We investigated relationships among immune, metabolic, and sleep abnormalities in mice with non-metastatic mammary cancer. Tumor-bearing mice displayed interleukin-6 (IL-6)-mediated peripheral inflammation, coincident with altered hepatic glucose processing and sleep. Tumor-bearing mice were hyperphagic, had reduced serum leptin concentrations, and enhanced sensitivity to exogenous ghrelin. We tested whether these phenotypes were driven by inflammation using neutralizing monoclonal antibodies against IL-6; despite the reduction in IL-6 signaling, metabolic and sleep abnormalities persisted. We next investigated neural populations coupling metabolism and sleep, and observed altered activity within lateral-hypothalamic hypocretin/orexin (HO) neurons. We used a dual HO-receptor antagonist to test whether increased HO signaling was causing metabolic abnormalities. This approach rescued metabolic abnormalities and enhanced sleep quality in tumor-bearing mice. Peripheral sympathetic denervation prevented tumor-induced increases in serum glucose. Our results link metabolic and sleep abnormalities via the HO system, and provide evidence that central neuromodulators contribute to tumor-induced changes in metabolism.

Cancer cells produce and secrete a number of factors, including inflammatory molecules and metabolic “waste,” which contribute to inflammation (). These factors are capable of altering the function of organs locally, or at a distal site (). Indeed, a prevalent hypothesis linking cancer to metabolic deregulation is that tumor-induced elevations in interleukin-6 (IL-6) promote hepatic insulin resistance via signal transducer and activator of transcription 3/suppressor of cytokine signaling 3 (STAT3/SOCS3) signaling (), and a similar inflammatory mechanism has been proposed to underlie changes in sleep (). Sleep is essential for proper metabolic regulation, as chronic sleep disruption promotes weight gain and the development of a chronic inflammatory state which may contribute to cancer initiation or progression (). Because the relationships among metabolism, sleep, and inflammation are poorly understood in the context of cancer, we sought to examine how these components interact during the course of tumor development in a mouse model of non-metastatic breast cancer. We hypothesized that tumor-induced inflammation contributes to metabolic and sleep abnormalities. To test this, we took a multifaceted approach yielding data on metabolic function, immune activation, and sleep throughout tumor growth. We demonstrate that syngeneic non-metastatic tumors (derived from 67NR cells) promote alterations in satiety hormone signaling and hepatic glucose processing coincident with peripheral IL-6-driven inflammation. These phenotypes were associated with disrupted and fragmented sleep, and aberrant activity of wake-stabilizing hypocretin/orexin (HO) neurons. Attenuation of HO signaling (via dual HO-receptor antagonism), but not blockade of IL-6, rescued metabolic abnormalities and enhanced sleep quality in tumor-bearing mice. Furthermore, peripheral sympathetic denervation (via 6-hydroxydopamine [6-OHDA] administration) prevented tumor-induced elevations in blood glucose concentrations. These data suggest that central neuromodulator systems contribute to impairments in glucose processing in tumor-bearing mice independent of the inflammatory milieu.

Among patients with cancer, metabolic dysfunction is a significant problem that is associated with decreased quality of life and increased mortality (). There is evidence of increased glucose concentrations at breast cancer diagnosis relative to values from matched controls without cancer (). Elevated glucose concentrations are linked to cancer progression (), and recent rodent studies suggest that cancer can also alter glucose homeostasis (e.g.,). A better understanding of the relationship between breast cancer and metabolism is critical given that elevated blood glucose among breast cancer survivors is associated with decreased survival independent of comorbid type II diabetes and body mass index (). Sleep problems are also prevalent in breast cancer survivors, and poor sleep is a strong predictor of subsequent mortality even when taking into consideration other risk factors including age, hormone receptor status, cortisol concentrations, depression, and metastasis (). Despite the prevalence of these problems, the underlying mechanisms mediating cancer-associated metabolic changes and sleep disruption are unknown.

If HO neurons contribute to tumor-induced changes in glucose metabolism and/or sleep, what then, is the signal that alters their activity? HO neurons are sensitive to metabolic signals from the periphery including ghrelin, leptin, and glucose (). We measured acyl-ghrelin in ad libitum fed mice during the mid-day and mid-night. Both tumor- and non-tumor-bearing mice showed the expected differences in acyl-ghrelin concentrations between the day and night, but no group differences were observed ( Figure 7 J). To investigate whether tumor-bearing mice were more sensitive to the effects of exogenous ghrelin, we administered ghrelin (IP; 30 μg/mouse) shortly before the active (dark) phase to fasting mice and measured food intake for 1 hr. Tumor-bearing mice showed enhanced feeding responses to exogenous ghrelin ( Figure 7 K). In addition, we investigated leptin concentrations, because leptin inhibits HO neurons (); serum leptin was significantly reduced in tumor-bearing mice during the mid-light (ZT 7) phase ( Figure 7 L). Together, these findings suggest that hormonal changes in the periphery (i.e., leptin, ghrelin) could modulate HO neural activity to drive aberrant hepatic metabolism and sleep in tumor-bearing mice.

As HO neurons can alter hepatic metabolism via the sympathetic nervous system (SNS) (), we tested whether peripheral ablation of noradrenergic signaling (i.e., sympathetic nerve terminals) would attenuate metabolic abnormalities in tumor-bearing mice. To accomplish this, we used peripheral 6-OHDA administration in a protocol similar to that of Rolls and colleagues () ( Figure 7 A). Systemic injection of 6-OHDA resulted in the expected decrease in norepinephrine content in the spleen, suggesting successful SNS ablation ( Figure 7 B). 6-OHDA successfully attenuated metabolic abnormalities in tumor-bearing mice, as mice that received this treatment showed equivalent blood glucose concentrations and normalization of several genes involved in gluconeogenesis/glycolysis (ldha, gck, pklr) at tissue collection (ZT 6; Figures 7 C–7F). These data provide evidence that SNS signaling contributes to changes in glucose metabolism in tumor-bearing mice.

(J and K) Serum acyl-ghrelin concentrations were unchanged between groups (n = 18–19/group) (J); however, tumor-bearing mice increased feeding responses to exogenous ghrelin after a 14-hr fast (n = 14–15/group; ZT 14 injection; main effect of ghrelin F 1,55 = 15.67, p = 0.0002; main effect of tumor status F 1,55 = 5.738, p = 0.02; †the main effect of tumor, ∗ the main effect of ghrelin) (K). Tumor-bearing mice showed reduced serum leptin concentrations during the day (n = 15/group; ZT 7 t = 5.54, p < 0.0001). (L). Error bars represent SEM; ∗∗∗ p < 0.001.

(G–I) g6pc (no change) (G), slc2a4 (n = 14–15/group; main effect of 6-OHDA F 1,53 = 70.04, p < 0.0001) (H), and pck1 (no change) (I) (all 6-OHDA data collected at ZT 6) (two-way ANOVA, †the main effect of 6-OHDA, ∗ the main effect of tumor, §the interaction; different letters indicate p < 0.05 difference with Tukey's post-hoc test). Data that did not meet requirements for ANOVA were log 2 -transformed. In a separate cohort, tumor-bearing mice had altered satiety hormonal signaling.

(C–F) 6-OHDA treatment normalized blood glucose concentrations in tumor-bearing mice (n = 14–15/group; main effect of tumor F 1,55 = 6.923, p = 0.011; main effect of 6-OHDA F 1,55 = 47.86 p < 0.0001; interaction F 1,55 = 7.58, p = 0.008) (C). 6-OHDA normalized (D) liver ldha expression (n = 14–15/group; main effect of tumor F 1,54 = 16.73, p = 0.0001; main effect of 6-OHDA F 1,54 = 10.51, p = 0.002; interaction F 1,54 = 33.48, p < 0.0001), (E) liver gck (15/group; main effect of tumor: F 1,56 = 19.97, p < 0.0001; main effect of 6-OHDA F 1,56 = 37.54, p < 0.0001), and (F) pklr (n = 15/group; main effect of 6-OHDA F 1,56 = 11.2, p = 0.0014).

To assess whether ALX could improve sleep quality, we administered the drug (or vehicle) as above in tumor-bearing mice equipped with EEG/EMG biotelemeters. ALX promoted restorative delta-rich (0.5–4 Hz) NREM sleep without changes to REM sleep ( Figures 6 I–6K), suggesting that HO antagonism enhances sleep quality in association with improved glucose metabolism.

Given the ability of HO neurons to regulate metabolism and sleep, we further hypothesized that the aberrant HO signaling we observed in tumor-bearing mice contributed to metabolic dysfunction. To test this, we administered (oral) the dual HO-receptor antagonist Almorexant (ALX) at multiple time points during the course of tumor development (days 15, 18, 21, and 24). We then collected tissue during the mid-day and mid-night (ZT 6 and ZT 18, respectively) on day 25 and assessed metabolic and immune phenotypes. ALX treatment attenuated daytime hyperglycemia in tumor-bearing mice ( Figure 6 A), and partially rescued the expression of several genes controlling gluconeogenesis and glycolysis within the liver (ldha, gck, slc2a4) ( Figures 6 B–6D) independent of coincident IL-6 driven inflammation ( Figures 6 F–6H). However, not all changes in gene expression were attenuated by ALX (pck1, pklr, g6pc; data not shown), which is likely due to the long latency (∼18 hr) between drug administration and tissue collection. Importantly, repeated administration of ALX did not alter body or tumor masses or food intake, ruling out the idea that improvement in metabolism was purely due to reductions in feeding ( Figure S7 ). Pre-treatment with ALX improved measures of glucose tolerance in a separate cohort of mice ( Figure S7 ). These data provide evidence that central HO signaling contributes to tumor-induced changes in glucose metabolism.

(K) ALX treatment decreased wakefulness during the first 6 hr following injections (day 22 ZT 14 t = 4.139, p = 0.0016; ZT 16 t = 2.974, p = 0.013; ZT 18 t = 2.831, p = 0.016; day 23 ZT 22 t = 2.213, p = 0.049; day 25 ZT 14 t = 3.625, p = 0.004; ZT 16 t = 3.828, p = 0.003; ZT 18 t = 2.421, p = 0.034). ALX decreased wakefulness theta/alpha EEG frequencies following treatment (7.5 Hz: t = 2.298, p = 0.042; 8 Hz: t = 2.888, p = 0.015; 8.5 Hz: t = 2.799, p = 0.017; 9 Hz: t = 2.737, p = 0.019; 9.5 Hz: t = 3.335, p = 0.0067; 10 Hz: t = 3.129, p = 0.0096; 10.5 Hz: t = 2.612, p = 0.024; 11 Hz: t = 2.346, p = 0.039) ( ∗∗ p < 0.05 and 0.01). n = 6 tumor + veh, 7 tumor + alx. Error bars represent SEM; †the main effect of tumor, ∗ the interaction, and §the main effect of ALX treatment; different letter headings represent multiple comparisons at p < 0.05, two-way ANOVA; Tukey's multiple comparisons test.

(I) ALX treatment (V, vehicle; A, ALX) increased NREM sleep time during the first 6 hr following injections (day 22 ZT 14 t = 3.755, p = 0.0032; ZT 16 t = 3.126, p = 0.0096; ZT 18 t = 2.925, p = 0.014; day 25 ZT 14 t = 3.574, p = 0.0044; ZT 16 t = 3.995, p = 0.0021; ZT 18 t = 2.301, p = 0.042). This sleep was characterized by more restorative delta (0.5–4 Hz) frequencies in the EEG on day 25 normalized to the same time frame (ZT14-20) on a non-treatment day (day 24) (1.5 Hz t = 3.361, p = 0.0063; 2 Hz t = 2.825, p = 0.0165, 20.5 Hz t = 2.354, p = 0.0382).

(F–H) Liver stat3 (ZT 6 main effect of tumor: F 1,32 = 24.41, p < 0.0001; ZT 18 main effect of tumor: F 1,33 = 22.79, p < 0.0001) (F), liver il6 (ZT 6 main effect of tumor: F 1,32 = 21.43, p < 0.0001) (G), and liver socs3 (ZT 6 main effect of tumor: F 1,30 = 17.37, p = 0.0002; ZT 18 main effect of tumor: F 1,34 = 17.4, p = 0.0002; main effect of ALX F 1,34 = 4.778, p = 0.0358; interaction F 1,34 = 7.874, p = 0.0082) (H) showed enhanced expression in tumor-bearing mice regardless of ALX treatment (n = 8–10/group/time point).

(A–D) Blood glucose (ZT 6 two-way ANOVA main effect of tumor: F 1,33 = 8.903, p = 0.0053; interaction: F 1,33 = 4.381, p = 0.0441) (ZT 18 two-way ANOVA main effect of tumor: F 1,35 = 5.149, p = 0.03) (A), liver ldha expression (ZT 6 two-way ANOVA main effect of tumor: F 1,32 = 9.194, p = 0.0048) (B), liver gck expression (ZT 6 main effect of tumor: F 1,30 = 19.25, p = 0.0001) (C), and liver slc2a4 expression (ZT 6 main effect of tumor: F 1,31 = 8.089, p = 0.0078) (D) were altered by tumor status and attenuated by administration of ALX.

Because insulin receptor substrates 1 and 2 (IRS1 and -2), key components in the insulin-signaling pathway, are targeted for degradation via IL-6-STAT3-SOCS3 signaling (), and we observed elevated hepatic responses coincident with tumor-derived IL-6, we predicted that blockade of IL-6 signaling would restore normal metabolic function and sleep. To test this, we administered a monoclonal antibody (mAb) (0.1 mg) against IL-6 (intraperitoneal [IP]) or the immunoglobulin G1 isotype control to mice in a full-factorial design at multiple time points throughout tumor development; on day 15 (before hepatic abnormalities become apparent), 19, and 23, followed by tissue collection on day 26. We conducted a parallel experiment to test whether IL-6 blockade could rescue sleep deficits in tumor-bearing mice by administering the mAb at day 22 post-injection, and collected tissue at day 25 after measuring sleep throughout the later stages of tumor development. We used a single antibody injection protocol for this experiment as its efficacy at reducing IL-6 was comparable with the triple-dose administration procedure ( Figure S6 ). Despite successfully reducing IL-6 concentrations and pSTAT3-mediated gene transcription ( Figures 5 C–5E ), the metabolic alterations persisted ( Figures 5 G–5L) and tumor-bearing mice still displayed hyperglycemia ( Figure 5 A). In addition, IL-6 blockade did not improve (or alter) sleep in tumor-bearing mice, which still displayed reduced wakefulness and fragmented vigilance states ( Figure 5 M). Two additional doses of IL-6 mAb (0.02 and 0.5 mg) also failed to normalize serum glucose in tumor-bearing mice relative to non-tumor-bearing mice ( Figure S6 ). Together, these data demonstrate that IL-6 signaling is not necessary for the development of tumor-induced changes in sleep and glucose metabolism.

(C–M) Despite successfully knocking down IL-6 (C) (n = 9 no tumor IgG, 7 no tumor anti-IL6, 10 tumor IgG, and tumor anti-IL6; main effect of tumor F= 5.344, p = 0.027, main effect of antibody F= 7.016, p = 0.012, and an interaction among the two F= 4.391, p = 0.044) and IL-6-mediated socs3 expression (D) (n = 9 no tumor IgG and anti-IL6, 10 tumor IgG and anti-IL6, main effect of tumor, F= 12.34, p = 0.0013, main effect of antibody, F= 4.431, p = 0.043), and il1r1 (E) (n = 10/group except no tumor anti-IL6, main effect of tumor, F= 17.54, p = 0.0002), but not ifnγ (F), tumor-bearing mice still showed deregulated expression of ldha (G) (n = 9 no tumor IgG and anti-IL6, 9 tumor IgG and 10 tumor anti-IL6, main effect of tumor, F= 10.3, p = 0.003), slc2a4 (J) (n = 10/group except 9 for tumor anti-IL6, main effect of tumor, F= 11.79, p = 0.0015), and gck (K) (n = 10/group except 9 for tumor IgG, main effect of tumor, F= 11, p = 0.0021), suggesting that IL-6 is not required for tumors to alter hepatic metabolism. In addition, Anti-IL6 mAb treatment (single injection at day 22, denoted by “mAb” in figure) did not alter sleep in tumor-bearing mice (M), demonstrating that IL-6 is not required for tumor-induced sleep disruption (tissue collected at ZT 16). No changes were detected in pck1, g6pc, and pklr (H, I, L). Error bars represent SEM; †the main effect of tumor,the main effect of antibody treatment, different letter headings represent multiple comparisons at p < 0.05, two-way ANOVA; Tukey's multiple comparisons test. See Figures S8 and S9

(A and B) Tumor-bearing mice increased (A) blood glucose (n = 10/group, main effect of tumor, F 1,36 = 11.67, p = 0.0016) and (B) serum insulin (n = 8 no tumor immunoglobulin G [IgG], 9 no tumor anti-IL-6, 9 tumor IgG, 7 tumor anti-IL-6, main effect of tumor, F 1,29 = 16.46, p = 0.0003) regardless of whether they received anti-IL6 or the IgG1 isotype control (a total of three injections on days 15, 19, and 23).

Next, we investigated whether sleep changes were associated with the altered activity of neurons that control behavioral state switching, namely HO and melanin-concentrating hormone (MCH) neurons in the lateral and perifornical hypothalamus. Because HO neurons gate arousal depending on metabolic state (), we hypothesized that changes in their activity would further be associated with disrupted metabolism. We collected tissue at two time points coincident with the peak and trough of normal HO neural activity (ZT 7 and ZT 17) () and co-labeled hypothalamic sections with antibodies against hypocretin and the immediate-early gene cFos. We observed an active-phase-specific (ZT 17) increase in cFos+ hypocretin+ neurons in tumor-bearing mice ( Figures 4 A and 4G ), without simultaneous changes in adjacent MCH populations ( Figure 4 J). This change was observed throughout the extent of the hypocretin field, as a greater percentage of cFos+ hypocretin+ cells were detected in the dorsomedial, perifornical, and lateral hypothalamic areas ( Figure 4 I); this was not due to a generalized activation of the hypothalamus, as the total number of cFos+ cells was unchanged between groups ( Figure 4 A).

(J) No changes were observed in co-distributed melanin-concentrating hormone (MCH) neurons. DMH, dorsomedial hypothalamus; PFA, perifornical area; LH, lateral hypothalamus. Error bars represent SEM; ∗ p < 0.05, ∗∗ p < 0.01, Student's t test). Merged images (D, D′, G, and G′) are composites of (B, C, and E, F), respectively. Scale bar, 50 μm.

(H and I) Schematic of the location of hypocretin/orexin neurons in the hypothalamus (H), and quantification of double-labeling observed in each sub-region during the active phase (ZT 17) (I) (n = 9 no tumor and 8 tumor mice, PFA t = 2.722, p = 0.016; LH t = 2.237, p = 0.041).

(B–G′) Representative confocal images of hypocretin/orexin (Alexa 488, green) and cFos (Alexa 594, red) immunofluorescence in the hypothalamus. (D′) and (G′) show zoomed in sections of (D) and (G), respectively. Double-labeled cells are denoted by arrowheads.

(A) Tumor-bearing mice showed increased cFos immunoreactivity within hypocretin neurons during the active phase (ZT 17) (n = 9 no tumor and 8 tumor mice, t = 2.158, p = 0.047 for total cell numbers and t = 3.016, p = 0.0087 for percentage of OxA neuron co-labeling at ZT 17).

Because metabolic and immune factors contribute to changes in sleep and wakefulness (), we assessed whether tumors directly altered sleep-wake cycles. We monitored electroencephalography (EEG)/electromyography (EMG) biopotentials via wireless telemetry throughout the course of tumor development. Reductions in locomotor activity, indicative of fatigue, as well as changes in body temperature regulation, were detected toward the later stages (days 19–24) of tumor development ( Figure S2 ). This was accompanied by reduced time spent awake, and an increase in non-rapid eye movement (NREM) but not rapid eye movement (REM) sleep ( Figures 3 A–3I ). Sleep was fragmented ( Figures 3 M and S3 ), as tumor-bearing mice did not maintain long bouts of wakefulness during their normal active phase. However, no changes in the spectral components (0.5–25 Hz) of sleep were detected during this time, suggesting that there was no change in sleep microstructure despite increased and fragmented sleep. Tumor-bearing mice did exhibit altered temporal patterns of NREM delta (0.5–4 Hz) power, an index of sleep depth ( Figure S4 ). To test whether tumors impaired normal responses to a homeostatic sleep challenge, mice were sleep deprived for 6 hr at the start of the normal inactive phase (). Both tumor and non-tumor-bearing mice showed enhanced NREM delta power during recovery sleep that dissipated over time as sleep pressure resolved, suggesting an intact sleep homeostat ( Figure S4 ). Importantly, these sleep changes were evident at this time point without simultaneous changes in other behaviors classically associated with disrupted sleep, including depressive-like and anxiety-like behaviors ( Figure S5 ).

(A–M) Early during tumor development (i.e., 2–4 days post-induction, sleep is not different from non-tumor-bearing controls (A, D, and G). However, late during tumor development (3 days prior to euthanasia at endpoint criteria), mice with tumors spend less time awake (B and C) (n = 7/group for days −3, t = 2.401, p = 0.033, and −2, t = 2.595, p = 0.0234, and 6 no tumor and 7 tumor for −1, t = 2.984, p = 0.0124, days prior to tissue collection) and more time in NREM sleep (E and F), n = same as in (C), −2 days t = 2.317, p = 0.039; −1 day, t = 2.925, p = 0.0138. Smaller changes are evident in REM sleep, although the total amount of REM sleep remained similar between groups (H and I). Representative band-passed EEG/EMG signals from tumor and non-tumor-bearing mice in the (J) wake, (K) NREM, and (L) REM sleep vigilance states. Tumor-induced sleep was fragmented, as shown in (M), which shows representative hypnograms during the light and dark phases during final day of tumor growth. Aberrant periods of sleep during the normal active phase are marked with red arrows. Error bars represent ± SEM; ∗ p < 0.05, Student's t test.

Because inflammatory signaling can disrupt liver glucose metabolism (), we investigated gluconeogenesis and glycolysis pathway gene expression in the livers every 4 hr around the clock. We observed marked deregulation of primary enzymes responsible for glucose processing including gluconeogenesis enzymes: lactate dehydrogenase-A (ldha), phosphoenolpyruvate carboxykinase 1 (pck1), and glucose-6-phosphatase (g6pc); and glycolysis enzymes: glucokinase (gck) and pyruvate kinase isozymes L/R (pklr) ( Figures 2 H–2L ). These changes were associated with impaired glucose tolerance after an overnight fast and higher blood glucose when fed ad libitum concurrent with increased food intake, particularly during the active phase ( Figures 2 A–2C and 2E). We further assessed whether insulin signaling was normal by measuring a downstream kinase in the insulin receptor activation pathway, phosphorylated Akt (Ser473). We observed reduced pAkt and the insulin-dependent glucose transporter (GLUT4; slc2a4) expression in the livers of tumor-bearing mice ( Figures 2 D and 2M), suggesting that serum elevations in glucose were likely a result of impaired insulin-dependent glucose uptake. We additionally tested functional gluconeogenesis via pyruvate and lactate tolerance tests, and demonstrate that tumor-bearing mice enhanced glucose production upon pyruvate and lactate challenge ( Figures 2 F and 2G). Notably, metabolic abnormalities did not become apparent until the exponential phase of tumor growth, as no differences were observed when tumor burden was ∼0.05 g, at 15 days post-injection ( Figure 2 N).

(F–N) Tumor-bearing mice enhanced gluconeogenesis in response to pyruvate (n = 15/group, 30 min post-injection, t = 3.43, p = 0.0019) (F) and lactate (n = 15/group, 30 min time point t = 2.534, p = 0.0174) challenges (G), altered gck (n = 10/group for ZT 18, t = 2.365, p = 0.029) (H), pklr (n = 10 no tumor and 11 tumor for ZT 10, t = 2.23, p = 0.038; 9/group for ZT 18, t = 2.764, p = 0.014) (I), g6pc (n = 8 no tumor and 10 tumor at ZT 2, t = 2.258, p = 0.038; and 10 and 9 at ZT 14, t = 2.428, p = 0.0266) (J), pck1 (n = 10/group for ZT 2, t = 2.694, p = 0.015; 9/group for ZT 14, t = 2.792, p = 0.013) (K), and ldha (n = 9 no tumor and 12 tumor mice for ZT 10, t = 2.38, p = 0.028; 8/group for ZT 14, t = 5.105, p = 0.00016) (L), as well as reduced pAkt expression in the liver (M), indicating impaired insulin receptor signaling (ZT 10) (only relevant lanes of the western blot are shown). These changes in inflammation and metabolism were not evident until after day 15 following 67NR inoculation, as expression of all genes was equivalent between groups at this time (ZT 16) (N). Error bars represent SEM; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; Student's t test.

(D and E) This was accompanied by (D) reduced expression of the insulin-dependent glucose transporter (slc2a4) (ZT 10, n = 15/group, t = 3.78, p = 0.0008), and (E) increased food intake (N23 n = 40/group, t = 2.251, p = 0.027; N24 t = 2.497, p = 0.0146), night average intake t = 2.542, p = 0.013; total intake t = 2.359, p = 0.021), and altered expression of gluconeogenesis and glycolysis pathway genes “around the clock.”

(A–C) Tumor-bearing mice had impaired (A and B) glucose processing during a glucose tolerance test following an overnight fast (n = 10 no tumor, 9 tumor mice at 15 min post-injection, t = 2.599, p = 0.0187; n = 10/group for area under the curve [AUC], t = 2.134, p = 0.047), as well as (C) spontaneously higher glucose during the active phase (n = 19 no tumor, 18 tumor mice, t = 2.398, p = 0.0219).

To assess the inflammatory profile in tumor-bearing mice, we measured the expression (mRNA and/or protein) of several cytokines in tumors, serum, spleen, liver, and brain at multiple time points during tumor development. We observed increased spleen masses, macrophage (F4/80+) infiltration into the tumor, as well as high concentrations of IL-6 protein in serum and tumor parenchyma ( Figures 1 A–1D ). Because the liver orchestrates acute phase responses to elevations in IL-6 (), we examined whether tumor-induced inflammation was associated with altered liver function. We sampled livers from tumor-bearing mice “around the clock” for 24 hr and observed increased protein concentrations of the IL-6-regulated transcription factor pSTAT3 ( Figure 1 D), whose phosphorylation at tyrosine 705 is known to activate STAT3 and promote its transcriptional activity via nuclear translocation (), as well as increased mRNA expression of IL-6 target genes including stat3, socs3, interleukin-1 receptor-1 (il1r1), interleukin-6 receptor-alpha (il6rα), chemokine ligand 2 (ccl2), and C-reactive protein (crp) ( Figures 1 E–1J). Importantly, these changes were not detected at all circadian times, suggesting that circadian gating of immune responses may modulate the hepatic response to tumors at a distal site (). Inflammatory changes were not evident in the hypothalamus ( Figures 1 K and 1L), a key brain region involved in the regulation of metabolism and sleep, or in the hippocampus or cortex ( Figure S1 ). These data suggest that 67NR-derived tumors promote peripheral, but not central, inflammatory responses which are largely driven by IL-6.

(D–L) This is associated with increased pSTAT3 activation (western blot, ZT 10) in the liver of tumor-bearing mice (Tumor) compared with non-tumor-bearing (No Tumor) mice (only relevant lanes of the western blot are shown), and marked macrophage (F4/80+) infiltration into tumors (D). Tumor-bearing mice have increased expression of downstream targets of IL-6 signaling in the liver including (E) stat3 (n = 9/group for ZT 6, t = 2.937, p = 0.0097; 10/group for ZT 14, t = 2.241, p = 0.038; 9 no tumor, 10 tumor for ZT 22, t = 3.571, p = 0.0024), (F) socs3 (n = 9 and 8/group for no tumor and tumor groups ZT 6, t = 2.692, p = 0.0167; n = 9 and 10 for ZT 10, t = 2.683, p = 0.0157), (G) il1r1 (n = 10 no tumor, 9 tumor for ZT 10, t = 2.379, p = 0.029; 8 and 10 for ZT 14, t = 3.725, p = 0.0018), (H) crp (n = 8 no tumor 10 tumor for ZT 10, t = 2.79, p = 0.013; 9 and 8 for ZT 14, t = 2.087, p = 0.054), (I) il6rα (n = 9/group for ZT 6, t = 3.237, p = 0.0052; 10 no tumor and 8 tumor for ZT 10, t = 2.144, p = 0.0478; 9 and 10 for ZT 14, t = 3.139, p = 0.00599; 10 and 9 for ZT 18, t = 2.137, p = 0.0474; and 9/group for ZT 22, t = 2.642, p = 0.0177), and (J) ccl2 (n = 10 no tumor and 9 tumor for ZT 10, t = 2.247, p = 0.038), but not the hypothalamus (K and L). Error bars represent ± SEM; ∗ p < 0.05, ∗∗ p < 0.01, Student's t test.

(A–C) Tumor-bearing mice have increased (A) spleen mass (n = 12/group; t = 3.702, p = 0.0012), (B) high serum levels of IL-6 (n = 13 no tumor, n = 14 tumor for IL-6; t = 2.449, p = 0.022, n = 14/group for tumor necrosis factor-α [TNF-α], t = 2.322, p = 0.028), and (C) their tumors contain high levels of IL-6 protein.

Discussion

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White M.F. SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2. First, we investigated whether non-metastatic tumors in the periphery were able to induce systemic immune activation ( Figure 1 ), and demonstrate that 67NR-derived tumors promote a predominantly IL-6 driven response. IL-6 is a pleiotropic cytokine that has modulatory actions on metabolism. Specifically, IL-6 binding its receptor results in the downstream phosphorylation of the transcription factor STAT3. This protein regulates the expression of hundreds of genes, including its own negative regulator socs3 (). SOCS3 interacts and targets IRS1 and -2 for ubiquitin-mediated degradation (). IRS1 and 2 are major components regulating insulin-dependent glucose uptake, and their suppression results in insulin insensitivity concurrent with hyperglycemia, a phenotype we observed in the present study.

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Dedhar S. Epithelial–mesenchymal transition (EMT) is not sufficient for spontaneous murine breast cancer metastasis. The promotion of hyperglycemia is a strategy used by tumors to facilitate their proliferation and growth (), therefore, we examined genes involved in gluconeogenesis and glycolysis in tumor-bearing mice. In addition, recent evidence suggests that hyperglycemia can promote metastatic seeding by impairing neutrophil mobilization (). We observed impaired glucose tolerance, spontaneous hyperglycemia, as well as altered temporal expression patterns of genes responsible for glucose synthesis (ldha, pck1, g6pc) and breakdown (gck, pklr), with the former showing an increase and the latter showing a decrease in expression, suggesting a shift toward hepatic glucose production. Indeed, tumor-bearing mice showed enhanced hepatic gluconeogenesis in response to pyruvate and lactate challenges, which provide non-carbohydrate carbon substrates for glucose production ( Figures 2 F and 2G). Tumor-driven disruption of liver function has recently been demonstrated in metastatic mouse models of lung adenocarcinoma (), transplanted colorectal cancer, and spontaneous pancreatic ductal adenocarcinoma (). Importantly, our data provide the first evidence for tumor-induced liver dysfunction in a non-metastatic mouse model of mammary cancer, as 67NR cells fail to intravasate and do not leave their primary site (). In addition, in contrast to other models, 67NR-derived tumors do not cause behavioral deficits or cachexia ( Figures S5 and S7 ), which may independently contribute to metabolic and sleep changes. We observed a similar metabolic phenotype in the livers of mice inoculated with metastatic sister cell lines to 67NR (i.e., 4T1 and 4T07; data not shown), suggesting that our findings are not specific only to 67NR-generated tumors.