Moderate-intensity aerobic exercise training improves CD8+ tumor-infiltrating lymphocytes effector function by reducing mitochondrial loss

Abstract

Aerobic exercise training (AET) has emerged as a strategy to reduce cancer mortality, however, the mechanisms explaining AET on tumor development remain unclear. Tumors escape immune detection by generating immunosuppressive microenvironments and impaired T cell function, which is associated with T cell mitochondrial loss. AET improves mitochondrial content and function, thus we tested whether AET would modulate mitochondrial metabolism in tumor-infiltrating lymphocytes (TIL). Balb/c mice were subjected to a treadmill AET protocol prior to CT26 colon carcinoma cells injection and until tumor harvest. Tissue hypoxia, TIL infiltration and effector function, and mitochondrial content, morphology and function were evaluated. AET reduced tumor growth, improved survival, and decreased tumor hypoxia. An increased CD8⁺ TIL infiltration, IFN-γ and ATP production promoted by AET was correlated with reduced mitochondrial loss in these cells. Collectively, AET decreases tumor growth partially by increasing CD8⁺ TIL effector function through an improvement in their mitochondrial content and function.

Keywords: Biochemistry; Biological sciences; Cancer systems biology; Immunology; Natural sciences; Physiology; Systems biology.

Graphical abstract

Figure 1

Moderate-intensity aerobic exercise training increases survival and aerobic capacity in mice with colorectal cancer (A) Study experimental design, (B) survival rates, (C) body mass changes post-tumor cell inoculation, and (D) aerobic capacity represented as distance run in meters during an exhaustion test on day 13 after tumor cell inoculation, comparing sedentary animals (control), sedentary tumor-bearing mice (CT26 SED), and trained tumor-bearing mice (CT26 TR). Data represent mean ± SEM. Comparison of survival curves by log rank (Mantel-Cox) test (∗p = 0.0356). Repeated measures ANOVA, and one-way ANOVA, followed by Duncan’s post hoc. ∗p < 0.05, ∗∗p < 0.01 vs. control, and #p < 0.05 vs. CT26 SED.

Figure 2

Moderate-intensity aerobic exercise training decreases CT26 tumor growth in mice with colorectal (A) Tumor latency, (B) tumor volume measured for 13 days following CT26 cells inoculation; arrow indicates the time with the largest statistical difference between groups (9 days), (C) tumor volume measured up to day 9 after tumor cell inoculation, (D) ex vivo tumor mass at day 9, (E) representative images of ex vivo solid tumors at day 9, and (F) correlation between aerobic capacity evaluated after AET and before CT26 inoculation and tumor volume measured at day 9 comparing sedentary tumor-bearing mice (CT26 SED) and trained tumor-bearing mice (CT26 TR). Data represent mean ± SEM. Tumor latency curves by log rank (Mantel-Cox) test (∗∗p = 0.0039). Unpaired Student’s t test. #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001 vs. CT26 SED.

Figure 3

Aerobic exercise training decreases tumor hypoxia while increasing total tumor-infiltrating immune cells (A) Experimental protocol, (B) tumor hypoxic area quantitatively measured by fluorescence, (C) immunohistological images (100x) of tumor sections stained with DAPI (nuclear), a pimonidazole primary antibody (Hypoxyprobe), followed by a FITC-secondary antibody incubation, and (D and E) total tumor-infiltrating leukocytes percentage (CD45⁺) analyzed by flow cytometry comparing sedentary tumor-bearing mice (CT26 SED) and trained tumor-bearing mice (CT26 TR) at day 9 post-tumor cell inoculation. Data represent mean ± SEM. Unpaired Student’s t test. #p < 0.05 vs. CT26 SED.

Figure 4

Tumor-infiltrating T cell populations are modulated by aerobic exercise training (A and B) Tumor-infiltrating T cells evaluated in tumor histological sections stained with hematoxylin-eosin (200x), (C and D) total TILs (CD3⁺) evaluated by flow cytometry, (E) total tumor-infiltrating CD4⁺ T cells, and (F) regulatory T cells (Treg), comparing sedentary tumor-bearing mice (CT26 SED) and trained tumor-bearing mice (CT26 TR). Data represent mean ± SEM. Unpaired Student’s t test. #p < 0.05 vs. CT26 SED.

Figure 5

Aerobic exercise training increases the number and function of CD8⁺ tumor-infiltrating T cells (A and B) Total and activated tumor-infiltrating CD8⁺ T cells, and the populations of (C and D) IFN-γ⁺ and (E) PD-1⁺ CD8⁺ TILs, comparing sedentary tumor-bearing mice (CT26 SED) and trained tumor-bearing mice (CT26 TR). Data represent mean ± SEM. Unpaired Student’s t test. #p < 0.05 vs. CT26 SED.

Figure 6

Increased IFNγ production in CD8⁺ TILs promoted by aerobic exercise training is associated with improved mitochondrial density and function (A) Mitochondrial number per cell, (B–D) area, elongation, and circularity of CD8⁺ TILs isolated using magnetic beads, and (E) representative transmission electron microscopy images, (F) CD8⁺ TILs mitochondrial density evaluated by the MitoTracker Green fluorescent probe, (G) and compared to the mitochondrial density of inguinal lymph node CD8⁺ T cells harvested from healthy sedentary controls (white bars), (H and I) the ratio between CD8⁺ TILs with high and low mitochondrial membrane potential (healthy and unhealthy mitochondria, respectively) evaluated by the JC-1 fluorescent probe, and (J) ATP production by total tumor-infiltrating leukocytes comparing sedentary tumor-bearing mice (CT26 SED) and trained tumor-bearing mice (CT26 TR). (K and L) Production of IFNγ (median fluorescence intensity, MFI) by draining lymph node (dLN) CD8⁺ T cells under baseline condition, and in response to oligomycin (mitochondrial ATP synthase inhibitor) and FCCP (inducer of maximal oxygen consumption by mitochondria), comparing sedentary animals (control), sedentary tumor-bearing mice (CT26 SED), and trained tumor-bearing mice (CT26 TR). Data represent mean ± SEM. Unpaired Student’s t test, and one-way ANOVA, followed by Duncan’s post hoc. ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control, and #p < 0.05 vs. CT26 SED.