Department of Veterinary Public Health, College of Veterinary Medicine, Jeonbuk National University, Iksan 54596, South Korea.
Department of Veterinary Public Health, College of Veterinary Medicine, Jeonbuk National University, Iksan 54596, South Korea.
J Adv Res. 2024 Jul;61:83-100. doi: 10.1016/j.jare.2023.09.003. Epub 2023 Sep 7.
The limitations of conventional cancer therapies necessitate target-oriented, highly invasive, and safe treatment approaches. Hence, the intrinsic anti-tumor activity of Salmonella can offer better options to combat cancers.
This study aims to utilize attenuated Salmonella and deliver cytolytic protein cytolysin A (ClyA) under quorum sensing (QS) signaling for precise localized expression in tumors but not in healthy organs.
The therapeutic delivery strain was imposed with tryptophan auxotroph for selective colonization in tumors by trpA and trpE deletion, and lipid-A and O-antigen were altered by pagL and rfaL deletions using lambda red recombination method. The strain was transformed with the designed QS-controlled ClyA expression vector which was validated by western blot. The in vivo passaged therapeutic strain was used for treatment four times at a weekly interval, with a dose of 5 × 10 CFU/mouse for cancer therapy.
The attenuated strain induced minimal endotoxicity-related cytokines TNF-α, IL-1β, and IFN-γ and exhibited adequate colonization despite earlier exposure in mice. The QS-controlled ClyA expression was confirmed by western blot from bacterial cultures grown at different cell densities. The results demonstrated that the in vivo passaged strain preferentially colonized the tumor after vacating the spleen, liver, and lung, leaving no outward histological scars. The anti-cancer effect of the designed construct was evaluated in the murine CT26 colon cancer model. The expression of ClyA increased tumoricidal activity by 67 % compared to vector control.
Hence, the anti-tumor effect of the engineered Salmonella strain was improved by ClyA expression via QS activation after achieving the threshold bacterial cell density. Further, immunohistochemical staining of the tumor and other organs corroborated the QS-controlled tumor-specific expression of ClyA. Overall, the results imply that the developed anti-cancer Salmonella has low endotoxicity and QS-controlled expression of ClyA as beneficial safety elements and supports recurrent Salmonella inoculation by O-antigen deficiency.
传统癌症疗法的局限性需要靶向、高度侵袭性和安全的治疗方法。因此，沙门氏菌的内在抗肿瘤活性为对抗癌症提供了更好的选择。
本研究旨在利用减毒沙门氏菌，并在群体感应 (QS) 信号下传递细胞溶解蛋白细胞溶解素 A (ClyA)，以在肿瘤中进行精确的局部表达，但不在健康器官中表达。
治疗传递菌株通过 trpA 和 trpE 缺失赋予色氨酸营养缺陷型，以选择性定植于肿瘤中，并通过 lambda red 重组方法改变脂质-A 和 O-抗原缺失 pagL 和 rfaL。该菌株被转化为设计的 QS 控制的 ClyA 表达载体，通过 Western blot 进行验证。体内传代的治疗菌株每周一次进行四次治疗，剂量为 5×10 CFU/只用于癌症治疗。
减毒菌株诱导最小的内毒素相关细胞因子 TNF-α、IL-1β 和 IFN-γ，并表现出足够的定植能力，尽管在小鼠中更早暴露。通过在不同细胞密度下生长的细菌培养物的 Western blot 确认了 QS 控制的 ClyA 表达。结果表明，体内传代的菌株在离开脾脏、肝脏和肺部后优先定植于肿瘤，而不会留下外部组织学疤痕。在 CT26 结肠癌细胞模型中评估了设计构建体的抗癌作用。与载体对照相比，ClyA 的表达增加了 67%的肿瘤杀伤活性。
因此，通过达到细菌细胞密度阈值后 QS 激活来表达 ClyA，工程化沙门氏菌菌株的抗肿瘤作用得到了改善。此外，肿瘤和其他器官的免疫组织化学染色证实了 ClyA 的 QS 控制的肿瘤特异性表达。总体而言，这些结果表明，开发的抗癌沙门氏菌具有低内毒素毒性和 QS 控制的 ClyA 表达，这是有益的安全性因素，并支持 O-抗原缺陷的沙门氏菌反复接种。
