Genetic manipulation of autonomic nerve fiber innervation and activity and its effect on breast cancer progression
Authors: Kazuto Kobayashi
Ryosuke Kaneko
Yohsuke Hayama
Takahiro Ochiya
Katsumasa Irie
Takushi Shimomura
Atsunori Kamiya
Akihiko Shimomura
Shigeki Kato
Yuchio Yanagawa
Journal: Nature Neuroscience (2019)
Discipline: NEUROSCIENCE & BEHAVIOR
Keywords: autonomic innervation; tumor growth; breast cancer; sympathetic nerves; parasympathetic nerves
Affiliations: Department of Cellular Physiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. ., Department of Cardiovascular Dynamics, National Cerebral and Cardiovascular Center Research Institute, Suita, Japan. ., PRIME, Japan Agency for Medical Research and Development, Tokyo, Japan. ., Department of Cardiovascular Dynamics, National Cerebral and Cardiovascular Center Research Institute, Suita, Japan., Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima, Japan., Department of Breast and Medical Oncology, National Cancer Center Hospital, Tokyo, Japan., Cellular and Structural Physiology Institute (CeSPI), Nagoya University, Furo-cho, Chikusa, Nagoya, Japan., Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Okazaki, Japan., Department of Genetic and Behavioral Neuroscience, Gunma University Graduate School of Medicine, Maebashi, Japan., Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan., Institute of Medical Science, Tokyo Medical University, Tokyo, Japan.

Abstract:  The effects of autonomic innervation of tumors on tumor growth remain unclear. Here we developed a series of genetic techniques to manipulate autonomic innervation in a tumor- and fiber-type-specific manner in mice with human breast cancer xenografts and in rats with chemically induced breast tumors. Breast cancer growth and progression were accelerated following stimulation of sympathetic nerves in tumors, but were reduced following stimulation of parasympathetic nerves. Tumor-specific sympathetic denervation suppressed tumor growth and downregulated the expression of immune checkpoint molecules (programed death-1 (PD-1), programed death ligand-1 (PD-L1), and FOXP3) to a greater extent than with pharmacological α- or β-adrenergic receptor blockers. Genetically induced simulation of parasympathetic innervation of tumors decreased PD-1 and PD-L1 expression. In humans, a retrospective analysis of breast cancer specimens from 29 patients revealed that increased sympathetic and decreased parasympathetic nerve density in tumors were associated with poor clinical outcomes and correlated with higher expression of immune checkpoint molecules. These findings suggest that autonomic innervation of tumors regulates breast cancer progression. 

Retracted: No

PubPeer comments:
 The same p-values appeared in the paper and supplement. For example, 1.3u00d710-6 appeared totally more than 100 times; 1.4u00d710-6 also appeared many times. How do the authors explain these unusual results? ![file] ![file] , 
 There are very many repeats of statistical errors, which can be illustrated with reference to the image below (from Fig. 1f). ![file] The relevant section of the legend is as follows (note that the panels are identified after their description):  quantification of luciferase intensity by bioluminescence imaging of the primary tumor (*Pu2009 u20090.05 between groups; two-way ANOVA followed by Tukeyu2019s multiple-comparisons test) (f, middle) and distant metastasis (*Pu2009 u20090.05 between groups; one-way ANOVA followed by Tukeyu2019s multiple-comparisons test) (f, right) For the tumor growth curves, a repeated-measures instead of a two-way ANOVA should have been employed. In addition, the requirement for equal variances is spectacularly violated. Both errors tend to  reinforce significance , which might go some way to explaining the rather extreme p-values reported (the repeats I m not sure). The stars on the right might reflect more realistic direct tests of the final points, but this doesn t seem to be stated. For the Metastasis analysis, the authors again employ a (one-way) ANOVA while violating the requirement for (roughly) equal variances. , 
 #2 Thank you for your comments on the issue. Here is the count of the repeat of two p-values. ![file] , 
  In the MDA-MB-231-Luc xenograft model of Balb c nude mice (Fig. 5au2013f), immunofluorescence analysis of co-staining with the parasympathetic neuronal (cholinergic) marker vesicular acetylcholine transporter (VAChT) and the neuron-specific marker NF-L revealed there was parasympathetic innervation of tumors (Fig. 5b).  Intratumoral injection of AAV-ChAT-NaChBac T220A-2A-GCaMP3 vectors, which induce parasympathetic neurostimulation, that also carried a parasympathetic neuron-specific choline acetyltransferase (ChAT) promoter led to retrograde gene delivery into VAChT+ parasympathetic nerves of tumors with high transduction efficacy (based on GFP immunofluorescence staining (Fig. 5c)), increased nerve Ca2+ activity (Fig. 5d) The authors showed that parasympathetic innervation for the subcutaneous organ, the breast, or breast cancer in this paper. These  findings beyond current knowledge about parasympathetic nerve anatomy. Where are the parasympathetic nerves innervated on the breast came from? Vagal nerve or the spinal cord? , 
 Here is the count of the repeat of two p-values in the Supplement file. Total counts include the paper and supplement are 138 times for 1.3u00d710-6 and 31 times for 1.4u00d710-6. ![file] , 
 Repeat usage is not limited to the numbers; it also appeared in the figures of the paper. Here is the case in Figure 3 and Figure 5. Welcome the authors to check more errors. ![file] , 
 Figure 7 presented human breast cancer samples. I want to ask the authors again: Where are the parasympathetic nerves innervated on the human breast come from? Vagal nerve or the spinal cord? ![file] , 
 In the supplementary, flow cytometry results exist so many similar images. I want to ask who is the expert in the field, are these phenomena natural? ![file] , 
 #6 There might be a repeat use in the early paper of the first author. https: doi.org 10.1152 ajpheart.00340.2009 Each multifiber nerve activity recording is like a fingerprint. However, there were two similar (almost the same, except for the difference in amplitude) images of nerve activity recordings. ![file] downloaded from: https: journals.physiology.org na101 home literatum publisher physio journals content ajpheart 2010 ajpheart.2010.298.issue-6 ajpheart.00340.2009 production images large zh40061093610001.jpeg , 
 The picture was duplicated in those figures represent different interventions. ![file] , 
 Another duplicate ![file] , 
 It was not simply a duplicate. The animal had grown up (enlarged the picture?) after 60 days, while the tumor became smaller than 2 months ago. ![file] ,