Method for the determination of oxygen consumption rates and diffusion coefficients in multicellular spheroids

A method has been developed for the quantitative evaluation of oxygen tension (PO2) distributions in multicellular spheroids measured with O2-sensitive microelectrodes. The experimental data showed that multicellular tumor spheroids in stirred growth media were characterized by a diffusion-depleted zone surrounding the spheroids. This zone was elicited by an unstirred layer of medium next to the spheroid leading to a continuous decrease in the PO2 values from the bulk medium towards the spheroid surface. Theoretical considerations demonstrate that the volume-related O2 consumption rate, Q, in the spheroids can be assessed by measuring the PO2 gradient in the diffusion-depleted zone outside the spheroids. Accordingly, Krogh's diffusion constant, KS, in the spheroids can be determined through measuring the PO2 gradient within the spheroids. The results obtained suggest that multicellular spheroids represent useful in vitro tumor models for the experimental and theoretical analysis of the interrelationship among O2 supply to tumor cells, O2 metabolism in tumors tissue, and the responsiveness of cancer cells to treatment.     

ATP concentrations in multicellular tumor spheroids assessed by single photon imaging and quantitative bioluminescence

To evaluate the interrelationship among the cellular energy status and the development of necrosis in tumor microregions, local ATP concentrations and the extent of necrosis were determined in multicellular tumor spheroids, i.e., in spherical tumor cell aggregates. The spheroids were grown in rotated suspension cultures using EMT6 cells that were derived from a murine mammary sarcoma. The distribution of viable and necrotic cell areas was assessed by histological investigations. The regional distribution of ATP concentrations was measured with a novel technique using quantitative bioluminescence and single photon imaging. This method makes it possible to determine ATP concentrations in absolute terms with a spatial resolution at the level of a single cell. The results show that ATP concentrations in the center of EMT6 spheroids decrease from values of 1.0 to 1.5 mM in small spheroids with 300 microns in diameter to values close to or at the background level in 750 microns spheroids. Necrosis was detectable in spheroids larger than 300 microns, and virtually no spheroid without necrosis was found at sizes larger than 600 microns. Since the emergence of central necrosis precedes the drop in ATP to undetectably low values, the data suggest that energy metabolism is not or not directly involved in the development of necrosis in tumor spheroids under the growth conditions investigated.     

Distributions of oxygen,nutrient, and metabolic waste concentrations in multicellular spheroids and their dependence on spheroid parameters

The distribution of oxygen, nutrients and metabolic wastes in multicellular tumor spheroids and its dependence on the parameters characterizing the spheroid (i.e., spheroid geometry, diffusivity, and consumption/ production rates of biological substances) have been investigated by a theoretical analysis: 1. Parameter dependence is qualitatively demonstrated and visualized. 2. Reduction of the number of variables by specific coordinate transformations made it possible to generate nomograms from which concentration distributions for any choice of parameter values may easily be obtained. In particular, these nomograms may also be used for estimating concentration profiles of metabolic waste products, e.g. of lactate, which are expected to accumulate in the tumor spheroids. 3. An additional set of nomograms is given which is more convenient for determining time courses of these concentrations during spheroid growth. 4. A quantitative sensitivity analysis of parameter dependencies is performed to identify those parameters upon which a concentration of interest depends most critically in a given experimental situation. Offprint requests to: W Mueller-Klieser     

Use of an image restoration process to improve spatial resolution in bioluminescence imaging

To improve spatial resolution in in vivo bioluminescence imaging, a photon scattering correction, image restoration method was tested. The chosen algorithm was tested on in vivo bioluminescent images acquired on three representative tumor models: subcutaneous, pulmonary, and disseminated peritoneal. Tumor size was chosen as a quantitative criterion, such that the tumor reference measurements (determined photographically or by computed tomography) were compared to those derived from bioluminescent images, before and after restoration. This technique allowed a significant reduction to be achieved in the relative error between reference measurements and dimensions derived from bioluminescent images. In addition, improved delineation of the tumor foci was achieved. The restoration method allows spatial resolution in bioluminescence imaging to be improved, with interesting perspectives in terms of staging and quantitation in experimental oncology.     

Live cell division dynamics monitoring in 3D large spheroid tumor models using light sheet microscopy

Background

Multicellular tumor spheroids are models of increasing interest for cancer and cell biology studies. They allow considering cellular interactions in exploring cell cycle and cell division mechanisms. However, 3D imaging of cell division in living spheroids is technically challenging and has never been reported.

Results

Here, we report a major breakthrough based on the engineering of multicellular tumor spheroids expressing an histone H2B fluorescent nuclear reporter protein, and specifically designed sample holders to monitor live cell division dynamics in 3D large spheroids using an home-made selective-plane illumination microscope.

Conclusions

As illustrated using the antimitotic drug, paclitaxel, this technological advance paves the way for studies of the dynamics of cell divion processes in 3D and more generally for the investigation of tumor cell population biology in integrated system as the spheroid model.     

Engineering Lipid Vesicles of Enhanced Intratumoral Transport Capabilities: Correlating Liposome Characteristics with Penetration into Human Prostate Tumor Spheroids

Liposomes have been widely used delivery systems, particularly relevant to the development of cancer therapeutics. Numerous liposome-based drugs are in the clinic or in clinical trials today against multiple tumor types; however, systematic studies of liposome interactions with solid or metastatic tumor nodules are scarce. This study is describing the in vitro interaction between liposomes and avascular human prostate (LNCaP-LN3) tumor spheroids. The ability of fluorescently labelled liposomal delivery systems of varying physicochemical characteristics to penetrate within multicellular tumor spheroids has been investigated by confocal laser scanning microscopy. A variety of liposome characteristics and experimental parameters were investigated, including lipid bilayer composition, duration of liposome-spheroid interaction, mean liposome size, steric stabilization of liposomes. Electrostatic binding between cationic liposomes and spheroids was very efficient; however, it impeded any significant penetration of the vesicles within deeper layers of the tumor spheroid. Small unilamellar liposomes of neutral surface character did not bind as efficiently but exhibited enhanced penetrative transport capabilities closer to the tumor core. Polymer-coated (sterically stabilised) liposomes exhibited almost no interaction with the spheroid, indicating that their limited diffusion within avascular tissues may be a limiting step for their use against micrometastases. Multicellular tumor spheroids were used as models of solid tumor interstitium relevant to delivery systems able to extravasate from the microcapillaries or as models of prevascularized micrometastases. This study illustrates that interactions between liposomes and other drug delivery systems with multicellular tumor spheroids can offer critically important information with respect to optimizing solid or micrometastatic tumor delivery and targeting strategies.     

Engineering lipid vesicles of enhanced intratumoral transport capabilities: correlating liposome characteristics with penetration into human prostate tumor spheroids

Liposomes have been widely used delivery systems, particularly relevant to the development of cancer therapeutics. Numerous liposome-based drugs are in the clinic or in clinical trials today against multiple tumor types; however, systematic studies of liposome interactions with solid or metastatic tumor nodules are scarce. This study is describing the in vitro interaction between liposomes and avascular human prostate (LNCaP-LN3) tumor spheroids. The ability of fluorescently labelled liposomal delivery systems of varying physicochemical characteristics to penetrate within multicellular tumor spheroids has been investigated by confocal laser scanning microscopy. A variety of liposome characteristics and experimental parameters were investigated, including lipid bilayer composition, duration of liposome-spheroid interaction, mean liposome size, steric stabilization of liposomes. Electrostatic binding between cationic liposomes and spheroids was very efficient; however, it impeded any significant penetration of the vesicles within deeper layers of the tumor spheroid. Small unilamellar liposomes of neutral surface character did not bind as efficiently but exhibited enhanced penetrative transport capabilities closer to the tumor core. Polymer-coated (sterically stabilised) liposomes exhibited almost no interaction with the spheroid, indicating that their limited diffusion within avascular tissues may be a limiting step for their use against micrometastases. Multicellular tumor spheroids were used as models of solid tumor interstitium relevant to delivery systems able to extravasate from the microcapillaries or as models of prevascularized micrometastases. This study illustrates that interactions between liposomes and other drug delivery systems with multicellular tumor spheroids can offer critically important information with respect to optimizing solid or micrometastatic tumor delivery and targeting strategies.     

Recent advances in three-dimensional multicellular spheroid culture for biomedical research

Many types of mammalian cells can aggregate and differentiate into 3-D multicellular spheroids when cultured in suspension or a nonadhesive environment. Compared to conventional monolayer cultures, multicellular spheroids resemble real tissues better in terms of structural and functional properties. Multicellular spheroids formed by transformed cells are widely used as avascular tumor models for metastasis and invasion research and for therapeutic screening. Many primary or progenitor cells on the other hand, show significantly enhanced viability and functional performance when grown as spheroids. Multicellular spheroids in this aspect are ideal building units for tissue reconstruction. Here we review the current understanding of multicellular spheroid formation mechanisms, their biomedical applications, and recent advances in spheroid culture, manipulation, and analysis techniques.     

Bioluminescence imaging: progress and applications

Application of bioluminescence imaging has increased tremendously in the past decade and has significantly contributed to core conceptual advances in biomedical research. This technology provides valuable means for monitoring of different biological processes in immunology, oncology, virology and neuroscience. In this review, we discuss current trends in bioluminescence and its application in different fields with an emphasis on cancer research.     

Quantification of viability in organotypic multicellular spheroids of human malignant glioma using lactate dehydrogenase activity: a rapid and reliable automated assay.

Organotypic spheroids from malignant glioma resemble the biological complexity of the original tumor and are therefore appealing to study anticancer drug responses. Accurate and reproducible quantification of response effect has been lacking to determine drug responses in this three-dimensional tumor model. Lactate dehydrogenase (LDH) activity was demonstrated in cryostat sections of spheroids using the tetrazolium salt method. Calibrated digital image acquisition of the stained cryostat sections enables quantification of LDH activity. Fully automated image cytometry reliably demarcates LDH-active and LDH-inactive tissue areas by thresholding at specific absorbance values. The viability index (VI) was calculated as ratio of LDH-active areas and total spheroid tissue areas. Duplicate staining and processing on the same tissue showed good correlation and therefore reproducibility. Sodium azide incubation of spheroids induced reduction in VI to almost zero. We conclude that quantification of viability in cryostat sections of organotypic multicellular spheroids from malignant glioma can be performed reliably and reproducibly with this approach.     

肿瘤微环境应激因子乳酸在肿瘤发展中的作用

肿瘤微环境应激主要包括:缺氧、胞外酸环境、葡萄糖缺乏等。乳酸堆积也是肿瘤微环境应激中一个重要特征。长期以来,乳酸一直被认为是代谢废物,但随着研究深入,发现乳酸作为癌代谢物与肿瘤增殖、转移、血管生成、免疫逃逸以及放化疗效果等肿瘤生物学功能密切相关,此外乳酸还可促进缺氧诱导因子稳定、作为"替代燃料"供能等进一步促进肿瘤恶性进展。因此,本文就肿瘤微环境中乳酸代谢与调控、乳酸对肿瘤生物学功能的影响等方面作一综述,旨在为针对乳酸这一异常代谢特征的抗肿瘤药物开发及临床治疗提供必要依据。     

Increases in 1H-NMR mobile lipids are not always associated with overt apoptosis: evidence from MG-63 human osteosarcoma three-dimensional spheroids exposed to a low dose (2 Gy) of ionizing radiation

The metabolic changes that occur in MG-63 osteosarcoma three-dimensional tumor spheroids exposed to 2 Gy of ionizing radiation, a dose that is comparable to radiation therapy, were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of control and exposed MG-63 spheroids were compared. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. The spectra of whole MG-63 spheroids as well as the perchloric acid extracts of these systems were evaluated. Cell damage was also examined by lactate dehydrogenase release and changes in cell growth. No cell damage was observed, but numerous metabolic changes took place in spheroids after exposure to ionizing radiation. In particular, significant increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis and also present in MG-63 spheroids undergoing overt apoptosis, were observed in spheroids irradiated with 2 Gy. However, the chromatin dye Hoechst 33258 and DNA fragmentation assays showed no overt apoptosis up to 7 days after irradiation with this low dose. Thus it is evident that increases in mobile lipids do not always indicate actual cell death. A detailed analysis of the other metabolic changes observed appears to suggest that the cell death program was initiated but not completed. In fact, the completely different behavior of two important cellular defense mechanisms, reduced glutathione and taurine, in spheroids irradiated with 2 Gy and in those undergoing overt apoptosis seems to indicate that these systems are protecting spheroids from actual cell death. In addition, these data also suggest that (1)H-NMR can be used to examine the effects of low doses of ionizing radiation in spheroids, a cell model of great complexity that closely resembles tumors in vivo. The importance of this possibility in relation to reaching the ultimate goal of a better evaluation of the outcome of radiotherapy protocols should not be ignored.     

Spatio-temporal modeling of nanoparticle delivery to multicellular tumor spheroids

The inefficiency of nanoparticle penetration in tissues limits the therapeutic efficacy of such formulations for cancer applications. Recent work has indicated that modulation of tissue architecture with enzymes such as collagenase significantly increases macromolecule delivery. In this study we developed a mathematical model of nanoparticle penetration into multicellular spheroids that accounts for radially dependent changes in tumor architecture, as represented by the volume fraction of tissue accessible to nanoparticle diffusion. Parameters such as nanoparticle binding, internalization rate constants, and accessible volume fraction were determined experimentally. Unknown parameters of nanoparticle binding sites per cell in the spheroid and pore shape factor were determined by fitting to experimental data. The model was correlated with experimental studies of the penetration of 40 nm nanoparticles in SiHa multicellular spheroids with and without collagenase treatment and was able to accurately predict concentration profiles of nanoparticles within spheroids. The model was also used to investigate the effects of nanoparticle size. This model contributes toward the understanding of the role of tumor architecture on nanoparticle delivery efficiency.     

Protein profiles in human ovarian cancer cell lines correspond to their metabolic activity and to metabolic profiles of respective tumor xenografts

Many solid tumors show a large variability in glycolytic activity and lactate accumulation, which has been correlated with different metastatic spread, radioresistance and patient survival. To investigate potential differences in protein profiles underlying these metabolic variances, the highly glycolytic human ovarian cancer cell line OC316 was investigated and compared with the less glycolytic line IGROV-1. Extracellular acidification and oxygen consumption were analyzed with an extracellular flux analyzer. Glycolysis-associated proteins, including specific membrane transporters, were quantified through in-cell western analyses. Metabolic properties of corresponding tumor xenografts were assessed via induced metabolic bioluminescence imaging. Extracellular flux analyses revealed elevated bioenergetics of OC316 cells. Hexokinase II, pyruvate kinase, pyruvate dehydrogenase E1 beta subunit and pyruvate dehydrogenase kinase 1, as well as the glucose transporter 1 and the monocarboxylate transporter 4, were overexpressed in these cells compared with IGROV-1. When generating tumor xenografts in SCID mice, cells maintained their glycolytic behavior, i.e. OC316 showed higher lactate concentrations than IGROV-1 tumors. In summary, a congruency between protein profiles and metabolic properties has been demonstrated in the human ovarian cancer lines investigated. Also, a perpetuation of glycolytic characteristics during the transition from in vitro to the in vivo situation has been documented. This model system could be useful for systematic studies on therapeutic intervention by manipulation of tumor glycolysis and associated pathways.     

Molecular imaging in prostate cancer

Prostate cancer (PCa) is the most common non-cutaneous malignancy in men. New ways to diagnose this cancer in its early stages are needed. Unique genetic and biochemical changes in the cell pave the way for tumors to grow and metastasize. Novel imaging approaches attempt to detect pathological processes in cancer cells at the molecular level. This has led to the establishment and development of the field of molecular imaging. Positron emission tomography (PET), magnetic resonance spectroscopic imaging (MRSI), magnetic resonance imaging (MRI), and radiolabeled antibodies are a few of the modalities that can detect abnormal tumor metabolic processes in the clinical setting. Other imaging techniques are still in their early phase of development but hold promise for the future, including bioluminescence imaging (BLI), measurement of tumor oxygenation, and measurement of uptake of iodine by tumors. These techniques are non-invasive and can spare the patient undue morbidity, while potentially providing early diagnosis, accurate follow-up and, finally, valuable prognostic information.     

Modeling mechanical inhomogeneities in small populations of proliferating monolayers and spheroids

Understanding the mechanical behavior of multicellular monolayers and spheroids is fundamental to tissue culture, organism development, and the early stages of tumor growth. Proliferating cells in monolayers and spheroids experience mechanical forces as they grow and divide and local inhomogeneities in the mechanical microenvironment can cause individual cells within the multicellular system to grow and divide at different rates. This differential growth, combined with cell division and reorganization, leads to residual stress. Multiple different modeling approaches have been taken to understand and predict the residual stresses that arise in growing multicellular systems, particularly tumor spheroids. Here, we show that by using a mechanically robust agent-based model constructed with the peridynamic framework, we gain a better understanding of residual stresses in multicellular systems as they grow from a single cell. In particular, we focus on small populations of cells (1–100 s) where population behavior is highly stochastic and prior investigation has been limited. We compare the average strain energy density of cells in monolayers and spheroids using different growth and division rules and find that, on average, cells in spheroids have a higher strain energy density than cells in monolayers. We also find that cells in the interior of a growing spheroid are, on average, in compression. Finally, we demonstrate the importance of accounting for stochastic fluctuations in the mechanical environment, particularly when the cellular response to mechanical cues is nonlinear. The results presented here serve as a starting point for both further investigation with agent-based models, and for the incorporation of major findings from agent-based models into continuum scale models when explicit representation of individual cells is not computationally feasible.     

Metabolic imaging in multicellular spheroids of oncogene-transfected fibroblasts.

Four rat embryo fibroblast (REF) cell lines with defined oncogenic transformation were used to study the relationship between tumorigenic conversion, metabolism, and development of cell death in a 3D spheroid system. Rat1 (spontaneously immortalized) and M1 (myc-transfected) fibroblasts represent early nontumorigenic transformation stages, whereas Rat1-T1 (T24Ha-ras-transfected Rat1) and MR1 (myc/T24Ha-ras-co-transfected REF) cells express a highly tumorigenic phenotype. Localized ATP, glucose, and lactate concentrations in spheroid median sections were determined by imaging bioluminescence. ATP concentrations were low in the nonproliferating Rat1 aggregates despite sufficient oxygen and glucose availability and lack of lactate accumulation. In MR1 spheroids, a 50% decrease in central ATP preceded the development of central necrosis at a spheroid diameter of around 800 micrometer. In contrast, the histomorphological emergence of cell death at a diameter of around 500 micrometer in Rat1-T1 spheroids coincided with an initial steep drop in ATP. Concomitantly, reduction in central glucose and increase in lactate before cell death were recorded in MR1 but not in Rat1-T1 spheroids. As shown earlier, myc transfection confers a considerable resistance to hypoxia of MR1 cells in the center of spheroids, which is reflected by their capability to maintain cell integrity and ATP content in a hypoxic environment. The data obtained suggest that small alterations in the genotype of tumor cell lines, such as differences in the immortalization process, lead to substantial differences in morphological structure, metabolism, occurrence of cell death, and tolerance to hypoxia in spheroid culture.     

An on-lattice agent-based Monte Carlo model simulating the growth kinetics of multicellular tumor spheroids

PurposeTo develop an on-lattice agent-based model describing the growth of multicellular tumor spheroids using simple Monte Carlo tools.MethodsCells are situated on the vertices of a cubic grid. Different cell states (proliferative, hypoxic or dead) and cell evolution rules, driven by 10 parameters, and the effects of the culture medium are included. About twenty spheroids of MCF-7 human breast cancer were cultivated and the experimental data were used for tuning the model parameters.ResultsSimulated spheroids showed adequate sizes of the necrotic nuclei and of the hypoxic and proliferative cell phases as a function of the growth time, mimicking the overall characteristics of the experimental spheroids. The relation between the radii of the necrotic nucleus and the whole spheroid obtained in the simulations was similar to the experimental one and the number of cells, as a function of the spheroid volume, was well reproduced. The statistical variability of the Monte Carlo model described the whole volume range observed for the experimental spheroids. Assuming that the model parameters vary within Gaussian distributions it was obtained a sample of spheroids that reproduced much better the experimental findings.ConclusionsThe model developed allows describing the growth of in vitro multicellular spheroids and the experimental variability can be well reproduced. Its flexibility permits to vary both the agents involved and the rules that govern the spheroid growth. More general situations, such as, e. g., tumor vascularization, radiotherapy effects on solid tumors, or the validity of the tumor growth mathematical models can be studied.     

Modeling of self-organized avascular tumor growth with a hybrid cellular automaton

Pattern formation in multicellular spheroids is addressed with a hybrid lattice-gas cellular automaton model. Multicellular spheroids serve as experimental model system for the study of avascular tumor growth. Typically, multicellular spheroids consist of a necrotic core surrounded by rings of quiescent and proliferating tumor cells, respectively. Furthermore, after an initial exponential growth phase further spheroid growth is significantly slowed down even if further nutrient is supplied. The cellular automaton model explicitly takes into account mitosis, apoptosis and necrosis as well as nutrient consumption and a diffusible signal that is emitted by cells becoming necrotic. All cells follow identical interaction rules. The necrotic signal induces a chemotactic migration of tumor cells towards maximal signal concentrations. Starting from a small number of tumor cells automaton simulations exhibit the self-organized formation of a layered structure consisting of a necrotic core, a ring of quiescent tumor cells and a thin outer ring of proliferating tumor cells.     

Glycolytic pyruvate regulates P‐Glycoprotein expression in multicellular tumor spheroids via modulation of the intracellular redox state

ABC transporters like P‐glycoprotein (P‐gp/ABCB1) are membrane proteins responsible for the transport of toxic compounds out of non‐malignant cells and tumor tissue. Aim: To investigate the effect of glycolysis and the tissue redox state on P‐gp expression in multicellular tumor spheroids derived from prostate adenocarcinoma cells (DU‐145), glioma cells (Gli36), and the human cervix carcinoma cell line KB‐3‐1 transfected with a P‐gp–EGFP fusion gene that allows monitoring of P‐gp expression in living cells. During cell culture of DU‐145, Gli36, and KB‐3‐1 tumor spheroids P‐gp expression was observed as well as increased lactate and decreased pyruvate levels and expression of glycolytic enzymes. Inhibition of glycolysis for 24 h by either iodoacetate (IA) or 2‐deoxy‐D ‐glucose (2‐DDG) downregulated P‐gp expression which was reversed upon coincubation with the radical scavenger ebselen as shown by semi‐quantitative immunohistochemisty in DU‐145 and Gli36 tumor spheroids, and by EGFP fluorescence in KB‐3‐1 tumor spheroids. Consequently endogenous ROS generation in DU‐145 tumor spheroids was increased in the presence of either IA or 2‐DDG, which was abolished upon coincubation with ebselen. Exogenous addition of pyruvate significantly reduced ROS generation, increased P‐gp expression as well as efflux of the P‐gp substrate doxorubicin. Doxorubicin transport was significantly blunted by 2‐DDG and IA, indicating that inhibition of glycolysis reversed the multidrug resistance phenotype. In summary our data demonstrate that P‐gp expression in tumor spheroids is closely related to the glycolytic metabolism of tumor cells and can be downregulated by glycolysis inhibitors via mechanisms that involve changes in the cellular redox state. J. Cell. Biochem. 109: 434–446, 2010. © 2009 Wiley‐Liss, Inc.