Endurance training slows breast tumor growth in mice by suppressing Treg cells recruitment to tumors
Authors: Luma Melo
Stephanie Vargas
Amit Hagar
John Foley
Josua Aponte Serrano
Richard Carpenter
Zemin Wang
Sachiko Koyama
Journal: BMC Cancer (2019)
Discipline: CLINICAL MEDICINE
Keywords: CD8+/FoxP3+ ratio; Endurance exercise; Forced running wheels; Hypoxia; Murine mammary tumor

Abstract:  Background: Aerobic exercise has been shown to slow tumor progression in rodents and humans, but the mechanisms behind this effect are still unclear. Here we show that aerobic exercise in the form of chronic endurance training suppresses tumor recruitment of FoxP3+ Treg cells thus enhancing antitumor immune efficiency. Methods: Adult wild-type and athymic BALB/c female mice were endurance-trained for 8 weeks. Circulating leukocytes as well as muscle and liver mtDNA copy number were compared to aged-matched concurrent sedentary controls to establish systemic effects. 4 T1 murine mammary tumor cells were injected subcutaneously to the 4th mammary pad at the end of the training period. Tumor growth and survival rates were compared, together with antitumor immune response. Results: Exercised wild-type had 17% slower growth rate, 24% longer survival, and 2-fold tumor-CD+ 8/FoxP3+ ratio than sedentary controls. Exercised athymic BALB/c females showed no difference in tumor growth or survival rates when compared to sedentary controls. Conclusions: Cytotoxic T cells are a significant factor in endurance exercise-induced suppression of tumor growth. Endurance exercise enhances antitumor immune efficacy by increasing intratumoral CD8+/FoxP3+ ratio. 

Retracted: No

PubPeer comments:
 Multiple issues undermine the validity of this article. Issues A-C are annotated on the attached modified figures. A. The authors state in their methods under **Tumor inoculation and size measurements**:  Once tumor volume exceeded **1000u2009mm3** mice were sacrificed and blood, tumor and tissue were harvested. In Figures 2 and 5, animals are clearly allowed to pass this endpoint. B. In Figs. 2 5 there are multiple issues with axes labeling that prevent estimation of values. C. Fig. 2 presents data from immuno-competent mice, while Fig. 5 are severely immuno-compromised (athymic nude mice; lack functional T cells). The premise of the paper is that exercise reduces Treg-mediated suppression of anti-tumor immunity, thereby slowing tumor growth (Fig. 2). Their claim is that removal of Tregs masks this effect (Fig. 5), however, these mice lack all functional T cells, importantly CD8+. Thus, one would expect to see a more aggressive tumor phenotype in the nude mice (Fig. 5), but the opposite is presented as tumors grow more quickly in the immuno-competent animals (Fig. 2). This does not square with the doubling times, and is never addressed. As reported, it takes the SED group nearly twice as long to achieve 200mm^3, and the exercise groups are nearly identical at this time point. D. Sample size is not reported for Fig. 1a, and might be inadequate for reported statistical analysis. E. The cortisol measurements reported in Fig. 1c do not adequately account for circadian variation in cortisol. F. In Fig. 3 the authors report that mean neutrophil count is approximately tripled in the exercise group, and that there are virtually no monocytes in the sedentary group. Sample size for the EX group monocytes is also suspiciously different from other cell counts. Lymphocytes are not reported despite the subject of the paper (lymphocytes). **It is well-established in the field that this model (4T1) induces a very strong polymorphonuclear expansion in blood, spleen, bone marrow, etc.** Particularly one of the myeloid-derived suppressor cell subsets, which is morphologically indistinguishable from a neutrophil. Furthermore, Fig. S5 shows an increase in neutrophils in the SED group, but this is comparing to exercise before tumor cell inoculation; in fact the authors report higher neutrophil counts pre-tumor in both groups (Fig. S5) when compared to tumor-bearing mice (Fig. 3). The authors contracted out this work and do not describe exactly how these cells were identified. A detailed cytometric analysis using appropriate cell markers and control(s) is necessary. There are no non-tumor bearing controls reported in Fig. 3 as a benchmark (S5 might be such a benchmark but is lacking precise cell ID parameters), which potentially invalidates any conclusions from these data. The authors do not discuss the significant divergence of these findings from a rich literature reporting the exact opposite effect of the 4T1 model.   ,