Salmonella typhimurium co-expressing cytolysin A and hyaluronidase suppresses tumor growth and metastasis.

Recently, various attenuated bacteria have been studied as cancer therapies due to their unique characteristics, which include tumor-targeting bioactivity and immunogenicity. Previously, we reported a Salmonella typhimurium strain, CNC018, which is attenuated by 10⁵-10⁶-fold compared with the wild-type strain but retains tumor-targeting specificity. However, although these bacteria suppress tumors at the early stage in mice, the tumors often regrow at later stages. Therefore, to increase antitumor efficacy, we used a doxycycline-inducible system to engineer this strain (CNC018pCH) to secrete both cytolysin A (ClyA) and hyaluronidase (HysA), a pore-forming toxin that kills tumor cells and an enzyme that disrupts the tumor microenvironment, respectively. Local secretion of ClyA from CNC018pCH triggered tumor cell death through pyroptosis, apoptosis, and necrosis (PANoptosis) in a cholesterol-dependent manner, thereby releasing cellular contents and danger signals to activate the immune system. In addition, localized secretion of HysA degraded hyaluronic acid secreted by cancer cells, facilitated bacterial penetration into tumors, and inhibited metastasis by down-regulating the ribosomal S6 kinase (RSK)-related signaling pathway. These therapeutic payloads enhanced the ability of S. typhimurium to control tumor growth and metastasis in various murine tumor models. Notably, CNC018pCH also generated memory responses by protecting cured mice from tumor rechallenge. Taken together, these findings demonstrate that this engineered bacterium is a promising candidate for cancer treatment by reshaping the tumor microenvironment through the induction of tumor cell death and degradation of hyaluronic acid.