Mo1917 Preclinical Trial of Botulinum Toxin Type a Injection in the Treatment of Gastric Cancer

Abstract

Background/aim: Innervation plays an important role in the regulation of epithelial homeostasis as well as tumorigenesis. Botulinum neurotoxin type A (Botox) is known to block both afferent and efferent nerve fibers by binding to SNARE proteins. The aim of this trial was to evaluate the effectiveness and underlying mechanism of local injection of Botox in the treatment of gastric cancer. Methods: 268 genetically-manipulated INS-GAS male and female mice with spontaneous gastric cancer were subjected to Botox injection or subdiaphragmatic unilateral vagotomy (UVT), with or without chemotherapy (5-FU, oxaliplatin, or 5-FU + oxaliplatin). Botox was injected subserosally along the greater curvature on the anterior side of the stomach and the injection was repeated every 2nd month. Chemotherapy was given by intraperitoneal injection once per week for 4 weeks in a two-cycle course with a 1-week rest. Clinical endpoints included tumor size, histopathological score, and survival. Gastric organoid cultures with or without Botox, neurons, or a muscarinic receptor agonist or antagonist were used. Results: Botox treatment was given to INS-GAS mice at 6 months of age when gastric cancer started to develop. Six months later, tumor size, cell proliferation rate, scores of inflammation, epithelial defects, atrophy, hyperplasia, dysplasia and metaplasia were markedly reduced in comparison with the uninjected posterior area of the stomach. When Botox treatment was given at 14 months of age in combination with 5-FU+oxaliplatin, tumor size was significantly reduced as early as 2 months after starting treatment, particularly in the area of Botox injection or in the surgically denervated stomach following UVT. The combination of either Botox or UVT with chemotherapy led to a significant increase in survival compared to chemotherapy alone. In vitro studies showed that neurons stimulated gastric organoid growth when compared to gastric organoids cultured in the absence of neurons, and that the addition of either Botox or scopolamine inhibited this stimulatory effect, whereas pilocarpine increased organoid growth. Furthermore, pilocarpine upregulated expression of stem cell markers and Wnt target genes, such as Lgr5, CD44, and Sox9, and both pilocarpine and neurons could substitute for Wnt3a in gastric organoid cultures that were otherwise strictly dependent on the addition of Wnt ligands. Conclusions: This preclinical trial demonstrates the efficacy of local Botox injection with or without chemotherapy in the treatment of gastric cancer. The therapeutic effect is likely mediated by acetylcholinemediated Wnt signaling in gastric stem cells. We may further suggest that greater consideration should be given to novel denervation approaches in combination with other therapies for gastric cancer and likely other solid malignancies.