Exercise Training Improves Tumor Control by Increasing CD8+ T-cell Infiltration via CXCR3 Signaling and Sensitizes Breast Cancer to Immune Checkpoint Blockade

Depletion of CD8+ T cells prevents the antitumor effect of exercise training.

“CD8+ T-cell depletion prevented the antitumor effect of ExTr.”

• Also stated in: § CD8+ T cells are necessary for the antitumor effect of ExTr · hedged

  “CD8+ T-cell depletion completely abrogated ExTr-induced tumor growth delay (Fig. 5B–C; individual values and time to triple tumor volume in Supplementary Fig. S9A-D; comparison between tumor growth curve from anti-CD8 and IgG in Supplementary Fig. S9E), indicating that CD8+ T cells’ tumor-infiltration is necessary for the antitumor effect of ExTr.”

The antitumor effects of exercise training are abrogated in Cxcr3−/− mice.

“The recruitment of CD8+ T cells and the antitumor effects of ExTr were abrogated in Cxcr3−/− mice, supporting the causal role of the CXCL9/CXCL11-CXCR3 pathway.”

Exercise training induced metabolic reprogramming by reducing glucose uptake/insulin pathways.

“ExTr induced a metabolic reprogramming by reducing glucose uptake/insulin pathways and upregulated several gene sets associated with oxidative metabolism, such as oxidative phosphorylation (OXPHOS).”

Exercise training increased the percentage of tumor vessels associated with αSMA+ perivascular cells in E0771 and MCa-M3C models.

“ExTr led to an increased percentage of vessels associated with αSMA+ perivascular cells (Fig. 2C–D), an indicator of vascular normalization.”

Exercise training sensitizes immune checkpoint blockade-refractory breast cancers to treatment.

“ExTr also sensitized ICB-refractory BCs to treatment.”

• AssumptionRests on an assumption: An abnormal tumor microenvironment caused partly by poor perfusion excludes CD8+ T cells and reduces the effect of immunotherapies such as immune checkpoint blockade.

Exercise training increased the fraction of perfused vessels.

“ExTr also increased the fraction of vessels that were perfused (Fig. 2E–F), indicating improved vascular function.”

Exercise training did not change vessel density.

“ExTr did not change vessel density (Fig. 2A-B) but promoted tumor vessels’ maturation.”

Exercise training upregulated gene sets associated with oxidative metabolism, such as OXPHOS.

“ExTr induced a metabolic reprogramming by reducing glucose uptake/insulin pathways and upregulated several gene sets associated with oxidative metabolism, such as oxidative phosphorylation (OXPHOS).”

Exercise training downregulated several pathways involved with immunosuppression in breast cancer.

“It also enhanced gene expression linked to cytokine activation and downregulated several pathways involved with immunosuppression in BC (20,40,41), whose inhibition is demonstrated in the clinic to synergize with anti-PD-1 against solid tumors (42).”

Exercise training enhanced gene expression linked to cytokine activation.

“It also enhanced gene expression linked to cytokine activation and downregulated several pathways involved with immunosuppression in BC (20,40,41), whose inhibition is demonstrated in the clinic to synergize with anti-PD-1 against solid tumors (42).”

Exercise training reduced tumor hypoxia.

“More functional vessels resulted in reduced hypoxia (Fig. 2G–H), indicating normalization of TME.”

Exercise training increased Ki67 expression in CD8+ T cells.

“The proliferation marker Ki67 was elevated in ExTr groups (Fig. 4E–F).”

Exercise training-induced recruitment of CD8+ T cells is abrogated in Cxcr3−/− mice.

“The recruitment of CD8+ T cells and the antitumor effects of ExTr were abrogated in Cxcr3−/− mice, supporting the causal role of the CXCL9/CXCL11-CXCR3 pathway.”

Exercise training increased the fraction of CD8+ T cells expressing granzyme B.

“ExTr increased the fraction of effector CD8+ T cells expressing granzyme B (Fig. 4G–H and) and IFNγ (Fig. 4I–J).”

In that human dataset, the exercise group also showed increased expression of pathways indicating vessel normalization.

“We found via GSEA that in the ExTr group, there was upregulation of transcriptional pathways consistent with antitumor immune activity and vessel normalization in the BC patients (Fig. 3C–D), similar to what we found in mice.”

• cites [38] Ligibel, 2019, Impact of a pre-operative exercise intervention on breast cancer proliferation and gene expression: results from the Pre-Operative Health and Body (PreHAB) Study, Clin Cancer Res sourceNot checked yet

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Exercise training boosts infiltration of CD8+ T cells into the breast cancer tumor microenvironment.

“ExTr boosted CD8+ T-cell infiltration, with enhanced effector function.”

• Also stated in: § ExTr increases the infiltration of CD8+ T cells into BC TME · plain

  “we found that ExTr increased the infiltration of CD8+ T cells into the BC TME (Fig. 4A-D, Supplementary Fig. S6A–B).”

Exercise training increased the fraction of CD8+ T cells expressing IFNγ.

“and IFNγ (Fig. 4I–J).”

In mouse models of established breast cancer, exercise training slowed tumor growth.

“ExTr delayed tumor growth and induced vessel normalization, demonstrated by increased pericyte coverage and perfusion, and decreased hypoxia.”

• Also stated in: § ExTr delays tumor growth in established BCs · plain

  “Using different models, we showed that ExTr delayed tumor growth in BC.”

In the human breast cancer dataset from Ligibel et al., the exercise group showed increased expression of pathways indicating antitumor immune activity.

“We found via GSEA that in the ExTr group, there was upregulation of transcriptional pathways consistent with antitumor immune activity and vessel normalization in the BC patients (Fig. 3C–D), similar to what we found in mice.”

• cites [38] Ligibel, 2019, Impact of a pre-operative exercise intervention on breast cancer proliferation and gene expression: results from the Pre-Operative Health and Body (PreHAB) Study, Clin Cancer Res sourceNot checked yet

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Exercise training did not change CXCL10 in both models.

“There was no change in CXCL10 in both models (Fig. 6C).”

The groups did not differ in CD8+ T-cell IFNγ expression.

“Majority of the CD8+ T cells expressed IFNγ+, and there was no difference between groups (Fig. 6J).”

Removing CD8+ T cells eliminated ExTr's antitumor effect.

“The depletion of CD8+ T cells abolished the antitumor effect of ExTr, confirming that the mechanism of antitumor activity from ExTr is CD8+ T cell-dependent.”

Only the ExTr plus ICB group showed an increased fraction of granzyme B-positive CD8+ T cells.

“ExTr also increased the total number of intratumoral granzyme B+CD8+ T cells with ExTr, although the fraction of granzyme B+CD8+ T cells was increased only in the ExTr+ICB group (Fig. 7E–H).”

In Cxcr3−/− mice bearing E0771 tumors, the absence of Cxcr3 eliminated the tumor-control effect of ExTr, based on growth curves and tumor weight at day 11.

“Loss of Cxcr3 completely abolished antitumor effects of ExTr, as assessed by tumor growth kinetics and final tumor weight at day 11 (Fig. 6F–G).”

• Also stated in: § Discussion · boosted

  “ExTr antitumor effect was entirely abolished in Cxcr3−/− mice, demonstrating that the CXCR3 pathway participates in the antitumor effect of ExTr.”

The two ExTr groups had comparable CD8+ T-cell infiltration.

“Further analysis indicated similar infiltration of CD8+ T cells in both groups subjected to ExTr (Fig. 7D–G).”

A greater increase in maximal aerobic exercise capacity correlates with higher CD8+ TIL infiltration.

“The higher the increase in maximal aerobic exercise capacity (a biomarker of response to ExTr), the higher was the infiltration of CD8+ TILs (Supplementary Fig. S6D).”

In MCa-M3C tumors, exercise training raised the count of CXCR3-positive CD8+ T cells by over threefold.

“We also observed that ExTr increased the absolute number of CD8+ T cells expressing CXCR3 more than 3-fold in MCa-M3C (p<0.05) and 2-fold in the E0771 (p=0.07) tumor (Fig. 6E).”

CXCL9+ immune cells were predominantly macrophages.

“CXCL9+ immune cells were predominantly macrophages (Supplementary Fig. S10H–I).”

CXCL9 showed a trend towards increase in MCa-M3C.

“CXCL9 also showed a trend towards increase in MCa-M3C (Fig. 6B).”

ExTr caused a modest but significant rise in CD8+ T cells in Cxcr3-KO mice.

“ExTr induced a small but significant increase in CD8+ T cells in Cxcr3-KO mice, suggesting CXCR3-independent mechanisms of CD8+ T-cell recruitment (Fig. 6H–I).”

In E0771 tumors, exercise training raised the count of CXCR3-positive CD8+ T cells by about twofold, with p=0.07.

“We also observed that ExTr increased the absolute number of CD8+ T cells expressing CXCR3 more than 3-fold in MCa-M3C (p<0.05) and 2-fold in the E0771 (p=0.07) tumor (Fig. 6E).”

Relative exercise intensity differs between C57BL/6 and FVB female mice.

“Our study demonstrated that the relative exercise intensity for C57BL/6 versus FVB female mice are distinct.”

Exercise training increased the total number of CD8+ T cells.

“However, ExTr significantly increased the total number of CD8+ T cells, contributing to the increased number of exhausted PD-1+CTLA-4+ T cells within the tumors (Supplementary Fig. S8F).”

ExTr prevented fatigue caused by cancer in mice receiving ICB.

“ExTr also prevented cancer-induced fatigue in ICB-treated mice (Supplementary Fig. S11G).”

Preclinical models show considerable variation in ExTr dose.

“We found a significant variability of ExTr dose within preclinical models, even though it is usually assumed that the same ExTr velocity would fit all mouse strains and experimental conditions.”

• AssumptionThe authors state this as an assumption: A single exercise training velocity is suitable for every mouse strain and experimental condition.

Exercise training did not change the percentage of exhausted CD8+ T cells in MCa-M3C.

“We found that ExTr significantly reduced CD8+ T cells showing an exhausted phenotype in E0771 and did not change the percentage in MCa-M3C (Supplementary Fig. S8E).”

Exercise training reduced CD8+CTLA-4+ cells in MCa-M3C tumors.

“We found ExTr reduced the amount of CD8+CTLA-4+ in MCa-M3C tumors and CD8+LAG3+ in E0771 tumors.”

Exercise training increased the number of infiltrating CD8+ T cells per field.

“indicated by the increased number of infiltrating CD8+ T cells per field (Fig. 4D).”

In tumors from exercised mice, exhausted CD8+ T-cell numbers rose in proportion to the greater total CD8+ T-cell recruitment.

“However, due to the increased total CD8+ T-cell recruitment in ExTr mice tumors, the number of exhausted CD8+ T cells also increased proportionally.”

Exercise training increased relative and absolute numbers of CD8+ tumor-infiltrating lymphocytes.

“ExTr consistently boosted CD8+ T-cell infiltration, indicated by changes in relative (Fig. 4A) and absolute numbers (Fig. 4B) of CD8+ TILs.”

Immune checkpoint blockade alone did not affect orthotopic breast cancers.

“We found no effect of ICB alone in the orthotopic BCs (Fig. 7B-C, Supplementary Fig. S11).”

Proteome array identified CXCL9 and CXCL11 as potential candidates.

“we conducted a proteome profile array of bulk tumors, and identified CXCL9 and CXCL11 as potential candidates (Fig. 6A, Supplementary Fig. S10).”

The treadmill protocol involved 10-20 times less exercise volume than the 20 km per day possible in voluntary running.

“Unlike the voluntary running model, in which mice can run 20 km per day (51), our treadmill model’s exercise volume is 10-20 times less than this.”

• cites [51] Manzanares, 2018, Voluntary wheel running: patterns and physiological effects in mice, Braz J Med Biol Res sourceNot checked yet

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ExTr raised the total count of intratumoral granzyme B-positive CD8+ T cells.

“ExTr also increased the total number of intratumoral granzyme B+CD8+ T cells with ExTr, although the fraction of granzyme B+CD8+ T cells was increased only in the ExTr+ICB group (Fig. 7E–H).”

Exercise training reduced CD8+LAG3+ cells in E0771 tumors.

“We found ExTr reduced the amount of CD8+CTLA-4+ in MCa-M3C tumors and CD8+LAG3+ in E0771 tumors.”

Exercise training increased the number of exhausted PD-1+CTLA-4+ T cells within tumors.

“However, ExTr significantly increased the total number of CD8+ T cells, contributing to the increased number of exhausted PD-1+CTLA-4+ T cells within the tumors (Supplementary Fig. S8F).”

In E0771 tumors from Cxcr3−/− mice, CD8+ T-cell counts were ten times lower than in wild-type mice after seven days of ExTr.

“The number of CD8+ T cells in E0771 tumors grown in Cxcr3−/− mice was 10-fold lower than that observed in wild-type mice after 7 days of ExTr (Fig. 6H).”

Exercise training significantly increased CXCL9 in E0771 tumors.

“We found ExTr significantly increased CXCL9 in E0771 and CXCL11 in the MCa-M3C model (Fig. 6B–D).”

Exercise training reduced the percentage of CD8+ T cells with an exhausted phenotype (PD-1+CTLA-4+) in E0771.

“We found that ExTr significantly reduced CD8+ T cells showing an exhausted phenotype in E0771 and did not change the percentage in MCa-M3C (Supplementary Fig. S8E).”

Changes in tumor volume inversely correlate with CD8+ TIL density.

“Changes in tumor volume were inversely correlated with CD8+ TIL density (Supplementary Fig. S6C).”

Exercise training expanded the spread of CD8+ T cells inside the tumor microenvironment.

“we found that ExTr increased the intratumoral distribution of CD8+ T cells within the TME (Fig. 4C)”

Exercise training raised the count of PD-L1-expressing non-immune cells.

“ExTr also increased the number of non-immune cells expressing PD-L1 (Supplementary Fig. S8C).”

Exercise training significantly increased CXCL11 in MCa-M3C tumors.

“We found ExTr significantly increased CXCL9 in E0771 and CXCL11 in the MCa-M3C model (Fig. 6B–D).”

CD8+ T-cell frequency was reduced in Cxcr3−/− mice relative to wild-type mice.

“The frequency of CD8+ T cells was also lower in Cxcr3−/− mice (Fig. 6I).”

In our study, 12 m/min would have been about 35% of maximal running capacity for C57BL/6 mice, not 60%.

“This velocity would have represented ~35% of maximal running capacity for C57BL/6 mice in our study, instead of 60%.”

Many infiltrating CD8+ T cells in Cxcr3−/− mice expressed IFNγ.

“Indeed, a large number of infiltrating CD8+ T cells expressed IFNγ in Cxcr3−/− mice.”

ExTr and ICB together synergistically increase effector CD8+ T-cell activity, measured by IFNγ- or granzyme B-expressing CD8+ T cells.

“Our work shows that ExTr and ICB synergistically enhance the activity of effector CD8+ T cells (i.e., a fraction of IFNγ- or granzyme B-expressing CD8+ T cells).”

ExTr increased PD-L1-expressing cell numbers, a response biomarker in metastatic breast cancer.

“ExTr increased the number of cells expressing PD-L1, a biomarker of response for metastatic BC (25).”

• cites [25] Savas, 2018, Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis, Nat Med sourceNot checked yet

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After ExTr, many CD8+ T cells still express immune checkpoint receptors.

“ExTr increases CD8+ T cells by reprogramming the TME towards a less aggressive phenotype, but a large fraction of CD8+ T cells remain expressing immune checkpoint receptors, suggesting these tumors would benefit from ICB therapy.”

ExTr raises CD8+ T-cell numbers by reprogramming the TME toward a less aggressive phenotype.

“ExTr increases CD8+ T cells by reprogramming the TME towards a less aggressive phenotype, but a large fraction of CD8+ T cells remain expressing immune checkpoint receptors, suggesting these tumors would benefit from ICB therapy.”

Infiltrating CD8+ T cells can express multiple immune checkpoints, which causes them to become exhausted.

“A varying degree of infiltrating CD8+ T cells express multiple immune checkpoints (e.g., co-expression of programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)), which cause these cells to become exhausted (26).”

• cites [26] Beavis, 2018, Dual PD-1 and CTLA-4 checkpoint blockade promotes antitumor immune responses through CD4(+)Foxp3(-) cell-mediated modulation of CD103(+) dendritic cells, Cancer Immunol Res sourceNot checked yet

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A less hypoxic tumor microenvironment makes tumors less aggressive.

“As a consequence, the TME becomes less hypoxic, rendering tumors less aggressive (14).”

• cites [14] Incio, 2018, Obesity promotes resistance to anti-VEGF therapy in breast cancer by up-regulating IL-6 and potentially FGF-2, Sci Transl Med sourceNot checked yet

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Exercise affects the tumor microenvironment.

“Because exercise is a homeostatic physiological intervention, it also affects the tumor microenvironment (TME) (5-10).”

• cites [5] McCullough, 2014, Modulation of blood flow, hypoxia, and vascular function in orthotopic prostate tumors during exercise, J Natl Cancer Inst sourceNot checked yet

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• cites [6] Betof, 2015, Modulation of murine breast tumor vascularity, hypoxia and chemotherapeutic response by exercise, J Natl Cancer Inst sourceNot checked yet

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• cites [7] Schadler, 2016, Tumor vessel normalization after aerobic exercise enhances chemotherapeutic efficacy, Oncotarget sourceNot checked yet

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• cites [8] Pedersen, 2016, Voluntary running suppresses tumor growth through epinephrine- and IL-6-dependent NK cell mobilization and redistribution, Cell Metab sourceNot checked yet

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• cites [9] Miller, 2016, Perfusion pressure is a critical determinant of the intratumoral extravasation of oncolytic viruses, Mol Ther sourceNot checked yet

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• cites [10] Rundqvist, 2020, Cytotoxic T-cells mediate exercise-induced reductions in tumor growth, Elife sourceNot checked yet

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A transient rise in mean arterial pressure during exercise increases tumor perfusion.

“any transient increase in mean arterial pressure, as seen during exercise, increases tumor perfusion (13).”

• cites [13] Zlotecki, 1995, Pharmacologic modification of tumor blood flow and interstitial fluid pressure in a human tumor xenograft: network analysis and mechanistic interpretation, Microvasc Res sourceNot checked yet

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Poor prognosis in breast cancer is associated with restricted T-cell recruitment and function within the tumor microenvironment.

“Limited T-cell recruitment and function in the TME correlates with poor prognosis in BC (21,22).”

• cites [21] Chung, 2017, Single-cell RNA-seq enables comprehensive tumour and immune cell profiling in primary breast cancer, Nat Commun sourceNot checked yet

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• cites [22] Denkert, 2018, Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy, Lancet Oncol sourceNot checked yet

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Better tumor blood vessels support adaptive antitumor immunity by helping tumor-infiltrating lymphocytes traffic and relocate, which improves results of immunotherapies such as immune checkpoint blockade.

“An improved vascular network favors adaptive antitumor immunity by promoting trafficking and re-localization of tumor-infiltrating lymphocytes (TILs), leading to improved outcomes of various immunotherapies, including immune checkpoint blockade (ICB) (15-20).”

• cites [15] Huang, 2012, Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy, Proc Natl Acad Sci U S A sourceNot checked yet

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• cites [16] Ashcraft, 2019, Exercise as adjunct therapy in cancer, Semin Radiat Oncol sourceNot checked yet

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• cites [17] Zheng, 2018, Increased vessel perfusion predicts the efficacy of immune checkpoint blockade, J Clin Invest sourceNot checked yet

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• cites [18] Tian, 2017, Mutual regulation of tumour vessel normalization and immunostimulatory reprogramming, Nature sourceNot checked yet

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• cites [19] Schmittnaegel, 2017, Dual angiopoietin-2 and VEGFA inhibition elicits antitumor immunity that is enhanced by PD-1 checkpoint blockade, Sci Transl Med sourceNot checked yet

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• cites [20] Chen, 2019, Blocking CXCR4 alleviates desmoplasia, increases T-lymphocyte infiltration, and improves immunotherapy in metastatic breast cancer, Proc Natl Acad Sci U S A sourceNot checked yet

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Vascular normalization can increase immune cell infiltration into tumors.

“We previously demonstrated that vascular normalization could increase immune cells’ infiltration into tumors, and multiple studies have confirmed these findings in several tumor models (15,17-19).”

• cites [15] Huang, 2012, Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy, Proc Natl Acad Sci U S A sourceNot checked yet

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• cites [17] Zheng, 2018, Increased vessel perfusion predicts the efficacy of immune checkpoint blockade, J Clin Invest sourceNot checked yet

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• cites [18] Tian, 2017, Mutual regulation of tumour vessel normalization and immunostimulatory reprogramming, Nature sourceNot checked yet

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• cites [19] Schmittnaegel, 2017, Dual angiopoietin-2 and VEGFA inhibition elicits antitumor immunity that is enhanced by PD-1 checkpoint blockade, Sci Transl Med sourceNot checked yet

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Exercise training increases effector activity of CD8+ T cells.

“with enhanced effector function.”

• Also stated in: § ExTr enhances the effector function of CD8+ T cells · plain

  “ExTr enhances the effector function of CD8+ T cells”

Exercise training causes normalization of tumor blood vessels in mouse breast cancer models.

“induced vessel normalization, demonstrated by increased pericyte coverage and perfusion, and decreased hypoxia.”

• Also stated in: § ExTr normalizes the tumor vasculature and TME · plain

  “ExTr induced vessel normalization in both E0771 and MCa-M3C BC models.”

The CXCL9/CXCL11-CXCR3 pathway causally mediates exercise training's effects.

“supporting the causal role of the CXCL9/CXCL11-CXCR3 pathway.”

Preclinical studies show that tumor perfusion and oxygenation rise from the first exercise session.

“Preclinical studies indicate that tumor perfusion and oxygenation increase from the very first exercise session (5,9,11).”

• cites [5] McCullough, 2014, Modulation of blood flow, hypoxia, and vascular function in orthotopic prostate tumors during exercise, J Natl Cancer Inst sourceNot checked yet

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• cites [9] Miller, 2016, Perfusion pressure is a critical determinant of the intratumoral extravasation of oncolytic viruses, Mol Ther sourceNot checked yet

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• cites [11] Garcia, 2016, Blood flow responses to mild-intensity exercise in ectopic vs. orthotopic prostate tumors; dependence upon host tissue hemodynamics and vascular reactivity, J Appl Physiol (1985) sourceNot checked yet

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CD8+ T-cell infiltration into tumors is necessary for exercise training's antitumor effect.

“indicating that CD8+ T cells’ tumor-infiltration is necessary for the antitumor effect of ExTr.”

Poor control of exercise dose may explain the wide variability and lack of significant findings in clinical exercise trials.

“More importantly, the lack of proper control of exercise dose might account for the large variability and absence of significant results from exercise interventions in clinical trials (2).”

• cites [2] Iyengar, 2019, Development of exercise as interception therapy for cancer: a review, JAMA Oncol sourceNot checked yet

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Only supervised exercise training with planned duration and intensity has demonstrated benefit.

“It seems only supervised ExTr, accounting for planned interventions considering both duration and intensity, has been proven beneficial (3,53-55).”

• cites [3] Williams, 2014, Significantly greater reduction in breast cancer mortality from post-diagnosis running than walking, Int J Cancer sourceNot checked yet

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• cites [53] Park, 2015, The effect of oncologists' exercise recommendations on the level of exercise and quality of life in survivors of breast and colorectal cancer: a randomized controlled trial, Cancer sourceNot checked yet

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• cites [54] Buffart, 2017, Effects and moderators of exercise on quality of life and physical function in patients with cancer: an individual patient data meta-analysis of 34 RCTs, Cancer Treat Rev sourceNot checked yet

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• cites [55] Sweegers, 2018, Which exercise prescriptions improve quality of life and physical function in patients with cancer during and following treatment? A systematic review and meta-analysis of randomised controlled trials, Br J Sports Med sourceNot checked yet

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The running wheel model is not a reliable preclinical model for therapeutic ExTr.

“The problem is that, along with the much larger exercise volume that does not recapitulate human exercise behavior, the running wheel model does not provide a reliable preclinical model on the therapeutic ExTr either.”

Earlier therapeutic ExTr studies using treadmills failed to show increases in cytotoxic NK or CD8+ T cells.

“Previous therapeutic ExTr interventions (i.e., starting treatment when tumors were established) on running treadmills could not demonstrate enhancement of cytotoxic NK nor CD8+ T cells (7,52,56).”

• cites [7] Schadler, 2016, Tumor vessel normalization after aerobic exercise enhances chemotherapeutic efficacy, Oncotarget sourceNot checked yet

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• cites [52] Colbert, 2009, Exercise effects on tumorigenesis in a p53-deficient mouse model of breast cancer, Med Sci Sports Exerc sourceNot checked yet

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• cites [56] Glass, 2017, Differential response to exercise in claudin-low breast cancer, Oncotarget sourceNot checked yet

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Exercise dose substantially influences tumor physiology.

“Emerging data suggest that exercise dose plays a significant role in modulating tumor physiology (2,52).”

• cites [2] Iyengar, 2019, Development of exercise as interception therapy for cancer: a review, JAMA Oncol sourceNot checked yet

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• cites [52] Colbert, 2009, Exercise effects on tumorigenesis in a p53-deficient mouse model of breast cancer, Med Sci Sports Exerc sourceNot checked yet

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ExTr's antitumor mechanism depends on CD8+ T cells.

“The depletion of CD8+ T cells abolished the antitumor effect of ExTr, confirming that the mechanism of antitumor activity from ExTr is CD8+ T cell-dependent.”

This prior failure to enhance cytotoxic NK or CD8+ T cells is likely due to differences in exercise intensity.

“This is probably due to differences in exercise intensity.”

Those CD8+ T cells in Cxcr3-KO mice were insufficient for tumor control.

“However, these CD8+ T cells were not enough to induce tumor control.”

This CD8+ T-cell increase in Cxcr3-KO mice points to CXCR3-independent recruitment mechanisms.

“ExTr induced a small but significant increase in CD8+ T cells in Cxcr3-KO mice, suggesting CXCR3-independent mechanisms of CD8+ T-cell recruitment (Fig. 6H–I).”

The treadmill model more closely mimics supervised exercise training in patients.

“Thus, it better recapitulates ExTr in patients participating in clinically supervised ExTr programs.”

This strain difference in relative exercise intensity may partly explain why earlier preclinical studies found no antitumor activity with similar ExTr protocols.

“This may explain, at least in part, why previous preclinical studies failed to show antitumor activity under similar ExTr protocols (7,52,56).”

• cites [7] Schadler, 2016, Tumor vessel normalization after aerobic exercise enhances chemotherapeutic efficacy, Oncotarget sourceNot checked yet

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• cites [52] Colbert, 2009, Exercise effects on tumorigenesis in a p53-deficient mouse model of breast cancer, Med Sci Sports Exerc sourceNot checked yet

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• cites [56] Glass, 2017, Differential response to exercise in claudin-low breast cancer, Oncotarget sourceNot checked yet

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Most earlier preclinical work used voluntary, ad libitum activity models that poorly reflect human exercise behavior.

“Because most previous preclinical studies use ad libitum physical activity models (6,8,10), which do not properly capture human exercise behavior, we adapted a controlled exercise.”

• cites [6] Betof, 2015, Modulation of murine breast tumor vascularity, hypoxia and chemotherapeutic response by exercise, J Natl Cancer Inst sourceNot checked yet

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• cites [8] Pedersen, 2016, Voluntary running suppresses tumor growth through epinephrine- and IL-6-dependent NK cell mobilization and redistribution, Cell Metab sourceNot checked yet

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• cites [10] Rundqvist, 2020, Cytotoxic T-cells mediate exercise-induced reductions in tumor growth, Elife sourceNot checked yet

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The synergy between vessel normalization and CD8-mediated tumor control appears to require at least moderate-to-vigorous ExTr intensity.

“Thus, the synergism between induced vessel normalization and CD8-mediated tumor control seems to occur when sufficient intensity (i.e., from moderate-to-vigorous) of ExTr is reached.”

CD8+CXCR3+ T cells are necessary for an improved immunotherapy response.

“This may explain why CD8+CXCR3+ T cells are required for improved response to immunotherapy.”

Schadler et al. saw no changes in tumor growth or immune cell infiltration with low-intensity ExTr.

“These authors did not observe changes in tumor growth or immune cell infiltration with the low-intensity ExTr.”

• cites [7] Schadler, 2016, Tumor vessel normalization after aerobic exercise enhances chemotherapeutic efficacy, Oncotarget sourceNot checked yet

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Only moderate-to-vigorous exercise sessions increase naïve and memory CD8+ T-cell subsets.

“Naïve and memory subsets of CD8+ T cells are increased only beyond moderate-to-vigorous exercise intensity sessions (58).”

• cites [58] Campbell, 2009, Acute exercise mobilises CD8+ T lymphocytes exhibiting an effector-memory phenotype, Brain Behav Immun sourceNot checked yet

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The CXCR3 pathway contributes to ExTr's antitumor effect.

“ExTr antitumor effect was entirely abolished in Cxcr3−/− mice, demonstrating that the CXCR3 pathway participates in the antitumor effect of ExTr.”

The shift toward OXPHOS can result from improved tumor perfusion after vessel normalization.

“This shift can occur as a consequence of enhanced tumor perfusion due to vessel normalization (60).”

• cites [60] Navarro, 2016, Targeting tumor mitochondrial metabolism overcomes resistance to antiangiogenics, Cell Rep sourceNot checked yet

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A shift toward less hypoxic, more oxidative metabolism that supports CD8+ T-cell effector function may have enabled the enhanced effector function seen with ExTr.

“Thus, a metabolic switch towards less hypoxic and more oxidative metabolism that favors CD8+ T-cell effector function (62) might have facilitated the enhanced CD8+ T cell effector function with ExTr.”

• cites [62] Calcinotto, 2012, Modulation of microenvironment acidity reverses anergy in human and murine tumor-infiltrating T lymphocytes, Cancer Res sourceNot checked yet

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CXCL9, CXCL10, and CXCL11 are CXCR3 ligands that promote trafficking of CD8+CXCR3+ T cells into tumors.

“CXCL9/10/11 are ligands of the CXCR3 receptor involved in increased trafficking of CD8+CXCR3+ T cells to tumors (47,48,64).”

• cites [47] House, 2020, Macrophage-derived CXCL9 and CXCL10 are required for antitumor immune responses following immune checkpoint blockade, Clin Cancer Res sourceNot checked yet

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• cites [48] Tokunaga, 2018, CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation - a target for novel cancer therapy, Cancer Treat Rev sourceNot checked yet

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• cites [64] Chow, 2019, Intratumoral activity of the cxcr3 chemokine system is required for the efficacy of anti-PD-1 therapy, Immunity sourceNot checked yet

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Without reaching a threshold exercise intensity, mobilization and redistribution of antitumor cells should not be expected.

“Therefore, if a certain threshold of exercise intensity is not achieved, one should not expect mobilization and redistribution of antitumor cells.”

Such tumors would likely benefit from ICB therapy.

“ExTr increases CD8+ T cells by reprogramming the TME towards a less aggressive phenotype, but a large fraction of CD8+ T cells remain expressing immune checkpoint receptors, suggesting these tumors would benefit from ICB therapy.”

Our results support ExTr as a safer way to stimulate the antitumor CXCL9/11-CXCR3 system in solid tumors.

“Our findings support ExTr as a safer strategy to stimulate the antitumor CXCL9/11-CXCR3 system in solid tumors.”

Supervised ExTr may unlock antitumor responses from immunotherapy in patients.

“Our data indicate that, supervised ExTr has the potential to unleash antitumor response from immunotherapy in the clinic.”

ICB has shown only modest efficacy in breast cancer.

“ICB has revolutionized the treatment of multiple malignancies. However, its efficacy in BC has been modest (27,70).”

• cites [27] Zhang, 2020, Immunotherapeutic strategies in breast cancer: a clinical update, J Surg Oncol sourceNot checked yet

  Not checked in the free run.

• cites [70] Adams, 2019, Current landscape of immunotherapy in breast cancer: a review, JAMA Oncol sourceNot checked yet

  Not checked in the free run.