Taken together, we find marked reductions in tumor incidence and growth with voluntary wheel running across 5 different tumor models. The delay in B16 melanoma progression required a 4-week pre-training period prior to tumor inoculation. During this period, the mice were habituated to wheel running, and the immune system was primed for the tumor challenge, suggesting that when using fast-growing transplantable tumor models, part of the exercise effect could potentially be killing of cancer cells at the inoculation site. In contrast, initiating running after tumor challenge in our slow-growing DEN-induced and Tg(Grm1)Epv models was sufficient to control tumor incidence and progression.

The average wheel running distance was 4.1 km/mouse/day for the B16-inoculated female mice and 6.8 km/mouse/day for the male DEN-injected mice. The presence of B16 tumors did not induce weight loss or cachexia ( Figure S1 E). In contrast, the presence of LLC tumors induced an average weight loss of −1.27 ± 1.74 g in the tumor-bearing control group. This weight loss was completely prevented in the running tumor-bearing mice (p < 0.05, Figures S1 E and S1G).

First, we evaluated the effect of wheel running before and/or during tumor challenge in a subcutaneous B16F10 melanoma model in female mice ( Figure 1 A). Four weeks of wheel running prior to tumor cell inoculation reduced tumor growth by 61% (p < 0.01, Figure 1 B). Similar reductions in tumor volume of 67% and 53% with running were verified in female adult (3 months, p < 0.05, Figure 1 C) and old mice (18 months, p < 0.001, Figure S1 A). Wheel running also dramatically reduced lung metastases after intravenous (i.v.) injection of B16F10 melanoma cells (p < 0.001, Figures 1 D and 1E). Next, the impact of wheel running on tumor growth was evaluated in three additional models. Male Naval Medical Research Institute (NMRI) mice were injected with diethylnitrosamine (DEN) at 4 weeks of age, which is known to cause liver tumors within 10 months. Here, wheel running reduced tumor incidence as only 31% of the running mice developed tumors, compared with 75% of the control mice ( Figure 1 F). Moreover, wheel running reduced tumor burden per mouse (p < 0.05, Figure 1 G). In a Lewis Lung carcinoma model (LLC) in female mice, running decreased tumor volume by 58% (p < 0.01, Figure S1 B) and tumor weight by 56% (p < 0.05, Figure S1 C), and in Tg(Grm1)EPv transgenic male mice, which spontaneously develops melanoma, wheel running tended to delay formation of malignant lesions (p = 0.08, Figure S1 D).

(G) Tumor burden assessed by MRI (n = 16 for both groups, two-way ANOVA with post hoc multiple t test). $: 11month: EX = 16, CON = 14. See also Figure S1 . Means ± SEM.p < 0.05,p < 0.01,p < 0.001.

To exclude that these differences in immune pathways were merely related to tumor size, we repeated the evaluation now based on B16 tumors of identical volume (approx. 150 mm Figure 2 F). Thus, the tumors were on average excised 2 days earlier in the control group ( Figure 2 G). In these similar sized tumors, upregulation of the pro-inflammatory cytokines (IL-1α and iNOS) and immune cell markers with exercise was verified ( Figures 2 H and 2I).

To identify differentially regulated pathways in tumors from running mice, we performed microarray analysis on B16 tumors from Figure 1 C ( Table S1 ). Of the 92 upregulated gene ontology (GO) pathways, the majority (52%) was related to immunological and inflammatory pathways ( Figure 2 A). qPCR analysis confirmed the increased expression of both pro- and anti-inflammatory cytokines ( Figure 2 B), as well as markers of the cellular innate and adaptive immune systems in B16 tumors from running mice ( Figure 2 C). Also in LLC tumors, pro-inflammatory cytokines (IL-1α and iNOS) and markers for NK and T cells were upregulated with running ( Figures 2 D and 2E).

(F–I) (F) Tumor volume of subcutaneous B16F10 tumors and (G) day of termination for tumors excised. qPCR analysis of (H) inflammatory cytokines and (I) immune cell markers from tumors in Figure 2 F (n = 10, multiple t testing). Black bars = CON, gray bars = EX. Means ± SEM.p < 0.05,p < 0.01,p < 0.001.

(A–E) (A) Distribution of upregulated GO pathways (n = 5). See also Table S1 . qPCR analysis of (B) inflammatory cytokines and (C) immune cell markers in B16 tumors from Figure 1 C (CON = 12, EX = 11, multiple t testing), and (D) inflammatory cytokines and (E) immune cell markers in Lewis Lung tumors (CON = 9, EX = 10, multiple t testing).

In addition to the mobilization of the NK cells during exercise, we found increased expression levels of NK cell-related activating receptor ligands, stimulatory cytokines, and chemoattractant chemokines in the tumors of running mice, suggesting that exercise works both on the mobilization of NK cells, and on the tumor microenvironment to generate a NK cell activating milieu. NK cells are regulated by a multitude of activating and inhibitory receptor-ligand interactions. Here, we show that ligands for the activating receptor NKG2D, MULT1 and H60a, as well as Clr-b, a ligand for NKR-P1B, which has proven important in the education of NK cells, were upregulated in the tumors from running mice (). Previously, it has been shown that B16F10 cells do not express Clr-b (). The methodology employed in this study did not allow for precise identification of whether Clr-b expression was attributable to tumor cells, infiltrating immune cells, or other cells in the tumor microenvironment. Furthermore, we found increased expression of the activating receptor NKp46 with wheel running. NKp46 has been shown to mediate control of B16 metastasis, which correlates well with our results ().

Taken together, these data point to a predominant role of NK cells in the training-dependent control of tumor growth. NK cells represent a critical component of the innate immune defense, recognizing transformed cells independently of antibodies or major histocompatibility complex (MHC) restriction (), while T cells are cytotoxic effector cells of the adaptive immune response. Both immune cell types are known to be regulated by exercise. During exercise, circulating lymphocytes increase in number and frequency and then fall below pre-exercise levels (). Of these lymphocytes, NK cells are the most responsive cells to the exercise-dependent mobilization, followed by CD8 T cells, CD4 T cells, and lastly B cells, which respond poorly to exercise (). Thus, the importance of NK cells in the training-dependent control of tumor growth follow their superior responsiveness to exercise.

Next, we investigated if running affected NK cell cytotoxicity, but determined per cell basis, NK cells from control and running mice were equally effective in killing NK-sensitive YAC-1 and B16 cells ( Figure 3 J). To exclude that cancer cell killing was mediated by T cell contamination, the NK-depleted fraction (NK cell content of 2%) was used as a negative control and showed no YAC-1 or B16 cell killing. In contrast, within the tumors from the running mice, we found increased mRNA expression of NK-cell-activating receptor ligands (H60a, MULT1, Clr-b), as well as cytokines (IL-2, IL-15, IFNγ) and chemokines (CCL3, CXCL10, CX3CL1, Chemerin) related to NK cell activation and chemotaxis ( Figure 3 K). No changes in the expression of markers of angiogenesis (i.e., CD31 and VEGF-A) were observed ( Figure 3 K).

We then evaluated the response to running in athymic mice, which lack functional T cells but retain NK cells. In these mice, a 66% reduction in B16 tumor volume persisted with 6 weeks of running (p < 0.05, Figure 3 H), showing that T cells were not responsible for the suppressive effect of running on tumor growth. However, the athymic nude mice in general had larger tumors than wild-type immune-competent mice (WT) ( Figures S3 C and S3D), indicating that T cells aside from the exercise situation play a role in control of tumor growth. To further document the role of NK cells, circulating NK cells were depleted by administration of anti-asialo-GM1 antibodies ( Figures S3 E and S3F). Depletion of NK cells completely abolished the suppressive effect of running on tumor volume, and both anti-asialo-GM1-treated groups (control and exercise, respectively) showed enhanced tumor growth compared with isotype IgG-treated mice (p < 0.001, Figure 3 I).

Next, we measured frequencies of immune cells in tumors by flow cytometry after 6 weeks of wheel running. In the subcutaneous B16 model, tumors from running mice showed markedly increased infiltration of NK cells (p < 0.001, Figures 3 A, 3C, and 3D ), as well as CD3 T cells and dendritic cells (p < 0.05, Figure 3 A). Of note, the CD3 gate includes CD3CD4CD8cells, and thus also includes cells such as gamma delta and NKT cells. In the i.v. B16 model, wheel running also increased NK cell infiltration in lung tumors (p < 0.05, Figure 3 B), but without enhancement of other immune cell subtypes. The level of NK cell infiltration correlated inversely with tumor burden (p < 0.01, Figures S2 A and S2B). Histological evaluation showed that exercise increased both NK1.1, CD8and CD4cells with the absolute numbers of CD8and CD4cells being higher than that for NK cells ( Figures 3 E and S2 C). In control mice, NK cells were rarely detectable. Infiltration of B cells did not change significantly with exercise ( Figure S2 D). In non-tumor-bearing mice, 6 weeks of running increased the frequencies of NK cells in bone marrow (p < 0.05), spleen (p < 0.01), and to a lesser extent peripheral blood mononuclear cells (PBMCs) (p = 0.17, Figure 3 F), indicating an overall increase in the basal pool of NK cells with exercise. In tumor-bearing mice, running did not alter the frequency of NK cells in these organs ( Figure 3 G), yet these mice showed pronounced accumulation of NK cells in their tumors ( Figure 3 C). The numbers of T cells did not increase in blood, bone marrow, or spleen with 6 weeks of running in neither tumor-bearing nor non-tumor-bearing mice ( Figures S3 A and S3B).

IL-6-Sensitive NK Cells Are Recruited by Exercise through β-Adrenergic Signaling

Pedersen and Febbraio, 2012 Pedersen B.K.

Febbraio M.A. Muscles, exercise and obesity: skeletal muscle as a secretory organ. −, CD27−) NK cells (p < 0.05, −, CD27+) NK cells (p = 0.11). In contrast, anti-IL-6 antibody treatment blocked the training-induced increase in cytotoxic (CD11b−, CD27+, p < 0.05) NK cells but increased the frequency of cytokine producing (CD11b+, CD27−, p < 0.05) NK cells ( Plasma IL-6 increases dramatically during exercise due to release from contracting muscles and might be the additional exercise factor, involved in tumor homing (). In our model, serum IL-6 increased from 4.3 ± 3.5 pg/ml (range: 1.6–12.4 pg/ml, n = 12) in the CON group to 29.3 ± 32.6 pg/ml (range: 5.7–106.5 pg/ml, n = 10, p < 0.05) during wheel running. In the spleen, 24.8% ± 8.4% of the NK cells expressed IL-6Rα and 63.4% ± 7.9% gp130 ( Figures 4 H and 4I). After EPI injection, the fraction of IL-6Rα-positive splenic NK cells decreased to 12.4% ± 5.3%, suggesting an EPI-dependent mobilization of IL-6-sensitive NK cells. Blocking of training-induced IL-6 by anti-IL-6 antibodies diminished the exercise-induced inhibition of tumor growth ( Figure 4 J) and inhibited the infiltration of NK cells into tumors ( Figure 4 K). In contrast, daily injections of 100 ng IL-6, which increased serum IL-6 from 2.7 ± 0.9 pg/ml to 301.1 ± 130.3 pg/ml and 263.8 ± 950 pg/ml at 30 and 60 min, respectively, did not mimic the training-induced reduction in tumor growth ( Figure S4 E) or enhanced NK cell infiltration ( Figure S4 E). Thus, with this IL-6 concentration, the redistribution of NK cells seen during running could not be mimicked, suggesting that the increment in NK cell infiltration is dependent on concurrent exercise-induced mobilization of immune cells. Both running and IL-6 injections decreased the frequency of immature (CD11b, CD27) NK cells (p < 0.05, Figure S4 E) and tended to increase the frequency of cytotoxic (CD11b, CD27) NK cells (p = 0.11). In contrast, anti-IL-6 antibody treatment blocked the training-induced increase in cytotoxic (CD11b, CD27, p < 0.05) NK cells but increased the frequency of cytokine producing (CD11b, CD27, p < 0.05) NK cells ( Figure S4 H).

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Born J. Selective mobilization of cytotoxic leukocytes by epinephrine. Steensberg et al., 2003 Steensberg A.

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Whiteside T.L. Response of human NK cells to IL-6 alterations of the cell surface phenotype, adhesion to fibronectin and laminin, and tumor necrosis factor-alpha/beta secretion. The epinephrine surge during exercise can mobilize NK cells to the blood stream through activation of their β-adrenergic receptors, increasing the NK cell frequency but not their cytotoxicity (). Early studies suggest that a relatively small increase in epinephrine level is sufficient to mobilize NK cells. Thus, to obtain the physiological changes, which we observe within the tumors, additional stimuli must be present. To this end, we observed a selective mobilization of IL-6Rα-positive NK cells after epinephrine injection. In further support of the role of IL-6 in NK cell redistribution and activation, recombinant human IL-6 infusion has been shown to mimic the acute and transient lymphopenia seen during the recovery from exercise (), and stimulation of human NK cells with IL-6 has been shown to increase their expression of adhesion molecules ().

Dethlefsen et al., 2013 Dethlefsen C.

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Hojman P. The role of intratumoral and systemic IL-6 in breast cancer. Mauer et al., 2015 Mauer J.

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Karin M. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Kraakman et al., 2015 Kraakman M.J.

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et al. Blocking IL-6 trans-signaling prevents high-fat diet-induced adipose tissue macrophage recruitment but does not improve insulin resistance. Scheller et al., 2014 Scheller J.

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Rose-John S. Interleukin-6: from basic biology to selective blockade of pro-inflammatory activities. Yet, the role of IL-6 in cancer is complex. Chronic elevated plasma levels of IL-6 have been associated with poor disease outcome across a number of cancer diagnoses, whereas increased IL-6 expression within tumors is a positive prognostic marker for overall and disease-free survival (). In particular for the DEN-induced liver model, used in this study, Naugler and colleagues showed that IL-6 KO mice were resistant to DEN-induced tumor formation (). In contrast to these models, plasma IL-6 displays a dynamic response during exercise. The exercise-induced surge in plasma IL-6 may signal directly to IL-6Rα-expressing cells or through its alternative pathway, IL-6 trans-signaling (). We show that about 25% of the splenic NK cells express IL-6Rα and are thus directly sensitive to classical IL-6 signaling. Regarding IL-6 trans-signaling, IL-6 binds the soluble IL-6 receptor and then this complex binds to membrane-bound gp130. The concentration of the soluble IL-6 receptor is about 40 ng/ml, thus there is a large buffer capacity for IL-6 binding, when IL-6 increases as seen during wheel running. Thus, the systemic IL-6 increase during wheel running can signal either directly through the classical IL-6 signaling or through trans-signaling to NK cells. In Figure 2 , we showed a 2.3- to 3.0-fold increase in IL-6 expression in the tumors of running mice, yet following on the importance of classical IL-6 signaling and IL-6 trans-signaling, this suggests that the exercise-induced increase in systemic IL-6 levels play a greater role than the intratumoral concentration of IL-6 in NK cell redistribution and activation.

Taniguchi and Karin, 2014 Taniguchi K.

Karin M. IL-6 and related cytokines as the critical lynchpins between inflammation and cancer. In further support of the beneficial role of IL-6 exclusively in the exercise setting, we found no protective beneficial effect of IL-6 injection alone on tumor growth or intratumoral NK cell infiltration, stressing the dependence on prior training-dependent mobilization of NK cells. IL-6 and exercise have previously been shown to increase tumor vascularization (), yet we did not find any effect of IL-6 or running on CD31 and VEGF-A mRNA expression levels, nor did we detect any marked increases in capillarization in our histological analyses (data not shown).