Use Of Spheroids Research Articles

Single and Combination Drug Screening with Aqueous Biphasic Tumor Spheroids

Spheroids of cancer cells represent a physiologic model of solid tumors for cancer drug screening. Despite this known benefit, difficulties with generating large quantities of uniformly sized spheroids in standard plates, individually addressing spheroids with drug compounds, and quantitatively analyzing responses of cancer cells have hindered the use of spheroids in high-throughput screening applications. Recently, we addressed this challenge by using an aqueous two-phase system technology to generate a spheroid within an aqueous drop immersed in a second, immiscible aqueous phase. Integrating this approach with robotics resulted in convenient formation, maintenance, and drug treatment of spheroids. Here, we demonstrate the feasibility of high-throughput compound screening against colon cancer spheroids using 25 anticancer compounds. Using a strictly standardized mean difference and based on a preliminary testing with each compound, we select effective compounds for further dose-response testing. Finally, we use molecular inhibitors to target upregulated protein kinases and use them for drug combination studies against spheroids. We quantitatively analyze the combination treatment results using statistical metrics to identify synergy between pairs of inhibitors in compromising viability of colon cancer cells. This study demonstrates the utility of our spheroid culture technology for identification of effective drug compounds, dose-response analysis, and combination drug treatments.

An Early Stone Age in Western Africa? Spheroids and polyhedrons at Ounjougou, Mali

Ounjougou stratigraphic sequence (Bandiagara, Dogon Country, Mali) is the most complete record in Western Africa for the Middle Pleistocene. This paper focuses on the lithic industry unearthed in the lowest levels of the sequence. Despite the impossibility to fix the dating of those layers, the assemblage clearly presents Oldowan features. A strong erosive process, combined to the absence of Acheulean industry, strengthens the idea of a probable ancient age for the lithic industry. Morphometric, Technological and techno-functional approaches were performed to study sandstone polyhedrons, spheroids and bolas, abundant in the collection, along with a flake production on quartz and quartzite pebbles.
 This study demonstrates that polyhedrons, spheroids and bolas were shaped from independent chaînes opératoires to realize specific tasks. The hypothesis of opportunistic knapping does not fit with these materials at Ounjougou. The evidence of shaping is highlighted by diacritic schemes showing that the flakes detached are not controlled for their usability, but for shaping the morphology of the spheroid. Apart from those polyhedrons and spheroids, flake production is also identified. While the shaping process is made on sandstone cobbles, sharp flakes are produced from quartz pebbles. This clear choice of different raw materials to produce shaped heavy tools in one hand and light flake tools in another hand strengthens the idea of a deliberate shaping of spheroid tools.
 The discussion compares this assemblage with other known assemblages like Ain Hanech and Olducai Gorge. Our point focus on the methodology used to study these specific artefacts, very different from one author to another. We stress out the fact that using the term ‘polyhedron’ is not accurate to define artefacts that can refer to shape tools or multifacial cores. We suggest to avoid using it and to focus on a technical identification to name the artefacts as cores or tools. The technological and techno-functional approaches are relevant to make this distinction.
 Then we propose some hypothesis about the use of spheroid and bolas and suggest experiments and traceological analyses in the future to confirm their functionality.
 Ounjougou lithic tool kit, composed of flake débitage, retouched flakes and shaped tools on pebbles, along with the spheroids and bolas component, gives an evidence of a diversified tool kit corresponding to the Oldowan Industrial Complex, and then represents the first Early Stone Age site in stratigraphy in Western Africa.

Liquid-based three-dimensional tumor models for cancer research and drug discovery.

Tumors are three-dimensional tissues where close contacts between cancer cells, intercellular interactions between cancer and stromal cells, adhesion of cancer cells to the extracellular matrix, and signaling of soluble factors modulate functions of cancer cells and their response to therapeutics. Three-dimensional cultures of cancer cells overcome limitations of traditionally used monolayer cultures and recreate essential characteristics of tumors such as spatial gradients of oxygen, growth factors, and metabolites and presence of necrotic, hypoxic, quiescent, and proliferative cells. As such, three-dimensional tumor models provide a valuable tool for cancer research and oncology drug discovery. Here, we describe different tumor models and primarily focus on a model known as tumor spheroid. We summarize different technologies of spheroid formation, and discuss the use of spheroids to address the influence of stromal fibroblasts and immune cells on cancer cells in tumor microenvironment, study cancer stem cells, and facilitate compound screening in the drug discovery process. We review major techniques for quantification of cellular responses to drugs and discuss challenges ahead to enable broad utility of tumor spheroids in research laboratories, integrate spheroid models into drug development and discovery pipeline, and use primary tumor cells for drug screening studies to realize personalized cancer treatment.

Methods: Using Three-Dimensional Culture (Spheroids) as an In Vitro Model of Tumour Hypoxia.

Regions of hypoxia in tumours can be modelled in vitro in 2D cell cultures with a hypoxic chamber or incubator in which oxygen levels can be regulated. Although this system is useful in many respects, it disregards the additional physiological gradients of the hypoxic microenvironment, which result in reduced nutrients and more acidic pH. Another approach to hypoxia modelling is to use three-dimensional spheroid cultures. In spheroids, the physiological gradients of the hypoxic tumour microenvironment can be inexpensively modelled and explored. In addition, spheroids offer the advantage of more representative modelling of tumour therapy responses compared with 2D culture. Here, we review the use of spheroids in hypoxia tumour biology research and highlight the different methodologies for spheroid formation and how to obtain uniformity. We explore the challenge of spheroid analyses and how to determine the effect on the hypoxic versus normoxic components of spheroids. We discuss the use of high-throughput analyses in hypoxia screening of spheroids. Furthermore, we examine the use of mathematical modelling of spheroids to understand more fully the hypoxic tumour microenvironment.

Autophagy Protects from Trastuzumab-Induced Cytotoxicity in HER2 Overexpressing Breast Tumor Spheroids.

Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance.

Abstract 696: ASS1, ANXA4 and MVP underlie acquired multicellular resistance of mesothelioma 3D cell cultures

Abstract Cancer cells are usually studied in flat 2D monolayers however, solid tumors, such as malignant mesothelioma, grow as 3D masses. When tumor cells are grown as 3D structures, they acquire multi-cellular resistance to apoptosis, resembling the stubborn chemoresistance seen in patients. In this study, we have identified a gene expression signature that is specific for 3D spheroids, possibly underlying multicellular resistance. By comparing this signature with the data from two sets of publicly-available gene expression datasets of patients’ specimens we have identified three genes in common, genes that may play a role in multicellular resistance: ASS1, ANXA4 and MVP. Moreover, we have studied the levels of the proteins encoded by the three genes and have begun investigating how their ablation by RNAi influences the response to chemotherapy. Furthemore, by Connectivity Analysis, we have found two safe and patent-free candidate drugs that can reduce the expression levels of the three genes and undermine the acquired chemoresistance of spheroids. Such drugs would have potential as adjuncts to chemotherapy. These studies will not only clarify the role of ASS1, ANXA4 and MVP in mesothelioma and provide therapeutic insights but also advance the use of spheroids as a more realistic model for studying tumor biology. Citation Format: Dario Barbone, Loes van Dam, Shu-Dong Zhang, Jithesh Puthen, Dean Fennell, V. Courtney Broaddus. ASS1, ANXA4 and MVP underlie acquired multicellular resistance of mesothelioma 3D cell cultures. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 696. doi:10.1158/1538-7445.AM2014-696

Optimization of Aqueous Biphasic Tumor Spheroid Microtechnology for Anti-Cancer Drug Testing in 3D Culture.

Tumor spheroids are three-dimensional clusters of cancer cells that exhibit characteristics of poorly perfused tumors and hence present a relevant model for testing the efficacy of anti-cancer compounds. The use of spheroids for drug screening is hindered by technological complexities for high throughput generation of consistent size spheroids individually addressable by drug compounds. Here we present and optimize a simple spheroid technology based on the use of an aqueous two-phase system. Cancer cells confined in a drop of the denser aqueous dextran phase are robotically dispensed into a microwell containing the immersion aqueous polyethylene glycol phase. Cells remain within the drop and form a viable spheroid, without a need for any external stimuli. The size of resulting spheroids is sensitive to volume variations of dispensed drops from the air displacement pipetting head of a commercial liquid handling robot. Therefore, we parametrically optimize the process of dispensing of dextran phase drops. For a given cell density, this optimization reproducibly generates consistent size spheroids in standard 96-well plates. In addition, we evaluate the use of a commercial biochemical assay to examine cellular viability of cancer cell spheroids. Spheroids show a dose-dependent response to cisplatin similar to a monolayer culture. However unlike their two-dimensional counterpart, spheroids exhibit resistance to paclitaxel treatment. This technology, which uses only commercially-available reagents and equipment, can potentially expedite anti-cancer drug discovery. Although the use of robotics makes the ATPS spheroid technology particularly useful for drug screening applications, this approach is compatible with simpler liquid handling techniques such as manual micropipetting and offers a straightforward method of 3D cell culture in research laboratories.

Multicellular Tumor Spheroids for Evaluation of Cytotoxicity and Tumor Growth Inhibitory Effects of Nanomedicines In Vitro: A Comparison of Docetaxel-Loaded Block Copolymer Micelles and Taxotere®

While 3-D tissue models have received increasing attention over the past several decades in the development of traditional anti-cancer therapies, their potential application for the evaluation of advanced drug delivery systems such as nanomedicines has been largely overlooked. In particular, new insight into drug resistance associated with the 3-D tumor microenvironment has called into question the validity of 2-D models for prediction of in vivo anti-tumor activity. In this work, a series of complementary assays was established for evaluating the in vitro efficacy of docetaxel (DTX) -loaded block copolymer micelles (BCM+DTX) and Taxotere® in 3-D multicellular tumor spheroid (MCTS) cultures. Spheroids were found to be significantly more resistant to treatment than monolayer cultures in a cell line dependent manner. Limitations in treatment efficacy were attributed to mechanisms of resistance associated with properties of the spheroid microenvironment. DTX-loaded micelles demonstrated greater therapeutic effect in both monolayer and spheroid cultures in comparison to Taxotere®. Overall, this work demonstrates the use of spheroids as a viable platform for the evaluation of nanomedicines in conditions which more closely reflect the in vivo tumor microenvironment relative to traditional monolayer cultures. By adaptation of traditional cell-based assays, spheroids have the potential to serve as intermediaries between traditional in vitro and in vivo models for high-throughput assessment of therapeutic candidates.

3D Spheroids’ Sensitivity to Electric Field Pulses Depends on Their Size

Dramatic differences of cells behavior exist between cells cultured under classical 2D monolayers and 3D models, the latter being closer to in vivo responses. Thus, many 3D cell culture models have been developed. Among them, multicellular tumor spheroid appears as a nice and easy-to-handle 3D model based on cell adhesion properties. It is composed of one or several cell types and is widely used to address carcinogenesis, or drugs screening. A few and recent publications report the use of spheroids to investigate electropermeabilization process. We studied the response of spheroids to electrical field pulses (EP) in terms of their age, diameter or formation technique. We found that small human HCT-116 colorectal spheroids are more sensitive to electric field pulses than larger ones. Indeed, the growth of spheroids with a diameter of 300μm decreased by a factor 2 over 4days when submitted to EP (8 pulses, lasting 100μs at a 1,300V/cm field intensity). Under those electrical conditions, 650μm spheroids were not affected. These data were the same whatever the formation method (i.e. hanging drop and nonadherent techniques). These observations point out the fact that characteristics of 3D cell models have to be taken into account to avoid biased conclusions of experimental data.

Accumulation and toxicity of antibody-targeted doxorubicin-loaded PEG–PE micelles in ovarian cancer cell spheroid model

We describe the evaluation of doxorubicin-loaded PEG–PE micelles targeting using an ovarian cancer cell spheroid model. Most ovarian cancer patients present at an advanced clinical stage and develop resistance to standard of care platinum/taxane therapy. Doxorubicin is also approved for ovarian cancer but had limited benefits in refractory patients. In this study, we used drug-resistant spheroid cultures of ovarian carcinoma to evaluate the uptake and cytotoxicity of an antibody-targeted doxorubicin formulation. Doxorubicin was encapsulated in polyethylene glycol–phosphatidyl ethanolamine (PEG–PE) conjugated micelles. The doxorubicin-loaded PEG–PE micelles (MDOX) were further decorated with a cancer cell-specific monoclonal 2C5 antibody to obtain doxorubicin-loaded immunomicelles (2C5-MDOX). Targeting and resulting toxicity of doxorubicin-loaded PEG–PE micelles were evaluated in three dimensional cancer cell spheroids. Superior accumulation of 2C5-MDOX compared to free doxorubicin or untargeted MDOX in spheroids was evidenced both by flow cytometry, fluorescence and confocal microscopy. Interestingly, even higher toxicity was measured by lactate dehydrogenase release and terminal deoxynucleotidyl transferase dUTP nick end labeling of targeted doxorubicin micelles in Bcl-2 overexpressing adriamycin-resistant spheroids. Overall, these results support use of spheroids to evaluate tumor targeted drug delivery.

Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy

Multicellular spheroids are three dimensional in vitro microscale tissue analogs. The current article examines the suitability of spheroids as an in vitro platform for testing drug delivery systems. Spheroids model critical physiologic parameters present in vivo, including complex multicellular architecture, barriers to mass transport, and extracellular matrix deposition. Relative to two-dimensional cultures, spheroids also provide better target cells for drug testing and are appropriate in vitro models for studies of drug penetration. Key challenges associated with creation of uniformly sized spheroids, spheroids with small number of cells and co-culture spheroids are emphasized in the article. Moreover, the assay techniques required for the characterization of drug delivery and efficacy in spheroids and the challenges associated with such studies are discussed. Examples for the use of spheroids in drug delivery and testing are also emphasized. By addressing these challenges with possible solutions, multicellular spheroids are becoming an increasingly useful in vitro tool for drug screening and delivery to pathological tissues and organs.

Abstract B49: Cancer cell spheroids as a model to study sequential chemotherapy protocols

Abstract This study aimed at comparing six combinations of priming drug / doxorubicin (DOX) in a spheroid model. Three-dimensional cultures of drug-resistant cancer cells (spheroids) are physiologically relevant[1] and have been used as models of limited drug penetration[2]. Enhanced tumor accumulation and therapeutic efficacy of liposomal DOX after apoptosis induction by paclitaxel (PTX) has been reported[3]. Moreover, mitochondrial depolarization is correlated with clinical response[4]. Here we proposed use of spheroids to evaluate sequential chemotherapy combinations for potentiation of DOX cytotoxicity after pretreatment with cytotoxic concentrations of clinically relevant drugs. To this aim, 400–500 μm NCI-ADR-RES (ovarian carcinoma) were used. We first incubated spheroids 48h with mitoxantrone (MXO), cisplatin (CIS), dexamethasone (DXM), methotrexate (MTX), sclareol (SCL) and PTX as first line (priming) agents at concentrations inducing a 1.5- to 2.5-fold lactate dehydrogenase (LDH) release compared to untreated cells. For DOX accumulation studies, these were further cultured in the presence of 25 μM of DOX during 24h before determination of DOX content (nanomoles DOX/mg of proteins) by fluorescence. For DOX cytotoxicity evaluation, pretreated spheroids were incubated 72h with 100 μM of DOX before assessment of viability with a Cytotox 96 nonradioactive cell viability kit (Promega). Three DOX forms were used: free drug (DOX), distearyl1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino(polyethylene glycol)-2000] / DOX micelles (MDOX, 13 nm) prepared according to [5, 6], and liposomal DOX (LDOX, Lipo-Dox®, 130 nm, Sun Pharma India). The accumulation pattern of these forms was DOX = MDOX > LDOX as previously reported[7]. We document enhanced DOX, MDOX and LDOX accumulation after spheroids pretreatment with mitoxantrone (2.8-; 1.8- and 2.5-fold, respectively) and increased distribution of DOX and MDOX in PTX primed spheroids (2- and 1.4-fold, respectively). CIS, DXM, SCL and MTX failed at enhancing free, micellar or liposomal DOX distribution in spheroids. Improved DOX accumulation obtained with MXO and PTX resulted in respectively 1.4- and 2.2-fold higher LDH release compared to spheroids treated only with DOX. Interestingly, although preincubation with SCL did not promote DOX, MDOX or LDOX accumulation, LDH release of spheroids cultured with SCL before DOX treatment was 2.5-fold higher than ones treated only with DOX. While LDOX toxicity was not increased by any pretreatment, viability of cells incubated with MDOX decreased further when pretreated with PTX and MXO (1.5- and 1.8-fold compared to MDOX respectively). Low LDOX toxicity is in agreement with [7] and may be due to insufficient DOX release[8]. We report for the first time use of spheroids to test DOX toxicity potentiation as free, micellar or liposomal forms. Out of the six inducers tested, three (PTX, MXO and SCL) enhanced DOX accumulation and/or toxicity. Results suggest different mechanisms of priming for these three drugs and support sequential chemotherapy with PTX, MXO or SCL and DOX. Acknowledgments: This work was supported by grant CCNE IUCA151881 to V.P. Torchilin.

Three-dimensional cell organization leads to a different type of ionizing radiation-induced cell death: MG-63 monolayer cells undergo mitotic catastrophe while spheroids die of apoptosis

The type of cell death occurring in the same cell line (MG-63 human osteosarcoma cells) grown in monolayer or as three-dimensional spheroids after exposure to 5 Gy of ionizing radiation was determined. Morphological analyses using the chromatin dye Hoechst 33258 demonstrated that spheroids showed the typical characteristics of apoptosis, while monolayer cells revealed those typical of mitotic catastrophe. In order to better characterize these two types of cell death, the role of caspases was examined in irradiated monolayer cells and spheroids using the broad spectrum caspase inhibitor zVAD-fmk. Death in monolayer cells was caspase-independent, whereas spheroid death was characterized by caspase dependence. Members of the Bcl-2 family of proteins and survivin involved in cell death processes were also studied by Western blot analysis. The pro-apoptotic protein Bax increased in spheroids, whereas this protein remained unchanged in monolayer cells after the same 5-Gy irradiation. The anti-apoptotic protein Bcl-2, on the other hand, remained unchanged in both monolayer cells and spheroids. Finally, survivin increased significantly after irradiation in both cells in monolayer and spheroids. The results presented suggest that three-dimensional cell organization leads to a different type of cell death after exposure to ionizing radiation. Thus, the use of spheroids, a cell model which mimics in vivo solid tumors more closely than cells grown in monolayer, is more appropriate when investigating the effects of antineoplastic treatments such as ionizing radiation.

Engineering Blood Vessels from Lumenized Vascular Tissue Spheroids

We have applied the principle of VEGF-mediated vascular fusion (Drake and Little, Proc Natl Acad Sci, 1995) to engineer blood vessels. Using the murine allantois as a source of endothelial precursor cells, spheroids were generated in hanging drop culture (HDC) that consist of an inner network of capillary-sized vessels (CSV) and outer layers of alpha smooth muscle actin-positive cells (SMAC). To transform spheroids into blood vessel-like structures (BVLS), VEGF was used to induce the fusion of the CSV. This resulted in the formation of BVLS having a single endothelial-lined lumen (dia. >300μm) surrounded by SMAC. Further, when two spheroids were exposed to VEGF in HDC, a single BVLS with an increased luminal diameter was formed. Essential to the use of spheroids in vascular engineering are 3D-matrices that are permissive for both spheroid formation and vascular fusion. In studies evaluating a collagen hydrogel (CH) versus a cross-linkable hyaluronan hydrogel (HH), we observed that HH but not CH was permissive for the formation of BVLS from spheroids. Finally, to demonstrate the utility of lumenized spheroids as modules to engineer blood vessels, we show that when multiple spheroids were exposed to VEGF in HH, they formed a single lumenized vascular tube-like structure surrounded by SMAC. These findings validate our mathematical model of lumenized vascular spheroid fusion and demonstrate the feasibility of a tissue self-assembly approach as a means of engineering blood vessels.

Direct Self-assembly of Hepatocytes Spheroids within Hollow Fibers in Presence of Collagen

Opposite to the established view that collagen is an extracellular substratum for only dispersed hepatocyte culture, hepatocyte spheroids were directly formed within hollow fibers by addition of moderate concentrations of soluble collagen. Morphologically, these spheroids indicated a close relationship with their in vivo structure of liver. The albumin and urea synthetic profiles confirmed that those spheroids maintained liver-specific functions for at least 8 days. Spheroid formation by addition of collagen not only presents a potential methodology for clinical use of spheroids in bioartificial liver device but also indicates a likely function of collagen for self-assembly of primary cells in tissue engineering.

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: II. nitric oxide synthesis and related changes.

Liver cells isolated from intact tissue can reaggregate to form three-dimensional, multicellular spheroids in vitro. During this process, cells undergo a histological and environmental change. How cells respond biochemically to this change has not been studied in detail previously. We have investigated some biochemical changes in rat liver cells during the formation and maintenance of spheroids. Liver cells were isolated from male Sprague rats and spheroids cultured by a gyrotatory-mediated method. Liver cells were shown to respond to the isolation procedure and the formation of spheroids triggered histological environmental changes that increased arginine uptake, nitric oxide (NO) and urea syntheses, as well as raised levels of GSH, GSSG, glutamic acid and aspartic acid secretion within the first couple of days after cell isolation. Levels were maintained at a relatively stable level in the mature spheroids (>5 days) over the 3 week period of observation. P450 1A1 activity was lost in the first 2 days and gradually recovered thereafter. This study, for the first time, shows that liver cells after isolation and during spheroid formation actively uptake arginine and increase NO and urea syntheses. A high level of NO is likely to play an important role in modulating a series of biochemical changes in liver cells. It is considered that liver cells actively respond to the 'challenge' induced by the isolation procedure and subsequent histological environmental changes, and biochemical modulation and instability result. The stable cell-cell contacts and histological environment in mature spheroids permit and support functional recovery and maintenance in vitro. This period of stability permits the use of spheroids in toxicity studies to establish acute and chronic paradigms.

Proton NMR microscopy of multicellular tumor spheroid morphology

We report proton NMR images obtained at microscopic (less than 30 microns) resolution of EMT6/Ro and HT1080 multicellular tumor spheroids 1.2-1.7 mm in diameter. T1-weighted images showed little contrast across a slice through the spheroid. There was also no difference between the inner and outer spheroid regions when signal intensity was measured as a function of the repetition time (TR), showing that T1 was the same across the spheroid. Conversely, T2-weighted and multi-echo images clearly revealed the central necrosis that occurs as the spheroids develop. Measurements of the thickness of the viable cell zone made on NMR images agreed with those made on standard histology sections for two different cell lines. The basis for the NMR discrimination of the necrotic region from the viable rim cells was found to be a shortened apparent T2 in the necrotic region (132 +/- 17 ms) with respect to that in the viable cells (173 +/- 9 ms). These results illustrate the applicability of NMR microscopy to assaying conditions inside intact tumor spheroids and suggest that this technology will allow the use of spheroids to investigate several important questions in tumor biology and pathophysiology.

Proton NMR Microscopy of Multicellular Tumor Spheroid Morphology

We report proton NMR images obtained at microscopic (<30 μm) resolution of EMT6/Ro and HT1080 multicellular tumor spheroids 1.2–1.7 mm in diameter. T1‐weighted images showed little contrast across a slice through the spheroid. There was also no difference between the inner and outer spheroid regions when signal intensity was measured as a function of the repitition time (TR), showing that T1 was the same across the spheroid. Conversely, T2‐weighted and multiecho images clearly revealed the central necrosis that occurs as the spheroids develop. Measurements of the thickness of the viable cell zone made on NMR images agreed with those made on standard histology sections for two different cell lines. The basis for the NMR discrimination of the necrotic region from the viable rim cells was found to be a shortened apparent T2 in the necrotic region (132 ± 17 ms) with respect to that in the viable cells (173 ± 9 ms). These results illustrate the applicability of NMR microscopy to assaying conditions inside intact tumor spheroids and suggest that this technology will allow the use of spheroids to investigate several important questions in tumor biology and pathophysiology.

Model of diffusion of oxygen to spheroids grown in stationary medium—I. Complete spherical symmetry

The use of spheroids as a tumor model has become commonplace since it was discovered that many cell lines can form spheroids when grown on a surface to which the cells cannot attach. This culture system complicates experiments which depend on oxygen supply because the oxygen concentration in the vicinity of a stationary spheroid has not been well defined. We present in this paper solutions to the oxygen diffusion equation for simple geometries: a spheroid in an infinite stationary medium and in a finite spherical stationary medium. Comparison of these solutions provides an estimate of the oxygen supply to a spheroid in a Petri dish. We show that typical spheroids can be expected to cause a substantial depletion of the oxygen in the nearby medium. Any disturbance of the medium or the spheroids will temporarily increase the oxygen supply. We provide a method for estimating the rate of return to equilibrium in the finite cases. These results indicate that the oxygen supply to stationary spheroids can be altered temporarily by small movements or changes in temperature which cause convection currents, or permanently by changes in the depth of the medium.