Australas Phys Eng Sci Med (2011) 34:3139 DOI 10.

1007/s13246-010-0048-y

SCIENTIFIC PAPER

A percolation-like model for simulating inter-cellular diffusion in the context of bystander signalling in tumour
C. R. Moulton A. J. Fleming M. A. Ebert

Received: 1 June 2010 / Accepted: 15 December 2010 / Published online: 28 December 2010 Australasian College of Physical Scientists and Engineers in Medicine 2010

Abstract Despite ongoing active research, the role of the radiation bystander effect in modifying local tissue response to an ionising radiation dose remains unclear. The present study aims to provide new insight by simulating the diffusion-mediated inter-cellular communication processes in 2D and 3D cell-like structures to calculate likely signal ranges in the diffusion limited case. Random walks of individual signalling molecules were tracked between cells with inclusion of molecule-receptor interactions. The resulting diffusion anomaly is a function of cell density, signal uptake probability and the spatial arrangement of cells local to the signal origin. Uptake probability effects dominate percolation effects in disordered media. Diffusion through 2D structures is more conducive to anomalous diffusion than diffusion through 3D structures. Values for time-dependent diffusion constants and permeability are derived for typical simulation parameters. Even at low signal uptake probabilities the communication range is restricted to a mean value of less than 100 lm owing to complete signal uptake by 600 s. This should be considered in light of the potential inuence of signal relaying, owdynamics or vasculature-mediated signalling.

Keywords Diffusion modelling Bystander effect Percolation theory Monte Carlo simulations

Introduction The radiation-induced bystander effect is the changed behaviour of unirradiated cells in response to molecular species released from irradiated cells [14]. Uptake of these species by unirradiated cells (inter-cellular signalling) can lead to enhanced killing or stimulated proliferation. Bystander effects have been demonstrated for a number of cell lines in vivo [58]. Previous investigations have looked at the inuence of inter-cellular signalling on the radiation response of cells [9, 10]. Studies suggest that molecular species with molecular weights in the order of 110 kDa such as cytokines are vital to diffusion-mediated bystander response [10, 11]. Diffusion processes for transport of signalling molecules are especially important in tumour environments where communication via gap junctions is suppressed [10, 12]. Previous studies have implemented diffusion simulations, with some developed specic to in vitro environments [9, 1317]. Simulations of tissue environments [1, 18, 19] have not included explicit transport calculations that may be inuenced by microscopic tissue characteristics. Previous theoretical investigations of diffusion processes in heterogeneous media representing cellular environments have been undertaken. Mixtures of immobile obstacles and mobile tracers have been examined [20, 21, 22] as have cell membrane-molecule binding [23], extracellular matrix constituents, cellular structure approximations and transport across cell membranes. It has been observed that diffusion in crowded media is heavily

C. R. Moulton (&) 21 Seventh Avenue, Bassendean, WA 6054, Australia e-mail: moultc02@student.uwa.edu.au; calynmoulton@gmail.com C. R. Moulton A. J. Fleming M. A. Ebert School of Physics M013, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia M. A. Ebert Department of Radiation Oncology, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, WA 6009, Australia

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restricted in comparison to that in aqueous solutions [24, 25]. These studies have found that the resultant effective diffusion constant depends on the size and mobility of the diffusing species as well as the size of the obstacles. Percolation theory [26, 27] is based on using lattices made up of voxels with a fraction q of these voxels occupied. Collections of neighbouring voxels that are occupied form clusters. One common assumption is that each voxel is populated independently of the occupation of neighbouring voxels and that the diffusing molecule can only move on empty voxels. As the occupation probability is increased a percolation threshold is eventually reached where for the rst time a cluster is large enough that it extends from one side of the lattice to the other. Above the percolation threshold the diffusing species becomes localized due to extensive cluster formation [27]. The problem of a particle trying to move through such a structure is the application of percolation theory to diffusion [2628]. General studies on diffusion in crowded media have found that below the percolation threshold there is anomalous diffusion over short times and normal diffusion over long times [21, 25]. These studies also report that uniform distributions of obstacles are more conducive to normal diffusion than random distributions and that two-dimensional (2D) media result in more anomalous diffusion than three-dimensional (3D) media. Results of such studies show that the effective diffusion coefcient is time and geometry dependent. The effective diffusion coefcient will determine the range of signals in vivo. The present study was motivated by a need for computational models to simulate the range of the bystander effect in tissue with the ability to vary parameters extensively and quickly. The impact of uptake by obstacles representing cells on Monte Carlo simulations of signal diffusion in 2D and 3D lattices was investigated. The aims of this study were to: 1. 2. Examine which aspects of simulation inuence signal diffusion. Examine the effective diffusion coefcient for a range of molecular species and simulation parameters.

constants used in the extra-cellular space (ECS) would also depend on the composition of the ECS and the proportion of total tissue occupied by the ECS would depend on tissue type and local dynamics. The assumption that there are irreversibly bound signals may not be valid for certain signalling species. In the case of Interleukin-6 (IL-6) for example, the signal is internalized and degraded [30]. Tumour micro-environments are observed to be inhomogenous in time and space [31, 32]. One aspect is that relative to normal tissue the vascularity and perfusion in tumours varies extensively [32]. The necrotic areas contain leaking blood vessels that create variable compositions of extracellular uid and blood constituents throughout the tumour environment [31]. But tumour structure for simulation durations of 600 s can be considered static relative to an average cell cycling duration of 12 h [33]. One benet of tumour environments is that transport via gap junction mediation is suppressed [10, 12]. The implications are that results of simulations depend on which tumour microenvironment is being considered and here results are most meaningful in the context of a slow-growing avascular tumor.

Theory Normal diffusion of particles results when the movement of particles is unobstructed, i.e. diffusion in homogeneous and isotropic space. Einsteins and Smoluckowskis work on Brownian motion in 1905 and 1906 respectively provided a diffusion equation for the mean-square displacement hr2 i of a diffusing particle as a function of time t, the dimensionality of the medium d and the diffusion coefcient D [24, 25, 34, 35]: hr 2 i 2dDt 1

It was intended to derive relationships between simulation parameters and resulting mean-square diffusion distances for input into macroscopic models of bystander effects (e.g. [1] and [29]). Given ongoing debate on the relevance of bystander signalling to radiotherapy dose delivery the model is presented in the context of radiationinduced bystander signalling, but could equally apply in other contexts (e.g. gene therapy). The scope of the applicability of results is determined by a number of additional factors in tumour environments including more complex cell shape, the dynamics near blood vessels and potential transport by vessels. Diffusion

The diffusion constant D is for particles in an innitely dilute solution and is related to the size of the particle and viscosity of the medium by the EinsteinStokes relation [36]. Anomalous diffusion of particles results when the movement of particles is hindered by obstacles. The meansquare displacement is proportional to a real-number power of time termed the anomalous diffusion exponent a, i.e. hr 2 i / ta . The relation is now non-linear with time [21]: hr 2 i 2dDtt=sc a1 2

where sc is a characteristic timescale. When a \ 1 it is termed sub-diffusion. An example of the conditions leading to sub-diffusion is diffusion in media containing dense regions of obstacles. For anomalous super-diffusion a [ 1. For normal diffusion, dividing both sides of (1) by 2dt gives an expression for the diffusion constant:

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D hr 2 i=2dt

An effective time-dependent diffusion coefcient is then dened in the sub-diffusion case from (2) by the same   procedure of dividing r 2 by the factor 2dt: : : 4 D Dthr 2 i=2dt Dt=sc a1 Alternatively, (2) can be dened as hr 2 i 2dD ta where D* can be considered as a cm2/sa transport coefcient (apparent diffusion constant) that ensures the equation is dimensionally correct [37]. Normal diffusion is the special case where the anomalous diffusion coefcient is one and the diffusion coefcient is constant with time. By taking the logarithm of (4) it is clear that a plot of the log of the mean-square displacement per time factor versus the log of time produces a line with a negative slope for sub-diffusion. Normal diffusion (a = 1) will have no dependence on time in this plot. The extra-cellular space (ECS) fraction b and tortuosity k summarise the diffusion relevant properties of complex media. The ECS fraction is the proportion of total tissue occupied by the extra-cellular space and is directly related to the tissue cell density. Tortuosity represents the hindrance provided by the complex media relative to an obstacle-free medium and is used to explain how diffusion relative to an obstacle-free environment depends on features of the environment, such as obstacle shape, media dimensionality, dead-end paths, channel widths and proportion of unoccupied space in the environment [38, 39]. It is related to the ratio of the effective and obstacle-free diffusion constants. In homogeneous and isotropic media, tortuosity is commonly dened as [38, 39]: k D=D 1=2 5

2.

3.

4.

5.

Diffusion permeability h is an alternative parameter with a simple relationship to the diffusion of molecules around obstacles and is commonly dened as [38]: h D =D 1=k2 6 6.

A diffusion permeability of zero or an innite tortuosity represents an impenetrable medium. A diffusion permeability of one or zero tortuosity represents obstacle-free media.

Method A Monte Carlo simulation algorithm was developed to simulate the diffusion of signalling molecules through an obstacle-rich medium representing tissue. The simulation was implemented in C?? in the following way: 1. The relevant diffusion constants were in the range 1 9 10-8 cm2/s B D B 1 9 10-6 cm2/s as larger

diffusion constants corresponded to small molecules less than 1 kDa in molecular weight [33, 40]. The diffusion constants were for species in obstacle-free ECS. The media were represented by grids of voxels dened on either a 2D or 3D lattice. Periodic boundary conditions were applied. In 2D simulations 5 9 5 squares were used to represent each cell occupying 25 voxels. For 3D simulations 5 9 5 9 5 cubes represented each cell occupying 125 voxels. The simplied 3D shape of cells was used to allow a clearly dened cellmolecule interaction boundary. Cells were distributed either randomly or uniformly to reect a certain tissue cell density. The uniform distribution had cells separated by a constant number of voxels. The random distribution had random spacing up to a maximum value. The 2D lower and upper limits on cell density were 1 9 104 cells/cm2 and 5 9 105 cells/cm2 [9, 41]. The 3D lower and upper limits on cell density were 1 9 108 cells/cm3 and 5 9 108 cells/cm3 [42]. The corresponding 2D upper and lower range ECS fractions were b = 0.99 and b = 0.5. The 3D upper and lower range ECS fractions were b = 0.90 and b = 0.4. Uniform spacing between cells ranged from 1 to 5 voxels for 2D environments and 1 to 3 voxels for 3D environments. Square lattice width ranged from 150 to 154 voxels and cubic lattice width ranged from 30 to 40 voxels. Simulations (trials) were made of individual molecules originally occupying voxels in extra-cellular matrix (ECM) adjacent to cell walls. Each molecule was tracked as it diffused from the signalling cell. Trials were uniformly sampled from all voxels adjacent to the walls of the signalling cells. Typically 20000 trials were made per starting position, with the total number of trials broken into 10 batches to allow generation of result statistics. The dimensionless parameters required in the algorithm were the number of time steps t*, step distance * equal to one and the displacement r*. The physical parameters related to these were the lattice constant (voxel size) , time step duration s, tracking time t, the molecular diffusion constant D in obstacle-free ECS and distance r from the starting position. They were related by the following Equations: r r t st

7 8 9

2 2dDs

The duration of each time step and the number of time steps were obtained from (8) and (9) as:

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s 2 =2dD t 2dDt=2

10 11

A value of * = 1 was used to allow diffusion across one voxel per time step. 7. At each time step a nearest neighbour voxel was selected using the Mersenne Twister random number generator [43]. If the selected neighbour voxel was empty then the molecule was moved there as it would under normal diffusion. If it was occupied by a cell then a cell-molecule interaction occurred. 8. For cell-molecule interaction there was a probability p of uptake by a cell, leaving a probability 1 - p that the molecule stayed where it was. Considering cytokine specic receptors cover 1% of cell surfaces [9], the lower limit for the uptake probability was 1%, based on a voxel size of 2 lm. Considering a capture radius of half a voxel width and a cell radius of 2.5 voxel widths with small molecule radius, an upper limit for the uptake probability was taken as 4%, based on the ratio of capture area to the area occupied by a cell [44]. 9. The component displacements x ; y ; z were updated at the end of each time step and the square distance for each trial was calculated r 2 x2 y2 z2 at the end of each time step. By averaging the total square distance over all trials and batches a mean square distance at the end of each time step was obtained. The time-dependent effective diffusion coefcient was obtained by dividing each time step result by the time dimensionality factor 2dn*, where n* is the number of time steps passed. To revert to physical units of cm2 and cm2/s for the mean square distance and the effective diffusion coefcient respectively, the former was multiplied by lattice constant squared 2 and the latter by 2/s. Variances were calculated across independent batches. 10. It was observed that results were dependent on the starting position and that molecular species with greater diffusion constants in free media replicate those species with lower diffusion constants played out at a faster rate. The diffusion constant was thus xed at 1 9 10-7 cm2/s. With a voxel size of 2 lm the time step durations from (10) were 0.1 and 0.067 s for 2D and 3D simulations respectively. The number of time steps from (11) for 2D and 3D simulations were 6000 and 9000 respectively for a 600 s tracking time. 600 s is sufcient for near complete uptake of all signalling molecules.

deviation expressed as a percentage of the mean. The CV across independent batches was less than 1%. The simulations of diffusion for uniformly and randomly spaced environments are shown in Fig. 1a for 2D media and in Fig. 1b for 3D media. Without molecular uptake the 3D simulations approached a normal diffusion regime faster at larger effective diffusion coefcients than 2D simulations (e.g. random b = 0.76 p = 0%). The random 2D media without molecular uptake show more anomalous diffusion than random 3D media with equivalent ECS fractions as shown in Fig. 1 (e.g. b = 0.76 p = 0%). In the absence of absorption the effective diffusion constant is seen to plateau at earlier times for uniform cases than for random cases. In the 1% uptake probability case, Fig. 2 shows the diffusion permeability asymptotically approaching 0 (e.g. 2D b = 0.49 p = 1% and 3D b = 0.42 p = 1%) as a consequence of increased hindrance provided through nearly complete molecular uptake. In low to medium ECS fraction regions (0 b 0:64) it takes up to 10 s for absorption to drive attentuation of the effective diffusion constant. Figure 3 shows the mean diffusion distance from starting locations at the end of simulations for various structures and uptake probabilities. In the absence of molecular uptake (p = 0%), the signalling distance is greater in uniform 2D media than in random 2D media, for b.0:7. Figure 3 shows that mean distance is very sensitive to uptake probability. Figure 4 shows the mean distance of signalling molecules from their starting locations over time. The high ECS fraction simulations do not become constant after 600 s and this is due to incomplete uptake of all signalling molecules by 600 s. Discussion The simulations show that at a microscopic level, the effective diffusion coefcient depends on time, starting position and the percolation threshold. Results also show a strong dependence on uptake probability, with increasing probability causing lower diffusion permeability and larger tortuosity as the structure provides more hindrance. In comparison to uniform media, Fig. 1a shows that signalling molecules in random 2D media experience more anomalous diffusion than in uniform 2D media with the same ECS fraction only when p = 0% (e.g. b = 0.49 p = 0%). From Fig. 2a the random 2D media tortuosity and permeability parameters asymptotically reach k = 1.4 and h = 0.49 respectively for the ECS fraction b = 0.49 with p = 0%, conrming that when p = 0% random 2D media hinder diffusion more than uniform 2D media with

Results We dene the coefcient of variation (CV) of the effective diffusion constant for a batch of trials to be the standard

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Australas Phys Eng Sci Med (2011) 34:3139 Fig. 1 a 2D results for the effective diffusion coefcient over 6000 time steps (600 s). Uniform refers to uniform spacing between cells and random indicates random spacing between cells. b is the ECS fraction, which is the proportion of extra-cellular space in the structure. The probability of molecular uptake in a cell-molecule interaction is given by p. b 3D results for the effective diffusion coefcient over 9000 time steps (600 s). Uniform refers to uniform spacing between cells and random indicates random spacing between cells. b is the ECS fraction, which is the proportion of total tissue occupied by extra-cellular space. The probability of molecular uptake in a cellmolecule interaction is given by p
Time Steps
1 1.00 0.70 10 100 1000 10 000 0 Uniform, Uniform, Uniform, 0.30 0.20 0.15 0.10 0.1 1 10 100 1000 Uniform, Random, Random, Random, Random, 0.69, p 0 0.69, p 1 0.49, p 0 0.49, p 1 0.76, p 0 0.76, p 1 0.49, p 0 0.49, p 1

35

D 10 7cm2 s

0.50

Time t s

Time Steps
1.5 1.00 0.70 15 150 1500 15 000 0 Uniform, Uniform, Uniform, Uniform, 0.20 0.15 0.10 0.1 1 10 100 1000 Random, Random, 0.76, p 0 0.76, p 1 0.76, p 0 0.76, p 1 0.42, p 0 0.42, p 1

D 10 7cm2 s

0.50

0.30

Time t s

asymptotic parameter values of k = 1.3 and h = 0.61 respectively. Figure 1 also shows that 3D media are more supportive of normal diffusion than 2D media (e.g. b = 0.76). These results for uptake-free diffusion are in agreement with previous reports [21, 25]. Figure 3 shows that 2D and 3D media results with molecular uptake have reduced signalling distances in comparison to results without molecular uptake, as is expected given the extra hindrance to diffusion that uptake introduces. Unlike the results without molecular uptake, it is now seen in Fig. 3 that the difference in signalling distances between 2D uniform media and 2D random media of similar ECS fractions are negligible. From percolation theory [27] it is known that above the percolation threshold diffusion should be anomalous for all times, due to clusters spanning from one side of the media to the other localizing signalling molecules in regions. The 2D percolation threshold [27] for the proportion of voxels occupied by cells is 0.59, which is equivalent to an ECS fraction of b = 0.41. Consistent with percolation theory, the 2D randomly-distributed media results without molecular uptake show that above this threshold the signalling

distance drops off faster than below the threshold. With molecular uptake these percolation effects are secondary to uptake effects, as shown in Fig. 3, where with the inclusion of uptake the signalling distance now drops off slower than below the threshold. The magnitude of the difference in signalling distance between random and uniform media increases with decreasing ECS fraction beyond the threshold. Figure 4 shows the mean distance of signalling molecules from their starting locations over time. The high ECS fraction simulations clearly do not become constant after 600 s and this is due to incomplete molecular uptake of all signalling molecules by 600 s. All the other curves except p p those modelling obstacle-free diffusion ( 4Dt and 6Dt) become constant by this time, due to complete molecular uptake of signalling molecules. Running simulations for greater time duration would result in the same mean distance. For the 4% uptake probability simulations the mean diffusion distance is restricted to less than 3 cell widths or 0.03 mm from the starting locations for low to medium ECS fraction ranges (0 b 0:64). With incomplete molecular uptake of all signalling molecules in the high

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36 Fig. 2 a 2D results for permeability h and tortuosity k over 6000 time steps (600 s). Uniform refers to uniform spacing between cells and random indicates random spacing between cells. b is the ECS fraction, which is the proportion of total tissue occupied by extra-cellular space. The probability of molecular uptake in a cellmolecule interaction is given by p. b 3D results for permeability h and tortuosity k over 9000 time steps (600 s). Uniform refers to uniform spacing between cells and random indicates random spacing between cells. b is the ECS fraction, which is the proportion of total tissue occupied by extracellular space. The probability of molecular uptake in a cellmolecule interaction is given by p
Time Steps
1 1. 10 100 1000

Australas Phys Eng Sci Med (2011) 34:3139

10 000 1 0 Uniform, 0.69, p 0 0.69, p 1 0.49, p 0 0.49, p 1 0.69, p 0 0.69, p 1 0.49, p 0 0.49, p 1

Diffusion Permeability

0.8

Uniform,

Tortuosity

0.6 0.4 0.2 0.

1.25 1.5 2 2.5 3 0.1 1 10 100 1000

Uniform, Uniform, Random, Random, Random, Random,

Time t s
Time Steps
1.5 1. 15 150 1500 15 000 1 0 Uniform, 0.76, p 0 0.76, p 1 0.64, p 0 0.64, p 1 0.42, p 0 0.42, p 1 0.76, p 0 0.76, p 1

Diffusion Permeability

0.8

Uniform,

Tortuosity

0.6 0.4 0.2 0.

1.25 1.5 2 2.5 3 0.1 1 10 100 1000

Uniform, Uniform, Uniform, Uniform, Random, Random,

Time t s

ECS fraction simulations (b ! 0:49), there is the possibility of the mean distance growing with further time duration. Uptake hinders the diffusion process and reduces the anomalous diffusion exponent. Fitting a curve to the data with uptake and extrapolating will at the extreme yield distances greater than those that would result if there were further hindrance via uptake. Performing the extreme tting procedure with 95% condence intervals for the 2D high ECS fraction simulations (b = 0.96) gives mean diffusion distance extrapolations of 2.01 and 1.05 mm (201 and 105 cell widths) after 72 h for the lower and upper uptake probability limits of 1 and 4% respectively. Experiments involving in vitro observation of bystander effects with plated cells involve signal diffusion through cell growth media which is largely unrestricted by signal uptake. This observation has been used to model bystander effects in experiments utilising clonogenic assays [45]. Figure 4 shows that the mean diffusion distance in the 1% uptake probability simulations is restricted to short ranges of less than 5.5 cell widths or 0.055 mm from the starting locations for low to medium ECS fraction ranges (0 b 0:64). The model does not allow for subsequent secretion of signalling species post uptake (signal relaying). Thus the results in medium and low ECS fraction

regions show that long range bystander signalling via damaged cells producing long range diffusible signals in the form of signalling proteins is unlikely. It has previously been hypothesised that bystander signalling could inuence tissue dose-response at the boundary (eld-edge) in radiotherapy treatments [10, 29]. With restricted diffusion distances in medium to low ECS fraction regions the importance of bystander signalling at the boundary of treatment is limited by other larger uncertainties, including organ motion or set-up error [46, 47]. Even in high ECS fraction regions, where results suggest that the mean diffusion range could reach up to 2.01 mm, the importance of bystander ranges in consideration of other uncertainties is limited. Inclusion of bystander signal relay from intermediate bystander cells would allow signals to move from closed-in regions to well-connected areas, where the diffusion process is much faster with less molecular uptake. A resultant increase in the mean diffusion distance would result in many more cells being indirectly affected by low dose radiation than would be predicted under high dose target theory. Long range diffusion distances bring additional factors into consideration, such as differential interstitial pressures between tumour and normal tissue, the dynamics near blood vessels and

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200

20

2D,

4 Dt , t 600s

150

15

100

10

Mean Distance r Cell Widths

Mean Distance r m

3D,

6 Dt , t 600s

50

0 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

ECS Volume Fraction

Fig. 3 Mean diffusion distances from starting positions after 600 s. Uniform refers to uniform spacing between cells and random indicates random spacing between cells. b is the ECS fraction, which is the proportion of total tissue occupied by extra-cellular space. The

probability of molecular uptake in a cell-molecule interaction is given p p by p. 4Dt and 6Dt indicate that distances are based on the diffusion equations after 600 s. The 2D percolation threshold is indicated by the dashed vertical line at b = 0.41

Distance r Cell Widths

Fig. 4 a The mean diffusion distance results over 6000 time steps for 2D simulations. Uniform refers to uniform spacing between cells and random indicates random spacing between cells. b is the ECS fraction, which is the proportion of total tissue occupied by extra-cellular space. The probability of molecular uptake in a cellmolecule interaction is given by p p. 4Dt indicates that the distance is based on the diffusion equation. b The mean diffusion distance results over 9000 time steps for 3D simulations. Uniform refers to uniform spacing between cells and random indicates random spacing between cells. b is the ECS fraction, which is the proportion of total tissue occupied by extra-cellular space. The probability of molecular uptake in a cellmolecule interaction is given by p p. 6Dt indicates the distance is based on the diffusion equation

Time Step
0 200 1000 2000 3000 4000 5000 6000 20 4 Dt

Distance r Cell Widths

Uniform, Uniform, Random, Random, Uniform, Uniform,

0.31, p 1 0.31, p 4 0.49, p 1 0.49, p 4 0.96, p 1 0.96, p 4

Distance r m

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0 0 100 200 300 400 500 600

Time t s
Time Step
0 200 1500 3000 4500 6000 7500 9000 20 6 Dt Uniform, Uniform, Random, Uniform, Uniform, Uniform, 0.42, p 1 0.42, p 4 0.64, p 1 0.64, p 4 0.76, p 1 0.76, p 4

Distance r m

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indirect radiation damage to remote regions via blood vessel transport of signalling molecules. The extent of cell damage due to bystander signalling also depends on the amount of signal required for cell response and the number of signalling molecules released from each signalling cell.

Tortuosity is proportional to the magnitude of the anomalous diffusion. The results presented here conrm that the degree of tortuosity is increased by decreasing channel width, smaller closed-in regions, narrower cul-desacs and greater cul-de-sac formation [38, 39]. A cul-de-

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Australas Phys Eng Sci Med (2011) 34:3139 Acknowledgments Adj. A/Prof. N. Suchowerska and Prof. D. McKenzie from the University of Sydney and A/Prof. B. Corry from the University of Western Australia are gratefully acknowledged for their useful discussions and insights. This work was supported by funding provided by the Cancer Council Western Australia.

sac is a region with only one entry point where the diffusion process gets slowed down and uptake probability becomes important. The pockets of well-connected regions are equally important for crowded and disordered media in decreasing (increasing) the tortuosity (permeability). This is evident in the difference between uniform and random media results, where the uniform media results become increasingly anomalous relative to the random media results as the uptake probability is increased. For uniform media there is consistent uptake due to uniform spacing in contrast to the reduced uptake observed for the well-connected regions of randomly oriented media. Random structure channel widths can be larger than in the uniform media with the presence of culde-sacs and closed-in regions ensuring the same ECS fractions as the uniform media. The ECS geometry could be better quantied beyond a simple fraction by the inclusion of fractions for the closed-in, cul-de-sac and well-connected regions.

References
1. Shuryak I, Sachs RK, Brenner DJ (2007) Biophysical models of radiation bystander effects: 1. Spatial effects in three-dimensional tissues. Radiat Res 168(6):741749. doi:10.1667/RR1117.1 2. Mothersill C, Seymour C (2001) Radiation-induced bystander effects: past history and future directions. Radiat Res 155(6): 759767 3. Mothersill C, Seymour C (2005) Radiation-induced bystander effects: are they good, bad or both? Med Con Surviv 21(2):101110. doi:10.1080/13623690500073398 4. Mothersill C, Seymour C (2006) Radiation-induced bystander and other non-targeted effects: novel intervention points in cancer therapy? Curr Cancer Drug Targets 6(5):447454. doi:10.2174/ 156800906777723976 5. Belyakov OV, Folkard M, Mothersill C, Prise KM, Michael BD (2003) A proliferation-dependent bystander effect in primary porcine and human urothelial explants in response to targeted irradiation. Br J Cancer 88(5):767774. doi:10.1038/sj.bjc. 6600804 6. Belyakov OV, Mitchell SA, Parikh D, Randers-Pehrson G, Marino SA, Amundson SA, Geard CR, Brenner DJ (2005) Biological effects in unirradiated human tissue induced by radiation damage up to 1 mm away. Proc Natl Acad Sci USA 102(40):1420314208. doi:10.1073/pnas.0505020102 7. Mothersill C, Rea D, Wright EG, Lorimore SA, Murphy D, Seymour CB, OMalley K (2001) Individual variation in the production of a bystander signal following irradiation of primary cultures of normal human urothelium. Carcinogenesis 22(9):14651471. doi:10.1093/carcin/22.9.1465 8. Mothersill C, Lyng F, Seymour C, Maguire P, Lorimore S, Wright E (2005) Genetic factors inuencing bystander signaling in murine bladder epithelium after low-dose irradiation in vivo. Radiat Res 163(4):391399. doi:10.1667/RR3320 9. Ballarini F, Facoetti A, Mariotti L, Nano R, Ottolenghi A (2009) Cellular communication and non-targeted effects: modelling approaches. Adv Space Res 44(8):917925. doi:10.1016/ j.asr.2009.05.021 10. Prise KM, OSullivan JM (2009) Radiation-induced bystander signalling in cancer therapy. Nat Rev Cancer 9(5):351360. doi: 10.1038/nrc2603 11. Facoetti A, Mariotti L, Ballarini F, Bertolotti A, Nano R, Pasi F, Ranza E, Ottolenghi A (2009) Experimental and theoretical analysis of cytokine release for the study of radiation-induced bystander effect. Int J Radiat Biol 85(8):690699. doi:10.1080/ 09553000903020016 12. Trosko J, Ruch R (1998) Cell-cell communication in carcinogenesis. Front Biosci 3:d208d236 13. Ballarini F, Alloni D, Facoetti A, Mairani A, Nano R, Ottolenghi A (2006) Modelling radiation-induced bystander effect and cellular communication. Radiat Prot Dosim 122(14):244251. doi:10.1093/rpd/ncl446 14. Brenner DJ, Little JB, Sachs RK (2001) The bystander effect in radiation oncogenesis: II. A quantitative model. Radiat Res 155(3):402408. doi:10.1667/0033-7587(2001)155[0402:TBEIRO] 2.0.CO;2 15. Khvostunov IK, Nikjoo H (2002) Computer modelling of radiation-induced bystander effect. J Radiol Prot 22(3A):A33A37. doi:10.1088/0952-4746/22/3A/306

Conclusion Static diffusion of a signal through media representing a cell aggregate is limited to a range of 55 lm at times greater than 10 mins for ECS fractions less than or equal to 0.69 with a signal uptake probability greater than 1% per interaction. This observation applies to signals such as the cytokine IL-6 diffusing through avascular tumours with ECS fractions of 20% like those that may be found in brain tissue [38]. In low ECS fraction regions beyond the percolation threshold, cluster formation by cells would create small closed-in regions that trap signals. Where signalling occurs further than 55 lm from the origin under these conditions we postulate that a relay process may occur, as has previously been suggested in attempts to model bystander signalling [19]. In this way closed-in regions trapping signals could be negated, increasing the mean diffusion distance. In long range signalling regions other environmental factors need to be considered, as transport could then occur via signal uptake by blood vessels. Implementation of the dynamics near blood vessels, a relay process and more accurate cell approximations would provide a means to further investigate the diffusion range and its implications for long range bystander signalling. Examination of other possibilities including reversibly bound signals or signals far in excess of receptors would extend the model. Our model of diffusion in the ECS is exaggerated with respect to hindrance owing to obstacles within the ECS, with viscous effects due to collagen decreasing macromolecule delivery [48].

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