Early Neutrophilia Marked by Aerobic Glycolysis Sustains Host Metabolism and Delays Cancer Cachexia

Abstract

An elevated neutrophil-lymphocyte ratio negatively predicts the outcome of patients with cancer and is associated with cachexia, the terminal wasting syndrome. Here, using murine model systems of colorectal and pancreatic cancer we show that neutrophilia in the circulation and multiple organs, accompanied by extramedullary hematopoiesis, is an early event during cancer progression. Transcriptomic and metabolic assessment reveals that neutrophils in tumor-bearing animals utilize aerobic glycolysis, similar to cancer cells. Although pharmacological inhibition of aerobic glycolysis slows down tumor growth in C26 tumor-bearing mice, it precipitates cachexia, thereby shortening the overall survival. This negative effect may be explained by our observation that acute depletion of neutrophils in pre-cachectic mice impairs systemic glucose homeostasis secondary to altered hepatic lipid processing. Thus, changes in neutrophil number, distribution, and metabolism play an adaptive role in host metabolic homeostasis during cancer progression. Our findings provide insight into early events during cancer progression to cachexia, with implications for therapy.

Keywords: aerobic glycolysis; cachexia; cancer; host; metabolism; neutrophils.

Figure 1

Immune changes during cancer progression in mouse models of cachexia. (A) The mice were injected with C26 colorectal cancer cells and sacrificed at three distinct time points: an early time point (9–10 days post-tumor inoculation), the pre-cachectic stage (15–16 days post-injection), and when cachexia occurred (≥21 days post-injection); (B–F) The body (B), tumor (C), spleen (D), quadriceps (E), and gonadal white adipose tissue (gWAT) (F) weights of mice that were sacrificed at the time points that are defined in Figure 1A; (G–I) Quantification of neutrophils (displayed as % of neutrophils out of all CD45+ cells), by flow cytometry in the blood (G), lung (H), and liver (I) of mice that were sacrificed at the time points that are defined in Figure 1A; (J) Quantification of neutrophils (displayed as % of neutrophils out of all CD45+ cells) in the spleen, quadriceps, gWAT, blood, lung, and liver of pre-cachectic KPC mice and the PC controls. The data are expressed as the mean ± SEM. A one-way ANOVA with Tukey’s correction for post hoc testing was used in (B–I). Statistical differences in (J) were examined using unpaired two-tailed Student’s t-test with Welch’s correction. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001, ns: not significant.

Figure 2

Quantification and FACS gating of progenitor cells, neutrophil precursors, and neutrophil populations in the KPC model. (A,B) Quantification of the HSC and progenitor cells (displayed as % out of all CD45+ cells) in the spleen (A) and bone marrow (B) of the pre-cachectic KPC and PC controls; (C,D) FACS gating strategy for neutrophils and neutrophil precursor populations in the spleen of the PC controls (C) and the pre-cachectic KPC mice (D); (E–G) Quantification of the neutrophil precursor populations (displayed as % out of all CD45+ cells) in the spleen €, bone marrow (F), and blood (G) of the pre-cachectic KPC mice and the PC controls. The data are expressed as the mean ± SEM. Statistical differences were examined using unpaired two-tailed Student’s t-test with Welch’s correction. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001.

Figure 3

Characterization of immune cell metabolism in the pre-cachectic KPC mice. (A) Extracellular acidification rate (ECAR) of circulating leukocytes from pre-cachectic KPC mice and PC controls that were measured in real-time by Seahorse assay at baseline, after glucose administration, and after treatments with oligomycin, phenylhydrazone (FCCP), and rotenone; (B–D) Normalized ECAR measurements (ratio of compared timepoints) in the circulating leukocytes from mice in Figure 3D at baseline (B), after glucose administration (C), and oligomycin treatment (D); (E) Representative MitoTracker and DAPI immunofluorescence staining in the sorted circulating neutrophils from pre-cachectic KPC mice and PC controls; (F) Quantification of the fluorescence intensity (AU) of MitoTracker staining relative to DAPI staining in circulating, hepatic, and pulmonary neutrophils from mice in (C). (G,H) Gene set enrichment analysis (GSEA) for glycolysis (G) and oxidative phosphorylation (H)-related genes in isolated neutrophils of the KPC compared to the wild-type C57BL/6J mice. The data are expressed as the mean ± SEM. Statistical differences in (B–D,F) were examined using an unpaired two-tailed Student’s t-test with Welch’s correction. Statistical analysis in (G,H) is described in the Methods section. ** p-value < 0.01, *** p-value < 0.001.

Figure 4

Effects of systemic inhibition of aerobic glycolysis on the severity of cancer cachexia. (A) Longitudinal tumor measurements of the C26 mice that were treated with heptelidic acid or the vehicle. The red arrow indicates start of treatment; (B) Overall survival of the C26 mice and littermates treated with heptelidic acid or vehicle; (C–E) The tissue weights of gonadal white adipose tissue (gWAT) (C), quadriceps (D), and spleen (E) of time-matched mice in (A); (F–K) Quantification of leukocyte (F,H,J) and neutrophil (G,I,K) counts in the circulation (F,G), the liver (H,I), and the lungs (J,K) of mice in (A). The data are expressed as the mean ± SEM. Unpaired two-tailed Student’s t-tests were performed at each time point in (A), with the Holm–Šidák method correction for multiple comparisons. Kaplan–Meier curves in (B) were statistically analyzed by using the log-rank (Mantel–Cox) test. A one-way ANOVA with Tukey’s correction for post hoc testing was used in (C–K). * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001.

Figure 5

Effect of acute neutrophil depletion during cancer-associated metabolic stress. (A,B) Body weight trajectories after TFR of littermates (A) and pre-cachectic C26 mice (B) that were treated with anti-Gr1 or isotype. (C,D) The glucose levels in C26 mice and littermates that were treated with anti-Gr1 or isotype, when fed ad libitum (C) and after 24 h of total food restriction (TFR) (D); (E,F) Lipid staining by Oil Red O in the liver of fed or fasted pre-cachectic C26 mice and littermates (E) and the fed pre-cachectic KPC mice and the PC controls (F); (G) Quantification of triglycerides by liquid chromatography-mass spectrometry in the liver of fed and fasted pre-cachectic C26 mice and littermates that were treated with anti-Gr1 or isotype. The data are expressed as the mean ± SEM. Unpaired two-tailed Student’s t-tests were performed at each time point in (A,B), with the Holm–Šidák method correction for multiple comparisons. One-way ANOVA with Tukey’s correction for post hoc testing was used in (C,D,G). * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, ns: not significant.