2023 07 04 547696v1 Full

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1 ON MINIMISING TUMOURAL GROWTH UNDER TREATMENT

2 RESISTANCE
3 MATTHIAS M. FISCHER1 AND NILS BLÜTHGEN1,†
1
4 Institute for Theoretical Biology, Charité and Humboldt Universität zu Berlin,
5 10115 Berlin, Germany

†
6 Corresponding author, E-Mail: nils.bluethgen@charite.de
Abstract. Drug resistance is a major challenge for curative cancer treatment,
representing the main reason of death in patients. Evolutionary biology suggests
pauses between treatment rounds as a way to delay or even avoid resistance
emergence. Indeed, this approach has already shown promising preclinical and
early clinical results, and stimulated the development of mathematical models
for finding optimal treatment protocols. Due to their complexity, however,
these models do not lend themself to a rigorous mathematical analysis, hence
so far clinical recommendations generally relied on numerical simulations and
ad-hoc heuristics. Here, we derive two mathematical models describing tumour
growth under genetic and epigenetic treatment resistance, respectively, which
are simple enough for a complete analytical investigation. First, we find key
differences in response to treatment protocols between the two modes of resis-
tance. Second, we identify the optimal treatment protocol which leads to the
largest possible tumour shrinkage rate. Third, we fit the ”epigenetic model” to
previously published xenograft experiment data, finding excellent agreement,
underscoring the biological validity of our approach. Finally, we use the fitted
model to calculate the optimal treatment protocol for this specific experiment,
which we demonstrate to cause curative treatment, making it superior to previ-
ous approaches which generally aimed at stabilising tumour burden. Overall,
our approach underscores the usefulness of simple mathematical models and
their analytical examination, and we anticipate our findings to guide future
preclinical and, ultimately, clinical research in optimising treatment regimes.
Keywords: Acquired Resistance, Cancer, Phenotypic Plasticity, Targeted
Therapy
Date: July 4, 2023.

1
bioRxiv preprint doi: https://doi.org/10.1101/2023.07.04.547696; this version posted July 4, 2023. The copyright holder for this preprint
MINIMISING TUMOURAL GROWTH UNDER RESISTANCE 2
7 1. Introduction
8 Despite tremendous advances in the field of cancer research, resistance to treatment
9 has remained a major challenge for the curative eradication of tumours and is consid-
10 ered the main reason of death in cancer patients (Vasan et al., 2019). Accumulating
11 evidence strongly suggests intratumoural heterogeneity, i.e. tumours being a mixture
12 of functionally distinct cancer cells differing either genetically or epigenetically, as a
13 major driver of drug resistance (Saunders et al., 2012; Pribluda et al., 2015). Under
14 the selective pressure exerted by treatments, resistance can then emerge as a result
15 of either the expansion of pre-existing resistant subpopulations or the emergence of
16 new drug-tolerant cells (Dagogo-Jack and Shaw, 2018).
17
18 From a perspective of evolutionary biology, it thus appears promising to reduce such
19 a selective pressure towards resistance by introducing strategically placed pause
20 phases into treatment protocols, during which no treatment is applied. Indeed, such
21 ”evolutionary strategies” have already shown promise in preclinical and early-stage
22 clinical trials (Silva et al., 2012; Enriquez-Navas et al., 2016; Zhang et al., 2017).
23 They have also stimulated the development and analysis of mathematical models aim-
24 ing to capture the essence of these processes and ultimately find optimal treatment
25 protocols (Smalley et al., 2019; Kim et al., 2021). While describing experimental
26 data with a high degree of accuracy and having a demonstrably large amount of
27 predictive power, these models do not lend themself to a rigorous mathematical
28 analysis due to the involved nonlinear terms which make deriving closed-form solu-
29 tions of the occuring dynamics impossible.
30
31 To close this gap in the literature, in this work we derive and study two mathemati-
32 cal models describing the population dynamics of a growing tumour for the cases
33 of a genetically and an epigenetically acquired treatment resistance, respectively.
34 Importantly, both models are simple enough to be entirely accessible to analytical
35 investigation. First, we compare the dynamics of these two models, finding key
36 differences in growth and response to treatment protocols. Subsequently, we identify

37 and characterise the optimal treatment protocol which leads to the asymptoti-
38 cally smallest tumour growth, i.e. the largest possible rate of tumour shrinkage.
39 Afterwards, we fit our model of phenotypically acquired treatment resistance to pre-
40 viously published experimental data from mouse experiments with patient-derived
41 melanoma xenografts harbouring acquired resistances to MEK -inhibitor treatment
42 (Hong et al., 2018). Doing so, we find excellent agreement between data and theory,
43 which underscores the predictive power of our modelling approach. Finally, we
44 use the fitted model to calculate the optimal treatment protocol for this specific
45 experiment and perform numerical simulations to demonstrate that the obtained
46 solution is indeed able to drive the tumour into complete extinction and hence
47 outperforms earlier ad-hoc approaches.
48 2. Materials and Methods
49 2.1. Genetic model. We assume a tumour consisting of two different cell types
50 S, R, denoting treatment-sensitive and treatment-resistant cells, respectively. During
51 treatment, the compartments grow exponentially at rates βS∗ , βR

∗
, respectively,
52 whereas in the absence of treatment their growth rates amount to βS , βR , respectively.
53 We decided to not introduce a carrying capacity because we assume that the overall
54 tumour size is sufficiently small compared to the maximum possible tumour size, and
55 thus growth to not be limited yet. We will revisit this assumption later. Resistance
56 to treatment emerges due to genetic mutations at a rate µ, which is small enough
57 for exact back-mutations to a sensitive cell state to be neglectable. Thus, during
58 treatment the system is governed by the following set of differential equations
dS(t)
= (βS∗ − µ)S(t)
dt
(1)
dR(t) ∗
= µS(t) + βR R(t),
dt
59 and during treatment pauses by
dS(t)
= (βS − µ)S(t)
dt
(2)
dR(t)
= µS(t) + βR R(t).
dt
60 2.2. Phenotypic plasticity model. The ”phenotypic plasticity model” is similar
61 to the genetic model described in the previous section. However, we assume that
62 resistance emerges as a consequence of treatment at a constant rate α, whereas in
63 the absence of treatment resistance is lost again at a rate δ. Thus, during treatment
64 the system is governed by the following set of differential equations
dS(t)
= (βS∗ − α)S(t)
dt
(3)
dR(t) ∗
= αS(t) + βR R(t),
dt
65 and during treatment pauses by
dS(t)
= βS S(t) + δR(t)
dt
(4)
dR(t)
= (βR − δ)R(t).
dt
66 2.3. Literature data, model fitting and illustrative model simulation. Tu-
67 mour size time-course data from mouse xenograft experiments have been extracted
68 from Figures 5a and 5b of Hong et al. (2018) using the WebPlotDigitizer soft-
69 ware (https://automeris.io/WebPlotDigitizer/), which has been found to be
70 a reliable and valid tool for extracting data from diagrams (Drevon et al., 2017).
71 Model fitting was performed using the optimize.curve fit routine from the scipy
72 package (Virtanen et al., 2020), version 1.10.1, for the Python programming lan-
73 guage (Van Rossum and Drake Jr, 1995), version 3.9.6. The routine implements
74 unconstrained non-linear least square optimisation via the Levenberg-Marquardt
75 algorithm. For illustrative purposes, the model has been simulated numerically using
76 the integrate.solve ivp routine from the same software package, which relies on
77 an explicit Runge-Kutta method with a fourth-order error control method and a
78 step size selection using a fifth-order accurate formula with local extrapolation. All
79 computations have been carried out on a 2023 MacBook Pro with an Apple M2
80 Max processor running macOS Ventura, version 13.3.

81 3. Results
82 3.1. Phenotypic plasticity requires a precisely timed treatment protocol.
83 We derived two simple mathematical models of tumoural resistance to treatment,
84 both of which distinguish between compartments S and R, denoting sensitive and
85 resistant cells, respectively. In both models, during treatment the two compartments
86 grow at rates βS∗ < 0 and βR

∗
, respectively. During treatment pauses, they grow at
87 different rates βS > 0, βR , respectively.
88
89 In the ”genetic model”, treatment resistance is acquired via random mutations at a
90 small rate µ regardless of whether a treatment is applied or not. Due to µ being
91 small, we neglect the possibility of back-mutations. In contrast, in the ”phenotypic
92 plasticity model”, resistance is induced at a rate α during treatment phases, and
93 lost again at a rate δ during treatment pauses. For an illustration refer to Figure
94 1A and Figure 1B.
95
96 For both models, we define y(t) := [S(t), R(t)]T and can write the system as


dy(t) Ty(t) Treatment Phase

=
dt 
Py(t)
 Pause Phase,
97 where we have in case of the genetic model (Equations 1 and 2)
   
∗
βS − µ 0 β
 S − µ 0
T= ,P =  .

∗
µ βR µ βR
98 and in case of the phenotypic plasticity model (Equations 3 and 4).
   
∗
βS − α 0 βS δ
T= ,P =  .

∗
α βR 0 βR − δ
99 We define a treatment protocol P as a sequence of alternating treatment and pause
100 phases. We write P = (τ1 , φ1 , τ2 , φ2 , . . . , τi , φi ), where τi represents the length of
101 the i-th treatment phase, and φi the length of the i-th pause phase.
102
Figure 1. Phenotypic plasticity requires a precisely timed treatment protocol. A

Schematic sketch of the model with treatment resistance emerging from random mutations (”genetic
model”) at a small rate µ. S: Sensitive subpopulation, R: Resistant subpopulation. βS ∗ , β ∗ denote
R
the growth rates of the compartments during treatment; βS , βR during treatment pause. B
Schematic sketch of the model with acquired treatment resistance due to phenotypic plasticity
(”phenotypic plasticity model”). Growth rates are the same as before, however during treatment
sensitive cells acquire resistance at a rate α, and in the absence of treatment resistant cells lose
resistance at a rate δ. C Final tumour size in the genetic model is not affected by the ordering of
the individual treatment and pause phases. Numerical illustration. D In the phenotypic model,
the order of the individual treatment and pause phases affects the final tumour size. Numerical
illustration.
103 For the genetic model (Equations 1 and 2), we assume that the mutation rate µ is
104 sufficiently small to be neglected. This decouples the two system compartments,
105 and thus the final compartment sizes SP , RP can be directly calculated as a product
106 of exponential growth terms:
∗ ∗ ∗ P P
SP = S0 eβS τ1 eβS φ1 . . . eβS τi eβS φi = S0 eβS i τi +βS i φi
∗ ∗ ∗ P P
RP = R0 eβR τ1 eβR φ1 . . . eβR τi eβR φi = R0 eβR i τi +βR i φi
107 Thus, given any initial condition (S0 , R0 ) and set of system parameters (βS , βS∗ , βR , βR
∗
),
P P
108 only the overall sum i τi of treatment phase lengths and the overall sum i φi of
109 pause phase lengths determine the final tumour size, but not their segmentation or
110 their order. We illustrate this finding in Figure 1C.
111
112 In contrast, for the phenotypic plasticity model (Equations 3 and 4), the final system
113 state yP = [SP , RP ]T is given by
yP = eφi P eτi T . . . eφ1 P eτ1 T y0
114 Since all matrix exponentials eφi P and eτ1 T are upper and lower triangular matrices,
115 respectively, they do not commute in general. Hence, the order of treatment and
116 pause phases will influence the final tumour size. We illustrate this finding in Figure
117 1D.
118
119 Taken together, from a biological point of view, these results show that the growth,
120 and thus the successful eradication of tumours with exclusively genetic resistance
121 mechanisms solely depend on the sum of time spent in treatment vs. in pause
122 phase. Conversely, the order of the individual phases of a treatment regimen does
123 not influence the final outcome. In contrast, tumours which show an acquired treat-
124 ment resistance stemming from phenotypic plasticity demand a precise segmentation
125 and ordering of the individual treatment and pause phases to be optimally controlled.
126
127 3.2. Change of variables identifies the optimal treatment protocol. In a
128 clinical setting, usually we can only monitor the overall tumour burden, for instance
129 by using medical imaging or by measuring the concentration of markers of overall
130 tumour burden. In contrast, generally we are not able to track the number of
131 sensitive vs. resistant cells. This motivates us to perform the following change of
132 variables: Let N (t) := R(t) + S(t) denote the observable overall tumour burden, and
133 Q(t) := R(t)/(R(t) + S(t)) denote the ”invisbile” fraction of resistant cells present
134 in the tumour (see Figure 2A for an illustration).
135
136 Both models then satisfy



dN (t) βS (1 − Q(t)) + βR Q(t) Pause Phase

r(t) := =
dt · N (t) 
βS∗ (1 − Q(t)) + βR
 ∗
Q(t) Treatment Phase,
137 which, importantly, shows that in both phases the overall relative tumour growth
138 r(t) is only a linear function of the tumour composition given by Q(t) (see Figure
139 2B for an illustration).
140
Figure 2. Rewriting the models in terms of overall population size N and fraction
Q ∈ [0; 1] of resistant cells reveals optimal treatment protocol. A Illustration of the model
transformation. B In both the treatment phase (blue line) and the pause phase (grey line), relative
local tumour growth Ṅ /N is a simple linear function of Q, determined by the subpopulation
growth rates. C Illustration of the analytically derived optimal treatment protocol: After a lead-in
treatment, during which Q reaches some Qa , we repeatedly switch between treatment (until Q
reaches some Qb > Qa ), and treatment pause (during which Q recedes back to Qa ). Upper diagram
shows log population size over time (continuous blue line during treatment, continuous grey line
during pause). Additionally, the sizes of the sensitive and resistant subpopulation are plotted with
a dashed and a dotted light grey line, respectively. Lower diagram shows fraction Q of resistant
cells over time.
141 It can easily be verified that during treatment Q follows a monotonically increasing
142 sigmoid function converging to unity, whereas during treatment pauses, Q follows
143 a monotonically decreasing function which converges to zero. Assuming that a
144 treatment-naive tumour mostly consists of sensitive cells, the relative tumour growth
145 rate before any treatment is approximately identical to βS , and the relative growth
146 rate directly after the onset of treatment approximately equals βS∗ . During treatment,
∗
147 r(t) will tend towards βR . Conversely, during treatment pause, r(t) will tend to βS
148 again. It should be noted that these results also imply that it is possible to infer the
149 current tumour composition by using sufficiently frequent measurements of overall
150 tumour burden alone. In particular, for the case of phenotypic plasticity knowledge
151 of the transition rates α, δ is not required.
152
153 We now want to find the optimal treatment protocol, defined as the treatment
154 protocol which, after transient behaviour has passed, leads to minimal relative
155 tumour growth r per unit of time. From the results of the previous section it follows
156 that any treatment protocol corresponds to a continuous trajectory through Q. As
157 per Equation 3.2, for any such trajectory θ, there exists a corresponding piecewise
158 continuous function rθ , describing the local growth rate of N over time. Accordingly,
159 the final tumour population Nθ at the end of θ is given by
R
rθ (t) dt
Nθ = e N0 ,
160 where the integral has to be broken into the individual treatment and pause phases.
161 Obviously, every finite trajectory through Q will only lead to a finite relative reduc-
162 tion in tumour size, as the exponential term is strictly greater than zero. Hence, we
163 wish to find a closed path through Q which we can follow over and over again in
164 order to force N (t) → 0.
165
166 In order to further characterise the optimal solution, we can prove (see Appendix
167 A) that among all closed trajectories, a trajectory can only be optimal if it is free
168 of any inner loops. In other words, the closed trajectory which minimises relative
169 tumour growth per unit of time consists of one treatment phase, where Q(t) grows
170 from Qa to Qb , followed by one pause phase, where Q(t) decreases back to Qa .
171
172 Accordingly, given any system parametrisation, it suffices to find the optimal switch-
173 ing points Qa , Qb , and the optimal treatment protocol consists of initial transient
174 phase where Q(t) approaches Qa from its initial value, followed by repeatedly apply-
175 ing treatment until Qb is reached and pausing treatment, until Qa is reached again
176 (see Figure 2C for an illustration).
177
178 3.3. Our approach accurately describes literature data and predicts the
179 optimal treatment schedule. After examining the models analytically, we fitted
180 the phenotypic model to time-course tumour burden data from mouse xenograft
181 experiments published in Hong et al. (2018). First, we used the data from Figure 5a,
182 where patient derived melanoma xenografts sensitive to MEK -inhibitor treatment
183 were implanted in mice and grown for 18 days. Afterwards, treatment was started
184 by giving 5 mg/kg MEK -inhibitor daily for additional 67 days. Because tumour
185 growth started to saturate and became subexponential around day 40, we discarded
186 all the data points from this point of time. This time-course describes the growth
187 of sensitive tumour cells in vivo, their reaction to the onset of treatment, and the
188 acquisition of treatment resistance (see Figure 3A, left panel).
189
190 Second, we also fitted the model to data from Figure 5b of the same paper, where
191 the same cell line was first made resistant to treatment by subjecting them to
192 inhibitor treatment in vitro. Resistant cells were then implanted into mice, which
193 were treated with 5 mg/kg MEK -inhibitor daily for 52 days, after which treatment
194 was stopped for additional 23 days. Accordingly, this time-course describes the
195 growth of resistant cancer cells during treatment, their reaction to the termination
196 of treatment, and the subsequent loss of resistance and re-emergence of the sensitive
197 phenotype (see Figure 3A, right panel). Note the excellent fit of the model to the
198 data in both cases, as well as very similar estimates of the two shared parameters
199 across the two fits.
200
Figure 3. Application of the phenotypic model to the data by Hong et al. (2018). A
Left: Tumour xenograft volume data (blue dots), model fit (continuous blue lines) and obtained
parameter estimates for the data from Fig 5A, sensitive population subjected to MEKi treatment.
Dashed grey line denotes the onset of treatment. Right: The same for Figure 5B, resistant cells
subjected to MEKi withdrawal. Dashed grey line denotes the termination of treatment. Observe
the very good fit for both data sets and the agreement between shared parameters. B Calculated
tumour growth rate per day as a function of chosen lower and upper switch points Qa , Qb . Cross
marks Qa = 0.25, Qb = 0.5 (i.e. 25% and 50% resistant cells), which will be examined in the rest
of this figure. C Relative local tumour growth rate r := Ṅ /N as a function of resistance fraction
Q for pause (continuous grey line) and treatment phase (continuous blue line). Arrows illustrate
the treatment protocol with switch points as chosen before. D Numerical simulation of a tumour
population over time treated as defined before (continuous blue line during treatment, continuous
grey line during pause phase). Note the convergence to tumour extinction, whereas continuous
treatment (dotted blue line) would cause relapse after ca. 10 days, and the ”50% stopping rule”
by Kim et al. (2021) (dashed blue line) would only lead to a stabilisation of tumour burden. Note
that the proposed ”20% stopping rule” from the same paper would not work here, as continuous
treatment cannot achieve a tumour reduction by 80%.
201 After parametrisation, we used the model to find the optimal treatment protocol
202 for this specific set of parameters, which means finding the optimal switching points
203 Qa > Qb in fraction of resistant cells (see previous section). Hence, we calculated
204 the realised tumour growth rate as a function of Qa and Qb , which is depicted
205 in Figure 3B, where blue colors denote areas of negative growth, i.e. a shrinking
206 tumour. Obviously, as Qa → Qb , the duration of treatment cycles shrinks, making
207 the realised growth rate diverge to −∞. However, in reality treatment cycles ob-
208 viously cannot become arbitrarily short. For this reason, we selected the scenario
209 of Qa = 0.25, Qb = 0.5 (marked with a white cross in the figure) as a reasonable
210 compromise between sufficient tumour shrinkage per day and feasibility in reality
211 for further investigation.
212
213 Analogously to Figure 2B, Figure 3C shows the relative tumour growth r as a
214 function of the fraction Q of resistant cells during treatment phase (blue line) and
215 pause phase (grey line) for the obtained parametrisation. Little blue and grey arrows
216 depict the treatment protocol we have selected in the previous paragraph, i.e. an
217 initial lead-in treatment from Q ≈ 0 to Qa = 0.25, followed by repeatedly applying
218 treatment until Q reaches Qb = 0.5 and pausing treatment until Q has reached
219 Qa = 0.25 again.
220
221 Numerically simulating this treatment protocol indeed confirms that it will lead
222 to tumour extinction (continuous line depicted in Figure 3D, where blue and grey
223 colours indicates treatment and pause phase, respectively). in contrast, classical
224 continuous treatment (dotted blue line) only causes an initial rediction in tumour
225 size, followed by tumour relapse and the onset of exponential growth. We also
226 simulated the ”50 % rule” mentioned and studied in Kim et al. (2021), where
227 treatment is applied until the tumour has reached half of its initial size, and then
228 paused until the tumour has reached its entire initial size again, where treatment is
229 then resumed (dashed blue line). This approach succeeds in permanently stabilising
230 tumour size, but cannot achieve curative treatment. Kim et al. (2021) also proposed
231 the ”20% rule”, where treatment is applied until the tumour has shrunk to a fifth of
232 its initial size instead. Of note, this rule would fail for this specific parametrisation,
233 as continuous treatment (see dotted line) is not able to cause such a drastic reduction
234 in the first place.
235
236 Taken together, we demostrated that, for the specific case of the experiments by
237 Hong et al. (2018) our model describes the data well and can be used to identify an
238 optimal treatment protocol that is superior to previously published approaches.
239 4. Discussion
240 In this work, we have derived and analysed two simple mathematical models de-
241 scribing tumoural resistance to treatment via genetic and epigenetic mechanisms,
242 respectively. We have used these models to solve the problem of introducing treat-
243 ment pauses to minimise tumour growth in the face of such resistances. In contrast
244 to preceding modelling efforts, we here aimed for utmost simplicity, in particular
245 by not including any growth limiting carrying capacities. While this simplifying
246 assumption is of course a departure from biological reality, we still considered it
247 necessary to build models accessible to a complete analytical examination, and thus
248 be able to obtain reliable insights independently of specific parametrisations and po-
249 tentially costly numerical simulations. The validity of this approach is demonstrated
250 by the fact that despite this simplification it was still able to describe experimental
251 data (Hong et al., 2018) with a high degree of accuracy.
252
253 Even without a carrying capacity, in our models it can be possible to achieve
254 complete tumour eradication, which is conceptually intriguing. This, however,
255 necessarily requires that resistant cells experience negative growth during treatment
256 pauses. Such a ”drug addiction” of resistant cells has been observed repeatedly
257 not only in cell culture (Suda et al., 2012; Sun et al., 2014; Moriceau et al., 2015)
258 and animal models (Das Thakur et al., 2013), but also in vivo (Dietrich et al.,
259 2013; Seifert et al., 2016; Dooley et al., 2016), and can thus be expected to be
260 a viable option to exploit therapeutically in some patients. Nonetheless, even if
261 during treatment pauses resistant cells grow only slower than sensitive cells but still
262 at a positive rate, in our models intermittent treatment will still minimise overall
263 tumour growth compared to continuous treatment protocols. All of these findings
264 agree with numerical results from a more complex competitive Lotka-Volterra-like
265 system (Strobl et al., 2021), in which additionally the role of cellular turnover was
266 found to be an important predictor of therapy success as it amplifies the effects of
267 competition and resistance costs. Accordingly, an interesting and straightforward
268 extension of our work might be the explicit introduction of a cellular turnover rate
269 (or ”apoptosis rate”) as opposed to using aggregated subpopulation net growth rates.
270
271 For the specific case of genetic resistances, we have shown that overall tumour
272 growth is only determined by the sum of time spent in treatment vs. pause phase.
273 In contrast, the exact segmentation and ordering of the individual treatment and
274 pause phases does not influence overall tumour size at the end of a treatment
275 protocol. From a clinical point of view, this implies that there is not much room for
276 improvement in treatment protocols apart from adjusting the fraction of time spent
277 in treatment pause. One could, however, argue that a relatively long initial lead-in
278 treatment before any treatment pauses might be advantageous, as it would reduce
279 the cumulative risk of resistance emergence via random mutations. Importantly, this
280 holds only if it can be guaranteed that the treatment does not induce the acquisition
281 of further resistance. As a direct consequence, this finding also underscores the
282 need for elucidating the exact mechanisms responsible for a given tumour becoming
283 resistant as these crucially affect the optimal treatment strategy (Housman et al.,
284 2014).
285
286 In stark contrast to genetically conferred resistance, in case of epigenetic mecha-
287 nisms, the order of the individual phases of a treatment protocol can profoundly
288 influence tumour growth, and thus the possibility of curative tumour eradication.
289 Hence, tumours harboring epigenetic resistances require a precisely tuned treatment
290 protocol to maximise chances of complete tumour extinction.
291
292 The main contribution of our work solves this problem by reducing this problem to
293 navigating along an one-dimensional axis which encodes the fraction of currently
294 resistant cells in the tumour. In this way, it becomes apparent that the optimal
295 protocol, which minimises overall tumour size, keep the fraction of resistant cells in
296 a range [Qa , Qb ] which can be calculated numerically. As shown with experimental
297 data (Hong et al., 2018), our approach can in some cases even cause complete
298 tumour eradication, which is a qualitative improvement over previous approaches
299 such as Kim et al. (2021) which generally aimed for stabilising tumour burden in a
300 pre-specified range. The drawback of our approach obviously lies in the fact that we
301 need to know the parameter values describing the tumour of a given specific patient
302 to be able to calculate the treatment protocol optimal for this specific patient. This,
303 in turn, requires sufficiently frequent and accurate measurements of tumour burden
304 over time, so that the model can be proeprly parametrised. In itself, this is not a
305 particularly strong requirement and similar to previous approaches such as Smalley
306 et al. (2019). Particlarly for leukemia, such a freqent monitoring of the tumor
307 burden is relatively straight forward and minimally invasive. However, the need to
308 perform curve-fitting on these data afterwards and interpret the obtained results is
309 likely to pose a challenge in a clinical setting.
310
311 Nonetheless, if this challenge is solved and such a real-time analysis of tumour
312 burden data is possible in the clinic, an idealised scenario of cancer treatment may
313 look as follows. Upon initial admission of the patient, data is collected as sketched
314 out in Figure 4. Before any treatment, tumour burden is measured multiple times,
315 so that the growth rate of the treatment-naive and hence -sensitive tumour cells
316 can be estimated (corresponding to the parameter βS in our model). Directly after
317 the onset of the first round of treatment, the tumour is still mostly comprised of
318 sensitive cells, so its rate of shrinking corresponds to the growth rate of sensitive cells
319 under treatment (βS∗ ). During treatment, sensitive cells continue to die, however
320 they may also switch to a resistant cell state at a rate we cannot measure directly
321 (α). Over time, the majority of tumour cells will have become resistant, and thus
322 the tumour growth rate will converge to the growth rate of resistant cells under
∗
323 treatment (βR ) until at some point the relapse becomes obvious. Treatment is then
324 halted, and directly afterwards the now mostly-resistant tumour approximately
325 follows the growth rate of untreated resistant cells (βR , which is less than zero
326 in case of drug addiction). Without treatment, however, resistant cells will also
327 lose resistance at another rate we cannot directly measure (δ). Accordingly, the
328 entirety of the collected data is now used for curve-fitting. Because the raw data
329 already provide good estimates or bounds for the four growth rates, the remaining
330 two parameters α, δ generally will be identifiable, as demonstrated in Section 3.3.
331 Now that all relevant parameters are estimated, the obtained parameters are used
332 to computationally identify the optimal course of treatment, which is initiated
333 afterwards. This optimal treatment is guaranteed to minimise overall tumour
334 growth and may, in some cases, even lead to curative treatment.
(1) Before treatment, the

tumour is ≈ 100% sensitive.
Thus, it grows at ≈ βS .
(2) Right at treatment onset,
the tumour shrinks at ≈ βS∗ .
(3) Treament kills sensitive cells,

but resistance starts to build.
Tumour Burden
(4) Resistance now dominates,

the tumour grows at / βR∗
(5) Treatment is stopped,

tumour shrinks at ' βR .
Before treatments First treatment First pause after relapse
Time
Figure 4. Data collection protocol for an idealised scenario of cancer treatment with
real-time monitoring of tumour burden. (1) Before treatment, the tumour mostly consists of
sensitive cells and thus follows the growth rate of untreated sensitive cells (βS > 0). (2) Directly
after treatment onset, the still sensitive tumour follows the growth rate of sensitive cells under
treatment (βS ∗ < 0). (3) During treatment, sensitive cells continue to die, however may also
switch to a resistant state at rate α, which is not directly observable and needs to be identified
via curve-fitting once all data have been acquired. (4) Once the majority of tumour cells has
become resistant, the tumour follows the growth rate of resistant cells under treatment (βR ∗ > 0).
(5) Treatment is halted, and directly afterwards the now mostly-resistant tumour approximately
follows the growth rate of untreated resistant cells (βR , which is less than zero in case of drug
addiction). Without treatment, resistant cells will also lose resistance at a rate δ only identifiable
via curve-fitting. Not shown: Once estimates of all six parameters have become available from
curve-fitting, they are used to computationally identify the optimal course of treatment, which is
initiated afterwards. This optimal treatment is guaranteed to minimise overall tumour growth and
may, in some cases, even lead to curative treatment.
335 Our analysis shows that optimal treatment protocols may differ for every individual
336 patient. This is in agreement with a general tendency against a ”one-size-fits-all
337 approach” in medicine and argues for true ”precision medicine” or ”personalised
338 medicine” where treatment is individually taylored to every given patient (King
339 and Robins, 2006). In contrast to the majority of approaches in precision medicine
340 (König et al., 2017), however, we here did not rely on any molecular data such
341 as genomic, transcriptomic, or proteomic profiling. Instead, we personalised our
342 model by fitting it to solely phenotypical data, i.e. tumour burden measurements
343 over time. In addition, instead of using involved and potentially hard to interpret
344 computational pipelines, our modelling approach aimed for utmost simplicity and its
345 success underscores the usefulness of simple mathematical models and their thorough
346 analytical examination. Thus, we anticipate not only our immediate results, but
347 also our general methodological approach to guide and inspire future preclinical and,
348 ultimately, clinical research in precision medicine in general, and optimising cancer
349 treatment regimes in particular.
350 5. Acknowledgements
351 MMF would like to thank Rosario Astaburuaga-Garcı́a for helpful comments on the
352 data analytic part of this work, and acknowledges funding by the DFG (Deutsche
353 Forschungsgemeinschaft, RTG2424, CompCancer).
354 Declarations
355 5.1. Author contributions. MMF carried out model derivations and analyses
356 with help from NB, and wrote the initial version of the manuscript. NB contributed
357 to the final version of the manuscript and supervised the project. All authors have
358 read and approve of the final version of this manuscript.
359 5.2. Competing interests statement. All authors declare that they do not have
360 any competing interests.
361 5.3. Code availability. The complete commented source code used for all numeri-
362 cal examinations presented in this manuscript are available in the following github
363 repository: https://github.com/Matthias-M-Fischer/Minimising.
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447 Appendix A. Optimality of trajectories without inner loops
448 First, we define a closed trajectory π through Q to be optimal, exactly if the relative
449 reduction of tumour size it causes per unit of time is greater than or equal to all
450 possible other closed trajectories through Q.
451
452 Now, we prove the following
453 Proposition 1. A closed trajectory through Q can only be optimal if it is free of
454 any inner loops.
455 Proof. Assume the opposite, that is, the optimal path π through Q consists of n
456 inner loops. Due to the commutativity of products of exponentials (see Equation
457 3.2), the final tumour volume does not change if we rearrange π into n + 1 separate
458 closed ”atomic” loops π1 . . . πn+1 without inner loops. Starting with an initial
459 tumour size N0 , one full traversal of the i-th loop takes tπi units of time, leading to
460 a relative tumour reduction of xi = (N0 − Nπi )/N0 . Let π ∗ denote the inner loop
461 which maximises xi /ti . Then, however, it is easy to show that π ∗ reduces tumour
462 growth more efficiently than π. Contradiction. □

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bioRxiv preprint doi: https://doi.org/10.1101/2023.07.04.547696; this version posted July 4, 2023.

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1 ON MINIMISING TUMOURAL GROWTH UNDER TREATMENT

3 MATTHIAS M. FISCHER1 AND NILS BLÜTHGEN1,†

5 10115 Berlin, Germany

Abstract. Drug resistance is a major challenge for curative cancer treatment,

representing the main reason of death in patients. Evolutionary biology suggests

pauses between treatment rounds as a way to delay or even avoid resistance

early clinical results, and stimulated the development of mathematical models

for finding optimal treatment protocols. Due to their complexity, however,

these models do not lend themself to a rigorous mathematical analysis, hence

so far clinical recommendations generally relied on numerical simulations and

ad-hoc heuristics. Here, we derive two mathematical models describing tumour

growth under genetic and epigenetic treatment resistance, respectively, which

differences in response to treatment protocols between the two modes of resis-

previously published xenograft experiment data, finding excellent agreement,

which we demonstrate to cause curative treatment, making it superior to previ-

ous approaches which generally aimed at stabilising tumour burden. Overall,

our approach underscores the usefulness of simple mathematical models and

their analytical examination, and we anticipate our findings to guide future

preclinical and, ultimately, clinical research in optimising treatment regimes.

Keywords: Acquired Resistance, Cancer, Phenotypic Plasticity, Targeted

Date: July 4, 2023.

MINIMISING TUMOURAL GROWTH UNDER RESISTANCE 2

8 Despite tremendous advances in the field of cancer research, resistance to treatment

11 evidence strongly suggests intratumoural heterogeneity, i.e. tumours being a mixture

12 of functionally distinct cancer cells differing either genetically or epigenetically, as a

15 of either the expansion of pre-existing resistant subpopulations or the emergence of

16 new drug-tolerant cells (Dagogo-Jack and Shaw, 2018).

18 From a perspective of evolutionary biology, it thus appears promising to reduce such

19 a selective pressure towards resistance by introducing strategically placed pause

21 ”evolutionary strategies” have already shown promise in preclinical and early-stage

27 predictive power, these models do not lend themself to a rigorous mathematical

29 tions of the occuring dynamics impossible.

33 of a genetically and an epigenetically acquired treatment resistance, respectively.

34 Importantly, both models are simple enough to be entirely accessible to analytical

36 differences in growth and response to treatment protocols. Subsequently, we identify

MINIMISING TUMOURAL GROWTH UNDER RESISTANCE 3

39 Afterwards, we fit our model of phenotypically acquired treatment resistance to pre-

40 viously published experimental data from mouse experiments with patient-derived

41 melanoma xenografts harbouring acquired resistances to MEK -inhibitor treatment

43 which underscores the predictive power of our modelling approach. Finally, we

45 experiment and perform numerical simulations to demonstrate that the obtained

47 outperforms earlier ad-hoc approaches.

48 2. Materials and Methods

50 S, R, denoting treatment-sensitive and treatment-resistant cells, respectively. During

51 treatment, the compartments grow exponentially at rates βS∗ , βR

52 whereas in the absence of treatment their growth rates amount to βS , βR , respectively.

56 to treatment emerges due to genetic mutations at a rate µ, which is small enough

57 for exact back-mutations to a sensitive cell state to be neglectable. Thus, during

58 treatment the system is governed by the following set of differential equations

MINIMISING TUMOURAL GROWTH UNDER RESISTANCE 4

60 2.2. Phenotypic plasticity model. The ”phenotypic plasticity model” is similar

62 resistance emerges as a consequence of treatment at a constant rate α, whereas in

64 the system is governed by the following set of differential equations

69 ware (https://automeris.io/WebPlotDigitizer/), which has been found to be

74 unconstrained non-linear least square optimisation via the Levenberg-Marquardt

77 an explicit Runge-Kutta method with a fourth-order error control method and a

80 Max processor running macOS Ventura, version 13.3.

MINIMISING TUMOURAL GROWTH UNDER RESISTANCE 5

82 3.1. Phenotypic plasticity requires a precisely timed treatment protocol.

83 We derived two simple mathematical models of tumoural resistance to treatment,

84 both of which distinguish between compartments S and R, denoting sensitive and