Multiple cancer cell types release LIF and Gal3 to hijack neural signals

Abstract

Neural signals can significantly influence cancer prognosis. However, how cancer cells may proactively modulate the nervous system to benefit their own survival is incompletely understood. In this study, we report an overlapping pattern of brain responses, including that in the paraventricular nucleus of the hypothalamus, in multiple mouse models of peripheral cancers. A multi-omic screening then identifies leukemia inhibitory factor (LIF) and galectin-3 (Gal3) as the key cytokines released by these cancer cell types to trigger brain activation. Importantly, increased plasma levels of these two cytokines are observed in patients with different cancers. We further demonstrate that pharmacologic or genetic blockage of cancer cell-derived LIF or Gal3 signaling abolishes the brain responses and strongly inhibits tumor growth. In addition, ablation of peripheral sympathetic actions can similarly restore antitumor immunity. These results have elucidated a novel, shared mechanism of multiple cancer cell types hijacking the nervous system to promote tumor progression.

Fig. 1. An overlapping pattern of brain responses in multiple mouse models of peripheral cancers.

a–d Mouse allograft models of LLC, RM1, MC38, or 4T1 cells and the MMTV-PyMT mouse model were utilized. Brain responses were assessed by the p-S6 immunostaining. Representative images of the PVN, SCN, Ve-L/-R, and Me5-L/-R (a) or the NTS, PBN-L/-R, and ARC (c) were shown. Scale bars, 100 μm. Neural activities in the indicated brain regions were quantified (b, d). Data are shown as mean ± SD. One-way ANOVA test; ns, not significant; *P < 0.05. e, f A multi-omic screening for the cancer cell-derived factor(s) triggering brain responses. The scheme of integrating RNA-seq data of different cancer types and secretomic analyses of cultured cancer cells was illustrated (e). The published scRNA-seq dataset of mouse trigeminal ganglionic neurons (GSE213105) was exploited to identify candidate proteins for functional examination (f).

Fig. 2. LIF and Gal3 are the cancer cell-derived factors that cooperatively activate specific brain regions.

a–e Non-tumor-bearing C57BL/6 wild-type mice were administered with the indicated amounts of recombinant mouse LIF or Gal3 protein. Brain responses were assessed by the p-S6 immunostaining. The scheme of the experimental procedure was illustrated (a). Representative images of the PVN, SCN, Ve-L/-R, and Me5-L/-R (b) or the NTS and PBN-L/-R (d) were shown. Scale bars, 100 μm. Neural activities in the indicated brain regions were quantified (c, e). Data are presented as mean ± SD. One-way ANOVA test; ns not significant; *P < 0.05. f Mouse allograft models of LLC, RM1, MC38, or 4T1 cells and the MMTV-PyMT mouse model were utilized. Plasma levels of LIF and Gal3 in different mouse models were examined by enzyme-linked immunosorbent assay (ELISA). Data are presented as mean ± SD. One-way ANOVA test; *P < 0.05. g Plasma levels of LIF and Gal3 in human patients with different peripheral cancers were measured by ELISA. Data are presented as mean ± SD. One-way ANOVA test; *P < 0.05.

Fig. 3. Cancer cell-derived LIF and Gal3 are essential for brain responses.

a–c LLC, RM1, MC38, or 4T1 cancer cells with LIF KO or Gal3 KO were generated by CRISPR/Cas9 and then tested in mouse allograft models. Brain responses were assessed by the p-S6 immunostaining. The scheme of the experimental procedure was illustrated (a). Representative images of the PVN, SCN, Ve-L/-R, and Me5-L/-R were shown (b). Scale bars, 100 μm. Neural activities in the indicated brain regions were quantified (c). Data are presented as mean ± SD. One-way ANOVA test; ns, not significant; *P < 0.05.

Fig. 4. Blockage of LIF or Gal3 signaling inhibits tumor progression.

a–c LLC, RM1, MC38, or 4T1 cancer cells with LIF KO or Gal3 KO were examined in mouse allograft models. Tumor growth rates under the indicated conditions were monitored (a). n = 8. Data are shown as mean ± SD. Two-way ANOVA test; *P < 0.05. MDSCs in the blood or tumors under the indicated conditions were quantified by fluorescence-activated cell sorting (FACS) (b). CD8⁺ T cells in the tumors under the indicated conditions were examined by FACS (c). Data are shown as mean ± SD. One-way ANOVA test; *P < 0.05.

Fig. 5. Cancer cell-derived LIF and Gal3 act via sympathetic signaling to facilitate MDSC generation.

a–d Th-Cre;TrkA^fl/fl (sympathetic ablation) and control Th-Cre;TrkA^+/+ littermates were utilized for LLC or RM1 allograft models. Tumor growth rates under the indicated conditions were monitored (a). n = 10. Data are presented as mean ± SD. Two-way ANOVA test; *P < 0.05. MDSCs in the blood or tumors under the indicated conditions were quantified by FACS (b). CD8⁺ T cells in the tumors of LLC (c) or RM1 (d) allograft models were examined by FACS. Data are presented as mean ± SD. Student’s t-test. e, f LLC (e) or RM1 (f) cancer cells with LIF KO or Gal3 KO were assessed in the allograft models with sympathetic ablation. Tumor growth rates under the indicated conditions were monitored. n = 10. Data are presented as mean ± SD. Two-way ANOVA test; *P < 0.05.

Fig. 6. Sympathetic signaling directly promotes the immunosuppressive function of MDSCs.

a–c C57BL/6 wild-type mice were subjected to spleen sympathectomy and then utilized for LLC or RM1 allograft models. Sympathetic axons in the spleen after sham surgery or sympathectomy were visualized by the immunostaining of tyrosine hydroxylase (a). MDSCs in the spleens (b) and the blood (c) under the indicated conditions were quantified by FACS. Data are presented as mean ± SD. Student’s t-test. d PMN-MDSCs or M-MDSCs were FACS-sorted from the spleens of tumor-bearing mice and in vitro treated with NE in combination with the β2-adrenergic receptor antagonist propranolol. mRNA levels of immunosuppressive genes were examined by qPCR analyses. Data are presented as mean ± SD. One-way ANOVA test. e Diagram of cancer cell-derived LIF and Gal3 hijacking the nervous system to promote tumor progression.