1. Cancer Biology
  2. Immunology and Inflammation
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Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

  1. Helene Rundqvist
  2. Pedro Veliça
  3. Laura Barbieri
  4. Paulo A Gameiro
  5. David Bargiela
  6. Milos Gojkovic
  7. Sara Mijwel
  8. Stefan Markus Reitzner
  9. David Wulliman
  10. Emil Ahlstedt
  11. Jernej Ule
  12. Arne Östman
  13. Randall S Johnson  Is a corresponding author
  1. Department of Cell and Molecular Biology, Karolinska Institutet, Sweden
  2. Department of Laboratory Medicine, Karolinska Institutet, Sweden
  3. Department of Surgery, Oncology, and Gastroenterology, University of Padova, Italy
  4. The Francis Crick Institute, United Kingdom
  5. Department of Physiology, Development, and Neuroscience, University of Cambridge, United Kingdom
  6. Department of Physiology and Pharmacology, Karolinska Institutet, Sweden
  7. Department of Oncology-Pathology, Karolinska Institutet, Sweden
Research Article
Cite this article as: eLife 2020;9:e59996 doi: 10.7554/eLife.59996
9 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Depletion of CD8+ T-cells abolishes the anti-cancer effects of exercise training.

(A) FVB mice were allowed to exercise voluntarily (in running wheels, runners) or left non-exercised (locked running wheels, non-runners) before and after being inoculated subcutaneously with 5 × 105 tumor cells of the breast cancer cell line I3TC. (B) Mean tumor volume and SEM over time (left) and survival (right). **p<0.01, *p<0.05, Log-rank (Mantel-Cox) survival test. (C–E) Flow cytometry determined frequency of lymphocytic populations within I3TC tumor, spleen and lymph nodes (LN) at day 55 after inoculation. *p<0.05 column factor combining all organs in a two-way ANOVA. ns = not significant. (F–G) Flow cytometry determined frequency of lymphocytic populations within spleen and I3TC tumors after CD8+ depletion and isotype control. (H–I) Same experimental setting as in (A) with (αCD8) or without (isotype ctrl) weekly antibody-mediated depletion of CD8+ T cells (arrows). Graphs show mean tumor volume and SEM over time (F) and survival (G). *p<0.05, Log-rank (Mantel-Cox) survival test.

Figure 1—figure supplement 1
Exercise trained MMTV-PyMT breast cancer mice show enhanced infiltration of GranzymeB+ cells.

(A) Wild type (WT) and MMTV-PyMT (PyMT) mice on the FVB background were allowed to exercise voluntarily (in running wheels, runners) or left non-exercised (locked running wheels, non-runners) between 4 and 12 weeks of age. (B) Running distance (km/day) in WT and PyMT mice. *p<0.05, two-tailed unpaired t test. (C) Tumor volume measured twice weekly for PyMT running and non-running mice. Mean and SEM (n = 10–11). (D) Survival of PyMT mice, runners, and non-runners. Survival curve ns = not significant, Log-rank (Mantel-Cox) test, (n = 10–11). (E) Tumor initiation as age (day) of first indication of a palpable tumor in running (Runners) and non-running (Non-runners) PyMT mice. n = 11, ns = not significant, two-tailed t test. (F) Tumor stage from histological scoring (1-4) in mammary glands of running and non-running PyMT mice at 12 (n = 6–10) and 8 (n = 4) weeks of age, ns = not significant, two-tailed t test. (G) Individual body weight for WT and PyMT running and non-running mice. n = 7–10, ns = not significant, one-way ANOVA with Tukey’s multiple comparison test. (H) Immunohistological characterization of PyMT tumors from non-running and running mice using CD3, F4/80, PCNA, Podocalyxin (PODXL), and Granzyme B (GZMB) antibodies, respectively. n = 8–15, ns = not significant, *p<0.05, Two-tailed unpaired t test. (I) Flow-cytometry-based frequency of macrophages within I3TC tumor. Ns = not significant, two-tailed t-test. (J) Flow-cytometry-based frequency of neutrofiles within I3TC tumor. Ns = not significant, two-tailed t-test.

Figure 2 with 2 supplements
Acute exertion alters the metabolic profile of plasma and lymphoid organs.

(A) FVB mice performed a single exhaustive treadmill exercise. Muscle, plasma, spleen, muscle draining, and non-draining lymph nodes were collected from exercising and control mice immediately after the exercise. Human subjects performed 30 min of endurance exercise at 75% of Wpeak and plasma samples were collected before (pre) and after (post) exercise. Samples were analyzed on the Precision Metabolomics mass spectrometry platform. Data is provided as Figure 2—source data 1. (B–G) Volcano plots of differentially induced metabolites per mouse tissue and human plasma. p-Values from the Student’s T-test and the log2FC (n = 6 and n = 8 for mouse and human samples respectively). TCA metabolites are colored blue. Glycolytic metabolites are colored orange. The dashed horizontal line is drawn at Y = -log10(0.05). (H) Heatmap of exercise-induced changes in metabolite concentrations in human and mouse plasma and tissues. Metabolites with a significant change (adjusted p<0.05) in at least one tissue following exercise were included and clustered using hierarchical clustering.

Figure 2—figure supplement 1
Acute exertion Increases the levels of TCA metabolites in muscle and plasma.

(A) Metabolic assessment (GC-MS) of muscle and plasma from FVB mice after a sprint exercise (n = 7), PCA plot. (B–C) Volcano plots of differentially induced metabolites in muscle and plasma from FVB mice after a sprint exercise (n = 7) measured by GC-MS. The adjusted p-values from the Student’s T-test with False Discovery Rate multiple comparison correction and the log2FC were used to generate these volcano plots. Selected metabolites (adjusted p-value<0.05) are colored blue (log2FC < 0) and red (log2FC > 0). (D) Acute exercise cause intramuscular reductions in glycolytic metabolites and an accumulation of TCA metabolites. TCA metabolites can also be found in plasma post-exercise. (E) Human subject characteristic (n = 8). Mean + / - SD. (F) Human subjects performed 30 min of endurance exercise at 75% of Wpeak, comparison of plasma samples collected before (pre-exercise) and 1 hr (1 hr post-exercise) after the exercise. (G) Murine lactate levels in tail vein blood after exhaustive treadmill exercise (n = 6 and 5, respectively).

Figure 2—figure supplement 2
Pathway analysis of post-exercise metabolic profiling.

(A) Pathway analysis of differentially available metabolites in exercising animals for muscle, spleen, plasma, muscle draining (dLN) and non-draining lymph nodes (ndLN) and for human plasma. The enrichment score (ES) was calculated using the ES = (k/m)/[(n–k)/(N–m)] where n is the total number of significant metabolites; m is the total number of metabolites detected in a specific pathway (count); N is the total number of metabolites detected and k is the number of significant metabolites in a specific pathway. Graph showing top 10 metabolites based on the -log10(p-value).

Figure 3 with 1 supplement
Central carbon metabolites alter the CD8+ T cell effector profile.

(A) Proliferation of activated murine CD8+ T cells in response to increasing concentrations of central carbon metabolites. The proliferation of activated murine CD8+ T cells was assessed by using CountBright counting beads on flow cytometry at day 3 of culture. (B) Flow-cytometry-based CD62L and CD44 expression of live CD8+ T cells expressed as frequency of cells at day 3 of culture with increasing concentrations of central carbon metabolites. Shaded areas represent mean and 95% confidence intervals at 0 mM. (C) Granzyme B median fluorescence intensity (MFI) at day 3 of culture with increasing concentrations of central carbon metabolites. Data from one representative experiment on CD8+ T cells purified and activated from pooled spleens of multiple mice. (D) ICOS median fluorescence intensity (MFI) at day 3 of culture with increasing concentrations of central carbon metabolites. Data from one representative experiment on CD8+ T cells purified and activated from pooled spleens of multiple mice. (E) Cytotoxicity against EL4-OVA tumor cells by OVA-specific OT-I CD8+ T cells activated for 3 days in the presence of 40 mM NaCl or NaLac. Graph represents specific cytotoxicity of n = 3 independent mouse donors at varying effector-to-target ratios. *p<0.01 repeated-measures two-way ANOVA with Sidak’s multiple comparison test.

Figure 3—figure supplement 1
Profiling of CD8+ T cell responses to central carbon metabolites.

(A) Reference values of central carbon metabolite availability in resting, human adult individuals. (B) Proliferation of activated murine CD8+ T cells was assessed with a resazurin assay after 3 days of cell culture. The response is expressed as fold change over the control (0 mM concentration). Error bars represents the standard error of the mean (SEM). CD8+ T cells purified and activated from pooled spleens of multiple mice in four separate experiments (n = 4). (C) Proliferation of activated murine CD8+ T cells in response to increasing concentrations of central carbon metabolites and sodium chloride control. The proliferation of activated murine CD8+ T cells was assessed by using CountBright counting beads on flow cytometry at day 3 of culture. CD8+ T cells purified and activated from four individual mice (n = 4). (D) Flow-cytometry-based CD62L and CD44 expression of live CD8+ T cells expressed as frequency of cells at day 3 of culture with increasing concentrations of central carbon metabolites. CD8+ T cells purified and activated from four individual mice (n = 4). (E) Granzyme B median fluorescence intensity (MFI) at day 3 of culture with increasing concentrations of central carbon metabolites. CD8+ T cells purified and activated from four individual mice (n = 4). Two-way ANOVA comparing sodium L-lactate and sodium chloride. *p<0.05, **p<0.01. (F) ICOS median fluorescence intensity (MFI) at day 3 of culture with increasing concentrations of central carbon metabolites. CD8+ T cells purified and activated from four individual mice (n = 4). (G) CTLA4 median fluorescence intensity (MFI) at day 3 of culture with increasing concentrations of central carbon metabolites. CD8+ T cells purified and activated from four individual mice (n = 4).

Figure 4 with 2 supplements
Acute exercise alters CD8+ T cell metabolism in vivo.

(A) Recipient mice received OT-I CD8+ T-cells followed by vaccination. On day 2 and 3 after vaccination, 10 mg of [U-13C6]glucose was introduced to the mice prior to a treadmill exercise. CD8+ T-cells were harvested from the spleen and incorporation of labeled carbons in cellular metabolites was assessed by GC-MS. Data is provided as Figure 4—source data 1. (B) Fraction of labeled metabolites in CD8+ T-cells 48 hr after vaccination. (C) Fraction of labeled metabolites in CD8+ T-cells 72 hr after vaccination. **p<0.01 and ***p<0.001 using a two-way ANOVA with Sidak’s multiple comparison test.

Figure 4—source data 1

Glucose derived carbon distribution in CD8+ T cells source data file.

https://cdn.elifesciences.org/articles/59996/elife-59996-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Glucose levels in CD8+ T cells of exercising mice.

(A) Recipient mice received OT-I CD8+ T-cells followed by vaccination. On day 2 and 3 after vaccination, 10 mg of [U-13C6]glucose was introduced to the mice prior to a treadmill exercise. CD8+ T-cells were harvested from the spleen and incorporation of labeled carbons in cellular metabolites was assessed by GC-MS (n = 4). Metabolic diagram showing total glucose fraction in in exercising (EXERCISE) and non-exercising (CTRL) animals at 48 and 72 hr after vaccination. Two-tailed t-test *p<0.05, **p<0.01. (B) Metabolic diagram showing labeled glucose fraction in in exercising (EXERCISE) and non-exercising (CTRL) animals at 48 and 72 hr after vaccination. Two-tailed t-test *p<0.05, **p<0.01,.

Figure 4—figure supplement 2
Exercise effects on glucose derived carbon distribution in CD8+ T cells.

(A) Metabolic diagram showing glucose-derived carbon incorporation normalized to labeled glucose in exercising (EXERCISE) and non-exercising (CTRL) animals at 48 and 72 hr after vaccination. Two-tailed t-test *p<0.05, **p<0.01.

Figure 5 with 1 supplement
CD8+ T cells transferred from trained mice show enhanced anti-tumoral capacity.

(A) OT-I mice carrying the congenic marker CD45.1 were given access to a locked or moving running wheel for 6 weeks. In parallel, C57Bl/6 (CD45.2) animals were inoculated with 5 × 105 ovalbumin (OVA)-expressing B16-F10 (B16-F10-OVA) melanoma and conditioned with 300 mg/kg cyclophosphamide (CPA). 4 × 105 OVA-reactive OT-I CD8+ T cells were isolated from running or non-running mice and adoptively transferred into tumor-bearing animals. Peripheral blood was sampled 10 days after adoptive transfer and tumor volume monitored. (B) Flow cytometry analysis of OT-I T cell expansion in peripheral blood 10 days after adoptive transfer. Adoptive cells were distinguished from endogenous immune cells by expression of the CD45.1 congenic marker. Histogram shows ICOS surface expression on adoptive and endogenous CD8+ T cells. (C) Frequency of adoptive OT-I T cells in peripheral blood. *p<0.05, two-tailed t-test. ns = not significant. (D) Median fluorescence intensity (MFI) of ICOS (bottom) of adoptive OT-I T cells in peripheral blood. *p<0.05, two-tailed t-test. ns = not significant. (E–G) Mean tumor volume and SEM (E), median IQR (F) and survival (G) over time. ↓ depicts time of OT-I T cell injection *p<0.05, Log-rank (Mantel-Cox) survival test.

Figure 5—figure supplement 1
OT-I mice carrying the congenic marker CD45.1 were given access to a locked or moving running wheel for 6 weeks.

In parallel, C57Bl/6 (CD45.2) animals were inoculated with 5 × 105 ovalbumin (OVA)-expressing B16-F10 (B16-F10-OVA) melanoma and conditioned with 300 mg/kg cyclophosphamide (CPA). 4 × 105 OVA-reactive OT-I CD8+ T cells were isolated from running or non-running mice and adoptively transferred into tumor-bearing animals. Peripheral blood was sampled 10 days after adoptive transfer. (A) Flow cytometry based frequency of CD62LlowCD44high cells in peripheral blood. ns = not significant. (B) Flow-cytometry-based frequency of CD62LhighCD44high cells in peripheral blood. ns = not significant.

Figure 6 with 1 supplement
Daily administration of sodium L-lactate delays tumor growth in vivo.

(A) Daily doses of PBS or 0.5, 1, or 2 g/kg Sodium L-lactate (NaLac) were administered i.p to FVB mice for 12 days before subcutaneous inoculation with 5 × 105 cells of the MMTV-PyMT-derived breast cancer cell line I3TC. Daily sodium L-lactate injections were continued throughout the experiment. Graphs show tumor volume (mean and SEM) over time and survival. *p<0.05, Log-rank (Mantel-Cox) survival test. (B) Flow cytometric characterization of I3TC tumor infiltrating immune cell populations. **p<0.01, *p<0.05, two-tailed t test. (C) Daily doses of PBS or 2 g/kg NaLac was administered i.p to C57BL/6J mice for 12 days before subcutaneous inoculation with 5 × 105 cells of the colon cancer cell line MC38. Injections were continued throughout the experiment. Graphs show tumor volume (mean and SEM) over time and survival. **p<0.01, Log-rank (Mantel-Cox) survival test. (D) Same experimental setting as in (A) with or without weekly antibody-mediated depletion of CD8+ T cells (arrows). Graphs show tumor volume (mean and SEM) over time and survival. ns = not significant, Log-rank (Mantel-Cox) survival test.

Figure 6—figure supplement 1
Characterization of tumor infiltrating CD8+ T cells after daily administration of sodium L-lactate.

(A) Peripheral blood lactate concentration (as measured with Accutrend Plus) following a single dose of 2 g/kg Sodium L-lactate (NaLac) administered i.p in FVB or C57BL/6J mice. (B) Daily doses of PBS or 3 g/kg NaLac were administered i.p to FVB mice for 12 days before subcutaneous inoculation with the breast cancer cell line I3TC. Injections were continued throughout the experiment. Graphs show tumor volume (mean and SEM) over time. *p<0.05, Log-rank (Mantel-Cox) survival test. (C) Flow-cytometrybased assessment of tumor infiltrating CD8+ T-cells (CD8+ cells as % of CD45+ cells) in I3TC tumors after daily lactate administration. *p<0.05, two-tailed t-test. (D) Flow-cytometry-based assessment of tumor infiltrating GzmB+CD8+ T-cells (% of CD45+ cells) in I3TC tumors after daily lactate administration. Ns = not significant, two-tailed t-test. (E) Flow-cytometry-based assessment of GzmB (left panel), PD1 (middle panel), and CTLA4 (right panel) MFI of CD8+ T-cells in I3TC tumors after daily lactate administration. Ns = not significant, two-tailed t-test.

Author response image 1
Author response image 2
Cytokine array (RayBiotech, Norcross, GA, USA), describing the cytokine profile in serum from WT and tumor bearing (PyMT) running and non-running mice.

Chemiluminiscens detection was done on the ImageQuant LAS 4000 (GE Healthcare, Buckinghamshire, UK).

Author response image 3
Left panel, mouse IL-6 plasma (RnD ELISA, heart puncture) levels after an acute treadmill exercise and in control animals.

Right panel, blood (tail vein) lactate in the same cohort (Accutrend device).

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (M. musculus)FVB/NJanvier LabsFVB/NFVB ‘WT’ mice
Strain, strain background (M. musculus)C57Bl/6JCharles RiverC57Bl/6J (JAX)C57Bl/6J ‘WT’ mice
Strain, strain background (M. musculus)OT-IThe Jackson Laboratory (JAX)Cat 003831TCR transgenic mice
Strain, strain background (M. musculus)CD45.1The Jackson Laboratory (JAX)Cat 002014CD45.1 congenic marker
Cell line (M. musculus)I3TCWeiland et al., 2012Murine breast cancer cell line derived from MMTV-PyMT FVB mice
Mycoplasma test data
RATIOS under 1 = clear.I3TC 0.3381995134
Cell line (M. musculus)B16F10ATCCCRL-6475Mouse melanoma cell line
Mycoplasma test data
RATIOS under 1 = clear.
B16 OVA 447 163 0.36465324
Cell line (M. musculus)LLCATCCCRL-1642Mouse lung carcinoma cell line
Mycoplasma test data
RATIOS under 1 = clear.
LLC 438 151 0.34474885
AntibodyaSINFENKLBioLegendClone 25-D1.16
Chemical compound, drugSodium L-lactateSigmaL7022Metabolite
Chemical compound, drugCyclophosphamideSigmaC0768Chemotherapy agent
Commercial assay or kitCD8a microbeadsMiltenyi Biotec130-117-044Beads for magnetic sorting
Chemical compound, drugCollagenase AFisher Scientific50-100-3278For single cell dissociation of tumors
Chemical compound, drugDNaseSigmaD5025For single cell dissociation of tumors
Commercial assay or kitDynabeads Mouse T-activatorThermo Fisher11456DDynabeads Mouse T-activator CD3/CD28
Chemical compound, drugIL-2Sigma11011456001IL-2
Chemical compound, drugCitric acidSigma251275
Chemical compound, drugMalic acidSigmaM1000
Chemical compound, drugSuccinic acidSigmaS3674
Chemical compound, drugFumaric acidSigma47910
Chemical compound, drugLactic acidSigmaL1750
Chemical compound, drugPyruvic acidSigma107360
Chemical compound, druga-Ketoglutaric acidSigmaK1750
Chemical compound, drugOxaloacetic acidSigmaO4126
Commercial assay or kitResazurin reduction assaySigmaR7017T-cell proliferation assessment
Commercial assay or kitDead cell stain kitThermo FisherL10119Fixable Near-IR Dead cell Stain Kit
AntibodyaCD44 (rat monoclonal)BD BiosciencesClone IM71:2000
AntibodyaCD45.1
(mouse monoclonal)
BD BiosciencesClone A201:200
AntibodyaCD45.2
(mouse monoclonal)
BD BiosciencesClone 1041:200
AntibodyCD8
(rat monoclonal)
BD BiosciencesClone 53–6.71:200
AntibodyaCTLA-4 (Armenian hamster monoclonal)BD BiosciencesClone UC10-4F10-111:200
AntibodyaCD62L
(mouse monoclonal)
BioLegendClone MEL-141:200
AntibodyaICOS
(Armenian hamster monoclonal)
BioLegendClone C398.4A1:200
AntibodyaGzmB
(mouse monoclonal)
Thermo FisherClone GB121:100
Commercial assay or kitCounting beadsThermo FisherC36950CountBright Absolute Counting Beads
SoftwareFlowJoTree StarVersion 8.8.7
SoftwarePrismGraphPadVersion 8
SoftwareRR Core TeamVersion 3.6.1
AntibodyCD3
(rabbit polyclonal)
Abcam#ab5690IHC 1:100
AntibodyF4/80
(rat monoclonal)
Abd Serotec#MCA497IHC 1:100
AntibodyPodocalyxin
(goat polyclonal)
R and D Systems#AF1556IHC 1:200
AntibodyPCNA
(mouse monoclonal)
Dako#M0879IHC 1:500
AntibodyGzmB
(rabbit polyclonal)
Abcam#ab4059IHC 1:100
Commercial assay or kitABC kitVectorPK6100Avidin-Biotin peroxidase Complex
Commercial assay or kitDABVectorSK4100Diaminobenzidine staining
SoftwareImage JNIH

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