Abstract
Anorexia is a common manifestation of chronic diseases, including cancer. Here we investigate the contribution to cancer anorexia made by calcitonin gene-related peptide (CGRP) neurons in the parabrachial nucleus (PBN) that transmit anorexic signals. We show that CGRPPBN neurons are activated in mice implanted with Lewis lung carcinoma cells. Inactivation of CGRPPBN neurons before tumor implantation prevents anorexia and loss of lean mass, and their inhibition after symptom onset reverses anorexia. CGRPPBN neurons are also activated in Apcmin/+ mice, which develop intestinal cancer and lose weight despite the absence of reduced food intake. Inactivation of CGRPPBN neurons in Apcmin/+ mice permits hyperphagia that counteracts weight loss, revealing a role for these neurons in a 'nonanorexic' cancer model. We also demonstrate that inactivation of CGRPPBN neurons prevents lethargy, anxiety and malaise associated with cancer. These findings establish CGRPPBN neurons as key mediators of cancer-induced appetite suppression and associated behavioral changes.
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Acknowledgements
We thank M. Chiang for maintaining the mouse colony, J. Carson for providing Apcmin/+ brain samples, T. Cole for assistance with PhenoTyper data and H. Nguyen for general assistance. We also thank G. Morton, J. Nelson and K. Ogimoto for their assistance with conducting the body composition and energy expenditure experiments. The research was supported by a fellowship grant from Hope Funds for Cancer Research to C.A.C., National Institutes of Health grants to M.W.S. (CA184630-01, DK090320, DK101997 and DK083042), R.D.P. (R01-DA24908) and the University of Washington CHDD Mouse Behavior Lab (U54 HD083091) and Nutrition Obesity Research Center (P30 DK035816).
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C.A.C., B.E.W., R.D.P. and M.W.S. conceived and designed the study. C.A.C. and A.J.B. performed and analyzed histological and feeding experiments. C.A.C., A.J.B. and S.H. performed and analyzed sickness behavior tests. R.D.P. and M.W.S. provided equipment and reagents. C.A.C. and M.W.S. wrote the manuscript with input from all authors.
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Integrated supplementary information
Supplementary Figure 1 Fos induction in CGRPPBN neurons of mice implanted with LLC tumors.
a-b, Representative images demonstrating coincidence of Cre:GFP-labeled CGRP neurons and Fos expression in tumor-bearing mice (a) or sham-treated mice (b) across the rostral-caudal extent of the PBN. Approximate coordinates were taken from the mouse Allen Reference Atlas. Scale bars are 50 μm. Related to Fig. 1.
Supplementary Figure 2 Body-weight changes and tumor size of mice with intact or TetTox-inactivated CGRPPBN neurons implanted with LLC tumors.
a, Change in body weight (minus tumor), from Day 0 to Day 14 after tumor implantation or sham treatment (GFP LLC, n = 9; GFP Sham, n = 8; TetTox LLC, n = 8; TetTox Unilat LLC, n = 4; GFP Sham PF, n = 8; TetTox LLC PF, n = 7; one-way ANOVA: F(5,45) = 3.05, p = 0.0200). b, Tumor size of various tumor-bearing experimental groups. Data points in graphs represent each individual animal (one-way ANOVA: F(3,29) = 4.77, p = 0.0088). * P ˂ 0.05, ** P ˂ 0.01. Related to Fig. 2.
Supplementary Figure 3 Individual food intake data, body-weight change and tumor size of mice with intact or hM4Di-inhibited CGRPPBN neurons.
a, Individual food intake data of mCherry tumor-bearing mice relative to CNO treatment. b, Individual food intake data of hM4Di tumor-bearing mice relative to CNO treatment. c, Sacrifice date (relative to day of tumor implantation) of sham-treated control groups and tumor-bearing experimental groups. d, Number of days receiving CNO treatment. e, Change in body weight calculated from body weight on day of tumor implantation and day of euthanasia, minus tumor weight (one-way ANOVA: F(3,30) = 7.15, p = 0.0011). f, Tumor size of experimental groups (two-tailed student’s t-test: t(13) = 0.25, p = 0.8072). Data points in graphs represent each individual animal. Sample size: mCherry LLC, n = 8; mCherry Sham, n = 8; hM4Di LLC, n = 7; hM4Di Sham, n = 8. * P ˂ 0.05. Related to Fig. 3.
Supplementary Figure 4 Individual food intake, body composition and tumor size data.
a-b, Individual food intake data of GFP and TetTox tumor-bearing mice. c-d, Individual fat mass of GFP and TetTox mice before and after tumor implantation. LLC1 and LLC2 measurements were taken before and after onset of anorexia, respectively. e-f, Individual lean mass of GFP and TetTox mice before and after tumor implantation. g, Tumor mass of GFP and TetTox mice. Sample sizes: GFP, n = 9; TetTox LLC, n = 9. Related to Figure 4.
Supplementary Figure 5 Representative images of the CeA and ovBNST of mice with intact or inactive CGRPPBN neurons.
a, Fos immunoreactivity and GFP fiber terminal labeling in the CeA of a tumor-bearing TetTox mouse with TetTox:GFP infection of CGRPPBN neurons. Sections are arranged caudal to rostral (top to bottom; bregma -2.055 to -1.055 mm), separated by approximately 60 μm between each section. This mouse was euthanized 21 days after tumor implantation, and food intake was 4.7 g and 5.4 g in final two days before euthanasia. b, Fos immunoreactivity and GFP fiber terminal labeling in the CeA of a GFP control mouse 21 days after tumor implantation. Food intake for this mouse was 1.6 g and 0.0 g in final two days prior to euthanasia. c, Fos immunoreactivity and GFP fiber terminal labeling in the caudal to rostral ovBNST (top to bottom; bregma 0.145 to 0.245 mm) of a tumor-bearing TetTox mouse. d, Fos immunoreactivity and GFP fiber terminal labeling in the ovBNST of a GFP mouse. Scale bars are 100 μm. Related to Fig. 4.
Supplementary Figure 6 Fos induction in CGRPPBN neurons of Apcmin/+ mice.
a-b, Representative images demonstrating coincidence of Cre:GFP-labeled CGRP neurons and Fos expression in Apcmin/+ mice (a) or cancer-free littermates (b) across the rostral-caudal extent of the PBN. Approximate coordinates were taken from the mouse Allen Reference Atlas. Scale bars are 50 μm. Related to Fig. 5.
Supplementary Figure 7 Inactivation of CGRPPBN neurons prevents cancer-induced weight loss in Apcmin/+ mice and activation of the CeA and ovBNST.
a-c, Quantification and representative histological examples of Fos expression in the CeA and ovBNST of wild-type or tumor-bearing mice with intact (GFP) or inactivated (TetTox) CGRPPBN neurons (GFP Apcmin/+, n = 7; TetTox Apcmin/+, n = 7; GFP WT, n = 5; CeA, one-way ANOVA: Interaction, F(2, 18) = 30.62, p < 0.0001; ovBNST, one-way ANOVA: Interaction, F(2, 18) = 17.84, p < 0.0001). d, Change in body weight (relative to week 12) of male Apcmin/+ mice (and cancer-free littermates) with intact (GFP) or inactivated (TetTox) CGRPPBN neurons (GFP Apcmin/+, n = 7; TetTox Apcmin/+, n = 7; GFP WT, n = 5; two-way ANOVA: Interaction, F(8, 76) = 7.10, p < 0.0001; Time, F(4, 76) = 2.85, p = 0.0296; Treatment, F(2, 76) = 77.26, p < 0.0001). e, Change in bodyweight (relative to week 12) of female experimental cohort (GFP Apcmin/+, n = 12; TetTox Apcmin/+, n = 7; GFP WT, n = 4; two-way ANOVA for weeks 12-21: Interaction, F(16, 149) = 3.88, p < 0.0001; Time, F(8, 149) = 1.63, p = 0.1210; Treatment, F(2, 149) = 72.15, p < 0.0001). f, Average daily food intake of the female experimental cohort (two-way ANOVA for weeks 12-21: Interaction, F(18, 168) = 0.90, p = 0.5807; Time, F(9, 168) = 0.58, p = 0.8149; Treatment, F(2, 168) = 13.98, p < 0.0001). † symbols represent euthanasia of GFP Apcmin/+ mouse. # symbols represent euthanasia of TetTox Apcmin/+ mouse. g, Food intake during 24-h period prior to euthanasia (female cohort; two-tailed student’s t-test: t(16) = 13.98, p < 0.0001). Line graphs show mean ± SEM. Box plots show mean (+), median, quartiles (boxes) and range (whiskers) for each treatment. * P ˂ 0.05, ** P ˂ 0.01, *** P ˂ 0.001. Scale bar is 100 μm. Related to Fig. 5.
Supplementary Figure 8 Time-course of sickness behavior analyses, change in body weight and tumor size of mice with LLC tumors.
a, Time course of behavioral analysis of mice with intact (GFP, n = 8) or inactivated (TetTox, n = 7) CGRPPBN neurons relative to day of tumor implantation. b, Change in body weight calculated from day of tumor implantation and day of euthanasia, minus tumor weight (two-tailed student’s t-test: t(13) = 3.77, p = 0.0023). c, Size of tumors take from GFP and TetTox mice ((two-tailed student’s t-test: t(13) = 1.80, p = 0.0946). Data points in graphs represent each individual animal. See Methods for detailed statistics. ** P ˂ 0.01. Related to Fig. 6.
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Campos, C., Bowen, A., Han, S. et al. Cancer-induced anorexia and malaise are mediated by CGRP neurons in the parabrachial nucleus. Nat Neurosci 20, 934–942 (2017). https://doi.org/10.1038/nn.4574
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DOI: https://doi.org/10.1038/nn.4574
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