Exercise Induced Metabolic Shield Blocks Cancer

CANCER RESEARCH | METABOLISM AND CHEMICAL BIOLOGY
An Exercise-Induced Metabolic Shield in Distant Organs

Blocks Cancer Progression and Metastatic Dissemination
Danna Sheinboim1, Shivang Parikh1, Paulee Manich1, Irit Markus2, Sapir Dahan1, Roma Parikh1,
Elisa Stubbs2, Gali Cohen2,3, Valentina Zemser-Werner4, Rachel E. Bell1, Sara Arciniegas Ruiz1,
Ruth Percik5,6, Ronen Brenner7, Stav Leibou1, Hananya Vaknine8, Gali Arad1, Yariv Gerber2,3,
Lital Keinan-Boker9,10, Tal Shimony10, Lior Bikovski11,12, Nir Goldstein2, Keren Constantini2, Sapir Labes13,
Shimonov Mordechai1,14, Hila Doron15, Ariel Lonescu16, Tamar Ziv17, Eran Nizri5,18, Guy Choshen5,19,
Hagit Eldar-Finkelman1, Yuval Tabach13, Aharon Helman20, Shamgar Ben-Eliyahu21,22, Neta Erez15,
Eran Perlson16,22, Tamar Geiger23, Danny Ben-Zvi24, Mehdi Khaled25, Yftach Gepner2, and Carmit Levy1
ABSTRACT
◥
Exercise prevents cancer incidence and recurrence, yet the in mice, exercise prior to cancer injection significantly protected
underlying mechanism behind this relationship remains mostly against metastases in distant organs. The protective effects of exercise
unknown. Here we report that exercise induces the metabolic were dependent on mTOR activity, and inhibition of the mTOR
reprogramming of internal organs that increases nutrient demand pathway with rapamycin treatment ex vivo reversed the exercise-
and protects against metastatic colonization by limiting nutrient induced metabolic shield. Under limited glucose conditions, active
availability to the tumor, generating an exercise-induced metabolic stroma consumed significantly more glucose at the expense of the
shield. Proteomic and ex vivo metabolic capacity analyses of murine tumor. Collectively, these data suggest a clash between the metabolic
internal organs revealed that exercise induces catabolic processes, plasticity of cancer and exercise-induced metabolic reprogramming
glucose uptake, mitochondrial activity, and GLUT expression. of the stroma, raising an opportunity to block metastasis by chal-
Proteomic analysis of routinely active human subject plasma lenging the metabolic needs of the tumor.
demonstrated increased carbohydrate utilization following exercise.
Epidemiologic data from a 20-year prospective study of a large Significance: Exercise protects against cancer progression and
human cohort of initially cancer-free participants revealed that metastasis by inducing a high nutrient demand in internal organs,
exercise prior to cancer initiation had a modest impact on cancer indicating that reducing nutrient availability to tumor cells repre-
incidence in low metastatic stages but significantly reduced the sents a potential strategy to prevent metastasis.
likelihood of highly metastatic cancer. In three models of melanoma See related commentary by Zerhouni and Piskounova, p. 4124
after tumor inoculation in vivo (3). This suggests that exercise provides
Introduction protection from tumor development; however, the mechanism under-
Clinical and preclinical studies have demonstrated that exercise lying this preventative effect has yet to be elucidated (4, 5).
plays a role in cancer prevention, as it reduces cancer incidence (1) and Clinically, it has been suggested that exercise can have an antitumor
recurrence (2). The effect of exercise prior to tumor detection appears effect through the regulation of the metabolic profile by increasing the
to be just as effective in inhibiting tumor growth as exercise before and body’s insulin sensitivity, thus contributing to glucose homeostasis (6),
1
Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Smith Faculty of Agriculture, Food and Environment, The Hebrew University,
Tel Aviv University, Tel Aviv, Israel. 2Department of Epidemiology and Rehovot, Israel. 21School of Psychological Sciences, Tel Aviv University, Tel Aviv,
Preventive Medicine, School of Public Health, Sackler Faculty of Medicine, and Israel. 22Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel. 23The
Sylvan Adams Sports Institute, Tel Aviv University, Tel Aviv, Israel. 3Stanley Weizmann Institute of Science, Rehovot, Israel. 24Department of Developmental
Steyer Institute for Cancer Epidemiology and Research, Tel Aviv University, Tel Biology and Cancer Research, Institute of Medical Research Israel–Canada, The
Aviv, Israel. 4Institute of Pathology, Tel Aviv Sourasky Medical Center, Tel Aviv, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel.
Israel. 5Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel. 6Institute 25
INSERM 1186, Gustave Roussy, Universite Paris-Saclay, Villejuif, France.
of Endocrinology, Chaim Sheba Medical Center, Tel Hashomer, Israel. 7Institute
of Oncology, E. Wolfson Medical Center, Holon, Israel. 8Institute of Pathology, D. Sheinboim, S. Parikh, P. Manich, and I. Markus contributed equally to this
E. Wolfson Medical Center, Holon, Israel. 9School of Public Health, Faculty of Article.
Social Welfare and Health Sciences, University of Haifa, Haifa, Israel. 10Israel
Center for Disease Control, Israel Ministry of Health, Ramat Gan, Israel. 11The Corresponding Authors: Carmit Levy, Human Molecular Genetics and
Myers Neuro-Behavioral Core Facility, Tel Aviv University, Tel Aviv, Israel. Biochemistry, Tel Aviv University, Tel Aviv, 69978, Israel. E-mail:
12
School of Behavioral Sciences, Netanya Academic College, Netanya, Israel. [email protected]; Yftach Gepner, E-mail: [email protected]; and
13
Department of Developmental Biology and Cancer Research, Institute of Mehdi Khaled, E-mail: [email protected]
Medical Research-Israel-Canada, The Hebrew University of Jerusalem,
Cancer Res 2022;82:4164–78
Jerusalem, Israel. 14Department of Surgery, E. Wolfson Medical Center, Holon,
Israel. 15Department of Pathology, Sackler Faculty of Medicine, Tel Aviv doi: 10.1158/0008-5472.CAN-22-0237
University, Tel Aviv, Israel. 16Department of Physiology and Pharmacology,
Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. 17The Smoler This open access article is distributed under the Creative Commons Attribution-
Proteomics Center, Technion, Haifa, Israel. 18Department of Dermatology, Tel NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.
Aviv Sourasky (Ichilov) Medical Center, Tel Aviv, Israel. 19Department of Internal
Medicine, Tel Aviv Sourasky (Ichilov) Medical Center, Tel Aviv, Israel. 20Robert H. 2022 The Authors; Published by the American Association for Cancer Research
AACRJournals.org | 4164
Exercise-Induced Metabolic Shield Blocks Cancer Progression
decreasing sex-steroid hormone levels (7), ameliorating the immune adjacent melanoma, whereas the stroma of control mice demonstrated
response (8), including reduction of inflammation (9), or secretion of the opposite trend. This suggests that the cancer–stroma cross-talk
skeletal muscle myokines that inhibit tumor growth (10). However, induced by exercise directly influences the tumor microenvironment,
whether these modifications contribute to the protective effect of therefore altering the metabolic capabilities of metastatic tumor cells.
exercise is unclear. Based on our data, we hypothesize that exercise reprograms the tumor
Following exercise, skeletal muscles’ metabolic adaptations are due microenvironment via the development of a stromal metabolic shield
to its increased energy demands (11, 12) and include the regulation of that protects the stroma from metastatic colonization by challenging
energy and glucose–insulin-related pathways by myokines, hepato- cancers metabolic demands.
kines, and adipokines (12), secreted by skeletal muscle, liver, and
adipose tissue, respectively, upon exercise (12). For example, myokine
interleukin 6 (IL6) increases lipid metabolism and glucose generation Materials and Methods
in hepatocytes and improves insulin secretion from the pancreas (13). Human study population
Similarly, metabolic alterations in cancer cells are a prominent The data set was a population-based cohort that constituted a
hallmark of tumor progression (14), which are determined by cell- random sample of the Israeli general population between the ages of
intrinsic characteristics such as tissue of origin (15), genetic muta- 25 and 64. The total study population included 2,734 participants; 243
tions (16), and disease stage. Aerobic glycolytic metabolism of cancer new cancer cases were recorded during the 20-year follow-up period.
cells, known as the Warburg effect, reflects a tumor’s intrinsic ability to The data were collected by the Israel Center for Disease Control and
alter its metabolism (14, 17), through the significant increase of its the Nutrition Department of the Israeli Ministry of Health. As our
glucose uptake, allowing cancer to proliferate uncontrollably via focus was on the relationship between exercise and cancer, we used a
increased anabolic processes, producing the carbon necessary for propensity score of multinomial logistic regression to control for key
proliferation (18). In addition to intrinsic changes, tumor interactions variables in the diet assessed using a validated questionnaire. A
with its microenvironment also have an impact on cancer metabo- schematic representation of all the experimental models of humans
lism (17). For example, the microenvironment provides metabolites, as used in this study appears in Supplementary Fig. S5.
was shown with cancer-associated fibroblasts that secrete lactate,
pyruvate, ketone bodies (19), glycogen (20), and cytokines (21) and SEER classification
can transfer mitochondria to cancer cells (21), enhancing cancer cell Each cancer case was classified according to the SEER summary
mitochondrial function, tricarboxylic acid cycle (TCA) activity, oxi- stage 2000, according to the following scale: 0, in situ; 1, localized only;
dative phosphorylation (OXPHOS; ref. 22), and glycolysis (20), which, 2, regional by direct extension only; 3, regional lymph nodes involved
together with extracellular matrix remodeling (23), leads to increased only; 4, regional by both direct extension and lymph node involve-
metastatic ability. Likewise, in ovarian cancer, lipids are transferred ment; 7, distant sites(s)/node(s) involved. Of the 243 subjects with
from adipocytes to tumor cells, promoting OXPHOS (24). cancer, 95 cancer cases had no SEER information and were excluded
Given the metabolic plasticity observed during cancer progres- from the analyses.
sion and the metabolic alterations in host organs following
exercise (11–14, 17, 19, 20, 25, 26), we speculate that these two Exercise assessment questionnaire
metabolic programs are clashing. We, therefore, hypothesize that The participants responded to two sets of questions from The
exercise-induced metabolic reprogramming of organs transforms Physical Activity Questionnaire, which aimed to assess exercise habits
them into metastatic-resistant metabolic microenvironments by such as the frequency of exercise (times per week) and the average
limiting nutrient availability to the cancer cells thus creating a amount of time spent on that activity. The first set of questions was
metabolic shield. asked regarding vigorous activity and the second set was asked about
To address these hypotheses, we subjected organs from active and moderate activity that lasted for a minimum of 10 minutes. Exercise
sedentary mice to proteomic analysis, primary cell metabolic capacity habits during leisure time were determined by a set of two questions.
tests, including mitochondrial activity and glycolytic function, and Participants reported the frequency (times per week) and average time
glucose uptake analyses of primary cells to reveal metabolic repro- they devoted to each of the following activities: walking outdoors or on
graming toward increased catabolic processes in the lungs, lymph a treadmill, jogging, swimming, bike riding or stationary cycling, light
nodes, liver, and muscle. We then performed a comparative proteomic exercise (i.e., yoga, the Feldenkrais method, the Alexander technique,
analysis of plasma collected from routinely active female and male light gymnastics), body shaping, and strength training; an “other
subjects before and after exercise that demonstrated a similar meta- activity” option was also offered. Based on the reported total weekly
bolic shift. Further, analysis of 20 years’ worth of follow-up data on a time of exercise and modalities intensity, participants were classified
prospective human cohort (n ¼ 2,734; 1,302 females and 1,432 males) into intensity categories according to the official American College of
revealed that high-intensity exercise significantly reduces the risk of Sports Medicine guidelines (27). Individuals with a history of cancer at
metastatic cancer. To understand how these findings directly affect baseline were excluded from the study. Data on the date of diagnosis
cancer progression, we established in vivo mouse models of forced and the diagnostic code, assigned according to the International
exercise via treadmill running both prior to and post tumor initiation. Classification of Diseases for Oncology, Third Edition, regarding only
We observed a significant reduction in melanoma dissemination into primary cancers (i.e., nonmetastatic), were obtained, and thereby
lungs, lymph nodes, and liver compared with sedentary (control) incident as well as previous cases of all-site cancer were identified
animals, in both models. Our results suggest that because exercise was (codes C00.0–C80.9).
performed prior to cancer initiation, exercise is altering the host organs
metabolic abilities, thus protecting them against cancer dissemination. Human cohort of steady-state intensity
Rapamycin treatment of active stroma abolished its metabolic advan- Population
tage, allowing for melanoma growth, ex vivo. We then show that the Fourteen apparently healthy, recreationally active male and female
stroma from active mice have a higher metabolic capability than the runners between the ages of 25 to 45 years of age were recruited to
AACRJournals.org Cancer Res; 82(22) November 15, 2022 4165

Sheinboim et al.
participate in this study. All participants performed endurance exer- (IACUC permit: 01-15-086 and 01-19-003). All mice were housed
cise on a weekly basis and were familiar with running exercise. in individually ventilated cages in reverse light with 22 þ 1 C
Exclusion criteria included smoking, prescribed medications, or a temperature and 32% to 35% humidity with ad libitum water and
self-reported history of chronic pulmonary, cardiac, metabolic, or food unless mentioned in the experiments. Six-week-old C57BL/
orthopedic conditions. Their physical characteristics, including weight 6JRccHsd female mice (Envigo) were habituated for 12 days prior to
(kg), height (cm), steady-state heart rate (beats/minute), and running experiment initiation. A schematic representation of all the exper-
pace (km/hour), are shown in Supplementary Table S4. The subjects imental models of mice used in this study appears in Supplementary
were instructed to avoid caffeine consumption for 12 hours, food Fig. S5.
consumption for 3 hours, and strenuous physical activity for at least
24 hours prior to arrival at the laboratory for testing. Exercise training
Female mice were chosen based on their increased metabolic
Steady-state running protocol response to exercise compared with males (28). One group of mice
Participants performed 30 minutes of steady-state running on a served as a control. The other group was subjected to an exercise
motorized treadmill (Saturn 100/300, hours/p/cosmos, Nussdorf- training protocol, modified from previously described (29). Mice were
Traunstein) using an individualized protocol. Speed was determined exercised every other day for the indicated time. Every day prior to the
by the highest speed that each participant can persist in for 30 minutes. start of the exercise, mice were habituated in the experimental room for
Ventilator and metabolic measurements were collected during the 20 minutes. On the first day, following the lighting habituation, mice
graded protocol using breath-by-breath analysis (Quark Cardiopulmo- were habituated on the treadmill (Panlab Harvard Apparatus) for 10
nary Exercise Testing, Cosmed) while subjects breathed through an oro- minutes and at a speed of 5 cm/s. From day 2 and beyond, the mice
nasal facemask (7450 Series, Hans Rudolph). Heart rate was contin- were habituated in the experimental room for 20 minutes followed by
uously monitored using a chest strap (Garmin, model Acc, HRM-Dual). treadmill exercise starting 18 cm/s for 5 minutes, increasing the speed
at 2 cm/s until 24 cm/s was reached and then sustained for 8 minutes.
Plasma extraction from the human cohort The speed was gradually decreased by 2 cm/s every minute until
Blood samples were collected for analysis prior to and immediately 18 cm/s. Total duration of the exercise session was 20 minutes per
after the exercise. For serum extraction, blood was allowed to sit at mouse. Following the exercise, the mice were returned to their
room temperature for 1 hour and then centrifuged at 1,200 g for 10 home cages.
minutes at 4 C. For plasma isolation, blood was placed in EDTA-
coated tubes (BD Biosciences) and centrifuged for 15 minutes twice at Tumor cell injections and tumor excision
1,200 g at 4 C. The serum or plasma fractions were stored at 80 C A schematic representation of all the melanoma experimental
until further downstream experiments. models of mice used in this study appears in Supplementary Fig. S5.
Subdermal injection: An aliquot of 1.5 105 low-passage (<p15)
Human RNA-seq data analysis Ret-melanoma mCherry–Luciferase melanoma cells were resus-
Human melanoma gene signature pended in sterile PBS (X1) and mixed at a 1:1 ratio with growth
RNA-sequencing gene expression from a melanoma cancer patient factor–reduced Matrigel (Corning) to a final volume of 50 mL. Mice
(different stages and metastatic tissues) was obtained from The Cancer were anesthetized using isoflurane, and melanoma cells were subder-
Genome Atlas (TCGA) and from the National Center for Biotech- mally injected on the right dorsal side, rostral to the flank, with a 29G
nology Information (NCBI) Gene-Expression Omnibus repository. insulin syringe (BD Biosciences).
Heat map of the normalized expression of genes (rows) that are Intracarotid injection: Mice were deeply anesthetized using intra-
separated between the in situ group [benign nevi, atypical nevi, vertical peritoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg).
growth phase melanoma (VGP), and in situ melanoma] compared 5104 Ret-melanoma mCherry–Luciferase melanoma cells suspended
with metastatic melanoma, denoted the disseminated group (lung to in 50 mL saline were injected into the internal carotid artery using
spleen), are shown in Supplementary Table S3. Color-coding repre- ultrasound guidance.
sents high expression, red, and low expression, blue, respectively. Intrasplenic injection: Mice were deeply anesthetized using
Melanoma metastatic genes were subjected to Kyoto Encyclopedia of intraperitoneal injection of ketamine (100 mg/kg) and xylazine
Genes and Genomes (KEGG) enrichment using the WebGestalt tool. (10 mg/kg). A small incision was made next to the spleen, and
1105 Ret-melanoma mCherry–Luciferase melanoma cells suspended
Cell culture in 100 mL PBS were slowly injected into the exposed hemi-spleen while
Ret-melanoma cells were cultured in RPMI (Biological Industries) the syringe was kept upright. The spleen and incision were sutured
supplemented with 10% FBS and 1% penicillin/streptomycin/ using Vicryl thread (Ethicon).
L-glutamine (Biological Industries). The Ret-melanoma stable cell line Tumor volumes were measured by a caliper three times per week
was generated by transfecting PLKO-mcherry-luc-puro plasmid every other day. The tumor volume was calculated using the formula
(Addgene) using the jetPEI-DNA transfection reagent (Polypus) and X2Y0.5 (X-smaller diameter, Y-larger diameter). Tumors were
after 48 hours the stable clones were selected using the mammalian excised when they reached a volume of 1 cm3. For the excision,
selection marker puromycin (Sigma-Aldrich, 10 mg/mL). The selected ketamine (100 mg/kg) and xylazine (10 mg/kg) were used as anes-
clones were expanded to be used for the experiments. thetics. The incision was made medial to the tumor. Tumors were
detached with margins, to prevent their recurrence. The incisions were
Mouse housing and exercise training then closed with Vicryl threads (Ethicon).
Mice
All animal experiments were performed in accordance with the Single-cell preparation
guidelines of the Tel Aviv University Institutional Animal Care and Lungs, lymph nodes, livers, and skeletal muscles (gastrocnemius)
Use Committee with institutional policies and approved protocols were harvested from mice following the indicated treatments. Organs
4166 Cancer Res; 82(22) November 15, 2022 CANCER RESEARCH

were washed with PBS and separated into single cells. Liver tissue was tion rate (OCR). In brief, the same number of cells originating from the
minced and filtered through a 70-mm cell strainer (Corning). The other muscle or liver was seeded on collagen 1 (BD Biosciences)–coated wells
tissues were minced and incubated at 37 C for 25 minutes in serum- of XF96 microplates. The same number of cells originating from the
free DMEM containing collagenase IV (Worthington) and deoxyri- lung was seeded on poly-D-lysine (Sigma)–coated wells, and cells
bonuclease I (Worthington). DMEM supplemented with 10% FCS was originating from the lymph were plated on gelatin (Sigma)-coated
used to stop the enzymatic reaction. The cell suspension was filtered wells. Cells were plated at 30,000 cells/well 24 to 48 hours prior to the
through a 70-mm cell strainer (Corning). The cells were then cen- assay. Total RNA was extracted using “RealTime Ready Cell Lysis
trifuged for 5 minutes at 500 g at 10 C. The pellet was reconstituted Buffer” (ROCHE) and subjected to qPCR. ECAR and OCR values were
in Red Cell Lysis Buffer (Sigma-Aldrich) according to the manufac- normalized to Gapdh mRNA for each sample. Each data point
turer’s protocol. The tissue-derived single cells were counted and represents mean SD (n > 4).
subjected to further assessments.
RNA purification and quantitative RT-PCR
Glucose uptake assay Extracted RNA was quantified and its quality was assessed by
For glucose uptake analysis by FACS, cells of lymph nodes, lungs, measuring the 260 nm/280 nm ratio. For the mRNA measurements
liver, and skeletal muscles from healthy mice were washed three times following TRIzol (Invitrogen) isolation, cDNA was first produced
with PBS, resuspended in 250 mL glucose-free DMEM (Biological using 500 ng RNA and cDNA SuperMix (QuantaBio) and then sub-
Industries), and starved for 30 minutes. The green fluorescent glucose jected to qRT-PCR using Blue SYBR low Rox (PCR Biosystems) and
analogue 2-NBDG (Thermo Fisher Scientific, 100 mmol/L) was added qRT-PCR primers. For the mRNA measurements following extraction
to the cell suspension and incubated for 30 minutes. Cells were using the RealTime Ready Cell Lysis Buffer (Roche), RNA was directly
immediately examined by FACS on a BD FACSAria Fusion Cell subjected to one-step qRT-PCR and reverse transcriptase (Invitrogen)
Sorter. 2-NBDG was detected using filters designed to detect fluores- was added to the SYBR mix. mRNA levels were normalized to
cein (excitation/emission ¼ 465/540 nm). endogenous Hprt, Rplp0, or Gapdh. The qRT-PCR primers used are
Lungs and lymph nodes were taken from intracarotid-injected listed in Supplementary Table S5.
control and active mice and dissociated into single cells. A single-cell
suspension containing a mixed population of melanoma and stromal Mitochondria membrane potential measurement
cells was incubated in glucose-free DMEM (Biological Industries). After Mitochondrial membrane potential was assessed with Tetra-
30 minutes, 2-NBDG (Thermo Fisher Scientific, 100 mmol/L) was added methylrhodamine Ethyl, Ester (TMRE; Thermo Fisher) dye. Murine
to the media. After another 30 minutes of incubation, the mixed primary single cells from control or active animals (lungs, lymph
population was sorted into cancer cells and stromal cells based on nodes, liver, and muscle) were incubated with a final concentration of
mCherry red fluorescence of melanoma cells using the BD FACSAria 100 nmol/L rapamycin for 30 minutes in a CO2 incubator. After
Fusion Cell Sorter. Green fluorescence of 2-NBDG was detected to washing three times with a complete medium to get rid of the
quantify glucose uptake. nonspecific dye. The cells were subjected to FACS analysis to deter-
mine the active and inert mitochondria in the cells at 561/610 nm.
In vivo bioluminescent assays
A fresh stock solution of D-luciferin, potassium salt (Biovision) Coculture assay
was prepared at 15 mg/mL in sterile PBS (X1) and sterilized Immunofluorescence
through a 0.2-mm filter. Mice were injected intraperitoneally with 1105 primary cells from the active and control groups were
150 mg luciferin/kg body weight 10 to 15 minutes prior to seeded in 24-well plates. After 24 hours, 1104 of Ret-melanoma
imaging. Mice were placed on a dark surface and imaged using mCherry–Luciferase melanoma cells were seeded on top of the
a Biospace photon imager. After the acquisition, a photographic primary cells. Following overnight incubation, cells were washed
image was taken. once with PBS and fixed using 4% paraformaldehyde (Electron
Microscopy Sciences). Samples were scanned at 20 magnification
Glycolysis and oxidative phosphorylation assays using a Nikon fluorescence microscope, and the mean fluorescence
Extracellular acidification rate intensity of mCherry was measured using ImageJ software. For the
Glycolysis in the same number of live cells was measured using flow cytometry analysis, primary and melanoma cell cultures
the XF Glycolysis Stress Test kit according to the manufacturer’s were suspended in PBS with 1% FBS (Biological Industries) and
instructions (Agilent). The glycolysis kit directly measures extracel- 5 mmol/L EDTA; the red fluorescent cells were quantified out of the
lular acidification rate (ECAR) and evaluates the glycolytic flux. In total population for each cell type.
brief, cells originating from the muscle or liver were seeded on wells
of XF96 microplates coated with collagen1 (BD Biosciences). Cells Mitochondrial potential
originating from the lungs were seeded on wells coated with poly-D- For FACS, 1 106 primary cells from active and control groups
lysine (Sigma-Aldrich), and cells originating from the lymph nodes were seeded in 6-well plates. After 24 hours, 1 105 of Ret-melanoma
were plated on wells coated with gelatin (Sigma-Aldrich). Cells were cells labeled with PKH67 Green Fluorescent Cell Linker Kit (Sigma-
plated at 3 104 cells/well 24 to 48 hours prior to the assay. Total Aldrich) were seeded on top of the primary cells. Following overnight
RNA was extracted using the RealTime Ready Cell Lysis Buffer incubation, cells were centrifuged at 400 g for 10 minutes and stained
(Roche) and subjected to qPCR. ECAR values were normalized to with the TMRE (Invitrogen) as per the manufacturer’s protocol. After
Gapdh mRNA in each sample. thoroughly washing three times to remove unbound dye residues,
samples were subjected to FACS analysis. Singlets were gated based on
Oxygen consumption rate DAPI (live cells), GFPþ (melanoma), and GFP cells (primary lung
The oxidative phosphorylation kit measures key parameters of cells) for mitochondrial activity. The number of active and inert
mitochondrial function by directly measuring the oxygen consump- mitochondria was calculated.

Sheinboim et al.
Proliferation ALDOA, and Complex 1) within and between areas (stroma and
For FACS, 0.02 106 primary cells from active and control groups tumor) for homoscedastic or heteroscedastic analyses.
were seeded in 96- well plates with the same amount of Ret-melanoma
mCherry cells labeled with CFSE (BioLegend) seeded on top of the Proteolysis and mass spectrometry analysis
primary cells. Following overnight incubation, cells were subjected to Human plasma or mouse internal organs after perfusion were
FACS analysis. Singlets were gated based on DAPI (live cells), subjected to a mass spectrometry (MS) analysis for the small proteins
mCherryþGFPþ (melanoma cells), and mCherryþGFP cells (mela- as previously done by us (30). Proteins from 10-mL aliquots of serum in
noma proliferated) for proliferation. The percentage of the mCherryþ 8 M urea were separated using a Microcon 30-kDa Centrifugal Filter
cells denoted as proliferated was calculated from the gated cells. Unit (Millipore). The resultant proteins were reduced with 3 mmol/L
DTT in 8 M urea and 400 mmol/L ammonium bicarbonate (60 C
Apoptosis for 30 minutes), modified with 12 mmol/L iodoacetamide (in the dark,
For FACS, 0.02 106 primary cells from active and control groups room temperature for 30 minutes) and digested in 1 M urea, 50 mmol/L
were seeded in 96- well plates with the same amount of Ret-melanoma ammonium bicarbonate with modified trypsin (Promega) at a
mCherry cells seeded on top of the primary cells with CellEvent 1:50 enzyme-to-substrate ratio, overnight at 37o C. The tryptic peptides
Caspase-3/7 Green Detection Reagent (Invitrogen). Following over- were desalted through C18 TopTips (Glygen), dried, and resuspended
night incubation, cells were subjected to FACS analysis. Singlets were in 0.1% formic acid. The peptides were resolved by reverse-phase
gated based on DAPI (live cells), mCherryþGFPþ (apoptotic mel- chromatography on 0.075 180 mm fused silica capillaries (J&W)
anoma cells) and mCherryþGFP cells (nonapoptotic melanoma packed with Reprosil reversed-phase material (Dr. Maisch, HPLC
cells) for apoptosis. The percentage of the mCherryþGFPþ or GmbH). The peptides were eluted with a 120-minute linear gradient of
mCherryþGFP cells shown for apoptosis was calculated from the 5% to 28%, 15 minutes linear gradient of 28% to 95%, and then 25
gated cells. minutes at 95% acetonitrile with 0.1% formic acid in water at a flow rate
of 0.15 mL/minutes. Mass spectrometry was performed with a Q
Long coculture experiment Exactive HF mass spectrometer (Thermo) in a positive mode using
For the direct coculture, cells were incubated up to 120 hours and a repetitively full MS scan followed by collision-induced dissociation of
analyzed by Incucyte. The intensity of mCherryþ melanoma cells was the 20 most dominant ions selected from the first MS scan. The MS
calculated using ImageJ. data from each sex (n ¼ 3 males and n ¼ 3 females) in the human
cohort or the indicated organs from eight mice (four in the active
Hematoxylin and eosin staining group, four in the control group) were analyzed using the MaxQuant
Mouse tissues were fixed with 4% paraformaldehyde at 4 C, software 1.5.2.8 and the human or mouse proteome from the Uniprot
dehydrated in a graded ethanol series, and embedded in paraffin wax. database (31) with 1% false discovery rate (FDR). The data were
The fixed tissues were sliced into 10-mm sections and dried overnight at quantified by label-free analysis using the same software. Statistical
37 C. Sections were stained with hematoxylin (Sigma-Aldrich) and analysis of the identification and quantization results was done using
eosin (Sigma-Aldrich) and mounted with DPX Mountant (Sigma- Perseus 1.6.10.43 software (32). Proteins were evaluated for KEGG
Aldrich), according to the manufacturer’s instructions, and imaged at pathway enrichment using the proteomaps too and the gene ontology
20 magnification with a Nikon brightfield microscope. (GO) functional annotation tool (33). For humans, the differential
expression of proteins after MS analysis appears in Supplementary
IHC analyses Table S2. For mice, the upregulated proteins in control (indicated in
Mouse tissues were fixed with 4% paraformaldehyde at 4 C, dehy- red) and active (indicated in blue) after MS analysis appear in
drated in a graded ethanol series, and embedded in paraffin wax. The Supplementary Table S1.
fixed tissues were then sliced into 10-mm sections and dried overnight
at 37 C, followed by deparaffinization in xylene and hydration in a Membrane labeling the melanoma cells
graded series of ethanol. After microwaving in sodium citrate buffer Na€ve Ret-melanoma cells were labeled either with mCherry
(pH 6.0) for antigen unmasking, tissue samples were blocked with 5% (PKH26) or GFP (PKH67) cell membrane labeling dye (Sigma-
BSA, 0.5% Tween-20 in PBS and then incubated with antibodies to Aldrich) following the manufacturer’s instructions.
GLUT1 (Abcam, ab40084), S100-beta (Abcam, ab52642), Complex I
(Abcam, ab109798), and aldolase A (Santa Cruz Biotechnology, Inhibiting the cellular metabolism using rapamycin
sc-377058), followed by incubation with fluorophore-conjugated Murine primary cells from control or active animals (lungs) were
secondary antibodies Alexa Fluor 488 (Invitrogen, A11008) and Alexa incubated with the final concentration of 100 nmol/L mTOR inhibitor
Fluor 594 (Invitrogen, A21203). Nuclear staining was performed with rapamycin (GoldBio) for 3 hours in a CO2 incubator followed by
DAPI (Vector Laboratories). Images were obtained at 40 magnifi- washing with a complete culture medium.
cation using a Nikon fluorescence microscope. For intensity quanti-
fication, 40 images were captured, and the stoma and tumor regions TMRE with and without rapamycin
were marked; at least 15 areas were quantified per image (stroma or For TMRE experiments, following the rapamycin treatment,
tumor), taken from at least three different tissue samples from each murine lung cells were stained to check the mitochondria membrane
mouse. Fluorescence images were split into separate channels and potential measurement using the TMRE as mentioned above followed
converted into 8-bit images using ImageJ software. A specific area in by FACS analysis.
each image was subjected to quantification using the ROI manager
function to precisely quantify the intensity from the same place in Melanoma survival with and without rapamycin
different channels simultaneously; each intensity was normalized to Ret-melanomas’ ability to survive in different primary cell envir-
DAPI from the same image to rule out discrepancies due to differences onments was measured via FACS. Na€ve Ret-melanoma cells were
in cell numbers. We performed t tests for each target (GLUT1, S100, labeled with PKH26, and 0.05106 cells were seeded into 24-well

culture plates. Single-cell preparations from the lungs of control and Proteins that were found to be differentially expressed from the MS
active mice were performed and 0.05 106 cells from each group were between the active and control groups (Supplementary Fig. S1C) were
treated with or without rapamycin (vehicle) and stimulated for 3 hours subjected to KEGG analysis and proteomaps revealing that there were
in the CO2 incubator. Rapamycin or control-treated cells were directly metabolic shifts in tissues from active versus control mice (Fig. 1B). In
cocultured with the mCherry-labeled Ret-melanoma cells for 24 hours active mice, there was upregulation of carbohydrate metabolism,
at 37 C and 5% CO2. Following the incubation, trypsinized cells were glycolysis, OXPHOS, and mitochondrial biogenesis in all tissues
subjected to FACS analysis for determining the survived melanoma examined (Fig. 1B). Some of the metabolic changes were organ
cells at 465/540 nm wavelength. dependent; for example, in both lymph node and liver tissues, there
was upregulation of glycolysis and mitochondrial biogenesis; however,
Primary mouse melanoma gene expression analysis the liver uniquely exhibited enrichment in the TCA cycle.
Downregulated genes from primary melanoma obtained from We subjected the differentially expressed proteins to GO analysis
B16F10 melanoma injected mice following exercise from (3) were without considering expression scores and compared the overlap of all
subjected to KEGG enrichment analysis using WebGestalt. the significantly enriched pathways from each organ (Fig. 1C). This
revealed that catabolic processes (defined as those with the term “GO
Statistical analysis and reproducibility biological process”) and mitochondrial processes (defined as those
All data are shown as means and standard errors of the mean. We with the term “GO cellular compartment”) were the most enriched in
used a random experimental design. For mouse experiments, Student the investigated tissues of active mice (Fig. 1C and D; Supplementary
t tests (two-tailed) for two-group comparisons were performed for the Fig. S1D). The blood glucose levels were the same before and after
indicated conditions. For the human cohort of 20-year follow-up in the exercise in both active and control mice (Supplementary Fig. S1E),
Israeli general population, a propensity score was constructed using a suggesting that systemic glucose homeostasis is maintained upon
multinomial logistic regression, through which was calculated the exercise, in line with the literature (35), and that the observed
probability of being classified into a specific physical activity category. metabolic alterations following exercise are due to changes in the
The propensity scores weighted model (w) included the following organs themselves.
variables as covariates: age, sex, ethnicity, neighborhood socioeco- Next, we measured the glucose uptake of primary single cells
nomic status, income, education, proportion of saturated fatty acids originating from the lymph nodes, lungs, livers, and skeletal muscles
out of the total energy intake, total energy intake, total alcohol of active and control mice. Cells were incubated for 30 minutes in
consumption, dietary fiber intake, self-rated health, anemia, osteopo- glucose-free DMEM followed by the addition of the green fluorescent
rosis, hypercholesterolemia, hypertriglyceridemia, diabetes, hyperten- glucose analogue 2-NBDG for 30 minutes. FACS analysis revealed
sion, stroke, coronary heart disease, occupational physical activity, significantly higher levels of 2-NBDG in cells from active lymph nodes,
marital status, self-definition of religious level, highest education lungs, livers, and skeletal muscles compared with the cells from control
certificate/academic degree, employment, body mass index and smok- mice (Fig. 1E). Because glucose is the main substrate of glycolysis and
ing year. For the MS data analyzed in the human cohort (n ¼ 6 of the OXPHOS cascade (22, 24) as well as the major source of energy
biologically independent humans) with 5% FDR or from the indicated during exercise (11), we next examined glycolysis in primary cells
organs from eight mice (n ¼ 4 from each group) with 1% FDR. originating from the lymph nodes, lungs, livers, and skeletal muscles of
active and control mice. Glycolytic function was assessed by the ECAR.
Data and materials availability In all the investigated tissue-derived cells from the active mice,
The accession number for the MS proteomics data reported in this glycolytic function was significantly higher than that in control mice
paper is deposited in the PRIDE repository under accession numbers (Fig. 1F; Supplementary Fig. S1F).
PXD035630 and PXD035648. The raw data associated with this paper To further understand metabolic differences between tissues from
can be available from the corresponding authors upon request. active and sedentary mice, we conducted a mitochondrial activity test.
Using tetramethylrhodamine ethylamine (TMRE; ref. 36), we analyzed
the number of active mitochondria in primary cells isolated from active
Results and control tissues via FACS. We found that primary cells originating
Exercise reprograms tissue metabolism in mice from control mice had significantly fewer active mitochondria com-
To explore the molecular changes that occur in murine internal pared with active primary cells (Fig. 1G). This significant reduction in
organs following exercise, we established an in vivo exercise model active mitochondria validates our proteomic analysis. Along the same
(Fig. 1A; Supplementary Fig. S5; ref. 29). We performed comparative line, OCR measurement was significantly higher in active animals
proteomics analyses, by MS, of typical metastasis host organs: lungs, compared with controls (Supplementary Fig. S1G) as well as the
lymph nodes, and livers from sedentary and active mice that were expression of mitochondrial-specific genes (TFAM, POLRMT,
subjected to an exercise regimen for 8 weeks (Fig. 1A; Supplementary TFB1M, TFB2M, Cyc1, and Mrps35; Supplementary Fig. S1H). Our
Table S1). Skeletal muscle was also analyzed, as they undergo signif- findings are in line with previous studies that showed that skeletal
icant metabolic changes upon exercise (34), and skeletal muscle is muscles and liver tissues have increased mitochondrial biogenesis
rarely a site for metastatic colonialization. Hematoxylin and eosin following exercise (37). However, to the best of our knowledge, this is
staining from lungs, lymph nodes, livers, and muscles from control and the first report of an elevation in mitochondrial activity following
active mice are shown (Supplementary Fig. S1A). Principal component exercise in the lymph nodes and lungs, which are not considered to be
analysis of organs from the control sedentary group was scattered, part of the direct response to exercise (37). Further, as glucose is unable
although organs from the active group demonstrated a grouped to cross lipid membranes (38), glucose transporters (GLUT) on the cell
pattern, suggesting that exercise synchronizes the organ’s biological surface are critical for glucose uptake. There are multiple GLUTs,
properties independently of an individual’s initial characteristics varying in expression (dependent on the cell type), in their affinities for
(Supplementary Fig. S1B). This finding suggests that the metabolic glucose, and in their abilities to transport fructose. GLUT1 is the most
alterations following exercise are common to all trained animals. universally expressed isoform of the GLUT receptors (38). GLUT2 is

Sheinboim et al.
A Excise organs Mass spectrometry

Proteomics
Control/ 8 weeks
Active
Lungs Lymph Liver Muscle Molecular characterization
nodes Single-cell preparation
B Lungs Lymph nodes Liver Muscle
Enriched KEGG Enriched KEGG Enriched KEGG Enriched KEGG

(n = 4) (n = 4) (n = 4) (n = 4)
pathways after exercise pathways after exercise pathways after exercise pathways after exercise
Scale
1.5
0
-1.5
Control Active Control Active Control Active Control Active
C Lungs
(205)
Lymph nodes
(1,004)
Cellular process
Nitrogen compound metabolic process D Lungs
(38)
Lymph nodes
(237)
Cellular anatomical entity
Organelle
Primary metabolic process
Muscle Muscle Intracellular organelle
Cellular compartment
Liver 136 808 Metabolic process Liver 18 173
(135)
Biological process
(693) (523) Cellular metabolic process (60) Intracellular anatomical structure

23 56 Organic substance metabolic process 6 32 Membrane-bounded organelle
10 0
491 328 21 70
1 Cellular amide metabolic process 2 1 Cytoplasm
0
Cellular catabolic process
Cytosol
13 Organonitrogen compound catabolic… 11
Organic substance catabolic process Organelle envelope
63 9 5 0
Organophosphate metabolic process Envelope
40 13 7 0
Catabolic process Mitochondrion
63 0 10 20 30 40 50 60 70 80 90 14 0 5 10 15 20
Fold enrichment (sum) Fold enrichment (sum)
E Glucose uptake F Glycolytic function G TMRE mitochondrial potential

Control Active
H qPCR analysis
Control Active Control Active TMRE+ TMRE+

Control Active TMRE− TMRE−
*
30 100%
2 1,000 10
* 80% **
1.5 800 8
TMRE- TMRE+ TMRE- TMRE+
Lungs
Lungs
Lungs
Lungs
20
% gated
% Gated
60%
SSC-A
600
**
6
1
400 40% 4 **
10 *
0.5 20%
200 2
0 0 0 0% 0
100 101 102 103
Control Active mCherry+TMRE+ mCherry+TMRE+ Control Active Control GLUT1 GLUT2 GLUT4
0 10 20 30 40 0 20 40 60 80
Mean green fluorescence intensity (fold change)
**
15 *** 800 100%
1.5 20
% Gated stroma cells with mitochondria
* *
Lymph nodes
Lymph nodes
80%
Lymph nodes
Lymph nodes
Relative mRNA levels (fold change)

% gated
600
% Gated
10 1 15 **
***
TMRE- TMRE+ TMRE- TMRE+ 60%
SSC-A
*
400 10
40%
5 0.5
ECAR (mpH/min)
200 20% 5
0 0 0 0% 0
0 1 2 103
0 10
10 10
20 10
30 40 Control Active 0 20 40 60 80 mCherry+TMRE+ mCherry+TMRE+ Control Active Control GLUT1 GLUT2 GLUT4
**
50 100%
2.5 *
120 6 ***
40 2 100 80% 5
TMRE- TMRE+ TMRE- TMRE+
% Gated
SSC-A
80
***
60% 4
Liver
30
Liver
Liver
Liver
1.5
60 3
20 1 40% *** **
40 2
10 0.5 20 20%
1
0 0 0 0% 0
100 101 102 103
Control Active mCherry+TMRE+ mCherry+TMRE+ Control Active
0 10 20 30 40 0 20 40 60 80 Control GLUT1 GLUT2 GLUT4
*
30 2 800 100%
10
** 80%
1.5 600 8 **
TMRE+ TMRE+
% Gated
20 TMRE- TMRE-
Muscle
Muscle
Muscle
Muscle
% gated
SSC-A
***
60%
1 6
400
40% *
10 4
0.5 200 *
20% 2
0 0 0 0% 0
100 101 102 103 Control Active mCherry+TMRE+ mCherry+TMRE+ Control Active
0 10 20 30 40 0 20 40 60 80 Control GLUT1 GLUT 2 GLUT4
FITC fluorescence intensity Time (min) mcherry 561__610-20-A / SSC-A TMRE
Figure 1.
Exercise causes a metabolic shift in tissues. A, Schematic representation of the exercise mouse model. B, Left, heatmaps showing proteins differentially expressed in
lungs, lymph, liver, and skeletal muscle of active mice versus control with red indicative of upregulation and green indicative of downregulation in the tissues of active
mice. Right, proteomaps of KEGG pathways enriched in differentially expressed proteins. C and D, Proteins differentially expressed in active mice enriched for GO
biological process (C) and GO cellular compartment (D) identified using GENEONTOLOGY tool. Left, Venn diagram of overlap of the GO terms for proteins
differentially expressed in the indicated organs for biological processes (C) and cellular compartment (D). Right, sum of fold enrichment of the GO terms for all the
indicated tissues. E, Left, glucose uptake in single cells originating from the indicated organs evaluated by analysis of fluorescence of 2-NBDG. Right, mean green
fluorescence intensity in single cells originating from the indicated organs of the control and active mice relative to intensity in control tissue. F, Glycolytic function of
single cells from the indicated organs determined using ECAR measurements. Samples were normalized to their Gapdh mRNA level. Error bars, SEM (n ≥ 4). G,
FACS analysis of mitochondrial activity in primary organ cells (lungs, lymph nodes, liver, and skeletal muscles) of control and active mice; TMRE expression is
indicative of active mitochondria. Left, representative image of the FACS data shows the TMRE and TMREþ cell populations for control and active mice. Right,
quantification (% gated cells) from the FACS for control and active mice. Statistical comparison between TMRE and TMREþ from each group (control and active) and
TMREþ between control and active groups is presented in the graphs (n > 3 animals in each group). H, qRT-PCR quantification of the mRNAs encoding the indicated
glucose transporters in tissues from active and control mice. Data were normalized to endogenous levels of Gapdh or Hprt. Error bars, SEM (n ¼ 3 independent
experiments). , P < 0.05; , P < 0.01; , P < 0.001.
expressed in the liver, kidney, and central nervous system, among four GLUT receptors as our panel for checking GLUT expression in
others, and is important in the expression of glucose-sensitive genes. mouse tissues after exercise, we were able to get a well-rounded
GLUT4, found in adipocytes, is stimulated by insulin levels, thus perspective from GLUTs differing in cellular locations and environ-
enhancing insulin sensitivity and glucose uptake (39). By using these mental triggers. We found that expression levels of Glut1, Glut2, and

Glut4 mRNAs, which exclusively transport glucose (38), were signif- the two. To investigate this relationship, we examined a prospective
icantly higher in cells from the active mice compared with control mice cohort (n ¼ 2,734: 1,302 females and 1,432 males), which was followed
(Fig. 1H), which is in line with GLUT1 and GLUT4 expression in for 20 years, with self-reported duration and intensity of exercise
skeletal muscle post-exercise (40). Taken together, these data indicate (Supplementary Fig. S5D). Data on exercise were obtained via a
that exercise causes metabolic reprogramming of various organs, personal interview at baseline, based on a standard questionnaire
creating a new microenvironment throughout the body. (further details can be found in Materials and Methods; refs. 42, 43).
The cohort study was linked to the Israel National Cancer Registry via
High-intensity exercise significantly reduces the risk of highly their national identification numbers. The registry covers the entire
metastatic cancers in humans Israeli population, with 97% estimated completeness of ascertainment
To examine the observed metabolic reprograming in mice upon for solid tumors (44), whereas all cases of malignant neoplasms,
exercise in humans, we performed a plasma proteomic analysis carcinoma in situ and high-grade intraepithelial neoplasia, and benign
(Supplementary Table S2). Using six subjects from a cohort of healthy, neoplasms of the brain and central nervous system have been
routinely active people (3 female and 3 male), we collected blood recorded (44).
samples before (following 48 hours of rest) and after they ran on a Our analysis revealed that exercise tends to lower the risk of
treadmill at a high intensity (75% heart rate) for 30 minutes (Fig. 2A; developing cancer in both men and women, with a greater association
Supplementary Fig. S5B). GO analysis of the upregulated proteins with highly metastatic cancers (SEER 7; Fig. 2E). Specifically, high-
following the exercise session revealed a significant enrichment in the intensity exercise significantly reduced the incidence of highly met-
IGF-I pathway in all subjects (Fig. 2B and C; Supplementary Fig. S2A). astatic cancers (73% risk reduction compared with the inactive group,
Like insulin, IGF-I promotes glucose uptake via the translocation of P < 0.05; Fig. 2E and F). This implies that high-intensity exercise may
glucose transporters such as GLUT1 and GLUT4 to the cell mem- prevent cancer dissemination to distant sites. Our epidemiologic study
brane (38, 41). Further, in a separate cohort of 14 subjects who ran at illustrates the unique and significant interaction between exercise
RER above 0.95 (high-intensity), we found that fat-to-carbohydrate intensity and metastatic cancer development in humans.
turnover (i.e., the ratio between glucose and fat utilization during These data suggest that our originally cancer-free participants had a
exercise) is affected by exercise intensity, with glucose utilization reduced likelihood of highly metastatic tumor formation. Additionally,
rising in prominence as the intensity of exercise increases (Fig. 2D; routinely active females have shifted their macronutrient utilization
Supplementary Fig. S5C). Therefore, the IGF-related pathway and enrichment of metabolic pathways, leading us to hypothesize that
regulating glucose homeostasis (41) from the first cohort is supported exercise is inducing systemic changes that are protecting against tumor
by the increased usage of carbohydrates (11) following high-intensity development in humans.
exercise in the second cohort.
Although metastases are the major cause of cancer mortality (14), Exercise inhibits melanoma metastasis formation in mice via
most human epidemiologic studies that have studied the role of metabolic shield
exercise in cancer outcomes have not considered the stage of cancer To evaluate the potential protective role of the exercise-induced
at diagnosis, the intensity of the exercise, and the interaction between increased metabolic ability of lymph nodes, lungs, and livers against
A Human exercise model B Upregulated pathways post exercise C Top 4 GO terms
Post- Subject 1
Exercise Negave regulaon of endopepdase
30 min treadmill acvity
running Subject 2 Subject 3 Anmicrobial humoral immune response
mediated by anmicrobial pepde
Collect blood Mass
16 Acute-phase response
Pre & Post spectrometry
exercise analysis Regulaon of insulin-like growth factor
Separate Subject 5 Subject 6 signaling pathway
plasma 0 10 20 30 40 50 60 70 80 90
Subject 4 Fold enrichment
D E Low–moderate intensity vs. high intensity F SEER 0-4 SEER 7

High intensity
Relative utilization of carbohydrate/fat
High intensity
100 High intensity 8.8%
% CHO
% FAT Low–moderate intensity 25.4%
SEER 7 – Weighted
80 Low–
20.6% moderate
SEER 7 – Age-adjusted
53.5% intensity
60 SEER 0-4 – Weighted Inactive
21.1% 70.6%
40 SEER 0-4 – Age-adjusted
Low– Inactive
All cancers – Weighted
20 moderate
All cancers – Age-adjusted intensity
0 0 1 2 3
Baseline Moderate High Fold enrichment (sum)
intensity intensity Hazard ratio
Figure 2.
High-intensity activities reduce metastatic cancer likelihood. A, Schematic representation of human exercise model. B, Venn diagrams showing an overlap of
16 pathways based on a GO enrichment analysis of differential proteins found in the plasma of the routinely active subjects (n ¼ 3 males and n ¼ 3 females) after a
30-minute run on the treadmill. C, The top four GO-enriched pathways of the 16 overlapped GO terms. D, The relative contributions of carbohydrates (CHO)
and fats to the total substrate utilization prior to exercise and during moderate and high-intensity during a graded exercise test performed on a motorized
treadmill. E, Fold enrichments of hazard ratio (HR) from a prospective cohort study among 2,734 cancer-free participants that were followed for 20 years, with
indicated SEER stages of cancer classified into inactive, low–moderate intensity (<6 metabolic equivalent, MET), and high-intensity exercise (>6 MET). F, Relative
exercise intensity of individuals with a cancer diagnosis during the 20 years of follow-up that was classified as SEER 0–4 compared with those with cancers
classified as SEER 7.

Sheinboim et al.
metastasis formation, we established a mouse cancer model in which exercised only pretumor cell injection compared with control mice
we tested the effects of exercise (Supplementary Fig. S5E). Because (Fig. 3G). The expression of mCherry and melanoma markers Mlana
cutaneous melanoma has a high probability of metastatic dissemina- and Tyrp1 was significantly lower in the lungs of the mice exercised pre
tion to most organs, including the lymph nodes, lungs, and liver, we tumor cells injection than in the controls (Fig. 3H). These results
used this cancer as our model. To follow tumor growth and spread, we suggest that exercise has a protective effect against metastatic dissem-
used a murine Ret-melanoma cell line derived from a spontaneous ination due to changes in the microenvironment prior to tumor arrival.
melanoma in Ret-transgenic mice, which was engineered to carry We then examined whether exercise could inhibit cancer formation
mCherry and luciferase reporters (Supplementary Fig. S3A and B). We ex vivo. Single cells were isolated from the lymph nodes, lungs,
used a forced treadmill running protocol, which enabled us to grad- and livers of control mice and active mice that were not injected
ually increase exercise intensity (5). Mice were divided into a sedentary with melanoma. These primary cells were then cocultured with Ret-
group (control) and a group subjected to the exercise protocol (active); melanoma cells for 24 hours (Fig. 3I; Supplementary Fig. S5G). The
each group contained at least 3 mice. The active group was subjected number of Ret-melanoma cells that survived during the experiment
to a training protocol (29) for 8 weeks prior to subdermal injection was significantly reduced when cocultured with cells that originated
of Ret-melanoma mCherry–Luciferase melanoma cells; after 4 days of from organs of active mice compared with those from control mice, as
recovery, the exercise regimen was continued for up to 4 additional shown by microscopy (Fig. 3J; Supplementary Fig. S4D) and by FACS
weeks (Fig. 3A). (Fig. 3K). Interestingly, melanoma cell proliferation was significantly
We found a significant reduction in the active group’s primary reduced in the presence of primary lung cells from active mice
tumor volume compared with tumors in the control group at the compared with controls whereas no significant changes in prolifera-
end of the second exercise period (Fig. 3B), which is in accordance tion were observed when in culture with liver and lymph nodes cells
with a previous study (3). We then investigated metastasis forma- (Supplementary Fig. S3G, left). On the other hand, melanoma apo-
tion after the excision of the primary tumor. This preclinical model ptosis showed an opposite trend (Supplementary Fig. S3G, right),
enables spontaneous metastasis formation, which allows us to trace indicating that active stroma may affect either melanoma proliferation
the multistep process of metastasis. The primary tumors were or survival, in a context-dependent manner.
excised when they reached 1 cm3 in size. Mice were then given Our findings thus far have pointed us toward metabolic alterations
10 days to recover before continuing with the training protocol. being a key player in making active tissues a hostile microenvironment
Four weeks after primary tumor excision training was finished, a for melanoma. To test this, we targeted the metabolic abilities of these
metastasis examination was completed (Fig. 3A). We then isolated cells directly with rapamycin, blocking the mTOR pathway (45), to see
single cells from the lymph nodes, lungs, and liver, and sorted if the protection against cancer progression can be reversed. To ensure
melanoma cells from stromal cells based on Ret-melanoma stably that rapamycin is in fact affecting the metabolism of the primary cells,
expressing mCherry fluorescence. The number of melanoma cells we used TMRE to test the mitochondrial activity in primary cells with
was significantly reduced in the lungs and liver and a reduced trend and without rapamycin treatment. We saw that a 3-hour treatment
in the lymph nodes of the active group compared with the control with rapamycin significantly reduced the number of active mitochon-
group (Fig. 3C). These results indicate that exercise inhibits both dria in primary lung cells from active mice (Supplementary Fig. S3H
primary tumor growth and spontaneous metastasis formation. and S4E). We then tested if the melanoma survival rate was affected by
Because the primary tumor volumes were significantly smaller in the change in the metabolism of the primary cells. We saw that
the active group than in the control group (Fig. 3B), we reasoned that melanoma that was cocultured overnight with rapamycin-treated
the number of circulating melanoma cells was decreased, and thus the primary cells survived and proliferate better than that in culture with
number of cells available for metastasis formation would be limited in untreated primary cells (Fig. 3L; Supplementary Fig. S3I).
the active group. Taking this into consideration, we implemented a Put together, these results imply that exercise reduces primary
model, specifically for metastatic formation, that bypasses the stage in melanoma volume and metastatic dissemination into lymph nodes,
which melanoma cells invade into the dermis for metastasis formation. lungs, and liver. The creation of a metabolic shield via the reprogram-
By using intracarotid injection of Ret-melanoma mCherry–Luciferase ming of key metabolic characteristics of these organs results in a tumor
melanoma cells (targeting the lymph nodes and lungs) or intrasplenic microenvironment that does not support tumor cell colonization and
injections (targeting the liver), we evaluated whether exercise per- therefore provides a systemic protective effect.
formed pre and post tumor injection had a protective effect against
metastases (Fig. 3D). Ex vivo examination of the metastases showed Exercise induces metabolic cross-talk between the tumor and
a significant reduction in bioluminescence in the lungs (Fig. 3E; its microenvironment ex vivo
Supplementary Fig. S3C) as well as in the lymph nodes and liver in One of the key hallmarks of cancer progression is its ability to make
the groups of mice that were exercised pre and post injection of tumor metabolic alterations, including enhanced tumor aerobic glycolytic
cells compared with control mice (Supplementary Fig. S3D and E). The metabolism, termed the Warburg effect (14). We performed a gene-
expression of mCherry and melanoma markers Mlana and Tyrp1 (34) expression clustering analysis of melanoma, organized according to
were significantly lower in the lungs (Fig. 3F) as well as lymph nodes the stage of progression, from benign nevi to metastasis, in various
and livers (Supplementary Fig. S3F) of mice exercised pre and post organs (Supplementary Table S3). This analysis revealed two distinct
tumor cell injection. This indicates that exercise pre and post tumor gene signatures related to primary versus metastatic human melanoma
inoculation has a significant protective effect against metastasis (Fig. 4A, left; Supplementary Fig. S4A). GO analysis of these genes
formation. found significant metabolic enrichment (i.e., metabolism of carbon,
To understand this protective effect of exercise and separate it from galactose, glycine, serine, amino sugar, and nucleotide sugar) in the
the effect of the tumor and stroma together, we altered our model to metastatic samples, whereas the immune-related pathways were more
training exclusively prior to the Ret-melanoma mCherry–Luciferase highly enriched in the primary tumors (Fig. 4A, right; Supplementary
melanoma injection (herein “pre”; Fig. 3D; Supplementary Fig. S5F). Fig. S4A), which is in line with the literature (14). Given the metabolic
In this model, there was significantly reduced bioluminescence in mice shift during cancer progression and the metabolic reprogramming

A G H
Subdermal injection Tumor

Metastasis
Photons/sec/cm2 (fold change)

Ret-melanoma mCherry-Luc excision examination Control Active
Tumor measurement Pre 1.2 1.2

*** ***
8 weeks 4 weeks 4–24 weeks
Lungs
0.8 0.8
Active : Treadmill
Control : Sedentary 0.4 0.4
0 0
Control Active Control mCherry MelanA Tyrp1
B Control Active
500 Control * I
Tumor volume (mm3)

400 Active
ve
** Melanoma and primary cell coculture (microscopy)
300
* Coculture:
200 Lymph nodes
Primary cells Primary + Melanoma cells
* Lungs single-cell
100
Control / Liver preparation 24 h Overnight
0 Melanoma quantification
Active
0 3 6
Measurement number Primary cells (organ single cells)
C Control Active J Control Active

Tumor cells (mCherry melanoma)
1.2 80
105
105
Melanoma - mCherry Melanoma - mCherry
Lymph nodes
1 60
Lymph nodes
104
104
0.8 40
SSC-A
SSC-A
*
0.6 20
103
103
% mCherry+ cells/Total count

0.4 100 μm 100 μm
0
Control Active
102
102
0.2 60
0 Melanoma - mCherry Melanoma - mCherry 50
102 103 104 105 102 103 104 105 Control Active 40
PE-Texas Red - A PE-Texas Red - A
Lungs
1.2 30
**
105
105
mcherry+ cells (fold change)
*** 20
1 10
100 μm 100 μm
104
0.8 0
104
SSC-A
SSC-A
Control Active
Lungs
0.6 50
103
103
Melanoma - mCherry Melanoma - mCherry

0.4 40
30
Liver
102
102
0.2
20 ***
0
102 103 104 105 102 103 104 105 10
Control Active
PE-Texas Red - A PE-Texas Red - A 100 μm 100 μm
0
1.2 Control Active
105
105
**
1
K
104
0.8
104
Control Active 50
SSC-A
SSC-A
Liver
0.6
103
40
103
104 105 106
106
melanoma cells
Percentage of
0.4
Lungs
30 **
104 105
102
0.2
102
SSC-A
SSC-A
20
0
102 103 104 105 102 103 104 105 Control Active 10
103
PE-Texas Red - A PE-Texas Red - A 103

0
D 102 103 104 105

GFP 488- C
106 102 103 104 105
GFP 488- A
106 Control Active
Intracarotid injection
Ret-melanoma mCherry-Luc
Metastasis
examination L 50 1.2
Vehicle Rapamycin
1
40
Pre & post
8 weeks 2–4 weeks 0.8

Melanoma survival (mCherry+)
30
0.6
Active : Treadmill
20 0.4 ***
Control : Sedentary 10 0.2
Fold change
Active : Treadmill 0 0
Count
Pre
3 4
10
0 101
10 202
10 10
30 40 Control Active
Control : Sedentary
50 1.2
1
E
40
F 30
20
0.8
0.6
Lungs Pre & post 0.4
10 0.2
Photons/sec/cm2 (fold change)
Control
1.2 1.2 0 0
** *** 0 10
10
1 10
20
2 10
30
3 10
40
4
Control Active
mCherry+ Fluorescence
0.8 0.8
0.4 0.4
Active
0 0
Control Active Control mCherry MelanA Tyrp1
Figure 3.
Exercise inhibits melanoma metastasis formation. A, Schematic of the subdermal melanoma mouse model. B, Left, representative images of mice from the
control and active groups. Right, calculated tumor volumes. Error bars, SEM (n ¼ 16 mice per group). C, Left, FACS analysis of single cells from the lymph
nodes, lungs, and liver of control and active mice subdermally injected with Ret-mCherry–labeled melanoma cells. Green, mCherry-negative stromal cells; red,
mCherry-positive melanoma cells. Right, fold change in mCherry-positive cells in active mice relative to controls based on the FACS analysis. Error bars, SEM
(n ¼ 8 group). D, Experimental design for intracarotid injection. Controls were sedentary. Active mice were exercised pre- and post-tumor cell injection or only
preinjection. E, Left, bioluminescence images of lungs taken from control and exercised mice. Right, photon quantification of melanoma metastases plotted as
fold change relative to control. Error bars, SEM (n ¼ 20 mice in control and active “pre and post” groups). F, qRT-PCR quantification of melanoma markers
mCherry, Tyrp1, and Mlana in the lungs of “pre and post” group. Data were normalized to Hprt and are plotted relative to quantities in control mice. Error bars,
SEM (n ≥ 3 independent experiments). G, Left, bioluminescence images of melanoma in the lungs taken from control mice and mice exercised only before
intracarotid injection of tumor cells. Right, photon quantification of melanoma metastases plotted as fold change relative to control. Error bars, SEM (n ¼ 8
mice in each group). H, qRT-PCR quantification of melanoma markers mCherry, Tyrp1, and Mlana in the lungs of control mice and mice exercised only before
intracarotid injection of tumor cells. Data were normalized to Hprt. Error bars, SEM (n ≥ 3 independent experiments). I, Experimental design for the coculture
of the melanoma cells and primary cells for microscopy analysis. J, Left, immunofluorescent images of melanoma cells that express mCherry seeded on
adhered primary cells from the lymph nodes, lungs, and livers of control and active mice. Right, percent melanoma cell numbers relative to total cell numbers
quantified in brightfield images. Error bars, SEM (n ¼ 3 independent experiments). K, FACS analysis of GFP-labeled Ret-melanoma cells cocultured with
primary lung cells of control and active mice. GFPþ (melanoma) and GFP (primary lung cells). Top, representational dot plots from FACS analysis. Bottom,
calculation of GFPþ cells based on the FACS analysis. Error bars, SEM (n ¼ 6 animals in each group). L, FACS analysis of mCherry-labeled Ret-melanoma
with primary lung cells from active and control animals. Top (vehicle treated) left, representative image of percent gated of mCherryþ cells with vehicle-
treated primary cells from active and control mice. Right, graph representing fold change of surviving melanoma cells cocultured with either active or control
primary cells. Bottom (rapamycin treated) left, representative image of percent gated of mCherryþ cells with rapamycin (100 nmol/L)-treated primary cells
from active and control mice. Right, graph representing fold change of surviving melanoma cells cocultured with either active or control primary cells.
Error bars, SEM (n > 3 animals in each group). , P < 0.05; , P < 0.01; , P < 0.001.

Sheinboim et al.
A Melanoma gene expression during progression (KEGG enrichment) B Primary melanoma gene expression following exercise (KEGG enrichment)
Protein digestion and absorption Amino sugar and nucleotide sugar metabolism
Th1 and Th2 cell differentiation Carbon metabolism
Amoebiasis Citrate cycle (TCA cycle)
T-cell receptor signaling pathway Oxidative phosphorylation
Glycine, serine and threonine Antigen processing and
Glycine, serine and threonine metabolism Antigen processing and presentation
metabolism presentation
NF-kappa B signaling
Galactose
Galactosemetabolism
metabolism NF-κβ signaling pathway
Mice data pathway
Cytokine-cytokine receptor
Carbon
Carbon metabolism
metabolism Cytokine–cytokine receptor interaction
interaction
VGP
Benign nevi
Atypical nevi
In situ
Lung
Lymph node
Soft tissue
Brain
Adrenal
Small intestine
Bowel
Bone
Spleen
Liver
Human data Amino sugar and nucleotide sugar
Amino sugar and nucleotide metabolism Hematopoieticcell
Hematopoietic celllineage
lineage
metabolism
-3 Log10 (P) −2 −1 0 1 2 3 Log10 (P) Log10 (P) −15 −10 −5 0 5 10 15 Log10 (P)
Immune related Metabolism related Metabolism related Immune related
C Melanoma and primary cell coculture E Glucose uptake by FACS

Coculture:
Primary cells Lungs Lymph nodes
Primary + melanoma cells
Organ single-cell
Control/ harvesting preparation 24 h Overnight
Active FACS
0.8
0.4
Lungs
Control : stroma
0.6
Primary cells (organ single cells)
0.3
% Gated
Active : stroma
% Gated
Stroma
Tumor cells (GFP melanoma)
0.4
0.2
Control
% Gated stroma cells with
Control Active 100

Active
90
0.2
0.1
80
106
106
mitochondria
70
60 * 102 103 104 105 102 103 104 105
104 105
104 105
50
SSC-A
SSC-A
FITC (530/30-502)
Stroma
FITC (530/30-502)
40
30
20
103
103
1
10
1
0 Control : tumor
Melanoma
102 103 104 105 106 102 103 104 105 106 Active Inert
Acve : tumor
% Gated
GFP 488- A GFP 488- A mitochondria mitochondria
% Gated
% Gated melanoma cells
100
0.5
Control
0.5
Active
106
106
with mitochondria
80
Melanoma
104 105
104 105
60
SSC-A
SSC-A
40 102 103 104 105 102 103 104 105
20
103
103
Control : tumor Active : tumor

0
102 103 104 105 106 102 103 104 105 106 Control : stroma Active : stroma
Active Inert
GFP 488- A GFP 488- A mitochondria mitochondria *
D
*
15,000 2,500
Glucose uptake of tumor and stroma
glucose analogue
Intensity of FITC
10,000 2,000
5,000 ** 1,500
Organs Single-cell Glucose analogue *
600 1,000
harvesting preparation Sorting Stroma cells FITC
Control/active FITC 400
(organ single cells) 30 min quantification 500
Subcutaneously injected Lymph Lungs 200
with Ret-melanoma nodes
incubation 0 0
Stroma Melanoma Stroma Melanoma
Tumor cells
(mCherry melanoma)
F G H
Lungs Lymph nodes Liver
Control Active Control : S100 Control Active Control : S100 Control Active Control : S100
Active : S100 Active : S100 Active : S100
H&E H&E H&E H&E H&E H&E
Control : Glut1/AldoA/complex1 Control : Glut1/AldoA/complex1 Control : Glut1/AldoA/complex1
T S
Active : Glut1/AldoA/complex1 Active : Glut1/AldoA/complex1 Active : Glut1/AldoA/complex1
Statistics (Stroma to tumor) Statistics (Stroma to tumor) Statistics (Stroma to tumor)
T
+
+
Control : s100 Control : s100
+
Control : s100
Active : s100 Active : s100 Active : s100
Control : Glut1/AldoA/complex1 S Control : Glut1/AldoA/complex1
+
+
Control : Glut1/AldoA/complex1
+
100 µm 100 µm Active : Glut1/AldoA/complex1 100 µm 100 µm
Active : Glut1/AldoA/complex1 100 µm 100 µm Active : Glut1/AldoA/complex1
Mean fluorescence intensity (marker / DAPI)
1.5
GLUT1 GLUT1 GLUT1 GLUT1 2.5 GLUT1 GLUT1 1.5
Mean fluorescence intensity (marker/DAPI)

Mean fluorescence intensity (marker/DAPI)
+++
S100 T
++
S100 S100 1 S100 S100 2 S100 S
1
+++
T *
+
DAPI T DAPI ** DAPI DAPI 1.5 *** DAPI DAPI ***

*** S
+++
1
+++
S 0.5 *** 0.5

T
S T T 0.5
0 S 0
0
S GLUT 1 S100 GLUT1 S100 GLUT1 S100 GLUT1 S100
GLUT1 S100 GLUT1 S100 S
50 µm 50 µm Stroma Tumor 50 µm 50 µm 50 µm Stroma Tumor
50 µm Stroma Tumor
ALDOA ALDOA 0.8 ALDOA 2.5
*** ALDOA ALDOA 1.5 ALDOA S
+++
*** ***
S100 S100 0.6 S100 S100 2
S100 S100
+++
+++
** S 1 1.5
DAPI T DAPI DAPI DAPI
+++
0.4 DAPI ** DAPI

+++
+++
S 1 **
0.2 T S T 0.5
S 0.5
T 0 0 T S 0
ALDOA S100 ALDOA S100 ALDOA S100 ALDOA S100 ALDOA S100 ALDOA S100
Stroma Tumor Stroma Tumor
S
50 µm 50 µm 50 µm 50 µm 50 µm 50 µm Stroma Tumor
1.5 2 2.5
COMPLEX1 COMPLEX1 COMPLEX1
++
COMPLEX1 COMPLEX1 COMPLEX1

1.2 2 ** **
S100 S100 S100 S100 1.5 S100 T
++
S *** S100
++
** S 1.5
++
DAPI DAPI 0.9 DAPI S *

+++
*** DAPI S 1 DAPI DAPI S

S 0.6 1
+++
TT T 0.5 S 0.5
S 0.3 T
T 0 0 T 0
OMPLEX I
S100
OMPLEX I
S100
OMPLEX I
S100
OMPLEX I
S100
COMPLEX I
S100
COMPLEX I
S100
C1 S100 C1 S100 C1 S100 C1 S100 C1 S100 C1 S100

50 µm 50 µm 50 µm 50 µm Stroma Tumor 50 µm Stroma
Stroma Tumor 50 µm Tumor
Figure 4.
Metabolic cross-talk between cancer cells and stroma. A, Left, heatmap of the normalized expression of genes from TCGA data set (rows) classified as benign nevi,
atypical nevi, vertical growth phase melanoma (VGP), and in situ melanoma compared with melanoma metastases of indicated tissues (Supplementary Table S3).
Red, high expression; blue, low expression. Right, KEGG enrichment analysis of the genes upregulated and downregulated in metastatic melanoma. B, KEGG
enrichment analysis if genes downregulated in primary melanomas of mice exercised pre- and post-melanoma cell injection compared with control mice (3). C, Top,
experimental design for the coculture of the melanoma cells and primary cells for mitochondrial activity (stained by TMRE) by FACS (as indicated). Bottom left, FACS
analysis of the mitochondrial activity in GFP-labeled Ret-melanoma cells cocultured with primary lung cells of control and active mice; TMRE expression is indicative
of active mitochondria. Bottom right, calculation of TMREþ cells in primary lung cells (top) and melanoma (bottom) based on the FACS analysis. Error bars, SEM
(n ¼ 6 animals in each group). D, Schematic representation of the glucose uptake assay. E, Top and middle, FACS analysis of the uptake of 2-NBDG by melanoma
(mCherryþ) and stromal (mCherry) cells originating from the lungs and lymph nodes. Bottom, intensity of the signal from 2-NBDG in stroma and melanoma from
lungs and lymph of control and active mice. F–H, Left, hematoxylin and eosin staining (20 magnification) and immunofluorescence analysis (40 magnification) of
S100-beta (green) and GLUT1, ALDOA, or COMPLEX I (red) in lungs (F), lymph nodes (G), and livers (H). Blue, DAPI-stained nuclei. White-dashed lines, tumor (T)–
stroma (S) boundaries. Right, quantification of the red and green mean fluorescence intensities in the tumor and stroma normalized to DAPI mean fluorescence
intensity using ImageJ. t test statistical comparison is represented: , black, a t test GLUT1, ALDOA, or COMPLEX I (red) comparison of the control and active animals in
stroma or tumor regions; þ, a t test GLUT1 (red), ALDOA (red), COMPLEX I (red) of S100-beta (green) comparison of the stroma and tumor regions of the control
animals; , t test GLUT1 (red), ALDOA (red), COMPLEX I (red), or S100-beta (green) comparison of the stroma and tumor regions of active animals. Error bars, SEM
*
[n > 15 fields from each area (stroma or tumor for each target) in at least three images]. þ or or , P < 0.05; þþ or or , P < 0.001; þþþ or or , P < 0.001. * ** ***

following exercise, we reasoned that the two metabolic programs might To examine melanoma metabolic adaptation further, we subjected
be clashing. biopsies of lymph nodes, lungs, and livers harboring metastases from
We then performed an enrichment analysis based on microarray active and control mice to pathology analysis. Melanoma and stroma
data collected in a previous study (3) and found downregulation of were defined based on distinct morphologic changes seen in hema-
metabolic pathways in the primary tumors of active animals compared toxylin and eosin staining and on staining for melanoma-specific
with tumors derived from control animals (Fig. 4B). This suggests that marker S100-beta. We also stained the tissues for GLUT1, ALDOA,
exercise may affect the metabolism of tumor cells directly or alter it and COMPLEX I. The latter takes part in the electron transport chain
indirectly through effects on the tumor microenvironment. In the case and is a marker of oxidative phosphorylation. To allow comparison
of an indirect effect, exercise could increase the uptake of nutrients by between stroma and tumor as well as between active and control
the active stroma microenvironment, thus changing the availability of samples, intensities of GLUT1, ALDOA, and COMPLEX I were
nutrients to tumor cells or exercise might induce stroma cells to secrete normalized to intensities of DAPI staining of the same areas. The
factors that affect the tumor phenotype. This type of competition, stroma of tissues from active mice had significantly higher expression
however opposite to our observations, between tumor and stroma has of metabolic markers than stroma of control animals, but tumor
been previously reported in the literature through the loss of T-cell metastases in tissues from active mice had significantly lower expres-
function due to enhanced glucose uptake by tumor cells leading to sion of these markers compared with metastases of control mice
reduced glucose consumption of tumor-infiltrating T cells. (Fig. 4F–H). When comparing the metastases to its stroma, we found
To evaluate how exercise challenges tumor metabolism, thus affect- significantly higher expression of the metabolic markers in the tumor
ing metastasis development, we examined the metabolism of mela- region compared with stroma in the control mice, whereas in the active
noma cells and primary stoma cells from active or control mice during animals we found the opposite trend (Fig. 4F–H). There were no
coculture. Primary lung cells from control and exercised mice were differences in S100-beta levels in the tumor area of the control and
cocultured with Ret-melanoma cells for 24 hours followed by mito- active mice, nor in the stroma area (Fig. 4F–H). Taken together, these
chondrial activity analysis using TMRE (Supplementary Fig. S5F). results support our hypothesis that exercise, which increases the
Lung stroma cells from active animals showed significant induction in microenvironmental metabolic demand, results in a microenviron-
mitochondrial activity compared with cells from control mice (Fig. 4C, ment that does not support melanoma colonization, thus protecting
top). Interestingly, no significant difference was observed in the against the metastatic spread.
mitochondrial activity of melanoma cells cocultured with the stroma
from active versus control mice (Fig. 4C, bottom).
To explore whether the stroma’s mitochondrial potential can affect Discussion
the tumor mitochondrial activity and growth, we repeated the exper- Mutation, selection, and adaptation processes during cancer pro-
iment (Fig. 4C) in the presence of mitochondria uncouplers and gression likely occur at both the primary tumor and at the metastatic
inhibitors of the ETC including FCCP and actinomycin D (Supple- site (14); however, the dynamics of these processes are poorly under-
mentary Fig. S2B, top). No significant differences were observed in stood. Tracking the metabolism of the tumor and its stroma might be
melanoma cells’ mitochondrial activity; however, melanoma growth an opportunity to reveal the dynamics of these three forces. Dupuy and
was significantly higher when cocultured with active stroma cells colleagues suggest that different metabolic traits that characterize the
treated with both drugs compared with vehicle (Supplementary heterogeneous population of breast cancer cells dictate their selective
Fig. S2B, bottom). This indicates that although only 50% of melanoma preferences to disseminate to specific sites via specific mutations or
cells succeeded in seeding in active tissue compared with control adaptations to metastatic microenvironments that support their met-
(Fig. 3K), melanoma mitochondrial status does not affect their ability abolic demands (46).
to seed in either active or control stroma. Our in vitro and in vivo data demonstrate that approximately 50% of
We next evaluated glucose uptake of primary cells isolated from the the cells succeed to disseminate in active tissue, suggesting that the
metastases in the lungs and lymph nodes of the active and control mice metastasis host organ transformed into a more highly metabolic organ.
(Fig. 4D). The cells were sorted based on the mCherry fluorescence of We challenged the heterogeneous population of the primary tumor,
the Ret-melanoma cells, resulting in two populations: cancer cells and the ability of certain clones to “choose” their metastatic site
(mCherryþ) and stromal cells (mCherry). Cells were then starved for according to their metabolic demands. This favors the selection
30 minutes in glucose-free DMEM, after 30 minutes 2-NBDG was model (14) by which cells from primary tumors metastasize to
added. FACS analysis of the 2-NBDG uptake performed after 30 favorable metabolic niches. Of the melanoma cells that were success-
minutes of incubation revealed significantly higher glucose uptake of fully able to metastasize, we showed that melanoma cells in active mice
the stroma from active mice than control mice (Fig. 4E). In contrast, had reduced their metabolic needs compared with cells in sedentary
melanoma cells from active animals had significantly higher glucose mice. This suggests that metastasis development stems from interac-
uptake than melanoma cells from the control animals (Fig. 4E). Sorted tions with the microenvironment (stromal cells, nutrients, and oxygen;
cells from active and control mice were also subjected to mRNA ref. 47). It is therefore clear that cancer cells’ metabolic state plays a
analysis of Glut4, a marker of glucose sensing, Aldoa, indicative of central role in both selection and adaptation during cancer progres-
glycolysis, and Hspa9, a marker of mitochondrial activity. Stromal cells sion, a hypothesis that requires further investigation to determine the
isolated from active mice had significantly higher expression of these precise dynamics.
metabolic markers compared with the control stroma (Supplementary The classic hallmarks of cancer, including immune response, tumor
Fig. S4B). Whereas, in most instances, tumor cells from active animals vasculature, hypoxia, pH, and autophagy (14), all can be affected by
expressed significantly lower levels of metabolic markers than tumor exercise thus helping to further create a hostile tumor microenviron-
cells from control mice (Supplementary Fig. S4C). These experiments ment against melanoma. We will expand here on two aspects, the
suggest that melanoma cells have less metabolic activity in tissues from immune response, and the extracellular matrix (ECM). Clinically,
exercised mice due to the high metabolic demand of the stroma in these melanoma patients who responded to two types of immunotherapies
animals. (TIL-based or anti–PD-1) had highly increased levels of metabolic

Sheinboim et al.
proteins, mitochondrial activity, and oxidative phosphorylation (48). altering drugs. Although we used melanoma as our preclinical model
Further, glucose consumption by cancer cells has been shown to as it is a highly metastatic cancer, we expect that our findings will
metabolically restrict T-cell activity in the tumor microenvironment translate to other cancers, given that our human studies were not
causing T-cell loss of function (49). Without proper levels of glucose, cancer specific, and our mouse findings refer to the metabolic shift
mTOR activity, as well as activity of other pathways, is reduced in prior to cancer arrival.
T cells (49). In our study, we targeted mTOR activity in the stroma and Glycogen metabolism is upregulated in many tumor types, suggest-
were able to significantly return active stroma cells to the control state, ing that it is an important aspect of cancer cell pathophysiology (56).
in terms of melanoma’s ability to grow when in coculture with treated Because muscle cells require a large amount of glycogen following
active stroma. This supports the hypothesis that exercise may induce bouts of prolonged or high-intensity exercise to return to preexercise
metabolic capabilities of the immune cells in the tumor microenvi- glycogen concentrations, muscles may take up to 24 hours to replace
ronment, thus enhancing the immune response to cancer. Future glycogen stores (57). This phenomenon, which is known as super-
studies should investigate the relationship between metabolic shifts compensation, might protect muscles from metastatic dissemination
and immune system efficacy. because the muscle cells compete with the malignant cells for available
Given that cancer progression and metastasis formation are com- glycogen resources. Further, the super-compensation process might
prised of many factors working together for melanoma to successfully also provide a metabolic shield again tumor seeding in internal
reach the metastatic phase, we recognize that factors such as prolif- organs (58). This should be further investigated. An additional way
eration inhibition may be at play in our model. Because we and to generate competition between normal and cancerous cells is by
others (3) found that the primary tumor is smaller upon exercise, we decreasing the available glucose resources. For example, low-
reasoned that there will be fewer circulating tumor cells and therefore carbohydrate diets, such as the ketogenic diet, have been proposed
fewer metastases; therefore, we bypass this hurdle, and we injected the to starve malignant growth by inhibiting the availability of glycogen
same amount of melanoma cells intercarotid. However, our inter- storage. Several long-term studies with large human cohorts and
cardiac injections also showed less growth and does not uncouple the studies using animal models have shown a correlation between cancer
proliferation defects from metastatic seeding defects. Further studies risk and dietary composition (59). Several factors related to nutrition
are required to understand the direct effect of exercise on in vivo tumor are associated with cancer incidence. For example, overfeeding leads to
proliferation separated from the tumors ability to seed in the metastatic obesity (60), which is associated with impaired whole-body metabo-
niche. lism including a decreased ability to quickly clear and store excess
Another aspect in tumor development is ECM remodeling of the calories. High levels of expression of the tumor progesterone receptor,
premetastatic niche (23). After exercise, the intermuscular ECM is as observed in obese humans and animals who are overfed, are
altered (23). It will be interesting to explore whether changes in associated with a glycolytic–lipogenic phenotype typical of aggressive
ECM-related genes and proteins induced by exercise are also tumors and with high metastatic rates (59). In addition, high-fat diets
observed in metastatic host locations (e.g., lung, liver, lymph and consumption of high amounts of saturated fatty acids increase gut
nodes), in addition to the muscle. We found that metabolic prop- permeability and induce colonic inflammation and mesenteric fat
erties such as glucose uptake and metabolic mitochondrial activity inflammation, which increase overall cancer risk (59). Other factors
marker expression were downregulated in melanoma cells from that are correlated with cancer risk are alcohol consumption, which is
active mice that had metastasized to stroma. Studies have shown detrimental, and fiber intake, which improves cancer immunosurveil-
that dormant cancer cells have lower metabolism than nondormant lance (59). In our human cohort analysis, we controlled for these key
cancer cells (50); thus, melanoma cells that survived in the active factors in the diet to determine the independent effect of the level of
microenvironment may have become dormant. Interestingly, exercise on cancer risk; however, a future study should investigate the
autophagy is activated during nutrient deficiency, and autophagy combination of both nutrition therapy and physical activity to limit
inhibition has been shown to result in the apoptosis of dormant metastatic growth.
breast cancer cells (51). Therefore, dormancy and autophagy should As a final note, it will be important for clinical application to
be explored with our exercise animal model to understand if explore how long the exercise effect lasts and how long-term resting
autophagy inhibition more efficiently induces apoptosis of meta- alters the dissemination of cancer cells. The existing literature is
static cells under an exercise protocol. contradictory. For example, in one study, a very limited amount of
Various agents have been identified that target the metabolism of strength and muscle fiber was retained after 8 weeks of detraining,
either the tumor or the stroma cells of the metastatic niche (14). Some whereas another showed that halting training did not negatively
metabolic enzyme inhibitors are currently being evaluated in clinical affect muscle fibers and breathing capacity benefits due to the initial
trials (52). Tumors can become resistant to such treatments due to exercise regimen (61, 62). Mouse studies are also contradictory,
their metabolic plasticity, allowing them to bypass the blockade of a which might be due to the use of various training methods, resting
specific metabolic pathway by adopting new metabolic traits (53). periods, and measures of exercise retention, as explained in the
Thus, like combined treatments of signaling pathway inhibitors for review by Gundersen and colleagues (63). To investigate the long-
melanoma (54), the simultaneous inhibition of multiple metabolic term effects of exercise and subsequent resting a future study is
pathways should be evaluated (55). Moreover, it has recently been needed. Further, because Olympic athletes and professional athletes
suggested that the immunotherapy resistance of melanoma patients are not immune from developing cancer, even with their high-
can be overcome by combining the treatment with metabolism-related intensity training regimens (64), and exercise intensity depends on
drugs (48). Here, we show that exercise reprograms the metabolic an individual’s ability, proposing a personalized exercise regime for
capacity of nonskeletal tissues known to be niches for metastatic each patient might provide better clinical outcomes.
melanoma as shown by the increased activity of the glycolytic pathway
and increased mitochondrial activity. This suggests that exercise
generates a metabolic shield and may be beneficial in combination Authors’ Disclosures
with immunotherapy as a replacement or in addition to metabolism- No disclosures were reported.

Authors’ Contributions supervision, funding acquisition, investigation, project administration, writing–

review and editing.
D. Sheinboim: Conceptualization, data curation, formal analysis, validation,
investigation, visualization, methodology, writing–original draft, project admin- Acknowledgments
istration. S. Parikh: Data curation, formal analysis, validation, visualization,
methodology, writing–review and editing. P. Manich: Data curation, formal C. Levy and Y. Gepner acknowledge grant support from the Israeli Cancer
analysis, validation, visualization, methodology, writing–review and editing. Association (01031005), the collaboration grant between schools at the Faculty of
I. Markus: Formal analysis. S. Dahan: Data curation, formal analysis, validation, Medicine (0601148551). C. Levy acknowledges grant support from the European
methodology. R. Parikh: Software, formal analysis, validation, methodology. Research Council (ERC) under the European Union’s Horizon 2020 research and
E. Stubbs: Data curation, formal analysis. G. Cohen: Data curation, formal innovation program (grant agreement no. 726225), the I-CORE Gene Regulation in
analysis. V. Zemser-Werner: Resources. R.E. Bell: Data curation, formal analysis, Complex Human Disease Center (no. 41/11), the Melanoma Research Alliance
methodology. S. Arciniegas Ruiz: Methodology. R. Percik: Resources, supervi- (MRA; grant 402792), and Israel Science Foundation (grant 129/13). C. Levy would
sion. R. Brenner: Resources. S. Leibou: Data curation. H. Vaknine: Methodology. like to thank Baruch the glazier from Givataim for his humble support in the technical
G. Arad: Formal analysis. Y. Gerber: Data curation, formal analysis, supervision obstacles of the primary experiment.
of the epidemiological data analysis. L. Keinan Boker: Formal analysis.
T. Shimony: Formal analysis. L. Bikovski: Resources. N. Goldstein: Data The publication costs of this article were defrayed in part by the payment of
curation, formal analysis. K. Constantini: Data curation. S. Labes: Methodology. publication fees. Therefore, and solely to indicate this fact, this article is hereby
S. Mordechai: Resources, formal analysis. H. Doron: Methodology. A. Ionescu: marked “advertisement” in accordance with 18 USC section 1734.
Methodology. T. Ziv: Formal analysis, methodology. E. Nizri: Visualization.
G. Choshen: Methodology. H. Eldar-Finkelman: Methodology. Y. Tabach: Note
Methodology. A. Helman: Methodology. S. Ben-Eliyahu: Methodology. N. Erez: Supplementary data for this article are available at Cancer Research Online
Resources, methodology. E. Perlson: Resources, methodology. T. Geiger: Formal (http://cancerres.aacrjournals.org/).
analysis, methodology. D. Ben-Zvi: Formal analysis, methodology. M. Khaled:
Conceptualization, supervision. Y. Gepner: Resources, data curation, formal analysis, Received January 25, 2022; revised June 16, 2022; accepted August 31, 2022;
supervision, writing–review and editing. C. Levy: Conceptualization, resources, published first September 9, 2022.
References
1. Moore SC, Lee I-M, Weiderpass E, Campbell PT, Sampson JN, Kitahara CM, 18. Liberti MV, Locasale JW. The Warburg effect: how does it benefit cancer cells?
et al. Association of leisure-time physical activity with risk of 26 types of cancer in Trends Biochem Sci 2016;41:211–8.
1.44 million adults. JAMA Intern. Med. 2016;176:816–25. 19. Fu Y, Liu S, Yin S, Niu W, Xiong W, Tan M, et al. The reverse Warburg effect is
2. Brown JC, Gilmore LA. Physical activity reduces the risk of recurrence and likely to be an Achilles’ heel of cancer that can be exploited for cancer therapy.
mortality in cancer patients. Exerc Sport Sci Rev 2020;48:67–73. Oncotarget 2017;8:57813–25.
3. Pedersen L, Idorn M, Olofsson GH, Lauenborg B, Nookaew I, Hansen RH, 20. Curtis M, Kenny HA, Ashcroft B, Mukherjee A, Johnson A, Zhang Y, et al.
et al. Voluntary running suppresses tumor growth through epinephrine- and Fibroblasts mobilize tumor cell glycogen to promote proliferation and metas-
IL-6-dependent NK cell mobilization and redistribution. Cell Metab 2016;23: tasis. Cell Metab 2019;29:141–55.
554–62. 21. Nocquet L, Juin PP, Souaze F. Mitochondria at center of exchanges between
4. Hojman P, Gehl J, Christensen JF, Pedersen BK. Molecular mechanisms linking cancer cells and cancer-associated fibroblasts during tumor progression.
exercise to cancer prevention and treatment. Cell Metab 2018;27:10–21. Cancers (Basel) 2020;12:3017.
5. Pedersen L, Christensen JF, Hojman P. Effects of exercise on tumor physiology 22. Gentric G, Mechta-Grigoriou F. Tumor cells and cancer-associated fibroblasts:
and metabolism. Cancer J 2015;21:111–6. an updated metabolic perspective. Cancers 2021;13:399.
6. Magkos F. Exercise and insulin sensitivity—where do we stand? you’d better run! 23. Nazemi M, Rainero E. Cross-talk between the tumor microenvironment,
US Endocrinol 2008;04:23. extracellular matrix, and cell metabolism in cancer. Front Oncol 2020;
7. Ennour-Idrissi K, Maunsell E, Diorio C. Effect of physical activity on sex 10:239.
hormones in women: a systematic review and meta-analysis of randomized 24. Nieman KM, Kenny HA, Penicka CV, Ladanyi A, Buell-Gutbrod R,
controlled trials. Breast Cancer Res 2015;17:139. Zillhardt MR, et al. Adipocytes promote ovarian cancer metastasis and
8. Koelwyn GJ, Wennerberg E, Demaria S, Jones LW. Exercise in regulation of provide energy for rapid tumor growth. Nat Med 2011;17:1498–503.
inflammation-immune axis function in cancer initiation and progression. 25. Karstoft K, Pedersen BK. Skeletal muscle as a gene regulatory endocrine organ.
Oncology 2015;29:908–22. Curr Opin Clin Nutr Metab Care 2016;19:270–5.
9. Woods JA, Wilund KR, Martin SA, Kistler BM. Exercise, inflammation and 26. Elia I, Schmieder R, Christen S, Fendt S-M. Organ-specific cancer
aging. Aging Dis 2012;3:130–40. metabolism and its potential for therapy. Handb Exp Pharmacol 2016;
10. McTiernan A. Mechanisms linking physical activity with cancer. Nat Rev Cancer 233:321–53.
2008;8:205–11. 27. American College of Sports Medicine, Riebe D, Ehrman JK. 1962-, Liguori G
11. Hawley JA, Hargreaves M, Joyner MJ, Zierath JR. Integrative biology of exercise. 1965-, Magal M. ACSM’s guidelines for exercise testing and prescription. 10th
Cell 2014;159:738–49. ed. Philadelphia: Wolters Kluwer; 2018.
12. Murphy RM, Watt MJ, Febbraio MA. Metabolic communication during exercise. 28. Priego T, Sanchez J, Pico C, Palou A. Sex-differential expression of metabolism-
Nat Metab 2020;2:805–16. related genes in response to a high-fat diet. Obesity (Silver Spring) 2008;16:
13. Ellingsgaard H, Hauselmann I, Schuler B, Habib AM, Baggio LL, Meier DT, et al. 819–26.
Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 29. Fainstein N, Tyk R, Touloumi O, Lagoudaki R, Goldberg Y, Agranyoni O, et al.
secretion from L cells and alpha cells. Nat Med 2011;17:1481–9. Exercise intensity-dependent immunomodulatory effects on encephalomyelitis.
14. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; Ann Clin Transl Neurol 2019;6:1647–58.
144:646–74. 30. Parikh R, Sorek E, Parikh S, Michael K, Bikovski L, Tshori S, et al. Skin exposure
15. Faubert B, Solmonson A, DeBerardinis RJ. Metabolic reprogramming and cancer to UVB light induces a skin-brain-gonad axis and sexual behavior. Cell Rep 2021;
progression. Science 2020;368:eaaw5473. 36:109579.
16. Levine AJ, Jenkins NA, Copeland NG. The roles of initiating truncal mutations in 31. The Uniprot Consortium. UniProt: a worldwide hub of protein knowledge.
human cancers: the order of mutations and tumor cell type matters. Cancer Cell Nucleic Acids Res. 2019;47:D506–15;
2019;35:10–5. 32. Tyanova S, Temu T, Sinitcyn P, Carlson A, Hein MY, Geiger T, et al. The Perseus
17. Pavlova NN, Thompson CB. The emerging hallmarks of cancer metabolism. computational platform for comprehensive analysis of (prote)omics data.
Cell Metab 2016;23:27–47. Nat Methods 2016;13:731–40.

Sheinboim et al.
33. The Gene Ontology Consortium. The Gene Ontology resource: enriching a GOld 51. Vera-Ramirez L, Vodnala SK, Nini R, Hunter KW, Green JE. Autophagy
mine. Nucleic Acids Res 2021;49:D325–34. promotes the survival of dormant breast cancer cells and metastatic tumour
34. Journe F, Id Boufker H, Van Kempen L, Galibert MD, Wiedig M, Sales F, et al. recurrence. Nat Commun 2018;9:1944.
TYRP1 mRNA expression in melanoma metastases correlates with clinical 52. Akins NS, Nielson TC, Le HV. Inhibition of glycolysis and glutaminolysis: an
outcome. Br J Cancer 2011;105:1726–32. emerging drug discovery approach to combat cancer. Curr Top Med Chem 2018;
35. Coker RH, Kjaer M. Glucoregulation during exercise : the role of the neuroen- 18:494–504.
docrine system. Sports Med 2005;35:575–83. 53. Nguyen T, Kirsch BJ, Asaka R, Nabi K, Quinones A, Tan J, et al. Uncovering the
36. Chen LB. Mitochondrial membrane potential in living cells. Annu Rev Cell Biol role of N-acetyl-aspartyl-glutamate as a glutamate reservoir in cancer. Cell Rep
1988;4:155–81. 2019;27:491–501.
37. Neufer PD, Bamman MM, Muoio DM, Bouchard C, Cooper DM, Goodpaster BH, 54. Greger JG, Eastman SD, Zhang V, Bleam MR, Hughes AM, Smitheman KN,
et al. Understanding the cellular and molecular mechanisms of physical activity- et al. Combinations of BRAF, MEK, and PI3K/mTOR inhibitors over-
induced health benefits. Cell Metab 2015;22:4–11. come acquired resistance to the BRAF inhibitor GSK2118436 dabrafe-
38. Cheeseman C, Long W. Structure of, and functional insight into the GLUT family nib, mediated by NRAS or MEK mutations. Mol Cancer Ther 2012;11:
of membrane transporters. Cell Health Cytoskelet 2015;167. 909–20.
39. Wang T, Wang J, Hu X, Huang X-J, Chen G-X. Current understanding of glucose 55. Kreuzaler P, Panina Y, Segal J, Yuneva M. Adapt and conquer: metabolic
transporter 4 expression and functional mechanisms. World J Biol Chem 2020; flexibility in cancer growth, invasion and evasion. Mol Metab 2020;33:
11:76–98. 83–101.
40. Sylow L, Kleinert M, Richter EA, Jensen TE. Exercise-stimulated glucose uptake 56. Zois CE, Harris AL 2016, Glycogen metabolism has a key role in the cancer
regulation and implications for glycaemic control. Nat Rev Endocrinol 2017;13: microenvironment and provides new targets for cancer therapy. J Mol Med. 94:
133–48. 137–54.
41. Clemmons DR. Involvement of insulin-like growth factor-I in the control of 57. Takahashi Y, Sarkar J, Yamada J, Matsunaga Y, Nonaka Y, Banjo M, et al.
glucose homeostasis. Curr Opin Pharmacol 2006;6:620–5. Enhanced skeletal muscle glycogen repletion after endurance exercise is asso-
42. Netz Y, Goldsmith R, Shimony T, Ben-Moshe Y, Zeev A. Adherence to physical ciated with higher plasma insulin and skeletal muscle hexokinase 2 protein levels
activity recommendations in older adults: an Israeli national survey. J Aging Phys in mice: comparison of level running and downhill running model. J Physiol
Act 2011;19:30–47. Biochem 2021;77:469–80.
43. Cohen G, Steinberg DM, Keinan-Boker L, Shaked O, Goshen A, Shimony T, et al. 58. Hingst JR, Bruhn L, Hansen MB, Rosschou MF, Birk JB, Fentz J, et al. Exercise-
Leisure-time physical activity and cancer risk among older adults: a cohort study. induced molecular mechanisms promoting glycogen supercompensation in
Mayo Clin Proc Innov Qual Outcomes 2020;4:115–25. human skeletal muscle. Mol Metab 2018;16:24–34.
44. Moore E, Silverman BG, Fishler Y, Ben-Adiva E, Davidov O, Dichtiar R, et al. An 59. Zitvogel L, Derosa L, Kroemer G. Modulation of cancer immunotherapy
assessment of the completeness and timeliness of the Israel national cancer by dietary fibers and over-the-counter probiotics. Cell Metab 2022;34:
registry. Isr Med Assoc J 2021;23:23–7. 350–2.
45. Ballou LM, Lin RZ. Rapamycin and mTOR kinase inhibitors. J Chem Biol 2008;1: 60. Giles ED, Wellberg EA, Astling DP, Anderson SM, Thor AD, Jindal S, et al.
27–36. Obesity and overfeeding affecting both tumor and systemic metabolism activates
46. Dupuy F, Tabaries S, Andrzejewski S, Dong Z, Blagih J, Annis MG, et al. PDK1- the progesterone receptor to contribute to postmenopausal breast cancer.
dependent metabolic reprogramming dictates metastatic potential in breast Cancer Res 2012;72:6490–501.
cancer. Cell Metab 2015;22:577–89. 61. Taaffe DR, Marcus R. Dynamic muscle strength alterations to detraining and
47. Vander Heiden MG, DeBerardinis RJ. Understanding the intersections between retraining in elderly men. Clin Physiol 1997;17:311–24.
metabolism and cancer biology. Cell 2017;168:657–69. 62. Meyer K, Schwaibold M, Westbrook S, Beneke R, Hajric R, G€ornandt L, et al.
48. Harel M, Ortenberg R, Varanasi SK, Mangalhara KC, Mardamshina M, Effects of short-term exercise training and activity restriction on functional
Markovits E, et al. Proteomics of melanoma response to immunotherapy capacity in patients with severe chronic congestive heart failure. Am J Cardiol
reveals mitochondrial dependence. Cell 2019;179:236–50. 1996;78:1017–22.
49. Pitt JM, Vetizou M, Daillere R, Roberti MP, Yamazaki T, Routy B, et al. 63. Gundersen K. Muscle memory and a new cellular model for muscle atrophy and
Resistance mechanisms to immune-checkpoint blockade in cancer: tumor- hypertrophy. J Exp Biol 2016;219:235–42.
intrinsic and -extrinsic factors. Immunity 2016;44:1255–69. 64. Luo H, Galv~ao DA, Newton RU, Fairman CM, Taaffe DR. Sport medicine
50. Phan TG, Croucher PI. The dormant cancer cell life cycle. Nat Rev Cancer 2020; in the prevention and management of cancer. Integr Cancer Ther 2019;18:
20:398–411. 1534735419894063.

Exercise Induced Metabolic Shield Blocks Cancer

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CANCER RESEARCH | METABOLISM AND CHEMICAL BIOLOGY

An Exercise-Induced Metabolic Shield in Distant Organs

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A Excise organs Mass spectrometry

B Lungs Lymph nodes Liver Muscle

Enriched KEGG Enriched KEGG Enriched KEGG Enriched KEGG

(693) (523) Cellular metabolic process (60) Intracellular anatomical structure

E Glucose uptake F Glycolytic function G TMRE mitochondrial potential

Control Active Control Active TMRE+ TMRE+

Relative mRNA levels (fold change)

FITC fluorescence intensity Time (min) mcherry 561__610-20-A / SSC-A TMRE

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A Human exercise model B Upregulated pathways post exercise C Top 4 GO terms

D E Low–moderate intensity vs. high intensity F SEER 0-4 SEER 7

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Relative mRNA levels (fold change)

Photons/sec/cm2 (fold change)

Tumor measurement Pre 1.2 1.2

Tumor volume (mm3)

C Control Active J Control Active

Melanoma - mCherry Melanoma - mCherry

% mCherry+ cells/Total count

mcherry+ cells (fold change)

Melanoma - mCherry Melanoma - mCherry

104 105 106

PE-Texas Red - A PE-Texas Red - A 103

D 102 103 104 105

8 weeks 2–4 weeks 0.8

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Immune related Metabolism related Metabolism related Immune related

C Melanoma and primary cell coculture E Glucose uptake by FACS

Control Active 100

40 102 103 104 105 102 103 104 105

Control : tumor Active : tumor

Control : s100 Control : s100

Mean fluorescence intensity (marker/DAPI)

DAPI T DAPI ** DAPI DAPI 1.5 *** DAPI DAPI ***

S 0.5 *** 0.5

0.4 DAPI ** DAPI

COMPLEX1 COMPLEX1 COMPLEX1

DAPI DAPI 0.9 DAPI S *

*** DAPI S 1 DAPI DAPI S

C1 S100 C1 S100 C1 S100 C1 S100 C1 S100 C1 S100

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Authors’ Contributions supervision, funding acquisition, investigation, project administration, writing–

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DAPI T DAPI DAPI DAPI 1.5 * DAPI DAPI ***