Anti-tumor Therapy Mediated By Endostatin-Expressing Attenuated Salmonella Typhimurium In Murine Tumor Models

As Gram-negative and facultative bacteria,Salmonella have been often used for delivering anti-tumor therapeutic drugs and mediating anti-tumor therapy in mouse tumor models and human clinical trials.However,because of the intrinsic pathogenicity of Salmonella bacteria,it is necessary to reduce bacterial virulence and to enhance its tumor targeting ability and capacity of inducing tumor cell apoptosis to maximize the anti-tumor effect of Salmonella.In previous work,we constructed a Salmonella Typhimurium mutant S633,which harbors hexa-acylated lipid A that has been considered to possess the most potent immunomodulatory activity and was with slightly lower virulence compared to the wild-type S.Typhimurium UK-1.In this study,in order to construct one Salmonella strain suitable for anti-tumor therapy,we knocked out aro A gene of the lipid A-modified strain S633 generating the strain S634.Then,we identified the phenotypes,virulence and tumor targeting of the newly constructed aro A mutant S634.Finally,we used this attenuated S.Typhimurium as a live vector to deliver the anti-angiogenic anti-tumor agent endostatin and evaluated its anti-tumor effects and toxic side effects in mouse tumor models.The main contents of this study are summarized as follows:1.Construction of an attenuated S.Typhimurium strain and identification of its phenotype,virulence and tumor-targeting specificityIn this study,an aro A-auxotrophic S.Typhimurium strain S634 was constructed in the context of Lipid A-modified strain S633.Results of in vitro experiments showed that the growth rate,the swimming ability and ability to invade tumor cells of S634were decreased compared to the parent strain S633 and wild-type strain UK-1;S634showed significantly decreased virulence upon intraperitoneal infection in mice;S634preferentially colonized in tumor tissue for at least three weeks,with tumor-to-liver ratio ranging from 1500:1 to over 100000:1.2.Evaluation of anti-tumor effects of endostatin-expressing attenuated S.Typhimurium in mouse tumor modelsWe constructed the endostatin expression plasmid"p ES"(asd~+)and introduced it into the strain S636(asd~-derivative strain of attenuated tumor-targeting S634)to form a plasmid-lethal balance lethal system(S636/p ES).In mouse models of CT26 colon carcinoma and B16F10 melanoma,we tested whether S636 is suitable for delivery of antitumor agents to elicit improved therapeutic efficacy.S636/p ES-treated tumor-bearing mice showed suppressed tumor growth and prolonged survival,compared to mice treated with either the bacteria carrying empty plasmids or PBS intraperitoneally.Immunohistochemical studies demonstrated that,when mice received the treatment of S636/p ES,the colonization of Salmonella bacteria and local expression of endostatin were accompanied by the increase of apoptosis level and suppression of tumor angiogenesis within tumor tissues.Besides,adverse side effects elicited by engineered S.Typhimurium on mice were also observed by monitoring the body-weight change of tumor-bearing mice,the pathological changes of the liver and spleen tissues and clinical chemistry indexes of blood in animal experiments.