routine study.

Rotating forms of suspension culture allow cells to aggregate into spheroids, prevent the de-differentiating influence of 2D culture, and, perhaps most importantly of all, provide physiologically relevant, in vivo levels of shear stress. Rotating suspension culture technology has not been widely implemented, in large part because the vessels are prohibitively expensive, labor-intensive to use, and are difficult to scale for industrial applications. Our solution addresses each of these challenges in a new vessel called a cell spinpod. These small 3.5 mL capacity vessels are constructed from injection-molded thermoplastic polymer components. They contain self-sealing axial silicone rubber ports, and fluoropolymer, breathable membranes. Here we report the two-fluid modeling of the flow and stresses in cell spinpods. Cell spinpods were used to demonstrate the effect of fluid shear stress on renal cell gene expression and cellular functions, particularly membrane and xenobiotic transporters, mitochondrial function, and myeloma light chain, cisplatin and doxorubicin, toxicity. During exposure to myeloma immunoglobulin light chains, rotation increased release of clinically validated nephrotoxicity cytokine markers in a toxin-specific pattern. Addition of cisplatin or doxorubicin nephrotoxins reversed the enhanced glucose and albumin uptake induced by fluid shear stress in rotating cell spinpod cultures. Cell spinpods are a simple, inexpensive, easily automated culture device that enhances cellular functions for in vitro studies of nephrotoxicity.

More than 70% of colorectal, prostate, ovarian, pancreatic and breast cancer specimens show expression of CD276 (B7–H3), a potential immune checkpoint family member. Several studies have shown that high CD276 expression in cancer cells correlates with a poor clinical prognosis. This has been associated with the presence of lower tumor infiltrating leukocytes. Among those, tumor-associated macrophages can comprise up to 50% of the tumor mass and are thought to support tumor growth through various mechanisms. However, a lack of information on CD276 function and interaction partner(s) impedes rigorous evaluation of CD276 as a therapeutic target in oncology. Therefore, we aimed to understand the relevance of CD276 in tumor-macrophage interaction by employing a 3D spheroid coculture system with human cells. Our data show a role for tumor-expressed CD276 on the macrophage recruitment into the tumor spheroid, and also in regulation of the extracellular matrix modulator PAI-1. Furthermore, our experiments focusing on macrophage-expressed CD276 suggest that the antibody-dependent CD276 engagement triggers predominantly inhibitory signaling networks in human macrophages.

Breast cancer stem cells (BCSCs) resist conventional treatments and cause tumor recurrence. Almost 25% of breast cancers overexpress human epidermal growth factor receptor-2 (HER2). Here we developed a novel multi-targeted nanosystem to specifically eradicate HER2+ BCSCs. Plasmids containing CXCR1 promoter, PE38 toxin, and 5′UTR of the basic fibroblast growth factor-2 (bFGF 5'UTR) were constructed. Polyamidoamine (PAMAM) dendrimers functionalized with anti-HER2 VHHs were used for plasmid delivery. Stem cell proportion of MDA-MB-231, MDA-MB-231/HER2+ and MCF-10A were evaluated by mammosphere formation assay. Hanging drop technique was used to produce spheroids. The uptake, gene expression, and killing efficacy of the multi-targeted nanosystem were evaluated in both monolayer and spheroid culture. MDA-MB-231/HER2+ had higher ability to form mammosphere compared to MCF-10A. Our multi-targeted nanosystem efficiently inhibited the mammosphere formation of MDA-MB-231 and MDA-MB-231/HER2+ cells, while it was unable to prevent the mammosphere formation of MCF-10A. In the hanging drop culture, MDA-MB-231/HER+ generated compact well-rounded spheroids, while MCF-10A failed to form compact cellular masses. The multi-targeted nanosystem showed much better uptake, higher PE38 expression, and subsequent cell death in MDA-MB-231/HER2+ compared to MCF-10A. However, the efficacy of our targeted toxin gene therapy was lower in MDA-MB-231/HER2+ spheroids compared with that in the monolayer culture. the combination of the cell surface, transcriptional, and translational targeting increased the stringency of the treatment.

Osteocytes are mechanosensory commander cells to regulate bone remodeling throughout the lifespan. While the osteocytes are known as terminally differentiated cells derived from mesenchymal stem cells, the detailed mechanisms of osteocyte differentiation remain unclear. In this study, we fabricated 3D self-organized spheroids using human mesenchymal stem cells (MSCs). Under the osteogenesis induction medium, the spheroid culture model exerted the osteocyte-likeness within 2 days compared to a conventional 2D monolayer model. Moreover, we showed that an inhibition of actin polymerization in the spheroid further up-regulated the osteocyte gene expressions. Notably, we represented that the cell condensed condition acquired in the 3D spheroid culture model determined a differentiation fate of MSCs to osteocytes. Taken together, we suggest that our self-organized spheroid model can be utilized as a new in vitro model to represent the osteocyte and to recapitulate an in vitro ossification process.

In recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells.

Three-dimensional cell spheroid models can be used to predict the effect of drugs and therapeutics and to model tissue development and regeneration. The utility of these models is enhanced by high throughput 3D spheroid culture technologies allowing researchers to efficiently culture numerous spheroids under varied experimental conditions. Detailed analysis of high throughput spheroid culture is much less efficient and generally limited to narrow outputs, such as metabolic viability. We describe a microarray approach that makes traditional histological embedding/sectioning/staining feasible for large 3D cell spheroid sample sets. Detailed methodology to apply this technology is provided. Analysis of the technique validates the potential for efficient histological analysis of up to 96 spheroids in parallel. By integrating high throughput 3D spheroid culture technologies with advanced immunohistochemical techniques, this approach will allow researchers to efficiently probe expression of multiple biomarkers with spatial localization within 3D structures. Quantitative comparison of staining will have improved inter- and intra-experimental reproducibility as multiple samples are collectively processed, stained, and imaged on a single slide.

The EGFR pathway plays a critical role in head and neck squamous cell carcinoma (HNSCC). Targeted therapies against the EGFR are utilized as a treatment for HNSCCC. However, patient response is heterogeneous and molecular biomarkers are lacking to predict patient response. Therefore, functional assays where drug response is directly evaluated in tumor cells are an interesting alternative. Previous studies have shown that experimental conditions modify the drug response observed in functional assays. Thus, in this work the influence of the culture environment on response to Cetuximab (EGFR monoclonal antibody) and AZD8055 (mTOR inhibitor) was evaluated. HNSCC UM-SCC-1 and UM-SCC-47 cells were cultured in 2D monoculture and compared with: 2D co-culture with cancer-associated fibroblasts (CAF); 3D culture in collagen hydrogels; and 3D culture in tumor spheroids. The results showed UM-SCC-1 drug response significantly changed in the different culture environments; leading to an increase in drug resistance in the CAF co-culture and the 3D spheroids. Conversely, UM-SCC-47 exhibited a more constant drug response across culture conditions. In conclusion, this work highlights the importance of culture conditions that modulate response to EGFR pathway inhibition.

Placental extravillous trophoblast (EVT) invasion is essential in establishing proper blood supply to the fetus during pregnancy. However, traditional 2D in vitro systems do not model the in vivo invasion process in an anatomically-relevant manner. Our objectives were to develop a 3D spheroid model that would allow better emulation of placental invasion in vitro and to characterize the transcriptomic and functional outcomes. HTR8/SVneo EVT cells were self-assembled into 3D spheroids using ultra-low attachment plates. Transcriptomic profiling followed by gene set enrichment and gene ontology analyses revealed major global transcriptomic differences, with significant up-regulations in EVTs cultured as 3D spheroids in canonical pathways and biological processes such as immune response, angiogenesis, response to stimulus, wound healing, and others. These findings were further validated by RT-qPCR, showing significant up-regulations in genes and/or proteins related to epithelial-mesenchymal transition, cell-cell contact, angiogenesis, and invasion/migration. A high-throughput, spheroid invasion assay was applied to reveal the dynamic invasion of EVTs away from the spheroid core into extracellular matrix. Lastly, lipopolysaccharide, dexamethasone, or Δ9-tetrahydrocannabinol exposure was found to impact the invasion of EVT spheroids. Altogether, we present a well-characterized, 3D spheroid model of EVT invasion and demonstrate its potential use in drug and toxin screening during pregnancy.

Knowledge of optical properties, such as the refractive index (RI), of biological tissues is important in optical imaging, as they influence the distribution and propagation of light in tissue. To accurately study the response of cancerous cells to drugs, optimised imaging protocols are required. This study uses a simple custom-built spectral domain optical coherence tomography (OCT) system to conduct RI measurements of multicellular spheroids, three-dimensional (3D) in-vitro culture systems, of the cell line HCT116. The spheroid RIs are compared to study the effect of growth over time. To improve confocal microscopy imaging protocols, two immersion media (glycerol and ScaleView-A2) matching the spheroid RIs were trialled, with the aim to reduce the RI mismatch between the spheroid and the immersion medium and thus improve imaging depth with confocal microscopy. ScaleView-A2 (n = 1.380) aided in achieving greater depths of imaging of the multicellular spheroids under confocal microscopy. This improvement in imaging depth confirmed the utility of our RI measurements, proving the promising outlook of OCT as a complementary tool to microscopy in cancer research.

Three-dimensional spheroids are widely used as cancer models to study tumor cell proliferation and to evaluate new anticancer drugs. Growth-induced stress (i.e., stress that persists in tumors after external loads removal) influences tumor growth and resistance to treatment. However, it is not clear whether spheroids recapitulate the tumor physical properties. Here, we demonstrated experimentally and with the support of mathematical models that, like tumors, spheroids accumulate growth-induced stress. Moreover, we found that this stress is lower in spheroids made of 5,000 cancer cells and grown for 2 days than in spheroids made of 500 cancer cells and grown for 6 days. These two culture conditions associated with different growth-induced stress levels also had different effects on the spheroid shape (using light sheet microscopy) and surface topography and stiffness (using scanning electron microscopy and atomic force microscopy). Finally, the response to irinotecan was different in the two spheroid types. Taken together, our findings bring new insights into the relationship between the spheroid physical properties and their resistance to antitumor treatment that should be taken into account by the experimenters when assessing new therapeutic agents using in vitro 3D models or when comparing studies from different laboratories.

A central challenge in cancer biology is the identification, longitudinal tracking, and -omics analysis of specific cells in vivo. To this aim, photoconvertible fluorescent dyes are reporters that are characterized by a set of excitation and emission spectra that can be predictably altered, resulting in a distinct optical signature following irradiation with a specific light source. One such dye, DiR, is an infrared fluorescent membrane probe that can irreversibly undergo such a switch. Here, we demonstrate a method using DiR for the spatiotemporal labeling of specific cells in the context of cancer cell monolayer cultures, 3D tumor spheroids, and in vivo melanoma xenograft models to monitor the proliferation of cellular subpopulations of interest over time. Importantly, the photoconversion process is performed in situ, supporting the pursuit of novel avenues of research in molecular pathology.

Radiotherapy (RT) and chemotherapy (CT) are the major therapeutics to treat cancer patients. Conventional in vitro 2D models are insufficient to study the combined effects of RT and CT towards optimized dose selection or drug screening. Soft-tissue sarcomas (STS) are rare cancers with profound social impacts as they affect patients of all ages. We developed a microfluidic device to form and culture STS spheroids to study the combined cytotoxicities of RT and CT. Uniformly-sized spheroids of two different cell lines, STS 93 and STS 117, were formed in the device. RT doses of 0.5 Gy, 2 Gy, and 8 Gy were used in combination with CT, doxorubicin at 2 µM and 20 µM. The spheroids culture chambers within the device were arranged in a 3 × 5 matrix form. The device was made “peelable”, which enabled us to collect spheroids from each treatment condition separately. Collected spheroids were dissociated into single cells and evaluated using flow cytometry and clonogenic assays. Through this workflow, we observed that STS 93 spheroids treated with doxorubicin die through apoptosis, whereas RT induced death through other pathways. Spheroids from the p53 mutant STS 117 cell line were more resistant to RT and doxorubicin. The developed device could be used for the discovery of new drugs and RT synergies.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Among the key challenges in developing effective therapeutics is the poor translation of preclinical models used in the drug discovery pipeline. This leaves drug attrition rates and costs at an unacceptably high level. Previous work has highlighted the discrepancies in therapeutic response between current in vitro and in vivo models. To address this, we conducted a comparison study to differentiate the carboplatin chemotherapy response across four different model systems including 2D monolayers, 3D spheroids, 3D ex vivo tumors and mouse xenograft models. We used six previously characterized EOC cell lines of varying chemosensitivity and performed viability assays for each model. In vivo results from the mouse model correlated with 2D response in 3/6 cell lines while they correlated with 3D spheroids and the ex vivo model in 4/6 and 5/5 cell lines, respectively. Our results emphasize the variability in therapeutic response across models and demonstrate that the carboplatin response in EOC cell lines cultured in a 3D ex vivo model correlates best with the in vivo response. These results highlight a more feasible, reliable, and cost-effective preclinical model with the highest translational potential for drug screening and prediction studies in EOC.

Organoids emulate many aspects of their parental tissue and are therefore used to study pathogen-host interactions and other complex biological processes. Here, we report a robust protocol for the isolation, maintenance and differentiation of rabbit small intestinal organoids and organoid-derived cell monolayers. Our rabbit intestinal spheroid and monolayer cultures grew most efficiently in L-WRN-conditioned medium that contained Wnt, R-spondin and Noggin, and that had been supplemented with ROCK and TGF-β inhibitors. Organoid and monolayer differentiation was initiated by reducing the concentration of the L-WRN-conditioned medium and by adding ROCK and Notch signalling inhibitors. Immunofluorescence staining and RT-qPCR demonstrated that our organoids contained enterocytes, enteroendocrine cells, goblet cells and Paneth cells. Finally, we infected rabbit organoids with Rabbit calicivirus Australia-1, an enterotropic lagovirus that—like many other caliciviruses—does not grow in conventional cell culture. Despite testing various conditions for inoculation, we did not detect any evidence of virus replication, suggesting either that our organoids do not contain suitable host cell types or that additional co-factors are required for a productive infection of rabbit organoids with Rabbit calicivirus Australia-1.

A series of stable and ready-to-use bioinks have been developed based on the xeno-free and tunable hydrogel (VitroGel) system. Cell laden scaffold fabrication with optimized polysaccharide-based inks demonstrated that Ink H4 and RGD modified Ink H4-RGD had excellent rheological properties. Both bioinks were printable with 25–40 kPa extrusion pressure, showed 90% cell viability, shear-thinning and rapid shear recovery properties making them feasible for extrusion bioprinting without UV curing or temperature adjustment. Ink H4-RGD showed printability between 20 and 37 °C and the scaffolds remained stable for 15 days at temperature of 37 °C. 3D printed non-small-cell lung cancer (NSCLC) patient derived xenograft cells (PDCs) showed rapid spheroid growth of size around 500 µm in diameter and tumor microenvironment formation within 7 days. IC50 values demonstrated higher resistance of 3D spheroids to docetaxel (DTX), doxorubicin (DOX) and erlotinib compared to 2D monolayers of NSCLC-PDX, wild type triple negative breast cancer (MDA-MB-231 WT) and lung adenocarcinoma (HCC-827) cells. Results of flow property, shape fidelity, scaffold stability and biocompatibility of H4-RGD suggest that this hydrogel could be considered for 3D cell bioprinting and also for in-vitro tumor microenvironment development for high throughput screening of various anti-cancer drugs.

Recent research indicated the potential of cold physical plasma in cancer therapy. The plethora of plasma-derived reactive oxygen and nitrogen species (ROS/RNS) mediate diverse antitumor effects after eliciting oxidative stress in cancer cells. We aimed at exploiting this principle using a newly designed dual-jet neon plasma source (Vjet) to treat colorectal cancer cells. A treatment time-dependent ROS/RNS generation induced oxidation, growth retardation, and cell death within 3D tumor spheroids were found. In TUM-CAM, a semi in vivo model, the Vjet markedly reduced vascularized tumors' growth, but an increase of tumor cell immunogenicity or uptake by dendritic cells was not observed. By comparison, the argon-driven single jet kINPen, known to mediate anticancer effects in vitro, in vivo, and in patients, generated less ROS/RNS and terminal cell death in spheroids. In the TUM-CAM model, however, the kINPen was equivalently effective and induced a stronger expression of immunogenic cancer cell death (ICD) markers, leading to increased phagocytosis of kINPen but not Vjet plasma-treated tumor cells by dendritic cells. Moreover, the Vjet was characterized according to the requirements of the DIN-SPEC 91315. Our results highlight the plasma device-specific action on cancer cells for evaluating optimal discharges for plasma cancer treatment.

X-ray irradiation of high Z elements causes photoelectric effects that include the release of Auger electrons that can induce localized DNA breaks. We have previously established a tumor spheroid-based assay that used gadolinium containing mesoporous silica nanoparticles and synchrotron-generated monochromatic X-rays. In this work, we focused on iodine and synthesized iodine-containing porous organosilica (IPO) nanoparticles. IPO were loaded onto tumor spheroids and the spheroids were irradiated with 33.2 keV monochromatic X-ray. After incubation in CO₂ incubator, destruction of tumor spheroids was observed which was accompanied by apoptosis induction, as determined by the TUNEL assay. By employing the γH2AX assay, we detected double strand DNA cleavages immediately after the irradiation. These results suggest that IPO first generate double strand DNA breaks upon X-ray irradiation followed by apoptosis induction of cancer cells. Use of three different monochromatic X-rays having energy levels of 33.0, 33.2 and 33.4 keV as well as X-rays with 0.1 keV energy intervals showed that the optimum effect of all three events (spheroid destruction, apoptosis induction and generation of double strand DNA breaks) occurred with a 33.2 keV monochromatic X-ray. These results uncover the preferential effect of K-edge energy X-ray for tumor spheroid destruction mediated by iodine containing nanoparticles., アインシュタインの光電効果をがん細胞の中で再現 放射線治療への新展開. 京都大学プレスリリース. 2021-07-14., Quantum physics helps destroy cancer cells. 京都大学プレスリリース. 2021-07-14.

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.

Drug combinations have been proposed to combat drug resistance, but putative treatments are challenged by low bench-to-bed translational efficiency. To explore the effect of cell culture format and readout methods on identification of synergistic drug combinations in vitro, we studied response to 21 clinically relevant drug combinations in standard planar (2D) layouts and physiologically more relevant spheroid (3D) cultures of HCT-116, HT-29 and SW-620 cells. By assessing changes in viability, confluency and spheroid size, we were able to identify readout- and culture format-independent synergies, as well as synergies specific to either culture format or readout method. In particular, we found that spheroids, compared to 2D cultures, were generally both more sensitive and showed greater synergistic response to combinations involving a MEK inhibitor. These results further shed light on the importance of including more complex culture models in order to increase the efficiency of drug discovery pipelines.

The aim of our study was to evaluate the influence of low-intensity pulsed US on the delivery of doxorubicin (DOX) into MDA-MB-231 triple-negative breast cancer and A549 non-small cell lung cancer cell 2D and 3D cultures. US with pulse repetition frequency of 10 Hz and 1 MHz center frequency was generated with peak negative pressure of 0.5 MPa and 50% duty cycle. SonoVue microbubbles were used. Spheroids were formed using 3D Bioprinting method. DOX delivery in 2D and 3D cultures was assessed using fluorescence microscopy. US without the addition of microbubbles did not enhance the penetration of DOX into monolayer-cultured cells and tumor spheroids. In the presence of microbubbles US improved the delivery of DOX into the edge end middle zones of A549 and MDA-MB-231 spheroids. Application of low-intensity pulsed US in combination with microbubbles may be a promising approach to enhance the delivery of DOX into tumor spheroids.

Three-dimensional (3D) spheroidal cell cultures are now recognised as better models of cancers as compared to traditional cell cultures. However, established 3D cell culturing protocols and techniques are time-consuming, manually laborious and often expensive due to the excessive consumption of reagents. Microfluidics allows for traditional laboratory-based biological experiments to be scaled down into miniature custom fabricated devices, where cost-effective experiments can be performed through the manipulation and flow of small volumes of fluid. In this study, we characterise a 3D cell culturing microfluidic device fabricated from a 3D printed master. HT29 cells were seeded into the device and 3D spheroids were generated and cultured through the perfusion of cell media. Spheroids were treated with 5-Fluorouracil for five days through continuous perfusion and cell viability was analysed on-chip at different time points using fluorescence microscopy and Lactate dehydrogenase (LDH) assay on the supernatant. Increasing cell death was observed in the HT29 spheroids over the five-day period. The 3D cell culturing microfluidic device described in this study, permits on-chip anti-cancer treatment and viability analysis, and forms the basis of an effective platform for the high-throughput screening of anti-cancer drugs in 3D tumour spheroids.

3D bioprinting of living cellular constructs with heterogeneity in cell types and extra cellular matrices (ECMs) matching those of biological tissues remains challenging. Here, we demonstrate that, through bioink material design, microextrusion-based (ME) bioprinting techniques have the potential to address this challenge. A new bioink employing alginate (1%), cellulose nanocrystal (CNC) (3%), and gelatin methacryloyl (GelMA) (5%) (namely 135ACG hybrid ink) was formulated for the direct printing of cell-laden and acellular architectures. The 135ACG ink displayed excellent shear-thinning behavior and solid-like properties, leading to high printability without cell damage. After crosslinking, the ACG gel can also provide a stiff ECM ideal for stromal cell growth. By controlling the degree of substitution and polymer concentration, a GelMA (4%) bioink was designed to encapsulate hepatoma cells (hepG2), as GelMA gel possesses the desired low mechanical stiffness matching that of human liver tissue. Four different versions of to-scale liver lobule-mimetic constructs were fabricated via ME bioprinting, with precise positioning of two different cell types (NIH/3T3 and hepG2) embedded in matching ECMs (135ACG and GelMA, respectively). The four versions allowed us to exam effects of mechanical cues and intercellular interactions on cell behaviors. Fibroblasts thrived in stiff 135ACG matrix and aligned at the 135ACG/GelMA boundary due to durotaxis, while hepG2 formed spheroids exclusively in the soft GelMA matrix. Elevated albumin production was observed in the bicellular 3D co-culture of hepG2 and NIH/3T3, both with and without direct intercellular contact, indicating that improved hepatic cell function can be attributed to soluble chemical factors. Overall, our results showed that complex constructs with multiple cell types and varying ECMs can be bioprinted and potentially useful for both fundamental biomedical research and translational tissue engineering.

Heterogeneity is an intrinsic characteristic of cancer. Even in isogenic tumors, cell populations exhibit differential cellular programs that overall supply malignancy and decrease treatment efficiency. In this study, we investigated the functional relationship among cell subtypes and how this interdependency can promote tumor development in a cancer cell line. To do so, we performed single-cell RNA-seq of MCF7 Multicellular Tumor Spheroids as a tumor model. Analysis of single-cell transcriptomes at two-time points of the spheroid growth, allowed us to dissect their functional relationship. As a result, three major robust cellular clusters, with a non-redundant complementary composition, were found. Meanwhile, one cluster promotes proliferation, others mainly activate mechanisms to invade other tissues and serve as a reservoir population conserved over time. Our results provide evidence to see cancer as a systemic unit that has cell populations with task stratification with the ultimate goal of preserving the hallmarks in tumors.

Drug combinations have been proposed to combat drug resistance, but putative treatments are challenged by low bench-to-bed translational efficiency. To explore the effect of cell culture format and readout methods on identification of synergistic drug combinations in vitro, we studied response to 21 clinically relevant drug combinations in standard planar (2D) layouts and physiologically more relevant spheroid (3D) cultures of HCT-116, HT-29 and SW-620 cells. By assessing changes in viability, confluency and spheroid size, we were able to identify readout- and culture format-independent synergies, as well as synergies specific to either culture format or readout method. In particular, we found that spheroids, compared to 2D cultures, were generally both more sensitive and showed greater synergistic response to combinations involving a MEK inhibitor. These results further shed light on the importance of including more complex culture models in order to increase the efficiency of drug discovery pipelines. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

In this manuscript, a series of amine tagged short cyclic molecules (cyclopropylamine, cyclobutylamine, cyclopentylamine and cyclohexylamine) were thermally grafted onto p-type silicon (111) hydride surfaces via nucleophilic addition. The chemistries of these grafting were verified via XPS, AFM and sessile droplet measurements. Confocal microscopy and cell viability assay was performed on these surfaces incubated for 24 hours with triple negative breast cancer cells (MDA-MB 231), gastric adenocarcinoma cells (AGS) endometrial adenocarcinoma (Hec1A). All cell types had shown a significant reduction when incubated on these ring-strain cyclic monolayer surfaces than compared to standard controls. The expression level of focal adhesion proteins (vinculin, paxilin, talin and zyxin) were subsequently quantified for all three cell types via qPCR analysis. Cells incubate on these surface grafting were observed to have reduced levels of adhesion protein expression than compared to positive controls (collagen coating and APTES). A potential application of these anti-adhesive surfaces is the maintenance of the chondrocyte phenotype during in-vitro cell expansion. Articular chondrocytes cultured for 6 days on ring strained cyclopropane-modified surfaces was able to proliferate but had maintained a spheroid/aggregated phenotype with higher COL2A1 and ACAN gene expression. Herein, these findings had help promote grafting of cyclic monolayers as an viable alternative for producing antifouling surfaces.

Neuronal injury leads to rapid, programmed disintegration of axons distal to the site of lesion. Much like other forms of axon degeneration (e.g. developmental pruning, toxic insult from neurodegenerative disorder), Wallerian degeneration associated with injury is preceded by spheroid formation along axons. The mechanisms by which injury leads to formation of spheroids and whether these spheroids have a functional role in degeneration remain elusive. Here, using neonatal mouse primary sympathetic neurons, we investigate the roles of players previously implicated in the progression of Wallerian degeneration in injury-induced spheroid formation. We find that intra-axonal calcium flux is accompanied by actin-Rho dependent growth of calcium rich axonal spheroids that eventually rupture, releasing material to the extracellular space prior to catastrophic axon degeneration. Importantly, after injury, Sarm1−/− and DR6−/−, but not Wlds (excess NAD+) neurons, are capable of forming spheroids that eventually rupture, releasing their contents to the extracellular space to promote degeneration. Supplementation of exogenous NAD+ or expressing WLDs suppresses Rho-dependent spheroid formation and degeneration in response to injury. Moreover, injured or trophically deprived Sarm1−/− and DR6−/−, but not Wlds neurons, are resistant to degeneration induced by conditioned media collected from wild-type axons after spheroid rupture. Taken together, these findings place Rho-actin and NAD+ upstream of spheroid formation and may suggest that other mediators of degeneration, such as DR6 and SARM1, mediate post-spheroid rupture events that lead to catastrophic axon disassembly.

In this report, a strategy for constructing three-dimensional (3D) cellular architectures comprising viable cells is presented. The strategy uses a redox-responsive hydrogel that degrades under mild reductive conditions, and a confluent monolayer of cells (i.e., cell sheet) cultured on the hydrogel surface peels off and self-folds to wrap other cells. As a proof-of-concept, the self-folding of fibroblast cell sheet was triggered by immersion in aqueous cysteine, and this folding process was controlled by the cysteine concentration. Such folding enabled the wrapping of human hepatocellular carcinoma (HepG2) spheroids, human umbilical vein endothelial cells and collagen beads, and this process improved cell viability, the secretion of metabolites and the proliferation rate of the HepG2 cells when compared with a two-dimensional culture under the same conditions. A key concept of this study is the ability to interact with other neighbouring cells, providing a new, simple and fast method to generate higher-order cellular aggregates wherein different types of cellular components are added. We designated the method of using a cell sheet to wrap another cellular aggregate the ‘cellular Furoshiki’. The simple self-wrapping Furoshiki technique provides an alternative approach to co-culture cells by microplate-based systems, especially for constructing heterogeneous 3D cellular microstructures.

In this work, the influence of parylene N film on the spheroid formation of osteoblast-like cells (MG-63) was determined and compared with that of high-hydrophilicity microenvironments, such as hydrophilic culture matrix and ultraviolet-treated parylene N film. To elucidate the change in cell properties due to the microenvironment of parylene N film, global gene expression profiles of MG-63 cells on parylene N film were analyzed. We confirmed the upregulated expression of osteoblast differentiation- and proliferation-related genes, such as Runx2, ALPL, and BGLAP and MKi67 and PCNA, respectively, using the real-time polymerase chain reaction. In addition, the differentiation and proliferation of osteoblast cells cultured on parylene N film were validated using immunostaining. Finally, the formation of spheroids and regulation of differentiation in human mesenchymal stem cells (MSCs) on parylene N film was demonstrated. The results of this study confirm that the microenvironment with the controlled hydrophobic property of parylene N film could effectively trigger the bone differentiation and maintains the proliferation of MSCs, similar to MG-63 cells without any scaffold structures or physical treatments.

PRK1 and PRK2 are two closely related AGC-family serine/threonine protein kinases. Here we demonstrate novel roles for them at cilia and in cancer biology. In both instances serum withdrawal leads to increased activating PRK1 and PRK2 phosphorylation (pPRK1/pPRK2) and their depletion results in reduced spheroid growth. pPRK1/pPRK2 localise to the transition zone of cilia and their co-depletion results in reduced cilia size, impaired planer polarity and impaired cilia associated signalling. High PRK2 (but not PRK1) expression correlates with poor outcome in patients with basal-like/Triple Negative (TN) Breast Cancer (BC) where there is also higher expression relative to other BC tumour subtypes. In agreement, depletion of PRK1 and PRK2 in mouse TNBC cells, or CRISPR/Cas9 mediated deletion of PRK2 alone, significantly reduces cell proliferation and spheroid growth. Finally proteomic analysis to identify PRK2 binding partners in mouse TNBC cells revealed proteins that are important for both cilia and BC biology. Taken together these data demonstrate novel roles for PRK1 and PRK2 at cilia and in BC biology and in the case of PRK2 in particular, identifies it as a novel TNBC therapeutic target.

Hepatocellular carcinoma (HCC), one of the most common malignant cancers worldwide, is associated with substantial mortality. Because HCCs have strong resistance to conventional chemotherapeutic agents, novel therapeutic strategies are needed to improve survival in patients with HCC. The multicellular tumor spheroid (MCTS) model is a powerful method for anticancer research because of its ability to mimic the complexity and heterogeneity of tumor tissue, the three-dimensional cellular context of tumor tissue, and the pathophysiological gradients of in vivo tumors. However, it is difficult to obtain meaningful results from the MCTS model without considering the conditions of clinical tumors. We, therefore, provided a proof of concept to determine whether spheroid models simulate in vivo tumor microenvironments. Through a high-throughput screening for HCC therapy using the MCTS model, we selected inhibitors of Na+/K+-ATPase (ouabain and digoxin) that could suppress cell growth and migration via inhibition of the epithelial-mesenchymal transition of HCC in vivo and in vitro. The results showed that this model provides a new paradigm for high-throughput drug screening and will significantly improve the efficiency of identifying new drugs for HCC treatment. Through utilization of MCTS models, here we found that inhibitors of Na+/K+-ATPase may be feasible as a novel target to sensitize HCC cells.

For multimodality therapies such as the combination of hyperthermia and radiation, quantification of biological effects is key for dose prescription and response prediction. Tumour spheroids have a microenvironment that more closely resembles that of tumours in vivo and may thus be a superior in vitro cancer model than monolayer cultures. Here, the response of tumour spheroids formed from two established human cancer cell lines (HCT116 and CAL27) to single and combination treatments of radiation (0–20 Gy), and hyperthermia at 47 °C (0–780 CEM43) has been evaluated. Response was analysed in terms of spheroid growth, cell viability and the distribution of live/dead cells. Time-lapse imaging was used to evaluate mechanisms of cell death and cell detachment. It was found that sensitivity to heat in spheroids was significantly less than that seen in monolayer cultures. Spheroids showed different patterns of shrinkage and regrowth when exposed to heat or radiation: heated spheroids shed dead cells within four days of heating and displayed faster growth post-exposure than samples that received radiation or no treatment. Irradiated spheroids maintained a dense structure and exhibited a longer growth delay than spheroids receiving hyperthermia or combination treatment at (thermal) doses that yielded equivalent levels of clonogenic cell survival. We suggest that, unlike radiation, which kills dividing cells, hyperthermia-induced cell death affects cells independent of their proliferation status. This induces microenvironmental changes that promote spheroid growth. In conclusion, 3D tumour spheroid growth studies reveal differences in response to heat and/or radiation that were not apparent in 2D clonogenic assays but that may significantly influence treatment efficacy.