Penetration of anti-melanoma immunotoxin into multicellular tumor spheroids and cell kill effects

Summary In order to gain a better understanding of the interaction between immunotoxins and tumor cells at the level of three-dimensional tumor mass, we evaluated the cell kill effects of monoclonal antimelanoma-antibody/ricin-A-chain immunotoxin (ITN) on melanoma cells in multicellular tumor spheroids (MTS) as well as the penetration of ITN into MTS. For Minor melanoma cells in monolayer the ITN exerted cytotoxic effects after as little as 1 h of exposure. Increasing exposure time resulted in progressive increases in cytotoxic activity. In contrast, the cell kill effects of ITN were markedly delayed and reduced when Minor cells were in MTS. The ITN cytotoxic effects on the melanoma MTS were more than 100 fold less than those in monolayer. Patterns of ITN-induced cytotoxicities for Minor and for another melanoma cell line, DND-1A, were comparable. The native ricin A was more active against PC-10 squamous lung cancer cells than Minor cells, whereas the ITN was more cytotoxic against Minor cells than PC-10 cells, thus exhibiting selectivity. An autoradiographic study revealed time-dependent penetration of radiolabeled ITN from the surface of Minor MTS into the core. Incubation for 1 h resulted in the penetration of ITN into only the two or three outer layers of the Minor MTS, and low grain counts. Prolonged exposure resulted in inhomogeneous penetration of ITN into almost the entire melanoma MTS. Penetration of ITN into PC-10 MTS was extremely poor. The reduced cytotoxicity of ITN on melanoma cells in MTS as compared to cells grown in monolayer appears to correlate with its inhomogeneous distribution in the MTS. The delayed cytotoxicity of ITN is also consistent with its slow penetration into the core of the MTS.     

Growth and characterization of multicellular tumor spheroids of human bladder carcinoma origin

Summary We have examined the MGH-U1 human bladder carcinoma cell line and 12 primary bladder carcinoma biopsies for their ability to form spheroids in suspension culture and in multiwell dishes. MGH-U1 cells formed tightly packed spheroids with a necrotic center and viable rim whereas three sublines formed loose aggregates only. Spheroids formed from as few as 100 MGU-U1 cells placed into multiwells. MGH-U1 cells derived from spheroids formed new spheroids more rapidly and consistently than cells derived from monolayer culture. Spheroid diameter increased at a rapid rate of ∼100 μm/d in multiwell dishes, and necrosis occurred only in spheroids of diameter >1 mm. Spheroids placed in spinner culture at a higher concentration (∼1.5 spheroids/ml) grew more slowly and developed necrosis at smaller diameters. The width of the viable rim of spheroids grown in spinner culture was maintained at ∼190 μm over a wide range of spheroid diameters (400 to 1000 μm). Sequential trypsinization of spheroids, which stripped layers of cells from the spheroids, demonstrated no difference in the plating efficiency of cells derived from varying depths into the spheroid. Only one of the 12 primary bladder biopsy specimens demonstrated an ability to form spheroids. This biopsy, designated HB-10, formed spheroids that grew linearly over 40 d, formed colonies in methylcellulose culture and grew as xenografts in immune-deprived mice. These studies characterize the MGH-U1 spheroids that are useful in vitro models to study the effects of various treatments for solid tumors and demonstrate the limited capacity of cells from primary human bladder biopsies to form spheroids. Supported in part by a grant from the National Cancer Institute of Canada and by grant CA29526 NCI through the National Bladder Cancer Project, U.S.A.     

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.     

Optimization of the formation of embedded multicellular spheroids of MCF-7 cells: How to reliably produce a biomimetic 3D model

To obtain a multicellular MCF-7 spheroid model to mimic the three-dimensional (3D) of tumors, the microwell liquid overlay (A) and hanging-drop/agar (B) methods were first compared for their technical parameters. Then a method for embedding spheroids within collagen was optimized. For method A, centrifugation assisted cells form irregular aggregates but not spheroids. For method B, an extended sedimentation period of over 24 h for cell suspensions and increased viscosity of the culture medium using methylcellulose were necessary to harvest a dense and regular cell spheroid. When the number was less than 5000 cells/drop, embedded spheroids showed no tight cores and higher viability than the unembedded. However, above 5000 cells/drop, cellular viability of embedded spheroids was not significantly different from unembedded spheroids and cells invading through the collagen were in a sun-burst pattern with tight cores. Propidium Iodide staining indicated that spheroids had necrotic cores. The doxorubicin cytotoxicity demonstrated that spheroids were less susceptible to DOX than their monolayer cells. A reliable and reproducible method for embedding spheroids using the hanging-drop/agarose method within collagen is described herein. The cell culture model can be used to guide experimental manipulation of 3D cell cultures and to evaluate anticancer drug efficacy.     

Redox-regulation of intrinsic prion expression in multicellular prostate tumor spheroids

The cellular function of the intrinsic prion protein (PrPc) remains largely unknown. In the present study PrPc expression was investigated in multicellular prostate tumor spheroids and was correlated to the intracellular redox state as evaluated using the fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In small tumor spheroids (diameter 100 +/- 20 microm) reactive oxygen species (ROS) levels were increased as compared with large (diameter 250 +/- 50 microm) spheroids. ROS generation was mediated by the mitochondrial respiratory chain and a NADPH oxidaselike enzyme, because carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, and diphenylene iodonium chloride (DPI) significantly reduced ROS levels. The elevated ROS were correlated to an increased expression of PrPc, Cu/Zn superoxide dismutase (SOD-1), and catalase in small as compared with large spheroids. In large tumor spheroids, PrPc was predominantly expressed in the peripheral cell layers and colocalized with SOD-1 and catalase. Raising intracellular ROS in large tumor spheroids by hydrogen peroxide, menadione, buthionine sulfoximine (BSO), and incubation in glutamine-reduced medium increased PrPc expression. In small spheroids PrPc was downregulated after incubation with the radical scavengers dehydroascorbate (DHA) and vitamin E. Our data indicate that PrPc expression in tumor spheroids is related to the intracellular redox state and may participate in antioxidative defense.     

A Monte Carlo method for the simulation of kinetie models

The purpose of this note is to illustrate the feasibility of simulating kinetic systems, such as commonly encountered in photosynthesis research, using the Monte Carlo (MC) method. In this approach, chemical events are considered at the molecular level where they occur randomly and the macroscopic kinetic evolution results from averaging a large number of such events. Their repeated simulation is easily accomplished using digital computing. It is shown that the MC approach is well suited to the capabilities and resources of modern microcomputers. A software package is briefly described and discussed, allowing a simple programming of any kinetic model system and its resolution. The execution is reasonably fast and accurate; it is not subject to such instabilities as found with the conventional analytical approach.Abbreviations MC Monte Carlo - RN random number - PSU photosynthetic unit Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Use of a laser diffraction particle sizer for the measurement of mean diameter of multicellular tumor spheroids

Summary Increasing use is being made of tumor cell lines cultured as cell aggregates (generally referred to as multicellular spheroids) in in vitro radiosensitivity and/or chemosensitivity tests. Conventional procedures for the determination of mean spheroid diameters for the construction of growth delay curves employ a microscope-image analyzer. However, this approach can prove excessively time consuming when large numbers of samples have to be, measured. We have, therefore, been exploring the use of a Laser Diffraction Particle Sizer, the Malvern 2600 long bench model, for the measurement of mean spheroid diameter and size distribution. We report here a direct comparison between measurements carried out by the instrument and under the microscope. Also a comparison of growth curves for six cell lines constructed from measurements by the microscope and by the instrument. A number of factors that might affect the accuracy of spheroid diameter measurement by the instrument have been investigated: The effect of stirring to maintain the spheroids in suspension during measurement. Sampling error due to removal of a series of spheroid samples from culture flasks for measurement. Optimum number of scans to be carried out by the instrument to reach a constant value for mean diameter, and minimum SE of the mean. This research was supported by the Yorkshire Cancer Research Campaign, Horrogate HG1 5LQ, United Kingdom, and by the Hospital Research Fund, Cookridge Hospitatl, Leeds LS16 6QB, United Kingdom.     

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     

Biofilm model calibration and microbial diversity study using Monte Carlo simulations

Mathematical models are useful tools for studying and exploring biological conversion processes as well as microbial competition in biological treatment processes. A single‐species biofilm model was used to describe biofilm reactor operation at three different hydraulic retention times (HRT). The single‐species biofilm model was calibrated with sparse experimental data using the Monte Carlo filtering method. This calibrated single‐species biofilm model was then extended to a multi‐species model considering 10 different heterotrophic bacteria. The aim was to study microbial diversity in bulk phase biomass and biofilm, as well as the competition between suspended and attached biomass. At steady state and independently of the HRT, Monte Carlo simulations resulted only in one unique dominating bacterial species for suspended and attached biomass. The dominating bacterial species was determined by the highest specific substrate affinity (ratio of µ/K_S). At a short HRT of 20 min, the structure of the microbial community in the bulk liquid was determined by biomass detached from the biofilm. At a long HRT of 8 h, both biofilm detachment and microbial growth in the bulk liquid influenced the microbial community distribution. Biotechnol. Bioeng. 2013; 110: 1323–1332. © 2012 Wiley Periodicals, Inc.     

A model for the growth of multicellular spheroids

Abstract. Based on biological observations and the basic physical properties of tri-dimensional structures, a mathematical expression is derived to relate the growth rate of multicellular spheroids to some easily measurable parameters. This model involves properties both of the individual cells and of the spheroid structure, such as the cell doubling time in monolayer, the rate of cell shedding from the spheroid and the depth of the external rim of cycling cells. The derived growth equation predicts a linear expansion of the spheroid diameter with time. The calculated growth rate for a number of spheroid cell types is in good agreement with experimental data. The model provides a simple and practical view of growth control in spheroids, and is further adapted to include parameters presumably responsible for the growth saturation in large spheroids.     

Using size‐controlled multicellular spheroids of murine adenocarcinoma cells to efficiently establish pulmonary tumors in mice

Previous studies demonstrated that multicellular spheroids developed using polydimethylsiloxane‐based microwells exhibited superior functions, such as insulin secretion from pancreatic cells, over suspended cells. To successfully apply these spheroids, the effect of spheroid size on cellular functions must be determined. In this study, using murine adenocarcinoma colon26 cells, the authors examined whether such spheroids were useful for developing tumor‐bearing animal models, which requires the efficient and stable engraftment of cancer cells at implanted sites and/or metastatic sites. The authors prepared microwells with widths of 360, 450, 560, and 770 μm through a micromolding technique, and obtained colon26 spheroids with average diameters of 169, 240, 272, and 341 μm, respectively. Small and medium spheroids were subsequently used. mRNA levels of integrin β1, CD44, and fibronectin, molecules involved in cell adhesion, increased with increasing colon26 spheroid size. Approximately 1.5 × 10⁴ colon26 cells in suspension or in spheroids were intravenously inoculated into BALB/c mice. At 21 days after inoculation, the lung weight of both colon26 spheroid groups, especially the group injected with small spheroids, was significantly higher than that of mice in the suspended colon26 cell group. These results indicate that controlling cancer cell spheroid size is crucial for tumor development in tumor‐bearing mouse models.     

Bayesian phylogenetic model selection using reversible jump Markov chain Monte Carlo

A common problem in molecular phylogenetics is choosing a model of DNA substitution that does a good job of explaining the DNA sequence alignment without introducing superfluous parameters. A number of methods have been used to choose among a small set of candidate substitution models, such as the likelihood ratio test, the Akaike Information Criterion (AIC), the Bayesian Information Criterion (BIC), and Bayes factors. Current implementations of any of these criteria suffer from the limitation that only a small set of models are examined, or that the test does not allow easy comparison of non-nested models. In this article, we expand the pool of candidate substitution models to include all possible time-reversible models. This set includes seven models that have already been described. We show how Bayes factors can be calculated for these models using reversible jump Markov chain Monte Carlo, and apply the method to 16 DNA sequence alignments. For each data set, we compare the model with the best Bayes factor to the best models chosen using AIC and BIC. We find that the best model under any of these criteria is not necessarily the most complicated one; models with an intermediate number of substitution types typically do best. Moreover, almost all of the models that are chosen as best do not constrain a transition rate to be the same as a transversion rate, suggesting that it is the transition/transversion rate bias that plays the largest role in determining which models are selected. Importantly, the reversible jump Markov chain Monte Carlo algorithm described here allows estimation of phylogeny (and other phylogenetic model parameters) to be performed while accounting for uncertainty in the model of DNA substitution.     

用蒙特卡罗方法模拟光在多层组织中的吸收特性

在讨论目前新颖的组织功能成像打骂能性（例如光声成像）时,光子在组织中的吸收和散射特性是一个很重要的问题,鉴于这一点,本文利用一个多层模型研究了光子在皮肤,脂肪和肌肉组织中的吸收和散射特性,得到了在组织中某一深度处光子在一个平面上的吸收分布,以及在不同吸收系数和散射系数的情况下,光子的反射,吸收和透射几率,结果表明在经过多次散射后,大部分的光子被吸收,在本文的模型中只有7.3%的光子从表面反射（包括镜面反射和漫反射）,还讨论了不同光学参灵敏对参流分布的影响。     

Monte Carlo simulation of protein folding in the presence of residue-specific binding sites

Ensemble growth Monte Carlo (EGMC) and dynamic Monte Carlo (DMC) simulations are used to study sequential folding and thermodynamic stability of hydrophobic-polar (HP) chains that fold to a compact structure. Molecularly imprinted cavities are modeled as hard walls having sites that are attractive to specific polar residues on the chain. Using EGMC simulation, we find that the folded conformation can be stabilized using a small number of carefully selected residue-specific sites while a random selection of surface-bound residues may only slightly contribute toward stabilizing the folded conformation, and in some cases may hinder the folding of the chain. DMC simulations of the surface-bound chain confirm increased stability of the folded conformation over a free chain. However, a different trend of the equilibrium population of folded chains as a function of residue-external site interactions is predicted with the two simulation methods.     

Monte Carlo Simulation of Nonionic Surfactants at the Oil-Water Interface

Abstract

A Monte Carlo simulation method has been developed for modelling amphiphiles at an oil-water interface. Properties are calculated for the mixture water, benzene and tetraoxyethylene glycol dodecyl ether.     

Monte Carlo Comparison of Multiple Comparison Procedures

Seven procedures of multiple comparisons: Tukey, Scheffé, Bonferroni, Studentized Maximum Modulus, Duncan, Newman-Keuls and F are compared with respect to the probability of the correct decision. Monte Carlo simulation shows that there is no the best procedure. AMS 1985 Subject Classification: 62 J 15.     

Visualizing the effect of tumor microenvironments on radiation-induced cell kinetics in multicellular spheroids consisting of HeLa cells

In this study, we visualized the effect of tumor microenvironments on radiation-induced tumor cell kinetics. For this purpose, we utilized a multicellular spheroid model, with a diameter of ∼500 μm, consisting of HeLa cells expressing the fluorescent ubiquitination-based cell-cycle indicator (Fucci). In live spheroids, a confocal laser scanning microscope allowed us to clearly monitor cell kinetics at depths of up to 60 μm. Surprisingly, a remarkable prolongation of G2 arrest was observed in the outer region of the spheroid relative to monolayer-cultured cells. Scale, an aqueous reagent that renders tissues optically transparent, allowed visualization deeper inside spheroids. About 16 h after irradiation, a red fluorescent cell fraction, presumably a quiescent G0 cell fraction, became distinct from the outer fraction consisting of proliferating cells, most of which exhibited green fluorescence indicative of G2 arrest. Thereafter, the red cell fraction began to emit green fluorescence and remained in prolonged G2 arrest. Thus, for the first time, we visualized the prolongation of radiation-induced G2 arrest in spheroids and the differences in cell kinetics between the outer and inner fractions.     

An evolutionary Monte Carlo algorithm for predicting DNA hybridization

Many DNA-based technologies, such as DNA computing, DNA nanoassembly and DNA biochips, rely on DNA hybridization reactions. Previous hybridization models have focused on macroscopic reactions between two DNA strands at the sequence level. Here, we propose a novel population-based Monte Carlo algorithm that simulates a microscopic model of reacting DNA molecules. The algorithm uses two essential thermodynamic quantities of DNA molecules: the binding energy of bound DNA strands and the entropy of unbound strands. Using this evolutionary Monte Carlo method, we obtain a minimum free energy configuration in the equilibrium state. We applied this method to a logical reasoning problem and compared the simulation results with the experimental results of the wet-lab DNA experiments performed subsequently. Our simulation predicted the experimental results quantitatively.