Mathematical Biosciences 199 (2006) 55–78

www.elsevier.com/locate/mbs

Mathematical modeling of cancer radiovirotherapy

David Dingli a, Matthew D. Cascino b, Krešimir Josić c,
Stephen J. Russell a, Željko Bajzer b,d,*
a
Molecular Medicine Program, Mayo Clinic College of Medicine, Rochester, MN 55905, United States
b
Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester,
MN 55905, United States
c
Department of Mathematics, University of Houston, Houston, TX 77204-3008, United States
d
Biomathematics Resource, and Department of Physiology and Biomedical Engineering,
Mayo Clinic College of Medicine, Rochester, MN 55905, United States

Received 20 April 2005; received in revised form 5 October 2005; accepted 10 November 2005

Communicated by Eberhard Voit

Abstract

Cancer virotherapy represents a dynamical system that requires mathematical modeling for complete
understanding of the outcomes. The combination of virotherapy with radiation (radiovirotherapy) has been
recently shown to successfully eliminate tumors when virotherapy alone failed. However, it introduces a new
level of complexity. We have developed a mathematical model, based on population dynamics, that captures
the essential elements of radiovirotherapy. The existence of corresponding equilibrium points related to
complete cure, partial cure, and therapy failure is proved and discussed. The parameters of the model were
estimated by fitting to experimental data. By using simulations we analyzed the influence of parameters that
describe the interaction between virus and tumor cell on the outcome of the therapy. Furthermore, we eval-
uated relevant therapeutic scenarios for radiovirotherapy, and offered elements for optimization.

2005 Elsevier Inc. All rights reserved.

Keywords: Cancer; Mathematical model; Virotherapy; Radioiodide; Therapy optimization; Gene therapy

*
Corresponding author. Address: Department of Biochemistry and Molecular Biology, Mayo Clinic College of
Medicine, Rochester, MN 55905, United States. Tel.: +1 507 284 8584; fax: +1 507 284 2053.
E-mail address: bajzer@mayo.edu (Ž. Bajzer).

0025-5564/$ - see front matter
2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.mbs.2005.11.001
56 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78

1. Introduction

Despite a remarkable increase in the understanding of cancer at the molecular level, with few
exceptions, this has not translated into more effective therapies. Thus, new therapeutic strategies
are required that can combat this disease at a different level. Over the last few years there has been
increasing interest in the use for replicating viruses for cancer therapy (tumor virotherapy).
Viruses such as Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), reovirus and
measles virus (MV) seem to have a natural tropism for tumor cells due to a variety of mechanisms
[1–7]. In addition, some of these viruses can be engineered to specifically infect, replicate and de-
stroy tumor cells [8–11]. We have focused our attention on attenuated strains of MV that prefer-
entially infect tumor cells due to high level expression of CD46, the receptor used by the virus for
attachment and entry to target cells [12]. MV is highly selective and has potent oncolytic activity
against a wide variety of tumors both in vitro and in vivo [5–7]. However, while some cell lines are
sensitive in vitro to the oncolytic effect of the virus, tumor xenografts in animal models can persist
despite repeated doses of the virus. In order to circumvent this problem, we engineered the virus to
induce expression of the human sodium iodide symporter (NIS) that allows infected tumor cells to
concentrate iodide isotopes such as 131I [13]. This virus, MV–NIS retains the natural oncolytic
activity of the parent virus but has the advantage that it can eliminate tumors resistant to the virus
when it is combined with radioiodide [13]. In general, patients with malignant disease have high
tumor burdens and it is often not possible to infect every single cell. Thus, a bystander effect is
considered a highly desirable feature for any type of virotherapy. NIS expression provides a by-
stander effect since iodide-131 undergoes beta particle decay and the emitted beta particles have a
path length of 0.8 mm with a significant bystander effect [14,15].
The interaction of a replicating virus with tumor cells and the potential immune responses to
both represent a complex dynamical system that can be described as a problem in population
dynamics [16–19]. Thus, the outcome of such therapy depends in a complex way on the intricate
interactions between the various populations involved. Modeling the kinetics of virotherapy can
help in the design of improved therapeutic protocols as well as an understanding of the outcome
of such therapies. There have already been some interesting modeling and observations in this
field [16–19]. In the case of MV–NIS, the dynamical system is somewhat more complex since ther-
apy requires not only the virus but the administration of a radioactive isotope. Radioiodide is in a
continuous state of flux between the tumor and the rest of the body and is continuously lost from
the system due to both physical decay as well as losses to the environment due to excretion [20].
We have developed a mathematical model which describes these interactions and fitted the model
functions to recently published experimental data obtained for immunocompromised mice [13].
Since these mice do not mount an immune response to either the virus or tumor, in our model
we do not explicitly include the immune system. Similarly, patients with multiple myeloma, the
disease for which this virus was designed, have profound defects in the immune system and a sig-
nificant fraction of these patients do not have circulating antibodies to the measles virus, making
systemic therapy with the virus feasible.
In Section 2 we present our mathematical model of radiovirotherapy. It is based on ordinary
differential equations for cell population dynamics and on a simple compartmental model for
iodine distribution. First we introduce a model for untreated tumor growth, and then present
the model for virotherapy, which is essentially the model proposed by Wodarz [19], except that
 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78 57

it includes a more general tumor growth model. The virotherapy model is then extended to include
the effects of radiovirotherapy. For both models we discuss possible equilibrium points and re-
lated parameter constraints. We also describe typical bifurcations of these equilibria, although
a complete bifurcation analysis is beyond the scope of this paper. Mathematical details and the
investigation of the stability of equilibrium points are presented in Appendices A,B,C,D. In Sec-
tion 3 we validate the model and estimate free parameters. Based on obtained parameters we then
perform simulations (Section 4) to investigate various therapy scenarios. We present simulations
which best characterize dynamical features of virotherapy and radiovirotherapy and offer ele-
ments for optimization. In Section 5 we discuss simulation results and present concluding
remarks.
The main goal of this paper is to develop the first mathematical model for radiovirotherapy and
to show its potential usefulness in predicting the outcome of this novel mode of cancer treatment.

2. Mathematical model

2.1. Growth of untreated tumor

To model the effects of any therapy on tumor growth, one should first model the kinetics of
growth of an untreated tumor. Most often the growth of untreated tumors is well described by
the Gompertz function [21–24], yet for some tumors the more general Bertalanffy–Richards
(or generalized logistic) model is required to describe data adequately [25]. We therefore use
the Bertalanffy–Richards model:


y 0 ¼ ðg=
Þy½1
ðy=KÞ ;
> 0; yð0Þ ¼ y 0 ; ð1Þ
where y(t) is the size of the tumor cell population, r = g/
> 0 is the effective growth rate constant
and K > 0 is the carrying capacity. If by fitting this model to growth data one obtains
 1, this
fit would be equivalent to the Gompertz model, since the two models are equivalent in the limit

! 0 [22,26]. The solution of Eq. (1) has the explicit form [26,27]:

1=

yðtÞ ¼ y 0 ½q
þ ð1
q
Þe
gt  ; q ¼ y 0 =K. ð2Þ

2.2. Tumor growth with virotherapy

We will assume that the virus treatment begins at time t = tv. Then, the population dynamics of
uninfected tumor cells y(t), of virus-infected tumor cells x(t), and of free infectious virus particles
v(t) can be modeled by differential equations:


y 0 ¼ ry½1
ðy þ xÞ =K

jyv; yðtv Þ ¼ y v ;
0
x ¼ jyv
dx; xðtv Þ ¼ 0; ð3Þ
0
v ¼ ax
xv; vðtv Þ ¼ v0 .

Model parameters r, K,
, j, d, a, x are considered positive. The total population of tumor cells
is now given by x + y, and this population can maximally reach the carrying capacity K
58 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78

(see Appendix A). The term jyv represents the rate by which the infected cells are leaving the pop-
ulation of uninfected cells and entering the population of infected cells. It is therefore assumed
that the rate of infection is proportional to the constant fraction of the number of all possible
encounters of uninfected cells and virus particles at time t given by y(t)v(t). The rate of death
of infected cells (either virus-inflicted or natural) is assumed to be proportional to the population
of infected cells and described by a rate constant d. Infected cells most probably do not proliferate
[28]. The model can accommodate modest proliferation with the rate proportional to population
size and to the constant a < d, while the effective death rate constant is d = d
a > 0. The popu-
lation of virus particles is growing due to replication of viruses in infected cells, which is modeled
by the rate ax(t), where a is the corresponding rate constant. Finally, the rate of virus elimination
is modeled as proportional to the virus population size, with the rate constant x. In principle, x
can effectively simulate virus elimination by any biological mechanism including the reaction of
the immune system. We assume that at the beginning of the virotherapy at t = tv, the tumor
has grown to the size yv, there are no infected cells, and the initial viral dose is v0.
The model given by (3) is very similar to the one proposed by Wodarz [19]. The main difference
is that our model features an exponent
5 1 which provides flexibility to describe growth kinetics
of various unperturbed tumors, and which was suggested by our data (see next section). In the
model of Wodarz an additional death rate term of the form (rate constant) · (population size)
was introduced in the first and in the second equation, i.e., his first two equations are of the form:
~
jyv;
y 0 ¼ ~ry½1
ðx þ yÞ=K
dy x0 ¼ jyv
ðd~ þ ~aÞx.
~ d ¼ d~ þ ~a we obtain equations given in (3) except
However if we introduce the notation r ¼ ~r
d,
for the exponent
. r > 0 and d > 0 are interpreted as effective proliferation and death rate con-
stants, respectively. r < 0 cannot occur because we assume that tumor grows.
There are three equilibrium points of system (3). The desired outcome of the therapy corre-
sponds to the equilibrium point at the origin:
y 1 ¼ 0; x1 ¼ 0; v1 ¼ 0; ð4Þ
which is, however, always a saddle for biologically relevant parameter values and hence unstable
(see Appendix B). The complete failure of therapy occurs when the tumor eventually grows to its
maximal size. It is represented by the equilibrium point:
y 2 ¼ K; x2 ¼ 0; v2 ¼ 0; ð5Þ
dx
which is either a saddle, or is stable if > K (see Appendix B). The partial success of therapy is
aj
represented by the equilibrium point:

x þ y 

dx rx 3 3 a
y3 ¼ ; x3 ¼ 1
; v3 ¼ x3 . ð6Þ
aj aj K x
This equilibrium is not given explicitly, but can be obtained by solving non-linear equation for x3
given in (6). We show in Appendix C that the unique solution in the positive octant exists under
the assumptions that
> 0, x3 > 0 and 0 < y3 < K. The constraint y3 < K is important because
y3 > K would yield x3 < 0 as can be seen from (6). When, y3 = K, x3 = 0 this point coincides with
the equilibrium point (5). As shown in Appendix B, when y 3 ¼ dx aj
¼ K, the two fixed points typ-
ically undergo a transcritical bifurcation, at which the point (y3, x3, v3) enters the positive octant.
 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78 59

General conditions for the stability of the point (y3, x3, v3) are not tractable. However, numer-
ical simulations, as well as general bifurcation theory, indicate that it becomes stable in this trans-
critical bifurcation. Since after this bifurcation
dx
y3 ¼ <K ð7Þ
aj
this indicates that the long term effects of the therapy first exhibit reduction in the number of unin-
fected tumor cells exactly at this bifurcation point. As it is shown in Section 4, for parameters of
interest the unstable manifold of (y2, x2, v2) asymptotes to (y3, x3, v3) until this point undergoes a
Hopf bifurcation. The periodic orbit arising in this bifurcation grows to a quasi-heteroclinic cycle
which will be described in Section 4.
The total tumor burden B = x3 + y3 at equilibrium (6) obeys the following inequalities:
xðr þ dÞ
B < K; B<  Bu . ð8Þ
ja
The first inequality follows from the fact that the total population cannot exceed K (see Appendix
A). It also directly follows from the equation for x3 rewritten in the following way:
 

B ja

1 ¼
lx3 ; l ¼ > 0. ð9Þ
K xr
Clearly, B > K would yield expression on the left-hand side greater than zero, which is in contra-
diction with the right-hand side. B = K implies x3 = 0, which represents the equilibrium point (5).
The second inequality in (8) follows from (B/K)
> 0 leading to x3 < 1/l, and from the expression
(6) for y3.
As one can expect upper limit Bu to B in (8) decreases as virus proliferation rate a and infection
rate j increase. Bu increases with the increase of tumor proliferation rate r, infected cells death
rate d and virus elimination rate x. The dependence of Bu on d is counterintuitive: it suggests that
virotherapy will be more successful if the virus is less cytopathic. This was already observed in
numerical simulations presented by Wodarz [18,19].

2.3. Tumor growth with radiovirotherapy

To model the effects of b-radiation from 131I, we first have to find the activity at the tumor site.
We assume that after the injection, radioactive iodine is distributed within two compartments: the
tumor site (T) and in the rest of the mouse (M) (see Fig. 1). We model the slower exchange and
decay dynamics established after rapid initial distribution which leads to the initial amounts of
radioactive iodine at the tumor site IT0 and in the rest of the mouse IM0 at time tr > tv when
the iodine was injected. The corresponding equations are:
I 0T ¼
kI T
k 1 I T þ k 2 I M ; I T ðtr Þ ¼ I T0 ;
ð10Þ
I 0M ¼
kI M
k 3 I M
k 2 I M þ k 1 I T ; I M ðtr Þ ¼ I M0 .

Here k is the decay constant, k1IT is the transition rate of iodine from compartment T to M, and
k2IM is the transition rate from compartment M to T. The excretion rate k3IM includes iodine
60 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78

Fig. 1. Schematic diagram of proposed model for radiovirotherapy. IT and IM are the amounts of radioactive iodine at
the tumor site and in the rest of the mouse, respectively. y denotes the population of uninfected cells which are also not
damaged by radiation. Proliferation of these cells is described by effective proliferation rate r, carrying capacity K and
parameter
which characterizes the shape of the sigmoidal growth curve. v denotes the population of virus particles. x
represents the population of infected cells which are not damaged by radiation, and u denotes the population of cells
damaged by radiation which is characterized by activity kIT. Indicated transfer rates are explained in the text.

excreted from the mouse, mostly by urination (Fig. 1). The total amount of iodine in the mouse at
any given time, which was actually measured, is simply given as I = IM + IT.
To model the effects of radiation on tumor cells, in addition to populations of virus particles,
uninfected cells and infected cells, we introduced a population of cells u(t) that are irreparably
damaged by radiation (Fig. 1). These cells do not proliferate, and are destined to die, but they still
occupy space and compete for nutrients, and therefore should contribute to the self-regulation of
growth. The rate by which tumor cells (both virus infected and uninfected) become irreparably
damaged by radiation is assumed to be proportional to the radiation dose D(t) absorbed by those
cells and to their respective number (cf. [29]). For higher doses this can be considered as conser-
vative assumption, since linear-quadratic model is used in modeling classical radiotherapy based
on discrete radiation intervals [30]. The absorbed radiation dose itself is proportional to the cumu-
lative activity [31]:
Z t
DðtÞ ¼ gk I T ðt0 Þ dt0 ; ð11Þ
tr

where g is a proportionality constant. Here we assume that only the radioactivity at the tumor site
contributes to the absorbed dose, as we can consider compartment T to include all iodine atoms
which can emit beta particles capable of reaching tumor cells.
 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78 61

Assuming that both infected and uninfected cells are equally affected by radiation, we modify
(3) to include radiation effects relevant after time t = tr (Fig. 1):
y 0 ¼ ry½1
ðx þ y þ uÞ
=K

jyv
bDy; yðtr Þ ¼ y r ;
0
x ¼ jyv
dx
bDx; xðtr Þ ¼ xr ;
0 m
ð12Þ
u ¼ bDðx þ yÞ
cu ; uðtr Þ ¼ 0;
v0 ¼ ax
xv; vðtr Þ ¼ vr ;
where according to (11) D(t) is given by the differential equation:
D0 ¼ gkI T ; Dðtr Þ ¼ 0. ð13Þ
Parameter b is the rate constant for tumor cells becoming irreparably damaged. The term cum
represents the effective death rate of damaged cells. Power-law dependence of rates is known to
represent general behavior of biological systems [34]. Because the fit to data was poor for
m = 1, the exponent m 5 1 was mandated by data (see next section). The values (yr, xr, vr) repre-
sents the solution of the system (3) at time t = tr. Since all tumor cells contribute to the self-reg-
ulation of growth, the corresponding term [1
(x + y + u)
/K
] includes the total population
x + y + u, which cannot exceed the carrying capacity K (see Appendix A).
Mathematically, Eqs. (10), (12) and (13) constitute an autonomous system of equations which
can be partially solved analytically. Indeed, the linear equations (10) can be solved to yield the
following expression for the dose given by (11):
gkI 0
DðtÞ ¼ ½c1 /ðt; s1 Þ
c2 /ðt; s2 Þ; ð14Þ
2S
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi
2
S ¼ ðk 1 þ k 2 þ k 3 Þ
4k 1 k 3 ; I 0 ¼ I T0 þ I M0 ; /ðt; sÞ ¼ ðesðt
tr Þ
1Þ=s; ð15Þ
c1;2 ¼ I T0 ðk 3
k 1
k 2  SÞ=I 0 þ 2k 2 ; s1;2 ¼ ð
2k
k 1
k 2
k 3  SÞ=2 < 0. ð16Þ
There are three equilibrium points of the dynamical system given by (10), (12), and (13). The suc-
cess of therapy is given by the equilibrium point:
gk½I T0 ðk þ k 3 Þ þ k 2 I 0 
I T ¼ I M ¼ 0;
y 1 ¼
x1 ¼

u1 ¼
v1 ¼ 0; D¼ 2
. ð17Þ
k þ kðk 1 þ k 2 þ k 3 Þ þ k 1 k 3
Note that subsystem (10) and (13) has the globally attracting equilibrium I T ¼ I M ¼ 0, so that
D ¼ limt!1 DðtÞ. Equilibrium (17) is neutrally stable if r < bD (see Appendix D). Here bD repre-
sents the maximal rate for radiation damage.
The partial success of therapy is represented by the equilibrium point

x2 ¼
v2 ¼ 0;
y 2 ¼ Kð1
bD=rÞ1=


y 1=m
2 ;
u2 ¼ b
y 1=m
2 ; b ¼ ðbD=cÞ1=m ; ð18Þ
and I T , I M , D given in (17). Here y2 is not given explicitly, but as the solution (unique, see Appen-
dix C) of the above non-linear equation which is defined under the condition:
r P bD. ð19Þ
When r ¼ bD the equilibria (17) and (18) coincide. In fact, with a decrease in the rate bD below
the proliferation rate r, the equilibrium (17) can be expected to lose its stability in a transcritical
62 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78

bifurcation (see Appendix D). The stable equilibrium (18), representing partial success of therapy,
enters the positive orthant after this bifurcation. Note that the equilibrium (18) exists in the ab-
sence of virotherapy, and is due only to the interaction between the tumor cells and radiation.
However, as will be demonstrated below, the stability of this point is changed in the presence
of virotherapy.
If condition (19) is satisfied, non-linear equation in (18) has a unique solution (see Appendix C)
leading to the tumor burden

u2 ¼ Kð1
bD=rÞ1=
< K.

B2 ¼
y 2 þ
ð20Þ
Compared to corresponding equilibrium point (5) representing the failure of the virotherapy, here
we see the benefit of additional radiation therapy in decreasing the tumor burden.
Finally, if the condition (19) is satisfied, and the following inequalities are valid
1=m
ð
y 3 þ b
y 3 Þ
< Kð1
bD=rÞ < K;
y 3 ¼ pðd þ bDÞ < K; p ¼ x=ðajÞ; ð21Þ

then there exists another equilibrium point defined by
y 3 and

1=m

l
x3 ¼ 1
bD=r
K

½
x3 þ
y 3 þ bð
x3 þ
y 3 Þ  ;
u3 ¼ bð
x3 þ
y 3 Þ1=m ;
v3 ¼ ax3 =x. ð22Þ
The solution of the above non-linear equation in x3 is unique (see Appendix C). Note that equi-
librium (22), corresponds to equilibrium (6) in the absence of radiotherapy. As discussed in Sec-
tion 2.2, equilibrium (6) collides with (5) in a transcritical bifurcation and they exchange stability.
Similarly, a transcritical bifurcation in which (18) and (22) exchange stability can be expected, and
is observed numerically, in a range of parameters.
Equilibrium (22) corresponds to a better outcome of the therapy than equilibrium (18), as the
corresponding tumor burden is (cf. Eq. (20)):

u3 ¼ K½1
l
x3
bD=r1=
.

B3 ¼
x3 þ
y 3 þ
ð23Þ
Since the expression in parenthesis needs to be positive, one obtains the upper bound for equilib-
rium population of infected cells: x3 < ðr
bDÞ=ðlrÞ.
The present analysis of equilibria shows that condition (19) is critical. When it is not satisfied,
the only allowable equilibrium point is (17) which represents the desired outcome of therapy.

3. Model validation and parameter estimation

The proposed model was validated by least-square fits to available data obtained for multiple
myeloma induced in SCID mice [13]. These data include tumor growth curve without treatment,
growth curve when virotherapy is introduced at day 15, iodine radioactivity I(t) detected in the
mouse, iodine radioactivity IT(t) detected at the tumor site, and the tumor growth curve when
in addition to virotherapy, the radiotherapy (131I) is introduced starting on day 24. Tumor size
was measured as volume (in mm3), while our model includes population of cells. In the following
we will assume that 1 mm3 corresponds to 106 cells and we will divide obtained cell populations y,
x, u and v with 106 to match the experimental data for tumor size.
 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78 63

The parameters were estimated by using weighted non-linear least squares method. Weighting
factors were chosen as 1=r2i , where ri is the experimentally determined standard deviation for the
ith data point. Technically, least-squares fitting was conveniently performed in MLAB (Civilized
Software Inc., Bethesda, MD, http://www.civilized.com) but in cases where the minimum was dif-
ficult to find, we used our powerful minimizer [32] in conjunction with custom made ODE solver.
Numerical solutions of differential equations with the best fit parameters were also checked by
several ODE-solvers available in MAPLE (Waterloo Maple Inc. Waterloo, ON) In case of un-
treated tumor the fitting was relatively simple, as the analytic form of the solution (2) is known.
A good fit was obtained with the exponent
= 1.65 rather than
 0, which would imply that the
growth follows the Gompertz function (see Fig. 2 for parameter estimates). We tried to fit the data
to the genuine Gompertz function and obtained a less adequate fit (Fig. 2) as confirmed by three
model selection criteria: Bayesian information criterion, modified Akaike information criterion
and Hannan–Quinn information criterion (see Fig. 2). The same criteria suggest the generalized
logistic model with
= 1.65 as more adequate then the logistic model (
= 1).
In case of virotherapy alone, we fitted x(t) + y(t) to data. The tumor size at the beginning of the
therapy, yv, was obtained from the best-fit growth curve for the untreated tumor. The values of
parameters r, K,
, are those obtained by fit to the untreated tumor (Fig. 2). The initial viral dose
v0 was known from the experiment. The fitting procedure was more involved, as it requires

Undisturbed Tumor Growth

Generalized Logistic Model (ε = 1.65)
Logistic Model (ε = 1.0)
2400 Gompertz Model
Tumor Size (mm 3 )

1600

800

0
0 10 20 30 40
Time (days)

Fig. 2. Weighted least-squares fitting the experimental tumor growth data by Gompertz (G), logistic (L), and
generalized
Pn logistic (GL) models. Bayesian information criterion for model selection defined as C = v2 + m ln n,
v ¼ i¼1 ½y i
yðti Þ2 =r2i (n – number of experimental data points (ti, yi) , m – number of free parameters; cf. [33] and
2

references therein) yielded: C(G) = 13.2, C(L) = 8.7, C(GL) = 7.2. Hannan–Quinn information criterion defined as
H = v2 + m ln(ln n) (cf. [33] and references therein) yielded: H(G) = 7.77, H(L) = 3.30, H(GL) = 2.32. Modified Akaike
information criterion A (cf. Scientist Handbook, MicroMath Scientific Software, 1995, p. 467; [33]) yielded:
A(G) = 7.66, A(L) = 8.80, A(GL) = 8.82. Note that higher A value implies more adequate model, while the reverse is
true for C and H values. The best fit parameters for GL model were: r = 0.206 d
1, K = 2139.3 mm3,
= 1.65.
64 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78

2400
Undisturbed Tumor Growth
Tumor Growth with MV-NIS
2000 Radioviro Therapy MV-NIS + I131

Tumor Size (mm3 )

1600

1200

800

400

0
0 10 20 30 40 50
Time (days)

Fig. 3. Weighted least-squares fitting of tumor growth data for virotherapy (MV–NIS) using model equations (3),
and for radiavirotherapy (MV–NIS + 131I) using model equations (10), (12) and (14). Obtained best fit parameters
were: j = 0.104 mm
3 d
1, d = 0.703 d
1, x = 0.285 d
1, ~
g ¼ 2:49 d
1, c = 18.57 mm
3 d
1, m = 0.176, with a set to

1
0.0015 d (see text).

simultaneous numerical solution of differential equations and minimization. While the best fit
curve follows the data and therefore validates the model (Fig. 3), due to the lack of data after
day 35, we were able to estimate by fitting only three of four free parameters which define the
model: j, d, a, x. Thus, parameter a was fixed to the value of 0.0015 for which the fit yielded re-
duced v2 closest to its theoretical value of 1. Lower and higher values of a yielded fits with lower
or higher v2 values, respectively.
For radiovirotherapy we first validated model (10) for iodine distribution in the mouse (Figs. 1
and 4). Counts from 156 keV photons resulting from 123I decay (k = 1/13.2 h
1) were measured
[13] by gamma camera both for the whole mouse and for the tumor site (Fig. 4). These two data-
sets were simultaneously fitted to model equations and the estimates for free parameters k1, k2, k3,
IT0/I0, I0 were obtained (cf. Fig. 4). The best fit yielded k2 = 0, i.e. negligible rate of iodine intake
by tumor, after the initial very fast intake which is modeled by IT0. This is compatible with the
recent findings which show net efflux of iodine from tumor cells [20]. By using obtained values
for these rate constants we evaluated function (11) for dose and subsequently fitted x(t) +
y(t) + u(t) to tumor growth data for radiovirotherapy (Fig. 3) by using Eqs. (12). First, we at-
tempted a fit with the simpler model which excludes the compartment of radiation damaged cells,
however the fit was not acceptable, reflecting the fact that radiation damaged cells do not die
immediately after being exposed to radiation. The death rate of damaged cells was found to be
proportional to um with m = 0.176. Attempted fits with m = 1 provided curves which poorly fol-
lowed the data. It should be noted that parameters g and b cannot be resolved by fitting. Thus
 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78 65

70
450

Counts (in thousands)

60 350

250
50
150
Counts (in thousands)

40 50

0 5 10 15 20 25
30 Time (hours)

20

10

0
0 5 10 15 20 25
Time (hours)

Fig. 4. Weighted least-squares fitting of 123I counts at tumor site and for the whole mouse (inset). Data were fitted
simultaneously and counts were calculated from model equation (10). Estimated rate constants: k1 = 3.64 d
1, k2 = 0,
k3 = 1.59 d
1. Other estimated parameters: I0 = 6.69 · 104, IT0/I0 = 0.145 (see text). Dotted line represents a pure
physical decay of the radioisotope.

we introduced a new variable DðtÞ e ¼ bDðtÞ ¼ ~g½c1 /ðt; s1 Þ
c2 /ðt; s2 Þ (cf. Eq. (14)), where
g ¼ bgkI 0 =ð2SÞ is the combined parameter which we estimated together with c and m by fitting
~
(see caption of Fig. 3).
In concluding this section, we wish to point out that more data in density and range would be
needed to make more robust estimates of parameters, especially for virotherapy. However, the
present results provide a good starting point for a numerical exploration of the model.

4. Simulations

We investigated the dynamics of myeloma growth under virotherapy and radiovirotherapy by

observing the effects of variations in model parameters. In particular, we considered changes ob-
tained by assuming different virus cytotoxicity (parameter d), different proliferation rates (param-
eter a), and variations of parameter ~ g which combines six model parameters including the rate
constant b for radiation damage of cells and the amount of iodine administered, I0. We also
considered the impact of changes in the time period between the start of virotherapy and iodine
treatment, as well as variations in the viral dosage on the effectiveness of radiovirotherapy.
To make the numerical simulations realistic our ODE solver sets any population variable to 0 if
it falls below one cell (or 1 virus particle). As an indicator of the effectiveness of therapy we intro-
duce the notion of ‘response time’ T, which is the time interval from the start of radiovirotherapy
66 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78

to the point when the tumor burden falls below an observable level, i.e. below one million cells (or
1 mm3 of tumor volume). Time T is measured in days, and approximated by integers. It is only
considered when there is numerical evidence that the tumor burden eventually falls below one cell.
In the following the sizes of cell populations and virus particles are expressed in units of millions.

4.1. Virotherapy

The model parameter which appeared to be difficult to estimate from available data is a – the
rate constant for viral proliferation. In Fig. 5, panel A, we show the tumor size as a function of
time elapsed after the start of the viral therapy. The value a = 0.0015 d
1 was accepted as the most
reasonable (see Section 3 and Fig. 3) for given data. The corresponding curve (solid line) shows
strongly damped oscillations terminating at equilibrium z3 = x3 + y3 = 1464. As a increases to
0.0024 d
1 we see less damped oscillations and a lower equilibrium value z3 = 976. At higher rates
of virus proliferation, the tumor burden is reduced, but does not lead to a cure.
If a is increased further, this equilibrium point undergoes a Hopf bifurcation. The stable peri-
odic orbit born in this bifurcation grows quickly in size, and becomes quasi-heteroclinic: It
approaches the invariant plane y = 0, tracks an orbit in this plane and thus converges to the
equilibrium at the origin. The orbit then leaves in the direction of the y-axis, which is the unstable
manifold of the origin (see Appendix B), and follows it to the vicinity of the point x2 = v2 = 0,
y2 = K. After a close approach to this equilibrium, the orbit is ejected along its unstable manifold,
and approaches y = 0, and the process repeats.
In approaching this periodic orbit y experiences oscillations with increasing peak values and
decreasing minimal values. This behavior can be observed for a = 0.05 d
1. After the second peak
the minimal value is well below the observable population size 1 i.e. z(55)  0.6. After the third
peak the minimal value drops below 10
6 which corresponds to less then one cell
(z(137) < 10
6) and, therefore, we can safely assume that the tumor is eradicated. However, the
third peak reaches the value of z(106) = 760. This implies that the desired results of viral therapy
can sometimes be expected after a long time period and with periodic high level relapses.
When a is sufficiently small, equilibrium (6) collides with the point (5) in a transcritical bifur-
cation. The point (5) becomes stable, and the therapy offers no reduction of the tumor burden in
the long run. Thus when a is such that y 3 ¼ dxja
¼ K, the model predicts a tumor burden B = K, and
x3 = 0. For the given set of parameters this happens for a  0.0008 d
1. When a becomes even
smaller, implying dxja
> K, model still predicts B = K. Fig. 5(A) shows such a scenario (dashed
line). Both the infected cell population and the virus population in this case drop to zero, as pre-
dicted by (5) (see panels B and C). On the other hand, for larger values of a these populations
oscillate in a manner similar to z(t), with a shift in time (panels B and C, Fig. 5).
As previously discussed by Wodarz [18,19] the cytotoxicity of the virus seems essential for the
success of viral therapy. In our model cytotoxicity is regulated by parameter d, which represents
the rate constant for net loss in a population of infected cells. In simulations shown in Fig. 6 we
were only changing d. In the case of higher cytotoxicity d = 1.2 d
1 (compared to d = 0.703 d
1
estimated from data) the therapy completely fails yielding the tumor burden at the level of carry-
ing capacity, since dx
aj
> K for this value of d. By lowering cytotoxicity we obtain damped oscilla-
tions (d = 0.3, z3 = 846), or oscillations with growing amplitudes (d = 0.07 d
1). We have to
further decrease cytotoxicity to obtain tumor eradication; this happens for d = 0.008 d
1 (curve
 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78 67

2400
A
2000

1600
z(t)

1200

800

400

0
500 25
B α = 0.0006
α = 0.0015
α = 0.0024
α = 0.05
400 20

300 15

x (t)
x (t)

200 10

100 5

0 0
2.4 36
C

2.0 30

1.6 24
v (t)
v (t)

1.2 18

0.8 12

0.4 6

0.0 0
0 20 40 60 80 100 120
t

Fig. 5. Simulations of cell population dynamics for virotherapy. Panel A: total tumor cell population z(t). Panel B:
virus-infected cell population x(t). Panel C: population of viral particles v(t). Viral proliferation rate constant a is
varied, while other parameters are kept at their estimated values (see legends of Figs. 2–4) Note that in panels B and C
we show curves in two different scales.
68 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78

2400

2000 δ = 1.20
δ = 0.703
δ = 0.30
δ = 0.07
1600
z(t)

1200

800

400

0
0 20 40 60 80 100 120
t

Fig. 6. Simulations of tumor cell population dynamics. Rate constant d of viral elimination is varied, while other
parameters are kept at their estimated values (see legends of Figs. 2–4).

not shown). However in this case the decrease in tumor size is very slow, yielding the response
time T = 924 d, i.e. z(924)  0.996. One would expect that this situation could be improved by
increasing the initial viral dose v0. Very little can be achieved by increasing v0 from 2 to 4 because
z(924)  0.95. Interestingly, further increase of v0 to 10 leads to an even worse situation:
z(924)  9.84.
A faster decrease in the tumor cell population leading to eradication can be obtained when a is
sufficiently large and d sufficiently small. Thus, for a = 0.05 d
1 and d = 0.07 d
1 we obtain the
response time T = 81 d. Complete eradication is expected after day 277 according to the obtained
result z(278) < 10
6. To understand this scenario better, we performed simulations starting with
these parameters and then changing first just one parameter among j, d, a, x, then two param-
eters in pairs, (such that dx
aj
stays constant), and finally all four parameters (Table 1). Parameters
were multiplied by factors 5 and 1/5. As a measure of the success of the therapy we calculated
corresponding response times and maximally obtained sizes Z. The results in Table 1 clearly indi-
cate that increasing d is beneficial, while decreasing it is detrimental to the therapy. This is differ-
ent from the situation described above, where decreasing d was beneficial. The reason is that the
dynamics of the system is now characterized by a quasi-heteroclinic cycle, rather then by equilib-
rium (y3, x3, v3). However, unlike for d = 0.703 d
1, a = 0.05 d
1 (Fig. 5(A)), the tumor cell pop-
ulation for considered parameters falls below one cell before the second peak appears.
Table 1 reveals that reduction in a and increase in x are detrimental to the therapy, as one can
expect. However the influence of these parameters is not as dramatic as the influence of d. The
results of Table 1 also suggest that in the considered case when tumor is eventually eliminated,
the characteristic parameter dx aj
is not related to response time T or maximal tumor burden Z.
 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78 69

Table 1
Response time and maximal tumor size for model parameters of virotherapy which lead to complete tumor elimination
dx/(aj) fj fd fa fx T Z
3.85 1 1 1 1 81 196
0.77 5 1 1 1 72 139
1 1/5 1 1 389 212
1 1 5 1 76 163
1 1 1 1/5 79 183
19.2 1/5 1 1 1 105 616
1 5 1 1 20 634
1 1 1/5 1 214 787
1 1 1 5 335 632
3.85 5 5 1 1 16 126
1/5 1/5 1 1 477 654
1 5 5 1 17 148
1 1/5 1/5 1 430 343
1 1 5 5 78 180
1 1 1/5 1/5 86 225
5 1 1 5 74 144
1/5 1 1 1/5 98 442
1/5 1/5 1/5 1/5 516 1048
5 5 5 5 15 121

4.2. Radiovirotherapy

While engineering a virus to become more or less lethal or to proliferate faster within the cells
could be a major undertaking, the change of timing and dose for radioiodide therapy, as well as
the initial viral dose are easily achievable. We will therefore consider simulations in which these
parameters are varied while the rest of model parameters are kept at estimated values. In all sim-
ulations tumors are eventually eradicated, i.e. equilibrium (17) is achieved. Two indicators of the
effectiveness of the therapy are the response time T and the maximal size of the tumor Z. Fig. 7
suggests that the start of radiotherapy after day 20 from the beginning of virotherapy (i.e. delay
time Dtr = tr
tv > 20 d) leads to a probably intolerable maximal size of the tumor. Earlier
administrations of iodine result in smaller maximal volume. The response time T, increases
approximately linearly with delay time Dtr. It is reasonable to assume that the observed kink
around Dtr = 17 d is more likely the result of chosen modeling functions than some biologically
caused effect; yet this would have to be experimentally investigated.
In experiments the initial viral dose was v0 = 2. From Fig. 8 it is clear that a twice smaller max-
imal size can be achieved if the initial viral dose is twice bigger, and that increasing v0 beyond 6
has little effect. Time T decreases linearly with v0. However, whether the tumor will be substan-
tially reduced in 32 or 25 days does not seem to warrant an increase of v0 from 6 to 10.
How much iodine should be given for treatment is the critical issue because of radiation damage
to the healthy tissue, in particular to the thyroid gland. In the model the initial iodine amount I0 is
effectively lumped into the parameter ~ g. To change I0 by a factor f we multiplied the value of ~g
70 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78

80

2000

70

1600
Z (in 106 cells)
60

1200

T (days)
50

800
40

400
30

0 20
0 7 14 21 28 35
tr-tv (days)

Fig. 7. Maximal tumor size Z and response time T (see text) for radiovirotherapy as a function of time elapsed after the
start of virotherapy until the iodide is introduced. Estimated model parameters presented in Figs. 2–4 were used.

700 44

600
40
Z (in 10 6 cells)

500
36
T (days)

400

32
300

28
200

100 24
0 2 4 6 8 10
v0 (in 10 6 virus particles)

Fig. 8. Maximal tumor size Z and response time T for radiovirotherapy as a function of the initial viral dose v0.
Estimated model parameters presented in Figs. 2–4 were used.

obtained from experimental data by f. Fig. 9 clearly shows that reduction of I0 below 80% is det-
rimental to the therapy especially in terms of T, while an increase above 120% does not lead to a
substantial improvement. Decrease to 50% leads to the equilibrium point (22) showing that
 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78 71

200

600

Z (in 106 cells) 150

550

T (days)
100
500

50
450

400 0
0.6 1.0 1.4 1.8
f

Fig. 9. Maximal tumor size Z and response time T for radiovirotherapy as a function of the fraction of radiation
activity actually administered in the experiments. Estimated model parameters presented in Figs. 2–4 were used.

600
Tumor Cell Populations (in 10 6 cells)

400

z (t)

y (t)
200

u (t)

x (t)
0
0 50 100 150 200
t (days)
Fig. 10. Simulation of cell population dynamics for radiovirotherapy in case that only 50% of actually administered
radiation activity is used. z(t) is the total tumor cell population, y(t) is the population of uninfected cells, x(t) is the
population of virus infected cells, and u(t) is the population of cells irreparably damaged by radiation which do not
proliferate. Estimated model parameters presented in Figs. 2–4 were used.
72 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78

radiovirotherapy can fail if the dose of iodine is equal to or below 50% of the dose applied in
experiments (Fig. 10). With 50% of the iodine dose and a five times increase in initial viral dose,
the corresponding simulations unfortunately still yielded a substantial tumor burden, i.e. an equi-
librium value not close to zero.

5. Discussion and conclusion

A previously proposed model of virotherapy [19] was investigated by simulations, but was not
validated by experimental data. Here we presented somewhat generalized version of this model
which is consistent with available data for multiple myeloma. We also presented more extensive
simulations and analysis of three equilibrium points. One equilibrium point represents a cure, as
the tumor burden monotonously decreases to zero, and the viral load decreases to zero. Another
equilibrium point represents the failure of therapy, because the tumor burden monotonously in-
creases and reaches carrying capacity. Finally, the third equilibrium point represents a partial suc-
cess of the therapy. In this case, after damped oscillations, the tumor burden eventually reaches a
level smaller than the carrying capacity. Our simulations show that for the lower equilibrium tu-
mor burden, oscillations are less damped and occur with higher frequency. Interestingly, for some
combination of model parameters we found yet another distinct dynamic behavior: a stable quasi-
heteroclinic cycle which corresponds to oscillations with sharp and limited growth of peaks both
in tumor burden and in viral population. In such a scenario each peak is followed by a dip which
increases as the peak grows. Eventually the dip reaches values smaller than one cell, which means
that the tumor has vanished. This could represent a successful therapy, if the organism can toler-
ate these fluctuations in tumor burden and viral load. In his paper Wodarz [19] has not reported
the possibility of such fluctuations, but instead provided an example where the dip after the peak
is shown and interpreted as sustained cancer remission (see Fig. 3 in [19]). In Table 1 we presented
a set of model parameters for which after the first peak tumor cell population falls below one cell
and is therefore considered eliminated. For this combination of parameters more cytotoxic virus
leads to faster elimination of the tumor, in contrast to the combination of parameters when more
cytotoxic virus is not beneficial for avoiding the equilibrium of persistent tumor population. These
behaviors have been also noted by Wodarz as a possible response of the model [19].
The advantage of replicating viruses for cancer therapy is the establishment of the persistent
infection with ongoing viral proliferation. One cannot assume that in vitro viral proliferation rates
represent the in vivo situation. However, it is clear from our simulations that this viral property
described by parameter a is an important determinant of the outcome. Technically, it is both dif-
ficult and inconvenient to determine a experimentally. The present model suggests that a is small
(in the range 0.0008–0.002). This can be interpreted as a very slow net rate of free virus produc-
tion. Biologically this is plausible because the measles virus is present mainly in cells and spreads
from cell to cell via membrane fusion between infected and uninfected cells. This is compatible
with in vitro studies showing that even in culture most of the virus is cell associated.
Safety considerations would suggest that the tumor and population of virus particles should be
eliminated concomitantly. This would ensure that there is no persistent viral infection in the
organism – a feature of virotherapy particularly important for patients with myeloma who are
immunocompromised. The presented model is designed to be compatible with the notion that
 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78 73

elimination of tumor substrate leads to elimination of virus, which was suggested by our experi-
ments (unpublished data).
The equilibrium points of the model for virotherapy are characterized by the complex param-
eter k = dx/(ajK) > 0. In the case when k P 1 therapy cannot be successful because the tumor
burden will inexorably increase to the carrying capacity. In the case when k < 1 there are two sce-
narios: (i) tumor cell population is always greater than 10
6 (one cell) and tumor burden at equi-
librium is smaller than K, decreasing as k decreases (up to a value which we were not able to
determine precisely); (ii) when k is of the order of 0.01, tumor cell population, after possible oscil-
lations eventually, falls below 10
6 (one cell). Our simulations indicate that in this latter case there
is no obvious relation between magnitude of k, response time T, and the maximal tumor cell pop-
ulation Z (see Table 1).
While the model parameters allow tumor cell population to fall below 10
6, in reality this may
not represent acceptable therapy, if the decrease in the number of tumor cells is too slow. For
example in one simulation we found that it would take over 900 days to reduce the tumor burden
below observable size. This time period is unacceptable when compared to the lifespan of a
mouse. Our simulations suggest that the values of rate constants describing viral infectivity, cyto-
toxicity and proliferation, which may produce a relatively fast and monotone decrease in tumor
burden, are confined to a somewhat limited range. This observation raises the question whether
oncolytic viruses can be genetically modified to favorably optimize these parameters. Short of
doing this, one can consider viruses as a source of targeted radiation therapy for sensitive tumors.
The model of virotherapy served as a basis for modeling radiovirotherapy. The latter model
incorporates description of iodine dynamics and radiation induced cell death. The proposed mod-
el for radiovirotherapy also exhibits three equilibrium points. However, in contrast to the viro-
therapy model, none of the equilibrium points represents a complete failure of therapy. The
critical parameter is q ¼ bD=r. For q > 1 only the zero equilibrium point (17) (complete cure) is
possible; however, when q 6 1 equilibrium points (18) and (22) are possible with the tumor burden
being smaller than K.
As available experimental data show, radiovirotherapy leads to a complete cure, while virother-
apy alone leads to a partial reduction of the tumor burden. To assess the effects of radiovirother-
apy we have performed a number of simulations keeping the model parameters at values found to
describe the experimental data, but we varied the time of the start of radiotherapy, the initial viral
dose, and the dose of iodine. These simulations lead to the following conclusions:

(a) It is best to start radiotherapy within 7 d of virus (MV–NIS) administration (Fig. 7). The
maximum benefit of radiation depends on the amount of iodide that is taken up and retained
by the tumor. This is a function of NIS expression that depends on viral proliferation and
gene expression [20]. If iodine is administered too soon after MV–NIS, NIS expression
may not be optimal. This suggest that there is an optimal window of opportunity for
radio-iodine administration that must be experimentally determined and adequately
modeled.
(b) The initial viral dose in part determines the maximum tumor burden and the response time.
However increasing the viral dose beyond 5 does not result in any substantial benefit (Fig. 8).
This has important implications, because higher doses of virus might be associated with
higher toxicity and increases the cost of therapy.
74 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78

(c) Similarly there is an optimal dose window for the radioactive iodide. Under the conditions of
the experiment this window is approximately between 80% and 120% (Fig. 9) of the exper-
imentally given activity (37 MBq). Higher iodide doses do not substantially decrease the
maximal tumor burden, or the response time (Fig. 8). Radioiodide doses less than 50% of
the experimental dose preclude the complete cure (Fig. 10).

It is clear from our investigations that virotherapy is a very sensitive dynamical system and the
outcome can be best understood through mathematical modeling. Radiovirotherapy appears to
be more complex and requires careful optimization. We have studied some of the potential opti-
mizations that need to be tested experimentally. Additional experimental data are needed to fur-
ther evaluate and refine our model.

Acknowledgments

We thank Mr. Ken Peters for his technical assistance in figure preparation. This work is sup-
ported in part by NIH grant CA 100634. K.J. was supported by NSF Grant DMS-0244529.

Appendix A. Upper bound K to tumor cell population

First we consider the model (3) for viral therapy, which we transform by introducing the var-
iable z(t) = x(t) + y(t):

z0 ¼ rðz
xÞ½1
ðz=KÞ

dx; yðtv Þ ¼ y v ;

x0 ¼ jðz
xÞv
dx; xðtv Þ ¼ 0; ð24Þ
v0 ¼ ax
xv; vðtv Þ ¼ v0 .

If for any finite t = tk, z(tk) = K, one would have z 0 (tk) =
dx(tk) implying that due to continuity
z(t) would decrease for t > tk in some interval (tk, tk + s). This remains true even when x(tk) = 0,
because then x 0 (tk) = jKv(tk) > 0 and therefore x(t) > 0 for t > tk in some interval (tk, tk + s1)
implying z 0 (t) < 0. Equilibrium point achieved when t ! 1 also cannot exceed K. Indeed, if
z(1) = K + n, n > 0 then r[K + n
x(1)][1
(1 + n/K)
]
dx(1) < 0 contradicts z 0 (1) = 0.
In the case of the model (12) for radiovirotherapy, an analogous argument holds. Helpful var-
iable transformation in this case is z(t) = x(t) + y(t) + u(t).

Appendix B. Stability of equilibrium points for the virotherapy model

We introduce non-dimensional variables: Y = y/K, X = x/K, V = v/K, s = at, and parameters:

r1 = r/a, j1 = jK/a, d1 = d/a, x1 = x/a. Then in terms of the rescaled time s model (3) becomes:

Y 0 ¼ r1 Y ½1
ðY þ X Þ

j1 VY ; X 0 ¼ j1 VY
d1 X ; V 0 ¼ X
x1 V . ð25Þ
 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78 75

Stability of equilibrium points can be determined by linearization. Calculation of Jacobian matrix

J1 at equilibrium point (Y1, X1, V1) = (0, 0, 0) yields
detðJ1
sIÞ ¼ ðr1
sÞðx1 þ sÞðd1 þ sÞ. ð26Þ
k11
The positive eigenvalue ¼ r1 implies that this point is unstable. The corresponding eigenvector
is e11 ¼ ð1; 0; 0Þ. Since the Y-axis is invariant, it follows that the unstable manifold of (Y1, X1, V1) is
the portion of the Y-axis connecting (Y1, X1, V1) and (Y2, X2, V2). The eigenvectors corresponding
to the eigenvalues k12 ¼
x1 and k13 ¼
d1 lie in the plane Y = 0, and therefore this plane contains
the stable manifold of (Y1, X1, V1).
The eigenvalues of the equilibrium point (1, 0, 0) are obtained from
detðJ2
sIÞ ¼
ðr1
þ sÞ½s2 þ ðd1 þ x1 Þs
j1 þ x1 d1  ¼ 0. ð27Þ
The eigenvector e21 ¼ ð1; 0; 0Þ corresponds to the eigenvalue k12 ¼
r1
. The portion of the Y-axis
connecting (Y1, X1, V1) and (Y2, X2, V2) is contained in the stable manifold of (Y2, X2, V2) hence
forming a heteroclinic orbit.
When d1x1 > j1, all eigenvalues are negative, and the equilibrium is stable. In the original
parameters this condition is dx/(aj) > K. In fact when dx/(aj) = K, the equilibria (y2, x2, v2)
and (y3, x3, v3) collide, typically in a transcritical bifurcation, and (y3, x3, v3) enters the biologically
relevant positive octant.

Appendix C. Existence and uniqueness of the solutions of Eqs. (6), (18) and (22)

For the proof of existence we use the Bolzano theorem which states that if a function f(x) is
continuous on [a, b] and f(a) < 0, f(b) > 0 then there exists a number x0 2 (a, b) such that
f(x0) = 0. In the case of Eq. (6) the corresponding function f is
f ðx3 Þ ¼ ½ðx3 þ y 3 Þ=K
þ lx3
1; ð28Þ


where l > 0 is defined in (9) and the interval is [0, K
y3]. Obviously, f(0) = (y3/K)
1 < 0, pro-
vided that y3 = dx/(aj) < K holds (cf. (7)), and f(K
y3) = l(K
y3) > 0. The uniqueness of the
solution of equation f(x3) = 0 is proved by assuming that there are two solutions s1 and s2. Then,
from (28) it follows:
f ðs1 Þ
f ðs2 Þ ¼ ½ðs1 þ y 3 Þ=K

½ðs2 þ y 3 Þ=K
¼
lðs1
s2 Þ. ð29Þ
Both s1 > s2 or s1 < s2 lead to contradiction and therefore s1 = s2.
In the case of (18) the function f is
1=m
f ð
y 2 Þ ¼
y 2 þ b
y 2
B2 ; ð30Þ
where B2 is defined in (20), and the interval is [0, B2], provided that B2 > 0, which implies r > bD.
The uniqueness of the solution of equation f ð
y 2 Þ ¼ 0 is proven in complete analogy as above.
Finally, for Eq. (22) the function f is given by (see (21) and (22)):
f ð
x3 Þ ¼
x3
pfr
bD
rK

½
x3 þ
y 3 þ bð
x3 þ
y 3 Þ1=m 
g ð31Þ
and the interval is ½0;
x, where
x satisfies the following equation:
76 D. Dingli et al. / Mathematical Biosciences 199 (2006) 55–78

/ð
xÞ 
x þ
y 3 þ bð
x þ
y 3 Þ1=m
K ¼ 0. ð32Þ

Under the condition (21), f(0) < 0, and f ð
xÞ ¼ l
x þ bD=r > 0, assuming that
x > 0 exists. This is
again true by means of Bolzano theorem applied to (32). We find that /(0) < 0 due to inequality
1=m
(21): ð
y 3 þ b
y 3 Þ < K; on the other hand /ðK
y
3 Þ > 0. This implies that
x 2 ð0; K

y 3 Þ. The
solution of f ð
x3 Þ ¼ 0 is unique because assuming that there are two solutions s1 and s2 would lead
to

s1
s2 ¼
rK

½s1 þ
y 3 þ bðs1 þ
y 3 Þ1=m 
þ rK

½s2 þ
y 3 þ bðs2 þ
y 3 Þ1=m 
; ð33Þ

which cannot be satisfied unless s1 = s2.

Appendix D. Stability of equilibria under radiovirotherapy

The analysis of the stability of (17) for Eqs. (10), (12) and (13) is complicated by the fact that
m < 1, so that the system is not Lipschitz, and solutions may only generate a semi-flow. However,
note that the system is in skew product form, with the subsystem described by IT, IM, and D pro-
viding a drive to the subsystem described by the variables y, x, u, and v. When r < bD, a simple
estimate shows that near the origin the fibers IT = C1, IM = C2 and D = C3 for constants Ci, are
contracted. Therefore there is an invariant manifold which is the graph of some function
/ : U ! V  R4 where ð0; 0; DÞ  U and (0, 0, 0, 0)  V are open sets [35] (this manifold may
not be unique due to the non-invertibility of the semi-flow, however the same results hold by con-
sidering a graph over the inverse limit of the driving system). Points starting in the vicinity of this
manifold approach it in forward time, and therefore approach the point (17), showing that it is
locally attracting. The equilibrium point ð0; 0; DÞ of the system given by (10) and (13) is only neu-
trally stable.
Restricting the equations to the y-axis it is easy to check that for r ¼ bD (17) collides with (18)
typically in a transcritical bifurcation. The two points therefore exchange stability, and for r > bD
the point (17) is unstable. The analysis of the bifurcation of fixed point (17) is complicated by the
fact that the solutions in general define only a semi-flow. However, numerical evidence suggests
that the bifurcation that occurs when points (17) and (18) coincide has the features of a transcrit-
ical bifurcation.

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