Engineering an arginine-nitric oxide circuit in Escherichia coli Nissle 1917 to remodel the tumor microenvironment for enhanced immunotherapy: a review.

Cancer immunotherapy, particularly immune checkpoint blockade (ICB), faces a major obstacle: the immunosuppressive tumor microenvironment (TME), characterized by abnormal vasculature, T cell exhaustion, and infiltration of suppressive immune cells including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). Nitric oxide (NO), a critical gasotransmitter, exhibits concentration-dependent dual roles in tumor biology. However, conventional NO delivery methods are limited by poor targeting specificity and systemic toxicity. Engineered Escherichia coli Nissle 1917 (EcN), with its century-long safety record, facultative anaerobic nature enabling tumor-selective colonization, and well-characterized genetic background, has emerged as a promising living therapeutic platform. This review systematically examines EcN engineered with a synthetic arginine-NO circuit (EcN-NO), in which deletion of argR eliminates feedback inhibition, while plasmid-encoded argAᶠᵐʳ, argG, argH, and Bacillus subtilis NO synthase (BsNOS) drive constitutive, sustained NO production after intratumoral colonization. We first provide a comparative analysis positioning EcN-NO within the historical landscape of engineered bacterial cancer therapy, including seminal work by Danino et al. (EcN tumor detection, 2015), Din et al. [synchronized lysis circuit (SLC), 2016], Chowdhury et al. (CD47 nanobody-mediated systemic antitumor immunity, 2019), and the framework of Gurbatri, Arpaia, and Danino [2022]. We then present a focused evidence-based analysis of EcN-NO's multidimensional mechanisms: tumor vascular normalization, dendritic cell (DC) recruitment and activation, reprogramming of immunosuppressive myeloid cells (TAM repolarization and MDSC reduction), direct cytotoxicity, and induction of immunogenic cell death (ICD). The review further details how this TME remodeling synergizes with αPD-L1 checkpoint blockade to expand functional CD8+ T cells, reverse T cell exhaustion, and establish long-term immunological memory across colorectal and breast cancer models. Finally, we outline unresolved translational questions regarding biosafety and biocontainment, optimal dosing and administration routes, good manufacturing practice (GMP) manufacturing, predictive biomarkers for patient stratification, and rational combination regimens. This review aims to provide a comprehensive theoretical foundation for overcoming immunotherapy resistance through engineered bacterial NO delivery.