Am J Cancer Res 2017;7(9):1926-1936
www.ajcr.us /ISSN:2156-6976/ajcr0059247

Original Article
Breast cancer pulmonary metastasis is increased inmice undertaking spontaneous physical training in
the running wheel; a call for revising beneficial
effects of exercise on cancer progression
Marta Smeda1, Kamil Przyborowski1, Bartosz Proniewski1, Agnieszka Zakrzewska1, Dawid Kaczor1, Marta
Stojak1, Elzbieta Buczek1, Zenon Nieckarz2, Jerzy A Zoladz2, Joanna Wietrzyk3, Stefan Chlopicki1,4
1
Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Bobrzynskiego 14, 30-348,
Krakow, Poland; 2Department of Physiology and Biochemistry, University School of Physical Education, Al. Jana
Pawła II 78, 31-571, Krakow, Poland; 3Department of Experimental Oncology, Hirszfeld Institute of Immunology
and Experimental Therapy, Polish Academy of Sciences, R. Weigla St. 12, 53-114, Wroclaw, Poland; 4Chair of
Pharmacology, Jagiellonian University, Medical College, Grzegorzecka 16, PL 31-531, Krakow, Poland

Received June 12, 2017; Accepted June 22, 2017; Epub September 1, 2017; Published September 15, 2017
Abstract: It has been repeatedly shown that regular aerobic exercise exerts beneficial effects on incidence and progression of cancer. However, the data regarding effects of exercise on metastatic dissemination remain conflicting.
Therefore, in the present study the possible preventive effects of voluntary wheel running on primary tumor growthand metastases formation in the model of spontaneous pulmonary metastasis were analyzed after orthotopic injection of 4T1 breast cancer cells into mammary fat pads of female Balb/C mice. This study identified that in the mice
injected with 4T1 breast cancer cells and running on the wheels (4T1 ex) the volume and size of the primary tumor
were not affected, but the number of secondary nodules formed in the lungs was significantly increased comparedto their sedentary counterparts (4T1 sed). This effect was associated with decreased NO production in the isolated
aorta of exercising mice (4T1 ex), suggesting deterioration of endothelial function that was associated with lower
platelet count without their overactivation. This was evidenced by comparable selectin P, active GPIIb/IIIa expression, fibrinogen and vWF binding on the platelet surface. In conclusion, voluntary wheel running appeared to impair,rather than improve endothelial function, and to promote, but not decrease metastasis in the murine orthotopic
model of metastatic breast cancer. These results call for revising the notion of the persistent beneficial effects of voluntary exercise on breast cancer progression, though further studies are needed to elucidate mechanisms involvedin pro-metastatic effects of voluntary exercise.
Keywords: Metastasis, 4T1 breast cancer, exercise, wheel running

Introduction
Breast cancer is one of the most prevalentmalignant diseases killing approximately
50,000 women worldwide each year. Although
there is a large body of epidemiologic evidence
suggesting reduced risk of developing themalignant disease by physically active women,
the impact of physical exercise on breast cancer patient prognosis (i.e. primary tumor growthand metastatic spread) along the disease continuum is less clear [1]. Based on the literature,it has been postulated that regular physicalexercise is beneficial in the treatment of various disorders ranging from psychiatric, meta-

bolic, cardiovascular and pulmonary diseases
as well as musculoskeletal disorders and, finally, cancer [2]. Indeed, regular physical exercise
was shown to improve quality of life, cardiorespiratory fitness, physical functioning andfatigue of breast cancer patients and survivors[3]. It has also been shown to reduce breast
cancer death rates and recurrence [4-7].
However, some controversy still exists whether
exercise is always beneficial with respect tobreast cancer [5, 8] since there are also reports
revealing no association between physicalexercise and breast cancer outcome [9, 10].
Similarly, although many pre-clinical studies
performed in animal models of breast cancer

Exercise increases lung metastasis in tumor-bearing mice
showed beneficial effects of exercise on disease progression [8, 11-13], there are also various reports indicating the lack of effect [13-15]
or even increased disease incidence and worse
prognosis [16].
With respect to pre-clinical studies, the differential effects of physical activity on diseaseprogression could be attributed to the mode of
exercise chosen. There is general agreementthat voluntary physical activity (i.e. wheel running by rodents) reflects better natural activity
patterns of animals and is associated withlower stress levels, than forced treadmill training [17]. It has certainly been demonstrated
that voluntary exercise slowed down the growthof primary tumor [12, 13, 18], although it did
not affect the metastatic burden [13-15], whileforced treadmill exercise increased tumor multiplicity and decreased animal survival [16].
Moreover, there is general agreement that thebest type of animal models to study antimetastatic effects of exercise are orthotopic models
that allow for complex microenvironmentalorganotypical interaction between tumor cells
and the surrounding stroma and that mimic primary tumor growth, differentiation, intravasation and metastatic spread in human patients[19]. Therefore, the orthotopic breast cancer
model based on the injection of 4T1 cells into
mammary fat pads of female Balb/C miceseemed to be the best choice to delineate theeffects of exercise on breast cancer dissemination in pre-clinical studies. This model was previously used by [18] to assess the effects ofvoluntary exercise on the primary tumor, but
not on metastasis. Given the fact that the vast
majority of cancer deaths results from formation of distant metastases, only the comprehensive preclinical studies addressing effects
of exercise not only on the primary tumor, butalso on metastatic dissemination are important [8]. Therefore, in the present study weinvestigated the effects of voluntary wheel running on formation of primary tumor and spontaneous lung metastasis in female Balb/C mice
injected orthotopically with 4T1 breast cancer
cells.
Materials and methods
Animals
Thirty-six female Balb/C mice were purchasedfrom the Mossakowski Medical Research
1927

Centre (Warsaw), injected orthotopically with 1
× 104 of 4T1 breast cancer cells and randomly
assigned to the 4T1 sedentary (4T1 sed; 20mice) or 4T1 exercising group (4T1 ex; 16 mice).
After injection, mice were placed in individual
cages. In the case of the 4T1 ex group, thecages were equipped with wheels (Columbus
Instruments, Columbus, OH, USA). The individual wheel count was recorded electronicallyevery 10 s throughout the 5-week disease progression and the weekly covered distance wascalculated based on the wheel count (the innerdiameter of the wheel was 8.9 cm) using thefollowing equation: D=(N * 8.9 * π)/100 [m]. These data was then used to calculate the mean
total distance achieved by all mice and themean velocity (V=D/t). The volume of the primary tumor was measured with calipers in theconsecutive weeks of disease progression andcalculated as described by [20]. Simultaneously,
the body mass of each mouse was recorded. At
the beginning of the 5th week of the disease, allanimals were euthanized (ketamine and xylazine, 100 and 10 mg.kg-1, respectively). The
spleens, primary tumors and lungs were excised
and weighed. The lungs were subsequently
fixed in formalin and pulmonary metastases
were counted with the magnifying glass.
Throughout the experiment, the animals were
kept in a temperature-controlled environment(22-25°C), maintained on a 12-hour light/day
cycle and given unlimited access to food (standard chow, AIN from Zoolab, Krakow, Poland)
and water. One mouse from the 4T1 ex group
was excluded from analysis since at the time ofeuthanization a primary tumor was not detectable. The experimental procedures involving
animals were compliant with guidelines andwere approved by the First Local EthicalCommittee at the Jagiellonian University (Krakow, Poland, permit no: 140/2013).
Cell culture
The mouse mammary adenocarcinoma 4T1
cells were obtained from the American Type
Culture Collection (ATCC, USA). Cells were cultured in RPMI 1640 (IIET, Poland) with OptiMEM® (Life Technologies, USA) (1:1 v/v) medium with 5% fetal bovine serum (HyClone,
Thermo Fisher Scientific Inc. UK), supplemented with 4.5 g/L glucose, 2 mM glutamine, 1.0
mM sodium pyruvate (all from Sigma-Aldrich,Germany) and antibiotics (penicillin and streptomycin-Polfa Tarchomin, Poland). Cell cultures
Am J Cancer Res 2017;7(9):1926-1936

Exercise increases lung metastasis in tumor-bearing mice
were maintained at 37°C in a humidified atmosphere with 5% CO2. Prior to the transplantations, cells were trypsinized (IIET, Poland), centrifuged (200 g, 4°C, 5 min) and counted. 4T1
cells were resuspended in Hank’s Balanced
Salt Solution (HBSS; IIET, Poland) such that asuspension of 1 × 104 4T1 cells in 0.050 ml of
HBSS was inoculated into the mammary fat
pad of female Balb/C mice.
Nitric oxide spin trapping and EPR detection
Krebs-Hepes buffer (consisting of, in mM: NaCl
99; KCl 4.7; MgSO4 1.2; KH2PO4 1.0; CaCl2 1.9;
NaHCO3 25; glucose 11.1; and Na-Hepes 20)
was filtered through a 0.22 µm paper syringe
filter and equilibrated to pH 7.4. Following thisstep, the filtered buffer was deoxygenized bybubbling argon gas for at least 30 minutes.
DETC (3.6 mg) and FeSO4 ·7H2O (2.25 mg) wereseparately dissolved under argon gas bubblingin two 10 ml volumes of ice-cold Krebs-Hepes
buffer and kept under gas flow on ice until
used. Freshly harvested thoracic aortas werecleaned of adherent fat, opened longitudinally,
placed in 24-well plates filled with 100 μl KrebsHepes and preincubated for 30 minutes at
37°C. DETC and FeSO4 ·7H2O solutions weremixed 1:1 (v/v) to obtain 250 μl Fe(DETC)2 colloid per well (final concentration 285 μM) and
immediately added to the aortas in parallel withcalcium ionophore A23187 (final concentration
1 μM) to stimulate eNOS and incubated at 37°C
for 90 min. After incubation, each aorta wasremoved from the buffer, drained on a piece of
kimwipe for 5 s and the wet mass was measured. Next, the aorta was frozen in liquid nitrogen into the middle of a column of Krebs-Hepes
buffer. The needle-end was cut from a 1 ml
insulin syringe, the plunger retracted 1 cm fromthe cut end, and 200 μl of the buffer was frozen
in liquid nitrogen. Subsequently, the buffer was
warmed for a few seconds to allow retraction
and the aorta was placed on top and refrozen inliquid nitrogen. The syringe was then warmed
once more to allow retraction of the column
and an additional 200 μl of the buffer was
pipetted on top of the frozen column, to allow
freezing of the aorta in the middle of a 400 μl
column of Krebs-Hepes buffer and was thenstored in -80°C until measured. The frozen column was pushed out directly into a finger Dewar(Noxygen, Germany) containing liquid nitrogen.
EPR spectra were obtained using an X-band
EPR spectrometer (EMX Plus, Bruker, Germany),equipped with a rectangular resonator cavity
1928

H102. Instrument settings were: centre-field(B0) 3276G, sweep 115G, microwave power 10
mW, modulation frequency 100 kHz, amplitude
modulation 8 G, sweep time 60 s, and numberof scans 4. Signals were quantified by measuring the total amplitude of the NO-DETC after
correction of baseline as previously performed[21].
Measurement of blood and plasma parameters
Blood samples were collected from the rightventricle into the syringe containing heparin (10U ml-1). The blood count was performed using
the animal blood counter Vet abc (Horiba
Medical, France) and cytometric analysis ofplatelet activation was performed using a flow
cytometer (LSRII and FACS/Diva ver. 6.0,
respectively, Becton Dickinson, Oxford, UK).
The rest of the blood was centrifuged at 1000
G/10 min/4°C to obtain plasma that was aliquoted to measure concentrations of 6-ketoPGF1α (Enzo Lifie Sciences, NY, USA) andP-selectin (Abcam, UK) with ELISA kits as wellas NO2- and NO3- concentrations with an ENO20 NOx Analyzer (Eicom Corp., Kyoto, Japan) as
previously described [22].
Flow cytometry
Prepared “washed blood” was incubated with
anti-mouse PE-conjugated GPIIb/IIIa or P-selectin antibodies for determination of their platelet surface expression and anti-mouse FITClabeled anti-fibrinogen or von Willebrand (vWF)
factor antibodies for determination of theirmembrane binding. Activation of circulatingplatelets was evaluated based on the measured expression/binding of surface membrane antigens. Platelets were identified bytheir forward- and side-scatter characteristics
and were gated on the basis of the expressionof platelet-specific antigen CD41/61. Isotype
matched FITC- or PE-conjugated control antibodies were used to assess non-specific binding. Flow cytometric analyses of platelet activation was performed using flow cytometer software (LSRII and FACS/Diva ver. 6.0, respectively, Becton Dickinson, Oxford, UK). Measurements were made under the logarithmic gain
and 10 000 events were collected. Appropriate
color compensation was determined in samples singly stained with either FITC-conjugatedanti-CD41/61 or PE-conjugated anti-CD41/61.
Unstained platelets were used to establish the
Am J Cancer Res 2017;7(9):1926-1936

Exercise increases lung metastasis in tumor-bearing mice

Figure 1. Mean distance covered in the consecutive weeks of breast cancer progression. Mice fromthe 4T1 ex group were placed individually in cages
equipped with wheels and the wheel count was recorded continuously throughout the four weeks ofthe disease progression as described in Materials
and Methods. The mean weekly distance in the 1st,
2nd, 3rd and 4th weeks of disease progression was
calculated as described in Materials and Methods.
At the beginning of the 5th week, the mice were euthanized. Statistical analysis was performed with
two-sided Student T test and only P<0.05 wereconsidered significant (n=15). The symbols # show
statistical significance vs 1st week while the symbols*show statistical difference between the 3rd and the
4th week: ##/** (P<0.01), ### (P<0.0001). The
data are presented as mean ± SEM.

level of autofluorescence set to fall within thefirst log order of brightness for each fluorescence channel. Suitable isotype controls wereused as appropriate to set up the backgroundnoise at less than 1 percent. Events appearing
above the background level were then recorded. Results were presented as the percentageof activation marker-positive events in platelets
population.
Statistical analyses
Statistical analysis was performed in GraphPad
Prism Software 5 (GraphPad Software, USA).
The data were analyzed with two-sided T test or
non-parametric Mann-Whitney test based on
the results of D’Agostino and Pearson omnibus
normality test. All data were presented as meanvalues ± SEM.
Results
Quantification of voluntary wheel running exercise along breast cancer progression
The mean total distance covered and the mean
velocity achieved by 4T1 ex mice during four
1929

weeks of voluntary running were 170.45±47.5
km and 17.45±1.8 m.min-1, respectively. In the
1st week following cancer cells injection, themice from the exercising group covered shorter
distance than in the subsequent two weeks(Figure 1) (29±4 km in the 1st week vs 48±5km, P=005, n=15 and 53±3 km, P<0.0001,
n=15 in the 2nd and 3rd week, respectively). Thiseffect was most likely due to progressive acclimatization of mice to the wheels. On the other
hand, in the week prior to mice euthanization,
the covered distance was lower than in theweek before (40±2.6 km vs 53±3 km,
P=0.0052, n=15) (Figure 1). This is likely to be
attributed to the mice attaining advanced stages of the disease (Figure 2A).
Effects of voluntary activity on the progressionof breast cancer
Primary tumors became palpable in all mice inthe 3rd week of the disease and their mass
increased afterwards (Figure 2A). There was nodifference in the primary tumor volume between
non-exercising 4T1 sed and exercising 4T1 ex
mice except in the 3rd week when it was slightly
higher in 4T1 sed mice (44.9±8 mm3 for 4T1
sed mice vs 24.2±3.5 mm3 for 4T1 ex mice,
n=15-20, P=0.03). There was also no difference between the weight of primary tumors
after their excision from 4T1 sed and 4T1 ex
mice in the 5th week of the disease (Figure 2B).
Interestingly, in the 5th week of the disease, thebody weight of 4T1 ex mice tended to be slightly
lower than the body weight of 4T1 sed mice
(P=0.0525, n=15-20) (Figure 2C). Although theweight of the lungs was not different between
4T1 sed and 4T1 ex mice (Figure 2D), the number of pulmonary metastases was significantly
higher in the 4T1 ex group as compared withthe 4T1 sed group (1.6±0.6 in 4T1 sed vs14.8±5.7 in 4T1 ex mice, P=0.035, n=9) (Figure2E). Analysis of blood count revealed that the
RBC number and the PLT number were lower inthe 4T1 ex mice (Table 1). No difference
between spleen weight, an indirect parameterof systemic inflammation [11], was found between the 4T1 sed and 4T1 ex mice.
Effects of voluntary activity on systemic endothelial function and NO bioavailability
In normal vascular physiology, endotheliumderived NO maintains the vascular wall in a quiescent state by inhibition of inflammation andcellular proliferation [23]. It was observed thatan increased number of pulmonary metastases
Am J Cancer Res 2017;7(9):1926-1936

Exercise increases lung metastasis in tumor-bearing mice

Figure 2. Effects of voluntary activity on breast cancer growthand pulmonary metastasis. A: Primary tumor volume wasmeasured once a week throughout the disease progression;
B: At the beginning of the 5th week, mice from the 4T1 sed
and the 4T1 ex groups were euthanized and primary tumors
were excised and weighed; C: Body mass was measured oncea week throughout the disease progression; D: Lungs were
excised and weighed at the beginning of the 5th week of disease progression; E: The number of pulmonary metastases
was counted in formalin-fixed lungs after their dissection into
the lobes. Statistical significance was assessed by two-sided
Student T or Mann-Whitney tests depending on the normalityof distribution that was assessed by D’Agostino & Pearson omnibus normality test and only P<0.05 were considered significant. The symbol * denotes statistical significance (P<0.05).
The data are presented as mean ± SEM.

in 4T1 ex mice (Figure 2E) was associated withdecreased production of NO in their aortas(45030±5636 AU for 4T1 ex vs 67860±4439
AU for 4T1 sed mice, P=0.0052, n=8-10) (Figure 3A) indicating more pronounced systemic
endothelial dysfunction in exercising mice. On
the other hand, the concentration of PGI2 stable metabolite 6-ketoPGF1α was increased in
the plasma of 4T1 ex mice (10112±1966
pg.ml-1 for 4T1 sed mice vs 20919±3144 pg.
ml-1 for 4T1 ex mice, P=0.021, n=8) (Figure 3B)
suggesting a compensatory activation of vascular PGI2 production in the setting of compro1930

mised endothelial NO production [24], as
reported also for other models [25].
Interestingly, the systemic NO bioavailability
was not lower in 4T1 ex mice compared with
4T1 sed mice as evidenced by similar plasma
concentrations of nitrite (NO2-) (Figure 3C) andnitrate (NO3-) (Figure 3D).
Effects of voluntary activity on platelet activation
It is known that platelets, once activated, shed
P-selectin rapidly from their surface into the
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Exercise increases lung metastasis in tumor-bearing mice
Table 1. Blood count and spleen weightExperimental groups
Parameter
4T1 sed
4T1 exRBC [M.µl-1]
10.7±0.16 (n=10) 10.2±0.18 (n=7) (P=0.018)*
HGB [g.dl-1]
14.6±0.9 (n=10)16.2±0.3 (n=9)
WBC [K.µl-1]93.42±21.8 (n=10)102.5±19 (n=9)
GRA [K.µl-1]
61.5±14.6 (n=10)72.1±0.5 (n=9)
LYM [K.µl-1]
21.4±4.8 (n=10)28±4.5 (n=9)
PLT [K.µl-1]
855±34 (n=8)
609±82 (n=6) (P=0.013)*
Spleen weight [mg] 454±50.5 (n=11)491±36 (n=9)

inhibit the signaling pathways
responsible for genome stability [29] and, thus, promotebreast cancer malignancy [30].

An increase of pulmonary
metastasis in mice voluntarily
running the wheels reported inthis study is surprising, considering the strong epidemiological evidence that exercise notthonly reduces breast cancer
At the beginning of the 5 week of the disease, mice from 4T1 sed and 4T1 ex
groups were euthanized. Blood count was performed and spleens were weighed.
risk [1, 31], but also exerts
Statistical significance was assessed by two-sided Student T test or Mann-Whitbeneficial effects in patientsney tests depending on the normality of distribution determined by D’Agostino &with previously diagnosed maPearson omnibus normality test and only P<0.05 were considered significant. The
mmary gland tumor [4-7];
symbol * denotes statistical significance (P<0.05). Data are presented as mean
though the studies showing no± SEM.
positive association between
physical activity and breast
cancer recurrence and mortality also exist [9,plasma [26]. Therefore, the plasma P-selectinconcentration (Figure 4A) and the platelet acti10]. Similarly to human studies, the data
vation ex vivo (Figure 4B-E) were measured toderived from pre-clinical experiments withevidence that neither the plasma P-selectinrodent models are also not consistent. In fact,
concentration nor the platelet activation markwhile some reports show retardation of breasters were increased in 4T1 ex compared withcancer progression in response to exercise [114T1 sed mice.
13, 18], others show no [14, 15, 32] or even the
opposite effect [16].
Discussion
An interesting study investigating effects of volThis study demonstrated that voluntary exeruntary wheel running on the primary tumor
cise increased formation of lung metastases ingrowth and perfusion after orthotopic implanorthotopic syngeneic mouse model of breast
tation of 4T1 breast cancer cells was conductcancer (Figure 2E). The pro-metastatic effects
ed by [18]. The authors found that voluntaryof voluntary exercise were associated withphysical activity reduced primary tumor growth,
lower NO production by isolated aortas of exerimproved perfusion of the tumor and increased
cising mice (4T1 ex) as compared with aortas
apoptosis of breast cancer cells within the priisolated from sedentary control mice (4T1 sed)mary tumor. However, this study did not refer tothat were also injected with 4T1 cells (Figure
the effects of exercise on the metastatic bur3A). However, no evident decrease in systemic
den. In view of the fact that the majority of canNO bioavailability was identified (Figure 3C andcer deaths results from distant metastasis [8],3D) probably due to the compensation of
the observation of increased spontaneous pulimpaired endothelial NO production by NO3-→
monary metastases in mice injected orthotopiNO2-→NO reductive pathway as reviewed bycally with 4T1 cells voluntarily running on the[27]. Moreover, similar plasma concentrations
wheels (Figure 2E), despite no effects on theof NO2- and NO3- in 4T1 sed and 4T1 ex mice inprimary tumor (Figure 2A and 2B), is even more
the terminal 5th week of disease progression
alarming with respect to the potential hazard
indicated that there was no increase in systemfor patients with breast cancer undergoing volic production of free radicals and oxidative
untary exercise. It is also worth noting thatstress in 4T1 ex mice (NO can undergo autoxithese authors investigated effects of exercise
dation to form NO2- and NO3- [28] at that stage
on the classical vessels lined with endothelialof the disease. However, it cannot be excluded
cells in the primary tumor. Interestingly, it hasthat at the beginning of the voluntary wheel
been recently shown that primary tumors comrunning ROS and RNS associated with exercise
prised of 4T1 cells are also capable of forming
were overproduced in breast cancer-bearing
pseudovessels, a phenomenon known as vasmice and exercise-induced oxidant stress could
cular mimicry [33]. Such pseudovessels, usu1931

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Exercise increases lung metastasis in tumor-bearing mice

Figure 3. Systemic endothelial function and NO bioavailability. Aortas were excised, cleaned from the surrounding tissues. A: NO production by the aorta was measured as described in Materials and Methods; B: Plasma 6-ketoPGF1α
production was measured by commercially available ELISA kit; C, D: Plasma NO2- and NO3- were determined as
described in Materials and Methods. Statistical significance was assessed by two-sided Student T or Mann-Whitney
tests depending on the normality of distribution assessed by D’Agostino & Pearson omnibus normality test and only
P<0.05 were considered significant. The symbol * denotes statistical significance (*P<0.05, **P<0.01). The data
are presented as mean ± SEM.

ally lined with tumor cells or a mixture of endothelial and tumor cells, converge with classicalendothelium-lined vasculature and support circulation within the tumor thereby facilitating
intravazation of cancer cells [34]. Moreover,[33] showed that improving perfusion in thepseudo-circulation within the primary tumor
comprised of 4T1 cells by injection of exogenous anticoagulant warfarin promoted pulmonary metastases. Similar pro-metastatic effectin this model of breast cancer was also
observed in the case of platelet depletion [35,36]. These results suggest that improved perfusion of the primary tumor comprised of 4T1
cells caused by voluntary exercise may be
responsible for increased metastatic spread in
Balb/C mice. Obviously, further studies areneeded to test such a hypothesis, but it is worth
noting that the number of circulating platelets

1932

was significantly reduced in 4T1 ex mice (Table1), concomitantly with increased number ofmetastatic foci in their lungs (Figure 2E).
As discussed above, regular exercise improves
perfusion of primary tumor [18] and, thus, canfacilitate intravazation of metastatic cancer
cells. Tissue perfusion can also be increased
by endogenous vasodilators such as prostacyclin (PGI2) [37, 38], that is also a potent inhibitorof platelet activation [39, 40]. Indeed, the plasma concentration of PGI2 stable metabolite
6-ketoPGF1α was higher in 4T1 ex mice (Figure
3B). Despite that fact, differences in basal
platelet activation between 4T1 ex and 4T1 sedmice were not observed (Figure 4). However,lower number of circulating platelets in 4T1 ex
mice (Table 1) suggested decreased probabilityof forming platelet aggregates in the circulation

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Exercise increases lung metastasis in tumor-bearing mice

Figure 4. Plasma P-selectin concentration andplatelet activation. (A) Plasma P-selectin concentration was measured with commercially available
ELISA kit. Platelet activation was measured by assessment of platelet activation surface markers
such as (B) P-selectin; (C) active form of GPIIb/IIIa
receptor; (D) von Willebrandt factor and (E) fibrinogen bound to platelet surface by flow cytometry.
Statistical significance was assessed by two-sided
Student T test providing the normality of distribution that was assessed by D’Agostino & Pearson
omnibus normality test or Mann-Whitney test. Data
are presented as mean ± SEM.

of exercising mice in comparison with their sedentary counterparts. Last but not least, higher
levels of circulating PGI2 in exercising mice
could also indicate the on-going angiogenesis[24], associated with increased endothelium
permeability [41], which would facilitate extravasation of circulating cancer cells and crossing
1933of the endothelial barrier during metastases[42].
It should be highlighted that in the present
experiment we used voluntary wheel running asa model to study the effects of physical training
on breast cancer progression. Such a model of
Am J Cancer Res 2017;7(9):1926-1936

Exercise increases lung metastasis in tumor-bearing mice
exercise is less stressful to animals than forced
running on a treadmill or other forced exercise
behaviors such as swimming. Thus, the exercise intervention imitates the self-paced voluntary exercise training sessions in humans,
which have a number of health-related benefits. It should be stressed however, that theexercise intervention was applied to breast
cancer-bearing animals with no history of training before the onset of illness. Our results
clearly suggest that even voluntary exercise
training intervention reported to improve healthstatus elsewhere [2], when applied to breast
cancer-bearing previously untrained animals,
could stimulate disease progression. It might
be argued that the differences between our
results and the other findings presented in theliterature concerning the effect of training on
breast cancer progression in animals or
patients [3, 4, 6, 7, 11-13, 18, 43] are due toincreased oxidant stress of the untrained animals placed on the wheels. Interestingly, as discussed above, no evidence of higher systemicoxidative stress was found in 4T1 ex mice vssedentary controls in the 5th terminal week ofthe disease (Figure 3C and 3D). However, itcannot be excluded that at the beginning ofwheel training, oxidative stress was more pronounced in yet untrained 4T1 ex mice and such
unbalanced exercise-induced increase in ROS
and RNS production in yet untrained individuals
previously reported by [44], could facilitate
effective metastases. Indeed, it is well documented that acute physical exercise increases
ROS production in skeletal muscles [45].
Furthermore, it has recently been demonstrated that eight weeks of endurance training
enhanced ROS production in non-phosphorylating (state 3) isolated rat skeletal mitochondria [46]. Therefore, increased number of pulmonary metastases in the previously untrained
animals could well be due to the local traininginduced enhancement of ROS and RNS production overreaching ROS and RNS defense mechanisms of cancer-bearing mice. However, further studies are needed to explain the role ofthe training-induced enhancement of ROS andRNS production on breast cancer progression
in previously trained vs. untrained animals.
In summary, this study has demonstrated thatspontaneous voluntary wheel running did notmodify the volume and size of primary tumor,
but promoted pulmonary metastasis. The

1934

effect was associated with decreased NO production in the isolated aorta, but not with activation of platelets suggesting that impaired
endothelial function could contribute to thatphenomenon. On the other hand, it might wellbe that exercise-induced improvement of tumor
perfusion could also be involved, particularly inthe case of aggressive breast cancer cells suchas 4T1 cells forming pseudo-vessels [33]. Last
but not least, it cannot be excluded that exercise-induced ROS and RNS production could
contribute to that phenomenon. However, toelucidate the underlying mechanisms, further
studies are warranted. Nevertheless, ourresults contradict the general belief that regular exercise always lowers breast cancer progression and call for revising the notion of thebeneficial effects of exercise on cancer metastasis.
Acknowledgements
This study was supported by the project
METENDOPHA (a grant coordinated by JCET-UJ,No STRATEGMED1/233226/11/NCBR/2015).
Disclosure of conflict of interest
None.
Address correspondence to: Stefan Chłopicki,
Jagiellonian Centre for Experimental Therapeutics(JCET), Jagiellonian University, Bobrzynskiego 14,
30-348, Krakow, Poland. Tel: +48 12 6645464;
Fax: +48 12 2974615; E-mail: stefan.chlopicki@
jcet.eu

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