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. 2023 Dec 15;133(24):e167725.
doi: 10.1172/JCI167725.

A brain-tumor neural circuit controls breast cancer progression in mice

Si-Yi Xiong  1 , Hui-Zhong Wen  2 , Li-Meng Dai  3 , Yun-Xiao Lou  2 , Zhao-Qun Wang  2 , Yi-Lun Yi  4 , Xiao-Jing Yan  5 , Ya-Ran Wu  6 , Wei Sun  7 , Peng-Hui Chen  2 , Si-Zhe Yang  1 , Xiao-Wei Qi  1 , Yi Zhang  1 , Guang-Yan Wu  4
Affiliations

A brain-tumor neural circuit controls breast cancer progression in mice

Si-Yi Xiong et al. J Clin Invest. .

Abstract

Tumor burden, considered a common chronic stressor, can cause widespread anxiety. Evidence suggests that cancer-induced anxiety can promote tumor progression, but the underlying neural mechanism remains unclear. Here, we used neuroscience and cancer tools to investigate how the brain contributes to tumor progression via nerve-tumor crosstalk in a mouse model of breast cancer. We show that tumor-bearing mice exhibited significant anxiety-like behaviors and that corticotropin-releasing hormone (CRH) neurons in the central medial amygdala (CeM) were activated. Moreover, we detected newly formed sympathetic nerves in tumors, which established a polysynaptic connection to the brain. Pharmacogenetic or optogenetic inhibition of CeMCRH neurons and the CeMCRH→lateral paragigantocellular nucleus (LPGi) circuit significantly alleviated anxiety-like behaviors and slowed tumor growth. Conversely, artificial activation of CeMCRH neurons and the CeMCRH→LPGi circuit increased anxiety and tumor growth. Importantly, we found alprazolam, an antianxiety drug, to be a promising agent for slowing tumor progression. Furthermore, we show that manipulation of the CeMCRH→LPGi circuit directly regulated the activity of the intratumoral sympathetic nerves and peripheral nerve-derived norepinephrine, which affected tumor progression by modulating antitumor immunity. Together, these findings reveal a brain-tumor neural circuit that contributes to breast cancer progression and provide therapeutic insights for breast cancer.

Keywords: Breast cancer; Neuroscience; Oncology.

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Figures

Figure 1
Figure 1. 4T1 tumor–bearing mice show obvious anxiety.
(A) Schematic of the experimental design. (B) Tumor growth of mice injected with 4T1-luc cancer cells (n = 14). (C) Representative tumors dissected from 4T1 tumor–bearing mice. (D) Tumor weights 4 weeks after inoculation with 4T1-luc cancer cells (n = 14). (E) Representative bioluminescence images of mice of the 2 groups. Scale bar: 20 mm. (F) The luciferase intensity of tumor 4 weeks after inoculation with 4T1-luc cells (n = 14). (G–I) LDT: representative heatmaps (G) and quantitative summary of the time spent in the light box (H) and the total number of transitions (I) in the vehicle (n = 17) and 4T1-luc (n = 14) treatment groups. (J–L) OFT: representative heatmaps (J) and quantification of the time spent in the center zone (K) and the distance traveled in the center zone (L) in the vehicle (n = 17) and 4T1-luc (n = 14) treatment groups. (M–P) EPM test: representative heatmaps (M) and quantification of the time spent in the open arms (N), entries into the open arms (O), and the anxiety index (P) in the vehicle (n = 17) and 4T1-luc (n = 14) treatment groups. (Q) NE content of tumor tissue 4 weeks after 4T1-luc cell inoculation (n = 14). (R–T) Correlation between tumor volume and tumor tissue NE content (R), tumor weight and tumor tissue NE content (S), and tumor luciferase intensity and tumor tissue NE content (T). Data are presented as the mean ± SEM, except in the box plot (K, L, and Q), in which the centerline indicates the median, box edges represent the first and third quartiles, and whiskers denote minimal and maximal values. ***P < 0.001, by 2-tailed, unpaired Student’s t test (H, I, K, L, and N–P) and 2-sided linear regression analysis (R–T). Max, maximum; Min, minimum.
Figure 2
Figure 2. Newly formed sympathetic innervation of 4T1 tumors connects to the brain.
(A) Schematic diagram of immunofluorescence staining for TH and NF-L in breast tumors (n = 4 for each group). (B) Representative images showing immunofluorescence staining for TH and NF-L at days 5, 7, and 9 after 4T1 cell inoculation. Scale bars: 100 μm. (C) Quantification of TH+ sympathetic nerve fibers in outer regions of the tumor (field surface = 0.15 mm2; n = 4 for each group). (D) Experimental scheme showing the transplantation of 4T1 cells and intratumoral injection of the neurotropic retrograde transpolysynaptic pseudorabies virus PRV-EGFP. (E–J) Representative images showing PRV-infected neurons (green) in the intermediolateral cell column (IML) (E) and CeM (F–J) from the mice 6 days after PRV-EGFP injection into the tumor tissue. Scale bars: 200 μm. (K) Quantification of PRV+ neurons in the left and right CeM (n = 5). (L) Representative images and quantification of PRV+CRH+ neurons among PRV+ neurons in the CeM (n = 5). (M) Representative images and quantification of PRV+TH+ neurons among PRV+ neurons in the LPGi (n = 5). Scale bars: 20 μm (L and M). Data are presented as the mean ± SEM, except in box and half violin plots (L and M), in which center lines indicate the median, box edges represent the first and third quartiles, and whiskers denote minimal and maximal values. ***P < 0.001, by 1-way ANOVA followed by Tukey post hoc test (C) and 2-tailed, unpaired Student’s t test (K). BLA, basolateral amygdaloid nucleus; CeL, central nucleus of the amygdala, lateral division; DH, dorsal horn; VH, ventral horn.
Figure 3
Figure 3. Activation of CeMCRH neurons increases the activities of local sympathetic nerves distributed in mammary tumors.
(A) Timeline for c-Fos and CRH immunofluorescence staining. (B) Representative bioluminescence images of mice 4 weeks after injection of vehicle or 4T1-luc breast cancer cells. Scale bar: 10 mm. (C and D) Representative images (C) and quantification (D) of c-Fos+ neurons colocalized with CeMCRH neurons from the vehicle and 4T1-luc treatment groups (n = 5 for each group). Scale bars: 50 μm. (E and F) Timeline and scheme for recording the activities of sympathetic nerves distributed in tumor stroma during optogenetic stimulation of CeMCRH neurons. (G and H) Typical image of virus expression in CeM (G) and tumor stroma (H). Scale bars: 200 μm (G) and 20 μm (H). (I and J) Comparison of the mean ΔF/F (0–5 s) (I) and peak amplitude of ΔF/F (J) between the GRABNE2h and EGFP groups (n = 5 for each group). (K) Average fluorescence change in the GRABNE2h and EGFP groups, with shaded areas indicating the SEM. (L and M) Heatmaps show the average fluorescence change in the GRABNE2h (L) and EGFP (M) groups. Data are presented as the mean ± SEM. ***P < 0.001, by 2-tailed, unpaired Student’s t test (D, I, and J).
Figure 4
Figure 4. Specific ablation of CeMCRH neurons significantly reduces cancer-induced anxiety and suppresses 4T1 tumor progression.
(A) Schematic of the experimental design. (B) Schematic showing bilateral injection of viruses into the CeM. (C) Representative images showing the successful ablation of CeMCRH neurons. Scale bars: 200 μm and 20 μm. (D–M) Representative heatmaps and summary data for the EYFP (n = 15) and taCasp3 (n = 12) groups in the LDT (D–F), the OFT (G–I), and the EPM test (J–M). (N) Ablation of CeMCRH neurons significantly slowed 4T1 tumor growth. (O) Representative images of 4T1 tumors dissected from mice of the 2 groups. Scale bar: 10 mm. (P) The ablation of CeMCRH neurons significantly reduced 4T1 tumor weight. (Q) Representative bioluminescence images of mice of the 2 groups. Scale bar: 20 mm. (R) The ablation of CeMCRH neurons significantly reduced the luciferase intensity of 4T1 tumors (P and R: EYFP, n = 15, taCasp3, n = 12). (S and T) The ablation of CeMCRH neurons significantly decreased NE content of 4T1 orthotopic mammary (S) and ectopic (T) tumors (S: EYFP, n = 15, taCasp3, n = 12; T: n = 6 for each group). (U) Timeline for immunofluorescence staining of 4T1 tumor tissues. (V and W) Representative images and quantification of Ki67+ cells (V) and TUNEL+ cells (W) within 4T1 tumors (n = 6 for each group). Scale bars: 50 μm. Data are presented as the mean ± SEM, except in box plots (H, I, S, and T), in which center lines indicate the median, box edges represent the first and third quartiles, and whiskers denote minimal and maximal values. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way, repeated-measures ANOVA followed by separate 1-way ANOVA (N) and 2-tailed, unpaired Student’s t test (E, F, H, I, K–M, P, R–T, V, and W).
Figure 5
Figure 5. Chemogenetic inhibition of CeMCRH neurons significantly attenuates both cancer-induced anxiety and 4T1 tumor progression.
(A) Schematic of the experimental design. (B) Schematic showing bilateral injection of viruses into the CeM. (C) Representative images showing hM4Di-mCherry expression in CeMCRH neurons. Scale bars: 200 μm and 20 μm. (D–M) Representative heatmaps and summary data of the mCherry (n = 15) and hM4Di (n = 14) groups in the LDT (D–F), the OFT (G–I), and the EPM test (J–M). (N) Chemogenetic inhibition of CeMCRH neurons significantly slowed 4T1 tumor growth. (O) Representative tumors dissected from mice of the 2 groups. Scale bar: 10 mm. (P) Chemogenetic inhibition of CeMCRH neurons significantly reduced 4T1 tumor weight. (Q) Representative bioluminescence images of mice of the 2 groups. Scale bar: 20 mm. (R) Chemogenetic inhibition of CeMCRH neurons significantly reduced the luciferase intensity of 4T1 tumors (P and R: mCherry, n = 15, hM4Di, n = 14). (S and T) Chemogenetic inhibition of CeMCRH neurons significantly decreased NE content of 4T1 orthotopic mammary (S) and ectopic (T) tumors (S: mCherry, n = 15, hM4Di, n = 14; T: n = 6 for each group). (U) Timeline for immunofluorescence staining of 4T1 tumor tissues. (V and W) Representative images and quantification of Ki67+ cells (V) and TUNEL+ cells (W) within 4T1 tumors (n = 6 for each group). Scale bars: 50 μm. Data are presented as the mean ± SEM, except in box plots (H, I, S, and T), in which center lines indicate the median, box edges represent the first and third quartiles, and whiskers denote minimal and maximal values. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way, repeated-measures ANOVA followed by separate 1-way ANOVA (N) and 2-tailed, unpaired Student’s t test (E, F, H, I, K–M, P, R–T, V, and W).
Figure 6
Figure 6. Chemogenetic activation of CeMCRH neurons significantly increases cancer-induced anxiety and accelerates 4T1 tumor progression.
(A) Schematic illustration of the experimental design. (B) Schematic showing bilateral injection of viruses into the CeM. (C) Representative image showing hM3Dq-mCherry expression in the CeM. Scale bar: 200 μm.(D–M) Representative heatmaps and summary data of the mCherry (n = 15) and hM3Dq (n = 15) groups in the LDT (D–F), the OFT (G–I), and the EPM test (J–M). (N) Chemogenetic activation of CeMCRH neurons significantly accelerated 4T1 tumor growth. (O) Representative image of tumors dissected from mice of the 2 groups. Scale bar: 10 mm. (P) Chemogenetic activation of CeMCRH neurons significantly increased 4T1 tumor weight. (Q) Representative bioluminescence images of mice of the 2 groups. Scale bar: 20 mm. (R) Chemogenetic activation of CeMCRH neurons significantly increased the luciferase intensity of 4T1 tumors (P and R: n = 15 for each group). (S and T) Chemogenetic activation of CeMCRH neurons significantly increased NE content of 4T1 orthotopic mammary (S) and ectopic (T) tumors (S: n = 15 for each group; T: n = 6 for each group). (U) Timeline for immunofluorescence staining of 4T1 tumor tissues. (V and W) Representative images and quantification of Ki67+ cells (V) and TUNEL+ cells (W) within 4T1 tumors (n = 6 for each group). Scale bars: 50 μm. Data are presented as the mean ± SEM, except in box plots (H, I, S, and T), in which center lines indicate the median, box edges represent the first and third quartiles, and whiskers denote minimal and maximal values. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way, repeated-measures ANOVA followed by separate 1-way ANOVA (N) and 2-tailed, unpaired Student’s t test (E, F, H, I, K–M, P, R–T, V, and W).
Figure 7
Figure 7. Optogenetic stimulation of the CeMCRH→LPGi circuit activates sympathetic nerves in 4T1 tumors.
(A) Schematic of the Cre-dependent retrograde transmonosynaptic rabies virus–tracing strategy in WT BALB/c mice. (B) EGFP-labeled neurons in the CeM traced from LPGiCA neurons. Scale bar: 200 μm. (C and D) Representative images showing colocalization of EGFP with CRH in the CeM (C) and summarized data (D; n = 5). Scale bar: 200 μm. (E and F) Timeline and scheme for recording the activities of sympathetic nerves distributed in the tumor stroma during optogenetic stimulation of the CeMCRH→LPGi circuit in the CeM. (G and H) Typical image of viruses (AAV and pLenti) expression in the CeM (G) and tumor stroma (H). Scale bars: 200 um (G) and 20 um (H). (I and J) Comparison of the mean ΔF/F (0–5 s) (I) and peak amplitude of ΔF/F (J) between the GRABNE2h and EGFP groups (n = 6 for each group). (K) Average fluorescence change in the GRABNE2h and EGFP groups, with shaded areas indicating the SEM. (L and M) Heatmaps show the average fluorescence change in the GRABNE2h (L) and EGFP (M) groups. Data are presented as the mean ± SEM, except in box and half violin plots (D), in which center lines indicate the median, box edges represent the first and third quartiles, and whiskers denote minimal and maximal values. ***P < 0.001, by 2-tailed, unpaired Student’s t test (I and J).
Figure 8
Figure 8. Chemogenetic inhibition of the CeMCRH→LPGi circuit significantly suppresses both cancer-induced anxiety and 4T1 tumor progression.
(A) Schematic of the experimental design. (B) Schematic showing bilateral injection of rAAV2/retro-CRH-Cre into the LPGi and of rAAV2/9-EF1α-DIO-hM4Di-mCherry or rAAV2/9-EF1α-DIO-mCherry into the CeM. (C) Representative images showing hM4Di-mCherry expression in the CeM. Scale bar: 200 μm. (D–M) Representative heatmaps and summary data of the mCherry (n = 14) and hM4Di (n = 13) groups in the LDT (D–F), the OFT (G–I), and the EPM test (J–M). (N) Chemogenetic inhibition of the CeMCRH→LPGi circuit significantly decelerated 4T1 tumor growth. (O) Representative tumors dissected from mice of the 2 groups. Scale bar: 10 mm. (P) Chemogenetic inhibition of the CeMCRH→LPGi circuit significantly reduced 4T1 tumor weight. (Q) Representative bioluminescence images of mice of the 2 groups. Scale bar: 20 mm. (R and S) Chemogenetic inhibition of the CeMCRH→LPGi circuit significantly reduced the luciferase intensity of 4T1 tumors (R) and tumor tissue NE content (S) (P, R, and S: mCherry, n = 14, hM4Di, n = 13). Data are presented as the mean ± SEM, except in box plots (H, I, and S), in which center lines indicate the median, box edges represent the first and third quartiles, and whiskers denote minimal and maximal values. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way, repeated-measures ANOVA followed by separate 1-way ANOVA (N) and 2-tailed, unpaired Student’s t test (E, F, H, I, K–M, P, R, and S).
Figure 9
Figure 9. Chemogenetic activation of the CeMCRH→LPGi circuit significantly increases cancer-induced anxiety and accelerates 4T1 tumor progression.
(A) Schematic illustration of the experimental design. (B) Schematic showing bilateral injection of rAAV2/retro-CRH-Cre into the LPGi and of rAAV2/9-EF1α-DIO-hM3Dq-mCherry or rAAV2/9-EF1α-DIO-mCherry into the CeM. (C) Representative images showing hM3Dq-mCherry expression in the CeM. Scale bar: 200 μm. (D–M) Representative heatmaps and summary data for the mCherry (n = 16) and hM3Dq (n = 13) groups in the LDT (D–F), the OFT (G–I), and the EPM test (J–M). (N) Chemogenetic activation of the CeMCRH→LPGi circuit significantly accelerated 4T1 tumor growth. (O) Representative image of tumors dissected from mice of the 2 groups. Scale bar: 10 mm. (P) Chemogenetic activation of the CeMCRH→LPGi circuit significantly increased 4T1 tumor weight. (Q) Representative bioluminescence images of mice of the 2 groups. Scale bar: 20 mm. (R and S) Chemogenetic activation of the CeMCRH→LPGi circuit significantly increased the luciferase intensity of 4T1 tumors (R) and tumor tissue NE content (S). (P, R, and S: mCherry, n = 16, hM3Dq, n = 13). Data are presented as the mean ± SEM, except in box plots (H, I, and S), in which center lines indicate the median, box edges represent the first and third quartiles, and whiskers denote minimal and maximal values. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way, repeated-measures ANOVA followed by separate 1-way ANOVA (N) and 2-tailed, unpaired Student’s t test (E, F, H, I, K–M, P, R, and S).
Figure 10
Figure 10. Alprazolam treatment significantly inhibits the activity of CeMCRH neurons and LPGiCA neurons and decelerates the progression of 4T1 breast tumors.
(A) Experimental protocol for treatment with alprazolam or vehicle and immunofluorescence staining. (B–E) Representative images and summarized data for c-Fos expression in CeM CRH neurons (B and C) and LPGiCA neurons (D and E) after treatment with alprazolam (Alp) or vehicle (n = 5 for each group). Scale bars: 50 μm (B and D). (F) Schematic overview of the experimental design. (G–P) Representative heatmaps and summary data for vehicle (n = 16) and alprazolam (n = 15) treatment groups in the LDT (G–I), the OFT (J–L), and the EPM test (M–P). (Q) Alprazolam treatment significantly decelerated 4T1 tumor growth. (R) Representative image of tumors dissected from mice of the 2 groups. Scale bar: 10 mm. (S) Alprazolam treatment significantly reduced 4T1 tumor weight. (T) Representative bioluminescence images of mice of the 2 groups. Scale bar: 20 mm. (U and V) Alprazolam treatment significantly reduced the luciferase intensity of 4T1 tumors (U) and tumor tissue NE content (V). (S, U, and V: vehicle, n = 16, alprazolam, n = 15). Data are presented as the mean ± SEM, except in box plots (C, E, K, L, and V), in which center lines indicate the median, box edges represent the first and third quartiles, and whiskers denote minimal and maximal values. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way, repeated-measures ANOVA followed by separate 1-way ANOVA (Q) and 2-tailed, unpaired Student’s t test (C, E, H, I, K, L, N–P, S, U, and V).
Figure 11
Figure 11. Alprazolam treatment significantly reduces cancer-induced anxiety and suppresses tumor progression in MMTV-PyMT mice.
(A) Schematic of the experimental design. (B–K) Representative heatmaps and summary data for the vehicle (n = 5) and alprazolam (n = 5) treatment groups in the LDT (B–D), the OFT (E–G), and the EPM test (H–K). (L) Alprazolam treatment significantly decelerated PyMT tumor growth. (M) Alprazolam treatment significantly reduced PyMT tumor weight. (N) Alprazolam treatment significantly reduced the NE content of PyMT tumor tissue (n = 5 for each group). Data are presented as the mean ± SEM, except in box plots (F, G, and N), in which center lines indicate the median, box edges represent the first and third quartiles, and whiskers denote minimal and maximal values. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-way, repeated-measures ANOVA followed by separate 1-way ANOVA (M) and 2-tailed, unpaired Student’s t test (C, D, F, G, I–K, and M).
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