VLM catecholaminergic neurons control tumor growth by regulating CD8+ T cells

Abstract

It is known that tumor growth can be influenced by the nervous system. It is not known, however, if tumors communicate directly with the central nervous system (CNS) or if such interactions may impact tumor growth. Here, we report that ventrolateral medulla (VLM) catecholaminergic (CA) neurons in the mouse brain are activated in tumor-bearing mice and the activity of these neurons significantly alter tumor growth in multiple syngeneic and spontaneous mouse tumor models. Specific ablation of VLM CA neurons by a dopamine-β-hydroxylase (DBH) promotor-activated apoptosis-promoting caspase-3 in Dbh-Cre mice as well as inhibition of these neurons by a chemogenetic method slowed tumor progression. Consistently, chemogenetic activation of VLM CA neurons promoted tumor growth. The tumor inhibition effect of VLM CA neuron ablation is mitigated in Dbh-Cre;Rag1^-/- mice, indicating that this regulatory effect is mediated by the adaptive immune system. Specific depletion of CD8⁺ T cells using an anti-CD8⁺ antibody also mitigated the tumor suppression resulting from the VLM CA neuron ablation. Finally, we showed that the VLM CA neuronal ablation had an additive antitumor effect with paclitaxel treatment. Collectively, our study uncovered the role of VLM CA neurons in the mouse brain in controlling tumor growth in the mouse body.

Keywords: CA neurons; neuroimmunology; tumor model.

Fig. 1.

Syngeneic mouse tumors activate VLM catecholaminergic neurons (VLM CA neurons) while depletion of VLM CA neurons slows tumor growth. (A) 1 × 10⁶ MC-38 murine colon adenocarcinoma cells or vehicle (PBS) were inoculated into the flank of C57BL/6 mice. The tumor group displayed activation of neurons; such activation was not observed in the PBS or naive group. TH in green is a marker for VLM CA neurons; and c-fos in red is a marker of neuronal activation. TH and c-Fos double positive cell number in VLM CA: 8 ± 1 (naive) vs. 9 ± 2 (PBS) vs. 213 ± 10 (MC-38). (B) Specific ablation of VLM CA neurons by rAAV-taCasp3 virus-induced apoptosis. Control mice were injected with the rAAV-mCherry virus. The colocalization of mCherry and TH shows the accurate stereotactic manipulation, and the absence of a TH signal in the Casp3 group confirmed the ablation. TH positive cell number in VLM CA: 876 ± 46 (mCherry) vs. 63 ± 17 (Casp3). (C) The ablation of VLM CA neurons by AAV-taCasp3 slows syngeneic tumor growth. (n_Casp3 = 7, n_mCherry = 7). (D) Images of the tumors from mCherry or Casp3 virus-injected mice. (n_Casp3 = 7, n_mCherry = 7). (E) The ablation of VLM CA neurons reduces tumor weight. (n_Casp3 = 7, n_mCherry = 7). (F) The ablation of VLM CA neurons extends the survival duration of mice. (n_Casp3 = 10, n_mCherry = 10). Results shown represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. See also SI Appendix, Fig. S1.

Fig. 2.

The activity of VLM CA neurons controls tumor growth. (A) Tumor growth curve. The direct activation of VLM CA neurons by AAV-hM3D(Gq) can promote tumor growth; however, direct inhibition by AAV-hM4D(Gi) can slow tumor growth progression (n_hM3D = 4, n_mCherry = 7, n_hM4D = 5). (B) The tumor size image, obtained from dissected tumor-bearing mice, showing that activation of VLM CA neurons can enhance tumor size, and that inhibition of VLM CA neurons can reduce tumor size (n_hM3D = 6, n_mCherry = 9, n_hM4D = 7). (C) The tumor weight showing the tumor promotion impact from the activation of VLM CA neurons and the tumor inhibition impact upon inhibition of VLM CA neurons (n_hM3D = 6, n_mCherry = 9, n_hM4D = 7). (D) IVIS imaging. In vivo imaging showing VLM CA neuron activation [by AAV-hM3D(Gq)] results in relatively stronger photon intensity, whereas inhibition of VM CA neurons by AAV-hM4D(Gi) results in relatively weaker photon intensity (n_hM3D = 3, n_mCherry = 3, n_hM4D = 3). (E) Histogram showing the quantified IVIS results (n_hM3D = 3, n_mCherry = 3, n_hM4D = 3). (F) Survival curve. Inhibition of VLM CA neuron activity by AAV-hM4D(Gi) can prolong survival while VLM CA neuron activation by AAV-hM3D(Gq) can accelerate the time to death for tumor-bearing mice (n_hM3D = 10, n_mCherry = 10, n_hM4D = 11). Results shown represent the mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

The adaptative immune system functions in VLM CA neuron-regulated tumor growth. (A) Quantification of FACS for CD4⁺ and CD8⁺ T cells in Dbh-Cre;Rag1^−/− mice. The Dbh-Cre;Rag1^−/− mice (n_Dbh-Cre = 3, n_Dbh-Cre;Rag1^−/−= 3) had no CD4⁺, CD8⁺ T cells, total T cells and B cells. (B and C) There were no differences observed in tumor weight between the AAV-mCherry– vs. AAV-Casp3–treated immunodeficient Dbh-Cre;Rag1^−/− mice (n_Casp3 = 8, n_mCherry = 14). (D) There was more leukocyte infiltration indicated by CD45⁺ cells after VLM CA neuron ablation compared with mCherry control mice (n_Casp3 = 6, n_mCherry = 6). There were more CD4⁺ and CD8⁺ T cells after VLM CA neuron ablation intratumor (E) and in spleen (F) (n_Casp3 = 6, n_mCherry = 6) after tumor inoculation. (G) The successful depletion of CD4⁺ and CD8⁺ T cells based on CD4⁺ or CD8⁺ T depletion antibodies, respectively (n_Casp3 = 3, n_mCherry = 3). (H) It was CD8⁺ T cells that functioned in VLM CA regulated tumor growth process mCherry + PBS vs. Casp3 + PBS, P = 0.004; mCherry + PBS vs. mCherry + CD4, P = 0.179; mCherry + PBS vs. mCherry + CD8, P = 0.027; Casp3 + PBS vs. Casp3 + CD4, P = 0.398; Casp3 + PBS vs. Casp3 + CD8, P = 0.000; mCherry + CD4 vs. Casp3 + CD4, P = 0.011; mCherry + CD8 vs. Casp3 + CD8, P = 0.618 (n_mCherry+PBS = 12, n_mCherry+CD4 = 12, n_mCherry+CD8 = 12, n_Casp3+PBS = 14, n_Casp3+CD4 = 14, n_Casp3+CD8 = 12). Results shown represent the mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

VLM CA neuron ablation slows down tumor progression spontaneously. There are fewer (A) and smaller (B) polyps in mouse intestine (including small intestine and colon) by using the Dbh-Cre;APC^min/+ mice model after VLM CA neuron ablation. SI, small intestine. (C) The smaller polyps (<3 mm) were dominant after VLM CA neuron ablation, and the larger polyps (>3 mm) were dominant in control mCherry-introduced mice (n_Casp3 = 6, n_mCherry = 9). (D) There was decreased tumor burden after VLM CA neuron ablation by using the APC^min/+ mice model. (E) There were fewer large polyps (>3 mm) in colons after VLM CA neuron ablation by using the AOM/DSS model. (n_Casp3 = 6, n_mCherry = 6). (F) There were additive effects of taxol (paclitaxel) and VLM CA neuron manipulation in the MC-38 inoculation mouse model. (n_mCherry+mock = 12, n_{mCherry+Paclitaxel} = 12, n_Casp3+mock = 12, n_{Casp3+Paclitaxel} = 14). Results shown represent the mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001.