Optimization Strategies and Synergistic Applications of Engineered Salmonella Typhimurium in Cancer Therapy: From Synthetic Biology to Nanobiotechnology

Abstract

The high incidence and mortality of cancer pose significant challenges to clinical treatment. Although established and emerging anticancer therapies have improved outcomes in selected settings, treatment resistance, limited penetration into poorly perfused tumor regions, and immunosuppressive tumor microenvironments remain important challenges in many advanced solid tumors. Salmonella enterica serovar Typhimurium (S. Typhimurium), as a live microorganism inherently endowed with tumor-targeting ability, has emerged as a research hotspot in tumor therapy owing to its unique properties including direct tumor cell killing and activation of anti-tumor immunity. This study systematically reviews the progress of its application in tumor therapy, dissects its intrinsic anti-tumor mechanisms, targeted modification strategies, and combinatorial therapeutic approaches, and discusses the core issues in clinical translation. This review summarizes four nanobiotechnology-based surface-engineering strategies: chemical conjugation, biomineralization, physical adsorption, and biomimetic or polymeric coating. The bacteria-nanomaterial hybrid systems constructed via these methods achieve synergistic complementarity with genetic editing engineering, optimizing the tumor-targeting capacity and therapeutic potential of strains under controllable biosafety conditions. Meanwhile, it summarizes combination regimens of this bacterium with chemoradiotherapy, immunotherapy, and multiple nanotechnology-mediated emerging therapies. Relying on multi-mechanism complementarity, such combinatorial modes break through the bottlenecks of monotherapy and boost the overall anti-tumor efficacy. Finally, we further analyze translational bottlenecks, including interspecies model gaps, administration limitations, and industrial production obstacles, and propose future directions to provide theoretical support for the development of precision bacterial cancer therapeutics.

Introduction

Literature Search Strategy

This review systematically retrieved relevant peer-reviewed literature from four core databases: PubMed, Web of Science, CNKI, and ClinicalTrials.gov. The retrieval time frame was set from January 1990 through July 2026. The combined search keywords were divided into three categories:

1. Bacterial carrier: Salmonella Typhimurium, attenuated Salmonella, engineered bacteria, live bacterial therapeutics;

2. Tumor therapy mechanism: bacterial oncolysis, tumor microenvironment remodeling, cancer immunotherapy, tumor hypoxia targeting;

3. Modification and combinatorial therapy: synthetic biology, nanobiotechnology, biomineralization, phototherapy, chemo-radiotherapy, immune checkpoint blockade, ferroptosis, calcium overload therapy.

Boolean operators (“AND”, “OR”) were used to combine the above terms. Manual screening was performed according to the following inclusion criteria: (1) original research articles and comprehensive reviews focusing on engineered S. Typhimurium for solid tumor treatment; (2) studies elaborating genetic engineering, nano-surface modification, or multimodal synergistic therapy; (3) preclinical in vitro/in vivo experimental studies and registered clinical trials. Exclusion criteria: conference abstracts without complete data, duplicate publications, irrelevant research on non-tumor fields, and studies using other bacterial chassis without S. Typhimurium. Reference lists of high-impact review articles were further manually back-searched to supplement missing classic literature. Finally, a total of 131 valid papers were included for systematic collation and discussion in this manuscript.

Research Background and Research Significance

Data from the Global Cancer Statistics 2024 report published by the International Agency for Research on Cancer (IARC) indicate that 20.6 million new cancer cases and 9.8 million cancer deaths were recorded worldwide in 2024.¹ Statistically, one in five individuals will develop cancer over their lifetime, while one out of nine men and one out of 13 women will eventually die from malignancies.¹ In 2021, cancer accounted for around 23% of all deaths attributable to non-communicable diseases (NCDs), rendering it the second leading global cause of mortality.¹ As a predominant contributor to morbidity and mortality across the globe, cancer poses an extremely severe challenge to global public health.

Although conventional anti-tumor modalities including surgical resection, chemoradiotherapy, and immune checkpoint inhibitors have undergone continuous iterative optimization, patients with advanced malignancies only achieve marginal improvements in short-term prognosis.² Meanwhile, a suite of novel anti-tumor technologies have emerged successively, such as molecular targeted agents, chimeric antigen receptor T (CAR-T) cell therapy, and nanomedicine delivery systems. Nevertheless, both conventional therapies and emerging technologies share inherent bottlenecks that remain difficult to overcome. Surgical resection fails to eradicate systemic micrometastases. Chemotherapy and radiotherapy are hampered by dense tumor stroma and hypoxic tumor microenvironments: insufficient accumulation of drugs or irradiation occurs within deep lesions, which readily triggers drug resistance and severe systemic toxicities. Monotherapy with immune checkpoint inhibitors suffers drastically attenuated therapeutic efficacy due to the immunosuppressive network constructed by regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment.³

With regard to novel technologies, molecular targeted agents are constrained by temporal and spatial tumor heterogeneity, which leads to rapid development of acquired drug resistance, and these agents lack the capacity to penetrate deep tumor tissues. CAR-T cells cannot efficiently infiltrate lesions owing to their poor penetration through the dense stroma of solid tumors. Nanocarriers merely rely on the enhanced permeability and retention (EPR) effect for passive tumor accumulation, making them incapable of reaching avascular necrotic tumor cores; additionally, they fail to confer sustained systemic immunomodulatory capacity.⁴ Taken together, none of the currently available anti-tumor regimens can simultaneously resolve two core clinical challenges: first, breaching the dense physical barriers of solid tumors to achieve effective delivery to deep hypoxic lesions; second, persistently remodeling the systemic immunosuppressive tumor microenvironment. Accordingly, there is an urgent clinical demand to develop innovative anti-tumor therapeutic strategies.

In this context, microorganism-based cancer therapy, which has a long tradition dating back to the late 19th century when the American scientist William Coley first applied killed bacteria (“Coley’s toxins”) to promote oncologic regression, is reemerging as a promising research direction by taking advantage of its tumor specificity and intrinsic properties.⁵ Based on a variety of microbial platforms, including Escherichia coli, Listeria monocytogenes, Bifidobacterium bifidum and others, each can exhibit distinct biological advantages in vivo for different therapeutic intents (Table 1).

Table 1.

Antitumor Engineered Bacteria: Properties and Applications

Bacterial Species	Tumor-Targeting Mechanisms	Core Antitumor Pathways	Advantages	Limitations	Application Scenarios
Salmonella Typhimurium	1. Chemotaxis to tumor metabolites6 2. Migrates and proliferates in hypoxic tumor regions7	1. Proliferation-mediated destruction of tumor tissues8 2. Flagellin activates TLR59 3. Downregulates VEGF to inhibit angiogenesis10	High targeting efficiency, multi-layered antitumor effects	Risk of sepsis, heterogeneous colonization	Deep-seated solid tumors
Escherichia coli	1. Colonizes hypoxic tumor regions; normal tissues cleared by complement system11 2. Proliferates utilizing tumor metabolites	1. Engineered to secrete ClyA to induce tumor cell apoptosis12 2. Engineered to produce L-asparaginase to deplete amino acids11 3. LPS activates TLR4 signaling pathway	High tumor specificity, excellent metabolic adaptability	Limited tumor penetration, susceptible to immune clearance	Hypoxic regions of solid tumors
Listeria monocytogenes	1. Enters tumor microenvironment via MDSCs13 2. Intracellular survival and colonization	1. Triggers ROS-mediated tumor cell death14 2. Activates DCs to induce CD8⁺ T cell immune response15	Unique intracellular targeting, strong induction of immune memory	Risk of systemic inflammation, rapid bacterial clearance	Metastatic tumors
Bifidobacterium bifidum	1. Exclusively colonizes hypoxic core of tumors16 2. Targets gastrointestinal tumor microenvironment	1. Activates antigen-presenting cells 2. Synergizes with photosensitizers to enhance ROS production17	High safety, ultra-high tumor targeting specificity	Restricted to tumor core region, poor monotherapy efficacy	Gastrointestinal tumors
Photosynthetic bacteria (PSB)	1. Colonizes hypoxic tumors and in situ oxygen production18 2. NIR light controls bacterial photosynthetic and therapeutic activity19	1. Light-triggered ROS/thermal effects for tumor killing20 2. Alleviates tumor hypoxia to enhance radiotherapy efficacy21	Unique tumor microenvironment modulation, spatiotemporal controllability	Limited light tissue penetration, poor bacterial stability	Superficial solid tumors
Clostridium butyricum	1. Spore germination in hypoxic tumor regions 2. Colonizes necrotic core of tumors	1. Produces butyric acid to mediate tumor‑suppressive and immunomodulatory effects 2. Engineered to enhance tumor radiosensitivity22	High colonization specificity for hypoxic‑necrotic tumor niches	Low spore germination efficiency, limited administration routes	Refractory solid tumors
Lactococcus lactis	1. No intrinsic tumor‑homing capacity 2. Persistence in tumor microenvironment through intratumoral delivery23	Genetically engineered chassis for sustained secretion of immunomodulatory payloads23	Ultra-high safety, potent in‑situ vaccination capacity	Narrow targeting range, dependent on local injection, prone to immune clearance	Intratumoral in‑situ vaccination for immune‑cold

Abbreviations: TLR, Toll-like receptor; VEGF, vascular endothelial growth factor; LPS, lipopolysaccharide; MDSCs, myeloid-derived suppressor cells; DCs, dendritic cells; ROS, reactive oxygen species; NIR, near-infrared; CD8⁺ T cell, cluster of differentiation 8⁺ T lymphocyte; PSB, photosynthetic bacteria.

Nevertheless, each chassis strain has inherent defects that restrict translational progress: Bifidobacterium bifidum only colonizes deep anoxic tumor cores with weak stromal infiltration and suffers strict requirements for in vitro culture and amplification; Listeria monocytogenes is an intracellular vector with strong antigen-presentation potential, whereas its pathogenicity requires stringent attenuation and safety control; Escherichia coli offers mature engineering tools and diverse probiotic or laboratory strains, although tumor accumulation and immune clearance vary substantially among strains.

Among these candidates, Salmonella enterica serovar Typhimurium (S. Typhimurium) combines facultative anaerobiosis, motility, immunostimulatory activity, and genetic tractability. Other bacterial chassis may remain preferable when different delivery routes, safety profiles, or intracellular antigen-presentation properties are required. (1) As facultative anaerobes, these bacteria exhibit intrinsic robust tropism toward hypoxic and necrotic tumor regions. The aberrant vasculature and locally immunosuppressive microenvironment within tumors enable Salmonella to evade immune clearance in peripheral normal tissues, facilitating stable colonization and massive proliferation in deep tumor lesions inaccessible to chemoradiotherapy and molecular targeted agents. This property effectively overcomes the delivery barrier imposed by the dense stroma of solid tumors. (2) Bacterial components including lipopolysaccharide and flagellin serve as pathogen-associated molecular patterns (PAMPs) with potent intrinsic immunoadjuvant activity. They fundamentally reverse local tumor immunosuppression and sustain the activation of systemic anti-tumor immune responses. S. Typhimurium has also demonstrated antitumor activity across several preclinical models. Experiments have shown its effectiveness against different types of solid tumors (melanoma, liver cancer, colorectal carcinoma) as well as some modeling leukemias (Table 2), suggesting the wide range of mechanisms and therapeutic possibilities of S. Typhimurium.

Table 2.

Intrinsic Antitumor Effects of Attenuated Salmonella Typhimurium in Preclinical Models

Tumor Category	Specific Tumor Type	Attenuated Strain	Core Antitumor Effect (Intrinsic Bacterial Activity)	References
Solid Tumors	Melanoma (B16F10)	VNP20009	Selective tumor colonization; inhibition of tumor growth and prolonged mouse survival; confirmed safety of msbB attenuating mutation.	[24]
	Breast Cancer (MDA‑MB‑231)	YB1 (Hypoxia-targeted)	Achieved tumor-specific proliferation via hypoxia-inducible promoter, resulting in retarded breast tumor growth.	[25]
	Colorectal Cancer (MC38)	BRD509E	Elevated MHC‑II expression on tumor‑associated myeloid cells and enhanced CD4⁺ T cell infiltration, exerting antitumor effects.	[26]
	Prostate Cancer (PC-3)	A1 (Leu/Arg auxotroph)	During in vitro infection, GFP-expressing bacteria proliferated within the cytoplasm of PC-3 human prostate cancer cells and induced nuclear destruction.	[27]
	Pancreatic Cancer (AsPC-1)	ΔppGpp‑deficient SL	Colonized and inhibited tumor growth in a pancreatic cancer xenograft model, providing a rationale for treating this dense-stroma refractory tumor.	[28]
	Malignant Peripheral Nerve Sheath Tumor (S462TY, STS26T)	VNP20009, ΔppGpp	Selectively colonized tumor tissues and significantly inhibited tumor growth.	[29]
Hematological Malignancies	B-Cell Lymphoma (A20)	LVR01	Following three intratumoral injections, significantly prolonged survival in tumor-bearing mice; induced intratumoral infiltration and activation of CD8⁺ T cells and neutrophils; elevated systemic IFN-γ responses, eliciting systemic antitumor immunity.	[30]

Abbreviations: GFP, green fluorescent protein; IFN-γ, interferon-γ; Leu, leucine; Arg, arginine.

However, inherent toxicity of wild-type strains and insufficient efficacy of single-agent regimens greatly undermine treatment safety and anti-tumour potency.³¹ To this end, relevant research has shifted from harnessing the innate physiological traits of bacteria to two complementary core engineering modification strategies (Table 3): the first is synthetic biology-guided genome editing engineering; the second is nanobiotechnology, a fast-growing technical system with irreplaceable strengths emerging in recent years. Nanobiotechnology modifies bacterial surfaces through non-genetic engineering tactics such as chemical covalent conjugation, biomineralization encapsulation, and electrostatic physical adsorption. This approach greatly expands the modification potential of S. Typhimurium, achieves precise spatiotemporal regulation responsive to the tumor microenvironment, and simultaneously improves the tumor-targeted accumulation efficiency, in vivo antitumor potency, and biosafety of bacterial strains. Current studies have also extensively investigated multimodal synergistic regimens combining engineered S. Typhimurium with other antitumor modalities, with the ultimate goal of eradicating deep solid tumors, reversing the immunosuppressive tumor microenvironment, and lowering the risks of tumor recurrence and metastasis.

Table 3.

Core Engineering Strategies for Salmonella Typhimurium Optimization in Cancer Therapy

Engineering Strategy	Technical Principles	Applications
Genetic Editing	Precise genomic modification using CRISPR-Cas9 and other genome-editing tools	1.Virulence gene knockout (eg, msbB) for attenuation24 2.Insertion of therapeutic genes (eg, IL-18, ClyA)32 3.Design of hypoxia/pH-responsive gene circuits33
Chemical Conjugation	Bacterial surface modification via covalent binding or bioorthogonal reactions (eg, SPAAC)	1.NHS-ester mediated biotin-streptavidin conjugation for PLGA nanoparticle loading on S. Typhimurium to enhance intratumoral nanomedicine delivery34 2.Copper-free SPAAC for site-selective conjugation of S. Typhimurium O-antigen to CRM19735
Biomineralization	Biologically mediated deposition of inorganic minerals or MOFs on bacterial surface	1.CaCO3 coating for pH-responsive shell degradation and bio‑agent release36 2.MnO2 mineralization for innate‑immune‑mediated tumor microenvironment remodelling37 3.In situ mineralization of FeS NPs on S. Typhimurium surface for photothermally-enhanced chemodynamic therapy (CDT) and H2S gas combination therapy38
Physical Interaction	Non-covalent interactions (electrostatic, hydrophobic, van der Waals forces)	1.Physical attachment of Au-Pt nanozymes39 2.Cationic polymer (PEI)-mediated nucleic acid delivery40
Biomimetic or Polymeric Coating	Encapsulation with biological membranes (macrophage, RBC) or synthetic polymers	1.Macrophage membrane camouflage for immune escape41 2.Polyserotonin (PST) coating for pH-responsive protection42 3.Glutathione (GSH)-responsive self-assembling peptide cloaking43

Abbreviations: CDT, chemodynamic therapy; CRM₁₉₇, cross-reacting material 197; GSH, glutathione; MOFs, metal-organic frameworks; PEI, polyethylenimine; PLGA, poly(lactic-co-glycolic acid); PST, polyserotonin; RBC, red blood cell; SPAAC, strain-promoted azide-alkyne cycloaddition; NHS, N-hydroxysuccinimide.

Importantly, beyond improving therapeutic efficacy, the clinical development of engineered bacterial therapeutics requires a careful balance between their intrinsic biological autonomy and the demands for safety, controllability, and translational feasibility. Based on this, this review will systematically elaborate on the intrinsic multi-dimensional anti-tumor mechanisms of S. Typhimurium; then focus on the strategies for its safety enhancement, functionalization, and intelligent modification through various modification approaches; subsequently provide a systematic review of the mechanisms and prospects of its synergy with a variety of anticancer regimens; and finally conduct a dialectical analysis of the prevailing translational hurdles and prospect the future development trajectories for its clinical implementation.

Antitumor Mechanisms and Inherent Limitations

The antitumor mechanism of S. Typhimurium is based on finely tuned interactions between the special biology of the bacteria and the unique aspects of the tumor microenvironment (TME). These effects involve four principal mechanisms: selective tumor colonization, direct tumor-cell cytotoxicity, activation of host antitumor immunity, and inhibition of tumor angiogenesis. Individually, the mechanisms constitute a well-coordinated and effective antitumor network. Nevertheless, these intrinsic therapeutic capacities are accompanied by a series of pathogenic drawbacks that restrict the efficacy of wild-type Salmonella monotherapy.

Tumor-Specific Colonization and Targeting

Enrichment and stable persistence within tumors is the essential first step in Salmonella’s antitumor activity, which is far from a passive process, but a conscious and step-by-step action consists of sequential physical entrapment, chemotactic sensing, directed penetration, and finally favored proliferation within the TME (Figure 1).

Figure 1.

Tumor-Specific Targeting and Colonization Mechanisms of Salmonella Typhimurium. Abnormal tumor vasculature enables Salmonella extravasation into tumor tissue. Tumor necrotic regions continuously release metabolites such as aspartate, serine, ribose, and galactose, forming chemoattractant gradients that rise progressively from tumor periphery to tumor center; downward arrows indicate the gradual decline of chemoattractant concentration from the tumor center toward the periphery. Extracellular chemical signals are specifically recognized by three transmembrane chemoreceptors Tar, Tsr, and Trg on the bacterial membrane. The horizontal rightward arrows forming the sequence CheW→CheA→CheY inside the bacterium denote the sequential intracellular Che signal transduction cascade triggered by ligand binding: CheW transmits conformational signals to CheA, and phosphorylated CheA further activates downstream CheY. Mot stands for flagellar motor complexes embedded in the membrane, and Fla refers to extracellular flagellar filaments. Through this intact Che signal transduction system, Salmonella continuously tracks the metabolite gradient, penetrates deep into poorly perfused hypoxic tumor regions, and finally achieves stable preferential proliferation and long-term colonization within the immunosuppressive tumor microenvironment. This figure was handcrafted in PowerPoint by the authors. ByteDance SeedEdit 3.0 merely optimized the definition of partial icons.

Passive Entrapment

The defective and leaky vasculature characteristic of solid tumors provides a theoretical physical escape path.⁷ Disordered endothelial cell adherences and incomplete basement membrane leave gaps of nano-to micro-size; the highly chaotic architecture of tumor vessels leads to bacterial entrapment within tumor vascular lumens.⁴ Impaired intratumoral lymphatic drainage may further hinder the clearance of these bacteria and increase time for subsequent intratumoral action. Passive trapping alone, however, cannot account for the extensive intratumoral bacterial accumulation observed experimentally.⁶

Directed Chemotaxis and Active Targeted Migration

Following initial passive entrapment, S. Typhimurium strains utilize flagella-mediated motility and chemotactic sensing systems to migrate toward favorable niches within the tumor microenvironment.

Motile Structures

The fundamental machinery is a motility apparatus comprising functional flagella and flagellar motors (fla and mot genes). Such cellular structures are critical for motility; mutants lacking these components cannot adequately colonize tumor masses, proving the requirement of active motility for penetration into the tumor stroma.^31,44

Chemotaxis

The tumor microenvironment provides unique chemical signal gradients for bacterial chemotaxis.³¹ Among these, hypoxia represents the most prevalent and critical feature, which generates potent spatial gradients of metabolic chemoattractants instead of serving as a direct chemotactic signal for Salmonella.⁴⁵ In addition, aberrant tumor metabolism and necrotic regions lead to the release or accumulation of specific chemical substances—such as aspartate, serine, ribose, and galactose—thereby establishing concentration gradients.³¹ Salmonella recognizes these signals via specific chemoreceptors embedded in its membrane. Specific chemoreceptors on the bacterial membrane, including Tar for aspartate, Tsr for serine, and Trg for ribose and galactose, detect these chemical gradients.³¹

This directed movement, initiated upon bacterial sensing of chemical signals, is executed by a highly conserved Che signal transduction system.³¹ The signaling cascade proceeds as follows: upon binding of specific ligands (eg, amino acids and carbohydrates released by tumors), chemoreceptors undergo conformational changes that transduce signals to the intracellular histidine kinase CheA. Activated CheA phosphorylates the response regulator CheY, yielding phosphorylated CheY (CheY~P). CheY~P subsequently binds to the flagellar motor switch complex, altering the direction of flagellar rotation and thereby modulating the balance between bacterial “run” and “tumble” motion patterns.³¹ By continuously sensing and comparing changes in chemical concentration within the surrounding microenvironment, Salmonella achieves directed migration toward hypoxic or necrotic tumor regions, although in vivo bacterial dissemination is further constrained by host‑derived microenvironmental barriers.⁴⁶

Deep Penetration

After administration, bacteria initially form larger colonies near blood vessels, while smaller colonies progressively penetrate deeper, poorly perfused tumor regions.⁴⁵ Researchers report the colonization of bacteria within both vascular peripheries as well as hypoxic cores.^45,47 A general pattern of colonization can be observed after the administration of the pathogen: a fairly fast penetration, leading to the proliferation into large colonies, close to blood vessels, and slower, steadier, penetration by smaller colonies penetrating the deeper parts of the metastasis, mostly poorly perfused areas.^45,47 This allows coverage of treatment-resistant areas lying in the midst of hypoxia. In the 4T1 murine mammary carcinoma model, Salmonella colonies exhibited a mean distance of ~310 μm from functional tumour vasculature at 48 h post‑injection, a feature that may confer potential advantages over most passively diffusing nanomedicines.⁴⁵

Preferential Proliferation

The tumor microenvironment provides unique survival advantages for S. Typhimurium, supporting the long-term colonization and sustained proliferation.⁴⁸ Under hypoxic conditions, the bacterium activates hypoxia-inducible promoters via the hypoxia-sensing factor FNR (fumarate and nitrate reduction regulator), driving expression of genes for adaptation to the low‑oxygen tumour niche.^48,49 Amino acids, carbohydrates, and other nutrients released from necrotic tumor regions provide ample energy sources for bacterial growth, while the immunosuppressive microenvironment reduces the risk of bacterial clearance by attenuating the cytotoxic activity of host immune cells.^47,48 As summarized by Sznol et al, VNP20009 establishes persistent intratumoral colonization at bacterial burdens exceeding 10⁸ CFU per gram of tumour tissue in multiple murine tumour models.⁴⁸

Direct Tumor Cell Cytotoxicity

Salmonella destroys tumor cells in various ways. This damage can be direct: pore-forming toxin activity; inhibition of death pathways; or the metabolism sabotage (Figure 2).

Figure 2.

Direct Tumor Cell Killing Mechanisms of Salmonella Typhimurium. Centered on Salmonella Typhimurium, this schematic diagram systematically illustrates three synergistic direct tumoricidal pathways targeting tumor cells. The upper-left blue panel depicts competition for nutrients and metabolic interference: rapidly proliferating engineered S. Typhimurium competitively consumes intratumoral methionine, ribose and galactose, sharply depleting intracellular ATP in tumor cells. Subsequent metabolic stress triggers cell-cycle arrest and inhibits malignant proliferation of tumor cells. The upper-right orange panel shows natural toxin-mediated cytolysis: engineered S. Typhimurium secretes ClyA, LPS and phospholipase C collaboratively. ClyA assembles transmembrane pores to raise membrane permeability, LPS further amplifies membrane leakage, and phospholipase C degrades membrane phospholipids; together they cause severe membrane disruption, osmotic imbalance and eventual tumor cell lysis. The bottom green panel displays induction of apoptosis and autophagy: bacterial infection induces ROS/Ca²⁺ overload, upregulates pro-apoptotic Bax while downregulating anti-apoptotic Bcl-2, and activates caspase-9/3 to initiate mitochondrial-dependent apoptosis; meanwhile, regulation of the TLR4/NF-κB signaling, accompanied by inhibition of the downstream Akt/mTOR pathway, enhances autophagic flux and induces lethal autophagic cell death. Collectively, these three independent yet mutually reinforcing pathways constitute the core direct cytotoxic system of S. Typhimurium against tumor cells. Note: The hypothetical TLR4/NF‑κB‑FAK‑Akt/mTOR signaling cascade illustrated in this diagram is derived from macrophage‑based studies; direct experimental evidence for this regulatory axis in tumour‑cell models is currently absent. This figure was generated using ChatGPT Images 2.0 (OpenAI; GPT Image 2) and was subsequently reviewed and verified by the authors for scientific accuracy. Symbols ↑ and ↓ within molecular or pathway labels indicate an increase and a decrease, respectively. Solid arrows connecting graphical elements indicate the direction of the mechanistic sequence.

Natural Toxin-Mediated Cytolysis

Genetically‑modified S. Typhimurium can produce cytotoxic agents that induce tumor cell lysis by directly disrupting the integrity of the tumor cell membrane or interfering with membrane-associated functions.⁵⁰ Among these, cytolysin A, lipopolysaccharide, and phospholipase C are the core effector molecules.

Cytolysin A (ClyA)

As a key toxin mediating tumor cell lysis (mostly introduced into S. Typhimurium via genetic engineering), ClyA binds to the tumor cell membrane and assembles into transmembrane pores. These pores disrupt the selective permeability of the cell membrane, leading to leakage of intracellular electrolytes and small organic molecules, accompanied by an influx of extracellular fluid.⁵¹ This cascade ultimately results in osmotic imbalance and cell lysis.

Lipopolysaccharide (LPS)

As a major outer‑membrane component of S. Typhimurium, LPS cooperates with ClyA to boost antitumour activity predominantly through host‑derived pro‑inflammatory signaling. LPS provides a TLR4‑dependent priming signal for inflammasome‑related gene transcription, whereas ClyA‑mediated pore‑formation acts as a second signal to activate the NLRP3 inflammasome; these two signals synergistically promote the maturation and secretion of pro‑inflammatory IL‑1β to exert antitumour effects.⁵²

Phospholipase C

Additionally, certain engineered S. Typhimurium strains secrete phospholipase C, which has the potential to degrade phosphatidylcholine and phosphatidylethanolamine on the tumor cell membrane, possibly compromising membrane structural integrity and accelerating cell death.⁵¹

Induction of Apoptosis and Autophagy

Apoptosis

Pathologic infection activates endogenous pathways in tumors which lead to apoptosis.⁵³ Bacterial contact and intracellular uptake impose oxidative stress (ROS) triggering caspase-9/-3 cascade and the apoptosis program in mitochondria.⁵³ Infections may also interfere with calcium homeostasis, potentially yielding calcium overload, and the imbalance in level of Bcl-2 family protein (upregulating Bax, down-modulating Bcl-2), thus promoting apoptotic programs even further.⁵³ In vitro studies in HepG2 cells demonstrated that attenuated SL7207‑lux treatment markedly induced caspase‑3 activation and apoptosis of hepatoma cells at 48 h post‑treatment.⁵⁴

Autophagy

Attenuated Salmonella infection can down‑regulate the Akt/mTOR signaling axis, a central regulator of autophagic survival in tumour cells.⁵⁵ This leads to excessive autophagic flux, which results in increased LC3-II/ LC3-I ratios and accumulation of autophagosome, and can further progress to autophagic cell death.^55,56 It has been postulated that tumor cells possess weakened antioxidant system functions (eg, Nrf2 activity), which may render the cells especially sensitive to this stress, while normal cells have greater resilience.

Competition for Nutrients and Metabolic Interference

Tumors can be auxotrophic for particular nutrients or can simply have a greater overall demand for those nutrients. Proliferating engineered Salmonella is an effective opponent in TME.

Amino Acid Deprivation: Engineered Salmonella consume methionine at high rates to deplete local methionine pools, thereby crippling protein synthesis and DNA methylation in tumor cells, and contributing to the arrest of those cell cycles.⁵⁷ Bacterial L-asparaginase further catalyzes deamination of asparagine and impairs proliferation of asparagine‑dependent tumor cells, including some leukemias, thereby starving them.⁵⁸

Energy Source Competition: Salmonella is capable of effectively scavening for sugars such as ribose and galactose from the TME. This competition for nutrients may induce a decrease in ATP content in tumor cells. This effects the metabolism catastrophe in combination with amino acid deprivation.³¹

Antitumor Immunity

Effective Salmonella infection can convert “cold” to “hot” tumors, strongly activating both the innate and the adaptive arms of the immune system, which is one of the keys to Salmonella’s potency.

Innate System Activation

Salmonella-associated molecular patterns, including lipopolysaccharide (a TLR4 agonist) and flagellin (a TLR5 agonist), serve as potent exogenous agonists for host pattern recognition receptors (PRRs) (Figure 3). LPS and flagellin activate NF-κB signaling in macrophages and dendritic cells (DCs) via TLR4 and TLR5, respectively, thereby triggering a cascade of pro-inflammatory cytokines (eg, TNF-α, IL-1β, IL-12) within the tumor microenvironment.⁴⁷ These cytokines subsequently activate natural killer (NK) cells and enhance their cytotoxic activity.

Figure 3.

Salmonella-Mediated Antitumor Immunity: Innate Immune Activation in the Tumor Microenvironment. Salmonella-derived pathogen-associated molecular patterns, including lipopolysaccharide and flagellin, engage TLR4 and TLR5 on dendritic cells (DCs), respectively. The downstream MyD88–IRAK4–TRAF6–IKK signaling axis activates NF-κB translocation into the nucleus to drive transcription of pro-inflammatory cytokines TNF-α, IL-1β, and IL-12. Intracellular Salmonella DNA is sensed by the cytosolic cGAS–STING pathway in macrophages, which synergistically activates IRF3 and NF-κB to trigger the production of type I interferons (IFN-α/β). The secreted pro-inflammatory cytokines and type I interferons collectively boost the tumoricidal cytotoxicity of innate effector cells, including natural killer (NK) cells and neutrophils. In addition, Salmonella infection mediates the functional reprogramming of immunosuppressive M2-type tumor-associated macrophages (TAMs) toward immunostimulatory M1 macrophages to remodel the immunosuppressive tumor microenvironment. This figure was handcrafted in PowerPoint by the authors. ByteDance SeedEdit 3.0 merely optimized the definition of partial icons.

Furthermore, this activation can induce NLRP3 inflammasome assembly, leading to the secretion of IL-18, which recruits and stimulates neutrophils and other innate effector cells.⁵⁹ Critically, Salmonella infection is capable of reprogramming tumor-associated macrophages (TAMs), converting them from an immunosuppressive phenotype toward a pro-inflammatory M1-like phenotype—as evidenced by the upregulated secretion of IL-12 and nitric oxide.^47,60

Adaptive Immune Memory Induction and Effector Functions

The endogenous immune activation elicited by Salmonella establishes an immunogenic microenvironment conducive to the initiation of tumor-specific adaptive immune responses (Figure 4). Tumor cell lysis—resulting from either direct bacterial cytotoxicity or host immune-mediated killing—leads to the release of tumor-associated antigens (TAAs).⁶¹ Concurrently, Salmonella infection triggers dendritic cell (DC) maturation and activation, thereby facilitating efficient antigen presentation and T cell priming.⁶¹

Figure 4.

Salmonella-Mediated Antitumor Immunity: Induction of Adaptive Antitumor Immune Memory. Salmonella induces oncolytic lysis of cancer cells via direct cytotoxicity and host immune killing, resulting in the release of tumor-associated antigens (TAAs). Released TAAs are captured and processed by dendritic cells (DCs), which undergo maturation and activation upon Salmonella infection. Activated DCs continuously secrete interleukin-12 (IL-12) to drive the differentiation of naïve T cells into tumor-killing effector T cells and memory T cells, while stimulating B cells to generate tumor-specific antibodies. Combined cellular and humoral immunity mediates tumor clearance and persistent anti-tumor immune memory to prevent tumor relapse. This figure was handcrafted in PowerPoint by the authors. ByteDance SeedEdit 3.0 merely optimized the definition of partial icons.

A salient advantage of this strategy lies in its capacity to induce durable, tumor-targeted immunological memory, which contributes critically to the prevention of tumor relapse. Activated DCs sustain IL-12 secretion, promoting the recruitment and maintenance of memory T cells with a CD44⁺CD62L⁻ phenotype.⁶¹ Salmonella‑mediated tumor therapy can trigger adaptive antitumor immunity characterized by expansion of CD8⁺ T‑cells and establishment of long‑term immunological memory, which contributes to protection against tumor recurrence.⁶¹ Besides the potential TAAs‑driven humoral antitumor response, humoral immunity further complements this process: B cells produce Salmonella‑specific natural IgM antibodies that restrict bacterial dissemination from tumor sites to healthy organs.⁶² However, Salmonella-induced immune activation is highly context-dependent: while acute bacterial stimulation can promote antitumor immunity, prolonged or excessive inflammatory responses may trigger compensatory immunosuppressive programs that limit therapeutic efficacy.

Anti-Angiogenic Suppression

Reprogrammed macrophages surrounding tumors limit new vessel formation and starve tumors of oxygen and nutrients. The main mechanism is the downregulation of the key pro-angiogenic mediator VEGF. Salmonella may downregulate the AKT/mTOR/HIF-1α pathway, leading to repression of HIF-1α-dependent VEGF expression.¹⁰ Concurrent with the reorganization of the vasculature upon invasion, infection upregulates connexin 43 (Cx43) in the tumor.⁶³ Cx43 might also further repress HIF-1α transcription and reinforce junctions in endothelial cells, lowering permeability and thereby inhibiting angiogenesis and metastasis.^64,65 Bacteria can also act directly to trigger apoptosis of endothelial cells and destroy pre-existing tumor vascularity.⁴⁴ Highly‑vascular tumours show more pronounced vascular disruption and exhibit earlier therapeutic responses following S. Typhimurium administration.⁶⁶

Inherent Limitations

Despite encouraging preclinical findings, antitumor activity and tolerability vary among strains, tumor models, doses, administration routes, and host conditions. While S. Typhimurium exerts multifaceted anti-tumor effects, it concurrently triggers certain adverse reactions, which compromise its safety profile and therapeutic efficacy to a certain extent.

Pathogen-associated molecular patterns such as bacterial lipopolysaccharide and flagellin act as initiators of the entire detrimental cascade response.⁶⁷ During the short-term intratumoral survival phase, these molecules activate toll-like receptor (TLR) family signaling pathways, trigger the secretion of pro-inflammatory cytokines including IL-1β and TNF-α, facilitate the maturation of dendritic cells and M1-type macrophages, and thereby exert tumor-suppressive effects.⁴⁷ Nevertheless, sustained bacterial proliferation continuously stimulates pattern recognition receptors and disrupts the intrinsic immune homeostasis of the host.⁶⁸ Excessive inflammatory mediators not only elicit inflammatory injuries in visceral organs such as the liver and spleen, but may also upregulate the HIF-1α/VEGF signaling axis to remodel the extracellular matrix (ECM), which subsequently facilitates tumor angiogenesis and distant metastasis.⁶⁸ Meanwhile, the type III secretion system (T3SS) exhibits temporally distinct functions: SPI‑1‑derived substrates support inflammasome activation in the early infection stage, whereas transcriptional reprogramming towards SPI‑2 expression mediates NLRC4‑dependent inflammasome evasion under long-term colonization, persistently exacerbating the adverse outcomes derived from inflammatory dysregulation.⁶⁷

Persistent intratumoral bacterial stimulation also drives robust secretion of suppressive factors including TGF-β and IL-10 by tumor cells and cancer-associated fibroblasts (CAFs). On the one hand, such factors induce massive accumulation of MDSCs within lesions; these MDSCs directly suppress the cytotoxic functions of NK cells and cytotoxic T lymphocytes via arginine depletion and reactive oxygen species release.⁶⁹ On the other hand, they prompt neutrophils to form neutrophil extracellular traps (NETs), a mesh-like physical barrier that impedes deep bacterial infiltration into tumor parenchyma and drastically reduces effective bacterial colonization density. The superposition of these two inhibitory effects further attenuates therapeutic efficacy.^70,71

The aforementioned inhibitory signals at the cellular and cytokine levels fail to resolve spontaneously following short-term bacterial clearance. As facultative intracellular bacteria, Salmonella exhibits latent residence within macrophages and tumor cells, a feature generating chronic low-grade infection that constructs an interconnected multi-layered immunosuppressive feedback loop: sustained TGF-β signaling drives regulatory T‑cell expansion; prolonged pathogen stimulation upregulates the enzymatic activity of IDO and TDO to disrupt tryptophan metabolism; persistent activation of the IL-10/STAT3 axis mediates antigen presentation tolerance in dendritic cells, with these multiple inhibitory pathways reciprocally reinforcing one another.⁷²

Sustained pressure from the multifaceted immunosuppressive microenvironment established during chronic infection drives progressive functional exhaustion of tumor-infiltrating lymphocytes (TILs). Asparaginase secreted by Salmonella blocks c-Myc-mediated metabolic reprogramming in T cells and suppresses the synthesis of effector molecules including IFN-γ and granzymes. Meanwhile, persistent inhibitory signaling upregulates multiple immune checkpoint receptors such as PD-1, TIM-3, and LAG-3. Collectively, these events render CD8⁺ T cells incapable of proliferation and cytotoxicity, impeding the formation of long-term tumor-specific immunological memory. This constitutes the core immunological trigger for the high recurrence rate of tumors following Salmonella administration.⁷³

Collectively, inflammatory dysregulation, infiltration of suppressive myeloid cells, multi-layered inhibitory circuits triggered by chronic infection, and T cell exhaustion represent intrinsic immunological drawbacks stemming from the native biological characteristics of Salmonella. To fundamentally mitigate the aforementioned adverse effects, diverse genetic engineering modification strategies and combinatorial regimens with alternative therapeutic modalities are indispensable to address this bottleneck.

Optimization Strategies for Salmonella Typhimurium-Mediated Tumor Therapy

The successful repurposing of S. Typhimurium from a pathogenic bacterium into a safe and efficient tumor therapeutic vector hinges on achieving a delicate balance between biosafety and therapeutic potential through interdisciplinary synergistic engineering via synthetic biology and nanobiotechnology.

Safety Engineering

The strong virulence and non-specific colonization of wild-type S. Typhimurium have limited its clinical translation. Therefore, a major challenge in engineering bacterial therapeutics is to achieve a balance between maintaining tumor-targeting and therapeutic functions while minimizing systemic toxicity and uncontrolled bacterial persistence. Biosafety engineering leverages precise genetic-level regulation and physical modification via nanotechnology to construct engineered bacterial strains with the properties of surviving in tumors, being eliminated in normal tissues, and exhibiting regulable functions. These engineering strategies are intended to reduce systemic toxicity and off-target colonization while preserving tumor-directed activity.

Gene-Edited Attenuation

Based on gene editing technologies such as CRISPR-Cas9 and homologous recombination, combined with the design of multi-target genetic circuits in synthetic biology, targeted attenuation is achieved from the dual perspectives of virulence regulation and metabolic dependence, thereby rendering bacterial survival strictly dependent on the TME.

Multi-Target Knockout of Virulence Genes

The systemic toxicity of S. Typhimurium is substantially reduced at the source by knocking out core genes associated with bacterial invasion and inflammation induction. For instance, the clinical strain VNP20009, generated by double gene knockout (ΔmsbB/ΔpurI), not only modifies the hexa-acylated lipid A of lipopolysaccharide into a penta-acylated form to attenuate the TLR4-mediated cytokine storm but also confers purine synthesis deficiency, restricting bacterial proliferation predominantly to the high-adenine microenvironment of necrotic tumor regions.^24,68,74

Construction of Auxotrophic Bacterial Strains

Taking advantage of the metabolic disparities between tumor and normal tissues, synthetic biology is employed to reprogram bacterial metabolic pathways for the generation of amino acid or nucleotide auxotrophic strains. For example, the A1-R strain (ΔleuC/ΔargH) is incapable of de novo synthesis of leucine and arginine, and thus can proliferate mainly by utilizing the high concentrations of free amino acids in necrotic tumor regions, with no persistent colonization in normal tissues in patient-derived orthotopic xenograft (PDOX) models.^75–77

Additionally, global regulatory gene modification represents another attenuation route. The ΔppGpp mutant strain (ΔrelA/ΔspoT) loses the ability to synthesize stringent‑response signaling molecules, resulting in pronounced virulence attenuation via down‑regulation of SPI‑dependent virulence‑gene expression, while it still achieves efficient colonization in the TME.^78–80 These targeted genetic modifications not only directly enhance biosafety but also indirectly optimize therapeutic selectivity. Attenuated strains are more readily cleared from immunocompetent normal tissues, yet can persist and proliferate within the immunosuppressive tumor microenvironment, thereby establishing a “colonization gradient” between tumor and normal tissues. Table 4 systematically summarizes representative engineered strains constructed based on different attenuation principles and their corresponding attenuation efficacy.

Table 4.

Genetic Engineering Strategies for Attenuation of Salmonella Typhimurium

Representative Strain	Key Genetic Modification	Core Molecular Mechanism	Attenuation Effect		References
			Biosafety	Efficacy
VNP20009	msbB− purI−	LPS lipid A penta-acylation attenuates TLR4‑driven pro‑inflammatory cytokine induction; purine auxotrophy renders survival dependent on tumor adenine	LD50 increased ≥10,000-fold vs wild type in mice	Mouse tumours‑to‑liver bacterial ratio ≥1000:1	[24,68,74]
A1-R	leu− arg−	Leucine/arginine auxotrophy; relies on free amino acids in necrotic tumor regions for proliferation	Markedly attenuated virulence; fails to establish persistent infection in normal tissues	Strong tumour‑selective enrichment; significant tumour‑growth suppression across multiple PDOX models	[76,77]
YB1	aroA⁻ parental background; chromosomally‑replaced asd under PpepT‑PsodA hypoxia‑responsive circuit	Essential‑gene circuit permits replication only under low‑oxygen conditions; normoxia‑induced bacterial lysis due to loss of diaminopimelate (DAP) biosynthesis	Markedly attenuated virulence; rapidly cleared from normoxic normal tissues after transient early‑phase detection in nude‑mouse studies	Strong preferential accumulation within tumour hypoxic regions; significant tumour‑growth delay in breast‑cancer xenograft models	[25,75]
SL7207	aroA−	Shikimate‑pathway defect; auxotrophic for host‑derived aromatic metabolites that mammals cannot produce	Attenuated in immunocompetent mice; retains severe virulence in immunocompromised nude mice, causing uncontrolled systemic infection	Tumour colonization observed in immunocompetent hosts; poor tumour‑to‑liver selectivity and lethal systemic proliferation in nude‑mouse models	[25,75]
SHJ2037 (ΔppGpp)	relA− spoT−	Loss of ppGpp synthesis capacity; down‑regulation of SPI‑encoded virulence genes	Markedly elevated LD50 (~105‑fold increase in immunocompetent BALB/c mice)	Suitable for use as a live bacterial delivery vector	[78–80]

Notes: The fold increase in LD₅₀ quantitatively reflects the reduction of systemic virulence.

Abbreviations: LD₅₀, median lethal dose; LPS, lipopolysaccharide; TLR4, Toll-like receptor 4; PDOX, patient-derived orthotopic xenograft; ppGpp, guanosine tetraphosphate; SPI, Salmonella Pathogenicity Island.

Refined Tumor-Homing

Following attenuation, targeted improvements can be implemented to refine Salmonella’s tumor-homing, improving the tumor‑to‑normal tissue colonization ratio and decreasing off-target toxicity. In contrast to genome-based engineering, nanobiotechnology provides a modular and flexible strategy for surface functionalization, enabling additional control over bacterial targeting, immune interaction, and in vivo behavior without directly altering the bacterial genome.

Antibody-Mediated Targeting

This approach involves presenting tumor-specific antibodies or antibody fragments on the bacterial surface, allowing for specific recognition of cognate antigens presented by cancer cells to drive selective colonization. Single-domain antibodies (VHHs or nanobodies), due to their small size, favorable tissue penetration, and low immunogenicity, are especially appropriate for this approach.⁸¹ Through genetic engineering, the presence of tumor-specific VHHs expressed on the outside of the membrane can substantially increase bacterial enrichment within tumors, coupled with a decrease in sequestration within normal organs.

As an example of this type of targeting strategy, a derivative of VNP20009 strain has been modified to display a carcinoembryonic antigen (CEA)-specific single-chain variable fragment (scFv). Indeed, this modification provides a significant increase in tumor-targeting efficacy and has been demonstrated to achieve preferential bacterial accumulation in colorectal cancer models.⁸² Another example, pertaining to CD20⁺ lymphoma, was accomplished with a Salmonella strain expressing a CD20-specific VHH. Not solely providing a specific binding mechanism to lymphoma cells, this strain also carries a prodrug converting enzyme. This design enables tumour‑restricted activation of systemically‑administered prodrug, achieving substantial anti‑tumour activity in human lymphoma xenograft models.⁸¹ Utilizing high-affinity antibody‑antigen interactions, this strategy introduces an accurate tumor-homing ability, ensuring the localized delivery of subsequent therapeutic payloads, as well as providing an avenue for customization, as it is possible to switch out the targeting moiety for different tumor antigens.

Peptide-Mediated Targeting

This targeting strategy leverages tumor-homing peptides that demonstrate specificity and high affinity for overexpressed receptors presented on tumor cell surfaces. Tumor-homing peptides, often small, easy-to-synthesize, and biocompatible, can be displayed onto Salmonella through genetic fusion or chemical conjugation. One of the most well-studied targeting ligands is arginine-glycine-aspartate (RGD). This peptide specifically binds αvβ3 integrin overexpressed on the surface of many tumor cells and neovasculature.⁸³ Presentation of the RGD peptide onto the outer membrane protein OmpA of the Salmonella strain produced a recombinant strain that successfully enhanced the tumor-targeting and elevated tumor‑resident bacterial loads in models of breast cancer and melanoma, achieving >1000‑fold higher intratumoral bacterial accumulation relative to isogenic control Salmonella strains.⁸³ However, such high tumor-to-normal accumulation ratios have mainly been demonstrated in preclinical models, and their reproducibility and clinical relevance in human patients remain to be further validated.

Beyond short peptide ligands, protein‑scale targeting moieties can also be harnessed in Salmonella‑based engineering. For instance, Salmonella can also be engineered to produce the TGFα‑PE38 immunotoxin, in which transforming growth factor alpha (TGFα) serves as an endogenous ligand for the epidermal growth factor receptor (EGFR). While this engineered Salmonella retains natural tumor‑colonizing capacity, it secretes the TGFα‑PE38 immunotoxin, whose TGFα moiety mediates EGFR‑dependent target recognition, whereas the PE38 domain confers cytotoxic effects against EGFR‑overexpressing tumors in colon and breast cancer preclinical models.⁸⁴ Additionally, other peptide-based targeting strategies against distinct tumor-specific receptors have also been investigated, offering a versatile platform for tumour‑targeted precision therapy across different cancer types.

Carrier-Mediated Targeting and Camouflage

Here, biological or synthetic nanomaterials are used to package and camouflage the bacteria in order to circumvent recognition and clearance by the host defense system, while also taking advantage of the tumor-homing potential of the carrier agents themselves. The major strategies have focused on: (i) “Trojan horse” delivery mechanism, (ii) “stealth” polymeric coating mechanism, and (iii) glutathione (GSH)-responsive self-assembling peptide cloaking mechanism.

Trojan Horse Delivery

A particularly promising approach uses host cells with innate tumor-homing potential as carriers. Since macrophages naturally chemotax toward tumors, they present themselves as ideal Trojan horses for delivery.⁴¹ For example, cryo‑shocked immortalized mouse macrophage (RAW264.7) cells were loaded with attenuated Salmonella VNP20009 to generate macrophage-Salmonella complexes (MACS/VNP), which maintained cellular viability while sheltering the bacteria.⁴¹ Exploiting macrophage homing, these complexes can achieve substantial accumulation within tumors. The intracellular bacteria were subsequently released in a delayed fashion within the TME, escaping immune clearance in the circulation while markedly increasing the final intratumoral bacterial load.⁴¹ Similarly, other tumor-homing cells such as neutrophils and mesenchymal stem cells have also shown promising potential as agents for targeted delivery of bacteria to various tumor models.⁸⁵

Polymeric “Stealth” Coating

Surface coating of bacteria by coating a biocompatible polymer film on the bacterial surface physically masks pathogen-associated molecular patterns, eluding immune detection. A polyserotonin (PST) coating is pH-responsive and forms on Salmonella (Sal@PST) via the oxidative self-polymerization of serotonin monomers.⁴² This dense coating is stable at physiological pH (7.4), protecting the bacteria from phagocytosis by neutrophils and macrophages. It can be degraded at pH 6 or lower to release the active bacteria predominantly within tumor tissue.⁴² Sal@PST exhibits prolonged circulating retention compared with uncoated bacteria, achieves enhanced tumor colonization in mouse models of melanoma, and significantly decreases non-specific accumulation in normal tissues.⁴²

GSH‑Responsive Self-Assembling Peptide Coating Layer

The uniquely high glutathione concentration within tumour cells forms the basis of another approach in which self-assembling peptides (eg, P1) are explored as cloaking materials.⁴³ They can coat bacteria via both electrostatic interaction and hydrogen bonding with LPS, forming a nanofibril covering that shields immunogenic epitopes and reduces premature immune clearance during systemic circulation. Upon reaching the tumor, the high concentration of GSH triggers disulfide bond cleavage, degrading the coat and releasing the bacteria.⁴³ This approach further significantly decreases serum concentrations of inflammatory cytokines (TNF-α, IL-1β) and mitigates elevations in liver/kidney function markers (AST, CREA), significantly boosting the biosafety of this treatment. This targeted and responsive approach provides a novel strategy for precise bacterial delivery.⁴³

Enhancement of Controllability

To ensure therapeutic safety and efficacy, it is critical to limit uncontrolled proliferation or off-target activity of attenuated strains, especially in normal tissues. The use of sophisticated gene regulatory methods to obtain spatiotemporal control of bacterial life cycle and therapeutic functions is considered the central direction of next-generation engineering. These approaches fall into two general categories: endogenous autonomous control and exogenously‑triggered control.

Tumor Microenvironment-Dependent Autonomous Control

These controls make use of tumor-specific physicochemical cues for intracellular activation (ie, hypoxia, low pH) to control bacterial viability, proliferation, or therapeutic protein expression. This allows potent activation within the intratumoral region without any outside intervention and greatly increases clinical usability.

Hypoxia Sensing Systems

The hypoxic zones of tumors are favored sites of bacterial colonization. Placing critical bacterial genes or therapeutic genes under the regulation of hypoxia-inducible promoters forms a “hypoxia-dependent” circuit.⁴⁹ The YB1 strain employs the wild‑type pepT hypoxia‑inducible promoter driving asd, coupled with an aerobic‑driven antisense‑asd cassette to suppress promoter leakage, to achieve DAP‑dependent conditional survival. This circuit results in bacterial lysis in normoxic tissues and permits bacterial survival and therapeutic‑gene expression within hypoxic tumors.^25,75 Another widely used hypoxia-inducible promoter, nirB, can drive preferential therapeutic‑gene expression within the hypoxic tumor microenvironment in engineered Salmonella strains.⁸⁶

Acidic pH Sensing Systems

Cancerous proliferation and tumor glycolysis produce a reduced extracellular pH within the tumor microenvironment (approximately 6.5), in contrast to the physiological pH of normal tissues (approximately 7.4). Exploiting tumor‑acidic‑pH‑responsive promoters represents a feasible strategy to achieve localized therapeutic gene expression. This selectivity is driven by the proton‑rich microenvironment generated by tumor metabolic reprogramming, whereas intact pH homeostasis in normal tissues keeps these promoters quiescent. This strategy illustrates how bacterial circuits can be precisely tuned to respond to intrinsic tumor microenvironmental cues.

Quorum Sensing (QS)-Mediated Autonomous Control

The quorum sensing mechanism, by which bacterial populations perceive their own density and self-organize group behavior, can be engineered to enable sufficient localized density of therapeutic molecules in the tumor vicinity while mitigating risks of bacterial overgrowth. Thus, for core regulation to occur, the concentration of the signal molecule is key. This property can be exploited to modulate the release of therapeutic molecules, aiming to achieve levels adequate to treat target cancers while limiting the risk of uncontrolled bacterial overgrowth.

The luxI/luxR system uses bacterial density rather than gene density to control the expression of therapeutic genes and has become one of the oldest and most well-understood QS modules. In this scheme, the luxI/luxR system’s dependence on bacterial density is mediated by the concentration of the signal molecule N-acyl homoserine lactone (AHL).⁸⁷ Upon proliferation of engineered bacteria to a sufficient local population within tumors, accumulated AHL can induce the transcription of downstream therapeutic genes.⁸⁷

The synchronized lysis circuit (SLC) is a more sophisticated example of QS that cleverly connects population sensing with lysis genes to create a loop of periodic bacterial lysis within a tumor. Specifically, when population density crosses a threshold, activation of lysis genes leads to lysis of majority of the bacterial population, causing intracellular therapeutic molecules to be released into the tumor, while the remaining bacteria continue to proliferate—creating a “proliferation-lysis” feedback loop.⁸⁸ Pre‑clinical in‑vivo studies confirm this oscillatory circuit constrains overall intratumoral bacterial burden, offering a conceptual solution to the recognized contradiction between the hazard of bacterial over‑proliferation and the demand for sustained therapeutic‑molecule release.⁸⁸

Exogenous Switches

Such switches enable tunable therapeutic intervention via an exogenous signal, providing clinicians’ adaptability in the form of “remote-control” means.

Small-Molecule Inducible Systems: Systems such as the arabinose-triggered P_BAD or tetracycline-triggered Tet systems provide precise and reversible control over therapeutic gene expression via the addition or removal of non-toxic/low‑toxicity small molecules. For example, intraperitoneal administration of arabinose to the engineered strain SMR2130-pS-GMCSF expressing GM-CSF resulted in significant increase in intratumoral GM-CSF levels to effectively turn on antitumor immunity, enabling “remote-switch‑like” control of therapeutic gene expression.⁷⁹

Physical Signal Induction: Extending control further, radiation-responsive systems are built on the bacterial SOS DNA damage repair pathway: ionizing radiation induces DNA damage within the engineered bacteria themselves to activate bacterial RecA protein. This in turn stimulates the auto-proteolysis of the LexA repressor to derepress synthetic SOS‑based radiation-responsive rRPE elements, triggering downstream therapeutic gene expression.⁸⁹ This system was successfully exploited to control Shiga toxin Stx1A release. In 4T1 breast cancer models, irradiation triggered robust induction of Stx1A expression; Stx1A produced by activated bacteria indirectly impairs tumour‑cell DNA‑damage‑repair capacity. The combination of radiotherapy and this bacterial therapy achieved potent tumour‑suppressive effects in mice.⁸⁹

Beyond radiation, another form of control system developed by optical means is light-controlled systems. Bacteria were metabolically labelled with an aggregation-induced emission (AIE) photosensitizer agent that is covalently incorporated into bacterial peptidoglycan; upon light irradiation it generates singlet oxygen (¹O₂), disrupting the bacterial membrane and thus inducing the release of therapeutic genes.⁹⁰ In 4T1 models, light irradiation drives the expression of target transgenes and suppresses tumour angiogenesis, with negligible gene expression detected outside the illuminated tumour region, enabling well‑defined spatial control over therapeutic effectiveness.⁹⁰

Biological Containment

Biological containment measures offer a safety net as the last line of defense for clinical deployment, effectively mitigating the risk of off-target proliferation. One of the integral measures of containment is retaining susceptibility to conventional antibiotics, which allows rapid clearance of misplaced bacteria after drug administration. On the other hand, synthetic conditional‑viability genetic circuits can be constructed so that bacterial survival strictly relies on externally supplied small‑molecule inducers. If off-target proliferation occurs, corresponding antibiotic treatment can ensure swift clearance. Such multi‑layered redundant containment mechanisms further attenuate the potential risks of bacterial therapies.

Enhancement of Antitumor Functionality

Having established a foundational level of safety and tumor-targeting capability, endowing Salmonella with enhanced direct cytotoxic functions or immunomodulatory capacity through genetic engineering is critical to improving therapeutic efficacy.

Enhancing Direct Tumor Cell-Killing Capabilities

Nutrient Depletion Strategy

This strategy exploits the high dependency of many cancers on certain essential nutrients. By engineering attenuated Salmonella to express metabolic enzymes within tumors, key nutrients can be locally depleted, inducing metabolic stress and death of cancer cells, while the bacteria’s tumor-targeting ability spares normal tissues.

One key target is methionine, a critical nutrient that fuels tumor DNA methylation and protein synthesis. The attenuated strain VNP20009 was employed as a starting point for introducing the L-methioninase gene derived from Pseudomonasputida, ultimately generating the engineered strain SGN1.⁵⁷ After colonizing tumors, SGN1 expresses methioninase intracellularly and causes significant intratumoral methionine depletion. This depletion downregulates the EZH2-H3K27me3 pathway and may potentially trigger ferroptosis‑related signaling, leading to apoptosis of tumor cells. SGN1 demonstrated significant tumor regression in models of prostate cancer, triple-negative breast cancer, and liver cancer, along with inhibition of metastatic growth, all without apparent systemic toxicity.⁵⁷

An alternative nutrient depletion approach targets asparagine utilization. Using an attenuated strain with ΔppGpp, the L-asparaginase (L-ASNase) gene was placed under the control of the E. coli araBAD promoter to generate the recombinant strain SL/pASN.⁵⁸ The enzyme produced upon induction with L-arabinose degrades asparagine in the TME and additionally exhibits glutaminase activity. This results in multifold nutrient depletion and inhibition of the mTORC1 pathway, enhancing apoptotic effects.⁵⁸ This strategy demonstrated potent killing efficacy against various cancer cells in vitro and in vivo while circumventing the common immune reactions and liver toxicity associated with intravenous administration of enzyme formulations.⁵⁸

Engineered Direct Delivery of Toxins

This approach involves genetically modifying attenuated Salmonella to express genes encoding tumor-killing toxins. Leveraging the bacterium’s tumor-targeting colonization effect, toxins are delivered locally, being expressed and secreted within the tumor site to maximize direct oncolytic activity with the aim of minimizing systemic toxicity.

MVIIA, a ω-conotoxin peptide, selectively blocks N-type calcium channels (Cav2.2). These channels are overexpressed in breast and prostate carcinomas and are linked to poor prognosis.⁹¹ Salmonella‑delivered MVIIA induces tumour‑cell apoptosis, putatively via perturbation of Cav2.2‑dependent calcium influx. The engineered strain exerted potent anti‑tumour activity across multiple solid tumour preclinical models.⁹¹

Targeted immunotoxin delivery has yielded highly specific oncolytic activity against EGFR-positive tumors, with TGFα-PE38 demonstrating efficient cytotoxicity and tumor selectivity.⁸⁴ Scientists fused the EGFR-binding ligand TGFα to a linker-containing fragment of Pseudomonas exotoxin A (PE38) and transformed this construct into a ΔppGpp attenuated strain under the control of the P_BAD promoter. Toxin release was regulated by phage lysis systems or the Psp secretion signal. Upon colonizing EGFR-positive tumors, TGFα-PE38 is secreted following L-arabinose induction. The TGFα moiety specifically recognizes EGFR on tumor cells and is internalized, where PE38 acts to inhibit protein synthesis, inducing apoptosis.⁸⁴ In vivo experiments demonstrated marked growth retardation of EGFR-positive tumors, including CT26 and 4T1.⁸⁴ This local‑delivery strategy is hypothesised to avoid the hepatotoxicity associated with systemic administration of free immunotoxin.

The dual-drug combinatorial delivery system incorporating cytolysin A and Vibrio flagellin B (FlaB) adds further antitumor efficacy and extended immunomodulatory activities.⁹² On a ΔppGpp backbone, the SAM-FC strain was engineered to express ClyA and FlaB together using a tetracycline-inducible expression system.⁹² ClyA is a pore-forming membrane toxin that disrupts tumor cell membrane integrity. It mediates immunogenic cell death (ICD) and releases damage-associated molecular patterns (DAMPs), including high mobility group box 1 (HMGB1) and calreticulin (CALR), which drive dendritic cell maturation. FlaB activates TLR5 signaling, promoting repolarization of M2-type macrophages to the more antitumor M1 phenotype, thereby inhibiting metastasis.⁹² The combined “direct killing + immune remodeling” effect rendered tumours undetectable in 24 of 32 (75 %) CT26‑bearing mice on day 12 after bacterial injection; animals remaining tumour‑free thereafter developed durable long‑term antitumour immunological memory.⁹²

Moreover, specialized regulatory systems have been optimized for toxic protein delivery. An inter-kingdom dual expression (IKDE) system drove sustained intratumoral expression of diphtheria toxin A chain (DTA) via a T7 RNAP autocatalytic positive-feedback loop. This therapeutic modality eradicated established 4T1 breast tumours in a subset of animals and suppressed lung metastasis, with complete tumour clearance achieved in 26 % of tumour‑bearing mice (5 out of 19).⁹³ By contrast, a synchronized lysis circuit (SLC) harnesses quorum‑sensing machinery to drive cyclic bacterial lysis for pulsed secretion of therapeutic payloads such as hemolysin E (HlyE). When the SLC‑3 multi‑strain cocktail was combined with chemotherapy, this therapeutic strategy produced roughly a 50 % increase in the mean survival time of mice bearing liver‑metastatic colorectal tumours, while keeping bacterial burden at a well‑tolerated level.⁸⁸

Enhancing Antitumor Immune Response

As a living delivery platform, attenuated Salmonella can be equipped to express and deliver cytokines and tumor-associated antigens, thereby constructing a multi-dimensional immune activation network. This significantly enhances the antitumoral immune response specificity and durability and compensates for the relative immunomodulators potency of parent strains.

Targeted Antitumor Immunomodulatory Delivery and Enhanced Immune Response

Engineered Salmonella serves as a tool for precise, sustained, and in situ delivery of immunomodulatory cytokines within tumors to dramatically boost antitumor immunity. Recombinant strains carrying genes for cytokines such as IL-12 and GM-CSF strongly enhanced dendritic cell maturation, Th1-type immune polarization, and cytotoxic T cell proliferation and activation. Engineered forms of such cytokines can retain much higher expression levels in vivo that correlate with enhanced antitumor responses.⁹⁴ The construction of an IL-18-secreting recombinant Salmonella induced proliferation of T cells and NK cells along with associated cytokine production, and also recruited large numbers of neutrophils into tumor tissue, thereby inhibiting tumor growth with minimal systemic toxicity.⁹⁵ Local expression of chemokines such as CCL21 is also highly efficient in chemoattracting T cells and dendritic cells to the tumor site, where they act cooperatively to inhibit primary tumor growth and lung metastasis development.⁹⁶ This effect was further evident when recombinant strain SL7207 was co-administered with a tumor-targeting albumin-IL-2 (Alb-IL-2) fusion protein, wherein the albumin’s tumor-accumulating property was exploited to elevate local IL-2 concentration within the TME, thereby inducing robust T cell-mediated antitumor immunity and significantly improving survival of tumor-bearing mice.⁹⁷

Specific Delivery of Tumor-Associated Antigens and Adaptive Immune Memory

Genetic engineering of Salmonella to insert genes encoding tumor-associated antigens represents a strategy that can facilitate highly specific antigen delivery to break immune tolerance and activate tumor-specific adaptive immunity.

For example, the genetically engineered attenuated strain SL7207 was constructed to display the melanoma antigen Melan-A on its surface using the AIDA-I autotransporter to activate CD4⁺ T cells, as well as to express recombinant DNA encoding MHC class I epitopes of TRP-1 and TRP-2 to activate CD8⁺ T cells. Simultaneous expression of tumor-associated antigens achieved tumor-specific CD4⁺ helper T cell and CD8⁺ cytotoxic T cell responses, inducing maturation and proliferation of T cells and the production of proinflammatory factors such as TNF-α and IFN‑γ.⁹⁸ For NY-ESO-1-positive tumors, recombinant Salmonella engineered to express NY-ESO-1 itself could unleash NY-ESO-1-specific T cell responses to induce tumor regression.⁹⁹

In addition to these strategies, further optimizations have modified the bacteria itself for improved adaptation to the immune microenvironment. For example, the DB1 strain, which elicits host‑derived IL‑10‑driven IL‑10R signaling responses in the tumour microenvironment, can enhance not only its survival within tumors but also its capacity to activate resident CD8⁺ T cells, thereby further boosting specific immune response intensity and persistence.⁷¹ Such genetic engineering approaches establish a comprehensive response circuit encompassing “innate immune activation-adaptive immune initiation-immune memory maintenance,” significantly enhancing the potency and durability of antitumor immunity.

Following the enhancement of the intrinsic antitumor efficacy of S. Typhimurium and the optimization of its biosafety profiles via synthetic biology, nanobiotechnology, and other interdisciplinary technologies, the bacterial platform is further engineered by integrating the core antitumor mechanisms and design paradigms of other therapeutic modalities. This rational engineering not only endows S. Typhimurium with novel antitumor functionalities that it natively lacks, but also leverages the robust tumor-targeting capacity of this strain to synergistically enhance the therapeutic efficacy of combined regimens while mitigating the off-target systemic toxicities of other therapeutic approaches. The comprehensive details of these synergistic therapeutic strategies are elaborated in Combination Therapy Strategies.

Combination Therapy Strategies

While engineered S. Typhimurium possesses independent antitumor capabilities, its antitumor efficacy as a monotherapy is frequently limited. There are, however, combination strategies that integrate such engineered Salmonella not only with existing therapeutic modalities including chemotherapy, radiotherapy, and immunotherapy, but also with emerging therapies such as photodynamic and sonodynamic treatment. These combinatorial regimens can generate remarkable synergistic effects through complementary mechanisms.

Combination with Chemotherapy

The dense extracellular matrix (ECM) and excessive interstitial fluid pressure (IFP) within tumors lead to a large physical barrier that blocks chemotherapeutic agents from entering the tumor core. Engineered Salmonella can actively reshape this barrier. To illustrate, the strain SL^lux/HAase was obtained by inserting the hyaluronidase gene (hysA), which was fused to the pelB signal peptide for secretion, into attenuated ΔppGpp S. Typhimurium.¹⁰⁰ After colonization of the tumor by this strain, it enzymatically degraded hyaluronic acid (HA), which was embedded within ECM. This HA degradation lowers tumor interstitial fluid pressure (IFP) to facilitate drug penetration, and interferes with interactions of HA and CD44 on tumor cells, leading to a decrease in cell adhesion and invasion.¹⁰⁰ In pre-clinical tumor models of 4T1 breast cancer and ASPC-1 pancreatic cancer, the combined therapy of SL^lux/HAase and chemotherapeutic agents improved intratumoral drug delivery and generated enhanced antitumor effects compared with chemotherapy alone.¹⁰⁰

In addition, the attenuated strain VNP20009 possesses specific benefits against deep-penetration barriers caused by collagen fibers.¹⁰¹ After colonization of the tumor, this strain remodels tumor stromal and vascular architectures and enhances tumor vascular permeability.¹⁰¹ In BxPC‑3 pancreatic cancer models, immediate prior administration of VNP20009 followed by liposomal doxorubicin injection achieved stronger antitumor efficacy than chemotherapy monotherapy.¹⁰¹

In addition to the enhancement of drug delivery, Salmonella may also attenuate tumor multidrug resistance (MDR). This is hypothesized to occur via the modification of resistance-related signaling pathways in tumor cells. It was discovered that combining the attenuated strain LVR01 with dacarbazine (DTIC) exerts synergistic effects in B16F10 melanoma models.¹⁰² We speculate that this mechanism may also be associated with the tumor microenvironment characterized by an inflammatory response that is induced by Salmonella. In this microenvironment, the action of cytokines such as IL-1β and TNF-α might modulate the activation of PI3K/Akt pathways, which may contribute to altered synthesis of resistance-relevant proteins, though complete experimental validation for this signalling axis is still lacking.

Meanwhile, Salmonella’s targeting attributes can localize the cytotoxic effects of therapeutic payloads more specifically within tumors. As an example, when the CD20‑targeted Salmonella strain 2G9 engineered to express the HSV‑TK prodrug‑converting enzyme was combined with the prodrug substrate ganciclovir to treat lymphoma, it could selectively bind CD20-positive lymphoma cells, increase intracellular bacterial invasion within antigen‑positive tumour cells and reduce non‑specific intracellular bacterial invasion in normal organs such as liver and spleen, and produce potent anti‑lymphoma effects in human lymphoma xenograft models.⁸¹

Combination with Radiotherapy

Radiotherapy plays a pivotal role in local tumor control, but its effect can be compromised by the improved repair of DNA damage in tumor cells, and the immunosuppressive microenvironment. By virtue of advanced technologies including nanobiotechnology, genetically engineered S. Typhimurium has the potential to act synergistically with radiotherapy primarily through two main mechanisms: by directly amplifying the damage to DNA caused by radiotherapy, and by providing additional complementary immune activation, which can increase the cytotoxicity locally, thereby facilitating more potent systemic antitumor effects.

Induction of DNA double-strand breaks (DSBs) is the central mechanism of radiotherapy. Abnormal activation of the functional DNA‑repair pathways in tumor cells is one of the major underlying causes of radioresistance.⁸⁹ The engineered Salmonella can act to directly impair these repair pathways, or to amplify the oxidative damage, by either delivering or secreting functional molecules.⁸⁹

First, based on the native bacterial SOS DNA-damage response pathway, an ionizing-radiation-responsive genetic circuit was constructed. The radiation-responsive VNP^R-S(wt) releases STx1A upon triggering by a radiotherapy-relevant dose of 6 Gy. Intracellularly delivered STx1A impairs tumour-cell DNA double-strand break (DSB) repair capacity, as evidenced by persistent γ‑H2AX foci accumulation. This combination strategy promotes tumour‑cell apoptosis and robust tumour growth inhibition in 4T1 breast cancer models and also induces systemic anti‑tumour immune responses with abscopal effects.⁸⁹

Second, the gold-based photosensitizer-loaded strain VNP@TBTP‑Au, in addition to increasing X-ray absorption efficiency through the Au atomic element in radiotherapy, can produce abundant reactive oxygen species (ROS). It then acts synergistically with the radiotherapy-related ROS, which amplifies more intense oxidative damage on the DNA and improves radiosensitization in B16F10 melanoma models.¹⁰³

Meanwhile, radiotherapy provokes multiple forms of danger‑associated molecular signals within tumor tissue, whose immunostimulatory effects can be augmented by Salmonella. For instance, the protamine‑modified strain VNP-PRM can enrich and deliver the cGAMP (a STING pathway agonist) locally released after radiotherapy to tumor‑associated macrophages, triggering STING-TBK1-IRF3 pathway and promoting TAM polarization toward the M1‑like phenotype.¹⁰⁴ M1-type TAMs can secrete cytokines including TNF-α and IL-12, and further induce and activate infiltrating CD8⁺ T cells and NK cells to boost post-radiotherapy antitumor immune responses.¹⁰⁴

Additionally, nattokinase‑secreting engineered Salmonella VNP^NKase remodels the tumor physical microenvironment by degrading the extracellular matrix components, restores radiotherapy‑compromised infiltration of CD103⁺ dendritic cells, and potentiates radio‑immunotherapy. In bilateral‑tumor models, it elicits prominent abscopal antitumor effects, revealing the synergy between local radiotherapy and systemic antitumor immunity.¹⁰⁵

Combination of Salmonella with Immune Checkpoint Inhibitors

Abnormal activation of the PD-1/PD-L1 pathway constitutes one of the core mechanisms underlying tumor immune escape. Relying on its unique tumor-targeting colonization property and the ability to reshape the tumor immune microenvironment, together with the precise delivery advantage of nanobiotechnology, genetically engineered S. Typhimurium can produce a remarkable synergistic effect when combined with PD-1/PD-L1 blockade strategies, thus providing a novel technical pathway for overcoming immunotherapy resistance.

Bacteria-Mediated Specific Delivery of PD-1 Nanobodies

Salmonella as a local nanobody factory is developed as a strategy. For example, VNP20009 strain was engineered to continuously express and secrete anti-PD-1 nanobodies (VNP-PD1nb) inside the tumor.¹⁰⁶ To maximize the delivery efficiency, macrophages were loaded with this bacterium as a Trojan horse. Then, the composite system of MF(VNP-PD1nb) was constructed.¹⁰⁶ This macrophage‑mediated specific delivery system, using anti‑PD‑1 nanobody‑expressing VNP‑PD1nb in melanoma model, utilized macrophage tumor tropism for efficient bacterial enrichment. This could help to minimize systemic toxicity for monotherapy with bacteria, enhance the infiltration of immune cells, remodel intratumoral M2‑like tumor‑associated macrophages toward an M1‑like phenotype, and could significantly improve the activation and killing ability of CD8⁺ T cells.¹⁰⁶

Non-Antibody PD-L1 Regulation (Gene Silencing)

Salmonella is also developed as another approach for the gene-level immune checkpoint regulation, achieving specific local‑tumor gene silencing.

As such, attenuated Salmonella LH430 was engineered to carry a PD-L1 short hairpin RNA (shRNA) plasmid. Such shRNA‑carrying LH430 was further surface‑modified with photosensitizer IR780 to develop the composite system shPD-L1@LHPS.¹⁰⁷ This system can kill tumor cells under near-infrared light irradiation and induce ICD via photothermal‑photodynamic effects, while bacterially delivered shRNA silences the PD-L1 gene expression specifically. Therefore, this “physical killing + immune gene regulation” strategy leads to a substantial increase in T‑cell proportion and IFN-γ secretion, and can effectively suppress the growth of distant abscopal tumors in bilateral tumor model.¹⁰⁷ Similarly, bacteria engineered to deliver PD‑1 siRNA can act on tumor microenvironment T cells specifically, therefore, reversing the T cells’ exhaustion and restoring antitumor immune function.¹⁰⁸ This gene‑silencing strategy based on bacterial tumor‑targeted precise delivery enables direct modulation of the PD‑1/PD‑L1 axis and offers a more flexible technical modality for combination therapy.

Nanobiotechnology-Mediated Combination of Salmonella Typhimurium with Emerging Therapies

In recent years, nanobiotechnology has undergone rapid advances, enabling precise regulation over the size, surface charge, biocompatibility, and stimuli-responsive degradability of nanomaterials. Such tunability facilitates the fabrication of multifunctional nano-delivery platforms, which serve as core tools for precision tumor therapy. Nanomaterials possess inherent merits including targeted cargo delivery, spatiotemporally controlled drug release, minimized systemic toxic side effects, and facile modification of physicochemical properties. Via surface engineering strategies such as chemical covalent conjugation, biomineralization, and electrostatic physical adsorption (Figure 5), hybrid bacteria-nanomaterial delivery systems can be fabricated.

Figure 5.

Nanoscale Surface Engineering Strategies of Salmonella Typhimurium. (A) Biomimetic or polymeric coatings can shield bacterial surface antigens and carry additional therapeutic components. (B) Biomineralization forms inorganic shells, such as CaCO₃ or MnO₂, on the bacterial surface. (C) Physical adsorption uses noncovalent forces, including electrostatic interactions, to associate nanomaterials with the bacterial envelope. (D) Covalent conjugation immobilizes functional materials through stable chemical bonds. These approaches generate bacteria–nanomaterial hybrids without directly altering the bacterial genome. This figure was handcrafted in PowerPoint by the authors. ByteDance SeedEdit 3.0 merely optimized the definition of partial icons.

Monotypic delivery carriers inevitably suffer from inherent, intractable limitations. Nonliving nanocarriers offer defined composition and stimulus-responsive cargo release, but their tumor distribution depends on physicochemical properties, vascular transport, and, where applicable, ligand-mediated targeting. Nonliving nanoformulations (liposomes, inorganic nanoparticles, polymeric nanocarriers, etc.) rely solely on the enhanced permeability and retention effect for passive tumor accumulation. Devoid of the hypoxia-tropic autonomous motility inherent to bacteria, these nanosystems fail to penetrate dense tumor stroma and reach hypoxic necrotic tumor cores.¹⁰⁹ Furthermore, non-proliferative nanocarriers only execute one-shot drug release and lack the capacity to continuously synthesize and secrete immunostimulatory moieties (eg, flagellin and cytokines) within tumor lesions. Their transient immunostimulatory effects thus preclude sustained remodeling of the immunosuppressive tumor microenvironment.¹¹⁰ In comparison, live bacterial vectors can provide active motility and in situ production of therapeutic molecules, but their pharmacokinetics, immune clearance, and biosafety are more difficult to control.

Attenuated Salmonella live bacteria exhibit intrinsic strengths in autonomous tumor tropism and in situ persistent immune activation. Nevertheless, highly immunogenic components including lipopolysaccharide and flagella are rapidly cleared by hepatic and splenic macrophages, resulting in short blood circulation half-lives and insufficient intratumoral bacterial accumulation. Additionally, systemic bacterial dissemination may trigger inflammatory responses or bacteremia, and residual viable bacteria impose long-term biosafety risks in vivo.⁶⁸

Hybrid delivery systems consisting of nanoparticle-functionalized Salmonella integrate four pivotal superiorities (Figure 6):

• (1)Prolonged systemic circulation and staged immune evasion. Nanocoatings composed of calcium carbonate, polydopamine, or metal-polyphenol complexes physically mask surface immunogenic epitopes (LPS and flagella) of Salmonella, blocking recognition by pattern recognition receptors such as Toll-like receptors and mitigating macrophage phagocytosis. This substantially extends blood circulation duration and elevates intratumoral bacterial accumulation efficiency. Upon arrival at the acidic tumor microenvironment, the nanoshell undergoes specific degradation, restoring the strain’s capacity for intratumoral colonization and proliferation to achieve temporally regulated targeted tumor engraftment.¹¹¹
• (2)Synergistic autonomous tumor colonization and long-lasting multi-modal immune activation. Leveraging the innate hypoxia-tropic motility of Salmonella, the hybrid construct actively penetrates deep tumor parenchyma. The nanocarrier simultaneously loads immune checkpoint inhibitors and immunoadjuvants, while bacteria continuously synthesize and secrete immunostimulatory factors. Degradation of the outer nanolayer synchronously releases immunomodulatory therapeutics. These dual cascades cooperatively drive M1-type macrophage polarization and dendritic cell maturation, ultimately establishing durable systemic anti-tumor immunological memory.³⁶
• (3)Compatibility with combinatorial photothermal, photodynamic, and sonodynamic therapy. Photothermal, photosensitizing, and catalytic nano-motifs can be stably anchored onto attenuated S. Typhimurium via biomineralization or covalent conjugation, constructing an all-in-one platform that integrates bacteria-mediated targeted delivery with PTT/PDT/SDT combinatorial therapy. This design addresses critical drawbacks of monomodal physical ablation, namely poor tumor specificity and limited tissue penetration depth.
• (4)Attenuated systemic toxicity of live bacteria and improved in vivo controllability. The outer nanoshell isolates encapsulated bacteria from normal tissues during circulation and restricts ectopic bacterial colonization and dissemination. Biomineralized layers (calcium carbonate, MnO₂, etc.) scavenge reactive oxygen species and neutralize lactic acid, alleviating bacteria-induced systemic inflammatory reactions. Combined with quorum-sensing responsive bacterial lysis modules, viable bacteria can be controllably eliminated after therapeutic intervention, drastically reducing biosafety hazards arising from residual live microbes.

Figure 6.

Antitumor Therapy Based on Nanoparticle-Functionalized Salmonella Delivery Systems. Unmodified Salmonella with exposed surface immunogenic epitopes is rapidly recognized and eliminated by macrophages in the bloodstream, resulting in insufficient tumor accumulation. In contrast, nanoparticle-coated Salmonella achieves effective immune evasion and prolonged systemic circulation, as the outer nanoshell masks bacterial lipopolysaccharides and flagellar antigens to avoid TLR-mediated immune clearance. Benefiting from the intrinsic hypoxic tropism, the engineered bacteria actively target and penetrate deep into tumor tissues. Once residing in the acidic tumor microenvironment, the outer nanoshell undergoes triggered degradation, thereby restoring the intratumoral colonization and proliferation capacity of Salmonella. The released bacteria further initiate robust antitumor immune responses by promoting dendritic cell maturation and M1 macrophage polarization, ultimately activating T cell and NK cell-mediated systemic immunotherapy. In addition, the nanoshell-anchored photothermal, photosensitizing, and sonocatalytic nano-motifs enable synergistic PTT/PDT/SDT combinatorial therapy under near-infrared irradiation, generating abundant reactive oxygen species and hyperthermia to induce profound tumor cell death and tumor regression. This figure was handcrafted in PowerPoint by the authors. ByteDance SeedEdit 3.0 merely optimized the definition of partial icons.

In summary, nanoparticle-decorated S. Typhimurium hybrid delivery platforms overcome the key bottlenecks of conventional monosystems: passive tumor enrichment of bare nanomaterials, rapid immune clearance of uncoated live bacteria, and systemic bacterial toxicity. By organically integrating deep-tumor autonomous targeting, sustained immune reprogramming, and multi-modal physical ablation functionality, this hybrid construct represents an optimal living delivery vector for combinatorial anti-tumor treatment.

Combining with Light-Mediated Therapies

Light-based therapies have become widely investigated approaches in cancer treatment. They possess great advantages in control of space and time, and minimal invasiveness but are also restrained by phototherapeutic agents’ lack of tumor targeting and poor light-penetration depth. The hypoxia-targeted colonization, deep penetration, and immune activation characteristics of S. Typhimurium can form efficient synergy with photothermal therapy and photodynamic therapy. The intervention of nanobiotechnology realizes the precise integration of phototherapeutic agents and S. Typhimurium through nanophotosensitizer modification, nanocarrier encapsulation, and other methods. Through the ordered actions of bacterial-mediated precise delivery, local killing by light effects, and amplified immune response, the therapeutic effect is significantly improved.

Combination of Salmonella with Photothermal Therapy (PTT)

PTT is based on near-infrared light for excitation of photosensitizers to achieve heat induction. Salmonella is used as a living cargo vehicle to overcome targeting and penetration limitations, while the photothermal response can remodel tumor stroma, improve the microenvironment, and further recruit bacterial enrichment to initiate a synergistic loop. Nanobiotechnology is the key means to achieve efficient combination of the two.

Photosensitizer‑Loaded Salmonella for Positive‑Feedback Photothermal Ablation: The hypoxia-targeted engineered strain YB1 was covalently conjugated to indocyanine green (ICG)-loaded nanoparticles to create the YB1-INPs hybrid system, combining hypoxia-targeting delivery by the bacterial strain YB1 with photothermal conversion and imaging capabilities provided by the loaded nanoparticles (INPs).¹¹² After intravenous administration, YB1 actively accumulated toward the hypoxic tumor core in MB49 bladder cancer models. Under NIR laser irradiation, the local tumor temperature was elevated to 63°C, directly inducing irreversible thermal ablation of tumor cells and disrupting the dense stromal structure.¹¹² Subsequent release of tumor cell debris and nutrients further recruited YB1-INPs for intratumoral widespread infiltration, achieving 14-fold higher accumulation than in the no-light control group.¹¹² In large solid tumor models (≥500 mm³) treated with two rounds of NIR irradiation following YB1-INPs administration, the target tumors were completely ablated without recurrence, and minimal off‑target toxicity was observed for free nanophotosensitizers at the tested dosage.¹¹²

A Photo‑Driven Salmonella with Biomimetic Camouflage for Systemic Delivery: To maximize systemic circulation efficiency and minimize off-target accumulation in organs such as the liver and spleen (ie, off-target toxicity), biomimetic camouflage was developed. This strategy constructs a macrophage membrane-camouflaged photothermal bacterial motor system (IP@VNP@M), which covalently anchors ICG-PLGA photothermal nanoparticles (IP) onto the surface of attenuated clinical strain VNP20009, followed by cloaking the entire IP@VNP construct with M1‑derived macrophage membranes.¹¹³ Such biomimetic membrane confers dual benefit: first, it can take advantage of the macrophage membrane α4/β1 integrins, favoring homing toward inflamed tumor vasculature, and second to physically mask bacterial pathogen‑associated molecular patterns (PAMPs), reducing recognition and phagocytosis by circulating innate immune cells and thereby significantly extending the in vivo circulation time.¹¹³ Under NIR irradiation, the rather mild photothermal action of IP@VNP in‑vitro suspension, elevating local temperature to 37.3 °C, activates bacterial motility and increases the migration speed by 4.0-fold. In combination with the intrinsic hypoxic chemotaxis delivered by VNP, the system achieves deep penetration within 4T1 breast cancer spheroids; near‑complete tumor eradication was observed in‑vivo tumor‑bearing mice.¹¹³ The system, through synergized photothermal and bacterial oncolytic effect, facilitated M2-to-M1 polarization of tumor‑associated macrophages and secretion of TNF-α and IL-6. It can greatly reduce the accumulation of VNP into liver by macrophage membrane camouflage system to keep the markers of liver injury for ALT, AST very well in the normal liver conditions, while extending the survival of tumor-bearing mice (to 55 days) and addressing the issue of off-target toxicity from classical bacteriotherapy.¹¹³

Combination of Salmonella and Photodynamic Therapy (PDT)

PDT is based on photosensitizers producing ROS to kill tumor cells and inducing immunogenic cell death. Salmonella can not only deliver the photosensitizer to the deep hypoxic tumor core accurately, but also help amplify the antitumor immune activity triggered by PDT by activating immune cells, overcoming resistance in low-immunogenicity tumors.¹⁰⁷

Exogenous‑Light‑Triggered shPD‑L1‑Loaded Photoresponsive Composite: The shPD-L1@LHPS composite utilizes attenuated S. Typhimurium LH430 (ΔphoPΔphoQ double knockout for reduced virulence with intact hypoxic targeting capacity) which is loaded with the photosensitizer IR780 and harbors a plasmid expressing shRNA targeting PD-L1.¹⁰⁷ Upon intravenous administration followed by local NIR tumor irradiation, IR780 generates ROS to induce ICD. Concurrently, the shPD-L1 plasmid is expressed in tumor cells, achieving sustained downregulation of PD-L1 expression and reversing immunosuppression.¹⁰⁷ In B16F10 melanoma models, this system markedly elevated intratumoral CD8⁺ T‑cell infiltration, exerted potent tumor‑suppressive effects, and significantly prolonged survival without obvious toxicity.¹⁰⁷

Bioluminescence-Driven Endogenous PDT Strategy: To overcome the limitations of external light source penetration depth, an endogenous PDT strategy was developed. A luciferase-encoding plasmid was introduced into attenuated ΔppGpp Salmonella (Luc-S.T. ΔppGpp) to produce an external light-free PDT system.¹¹⁴ Once bacteria colonize the tumor, injection of the substrate D-luciferin induces an enzymatic reaction inside the bacteria to produce 450–700 nm bioluminescence. This bioluminescence directly activates the co-delivered photosensitizer Ce6 to generate cytotoxic singlet oxygen, enabling internal light-free PDT.¹¹⁴ Alginate in situ gel fixation prolongs retention of the bacteria and photosensitizer in the tumor. This endogenous light is uniformly distributed, overcoming the unevenness of external light penetration, and effectively kills cells in CT26 colorectal cancer, B16 melanoma, and even large rabbit VX2 tumor models. It induces M2 to M1 macrophage polarization, CD8⁺ T cell infiltration and IFN-γ secretion, and activates systemic immunity. Doubled‑dose administration markedly improved therapeutic efficacy against large‑volume CT26 tumors, and tumor‑rechallenge assays confirmed the establishment of protective antitumor immune memory.¹¹⁴

Fluorogen‑Activated Photosensitization for Higher Specificity and Greater Safety: A fluorogen‑activation strategy for higher specificity and safety was used. An engineered attenuated ΔppGpp strain was constructed to express fluorogen activating protein (FAP), dL5** under L-arabinose induction (SLpdL5).¹¹⁵ In the CT26 colorectal tumor models, after intratumoral injection of the fluorogen MG-2I, dL5** and MG-2I combine to form a photosensitive complex that generates ROS under appropriate LED light irradiation.¹¹⁵ This treatment can directly kill tumor cells and tumor‑colonized bacteria, mitigating systemic bacterial‑related toxicity. Moreover, lysis of bacteria and tumor cells releases PAMPs and DAMPs, recruiting neutrophils, macrophages, and dendritic cells, accompanied by elevated intratumoral levels of pro‑inflammatory factors, namely TNF‑α and IL‑1β and reduced levels of the immunosuppressive cytokine IL‑10.¹¹⁵ One bacterial injection plus three illumination treatments achieved enhanced tumor‑suppressive effects relative to oncolytic bacterial monotherapy. The bacteria‑based FAP system enables real‑time fluorescence imaging to track bacterial colonization in tumors.¹¹⁵

Synergizing with Calcium Overload Therapy

Calcium overload induces tumor cell death by disrupting mitochondrial function and interfering with calcium signal homeostasis, which is a promising new therapeutic strategy. However, calcium-based nanomaterials have problems such as poor targeting and insufficient intratumoral accumulation. Nanobiotechnology provides a feasible path for the combination of S. Typhimurium and calcium overload strategy. By integrating calcium-based carriers with S. Typhimurium through nanomodification methods such as biomineralization and supramolecular assembly, precise tumor delivery of calcium-based materials can be achieved. Meanwhile, calcium overload forms synergy with the direct tumor-killing and immune activation effects of S. Typhimurium, significantly improving the therapeutic effect. In recent years, researchers have constructed composite systems through material modification, realizing the dual optimization of targeting and curative effect.

Calcium-Based Carriers with Acid-Responsive Release

Using tumor-specific Salmonella, we can deliver calcium-based materials to tumor with enhanced targeting; the acidic tumor microenvironment triggers calcium release to induce calcium overload (Ca²⁺), and materials can be loaded with functional molecules to amplify this calcium‑overload effect. Xie et al developed CaPCAV, a supramolecular conjugate system with attenuated S. Typhimurium VNP20009 as a vehicle, combined with polydopamine-coated CaCO₃ nanoparticles loaded with curcumin.¹¹⁶ After VNP20009 colonized 4T1 breast tumor tissue efficiently, the acidic milieu induced CaCO₃ degradation and Ca²⁺ liberation. Curcumin blocked Ca²⁺ efflux and facilitated endoplasmic reticulum calcium release. This synergism weakened tumor cell mitochondrial membrane potential, released cytochrome C, and caused the degeneration of tumor cells.¹¹⁶ The system resulted in prominent intratumoral CD8⁺ T‑cell infiltration, and serum biochemistry revealed hepatic and renal biomarkers remained within physiological reference ranges following treatment.¹¹⁶

CaCO₃-biomineralized Salmonella (Sal@CaCO₃/A23187): Guo et al mineralized CaCO₃ on the attenuated ΔppGpp strain’s surface and loaded it with the calcium ionophore A23187.³⁶ CaCO₃ breaks down in the acidic environment after intratumoral injection and A23187 facilitates Ca²⁺ influx into immune cells, working together with Salmonella to modulate the immune microenvironment.³⁶ In B16F10 melanoma animal models, this single-treatment system exerted potent tumor‑suppressive effects. When used in combination with the PD-L1 inhibitor, it achieved near‑complete suppression of pulmonary metastatic nodules and substantially elevated levels of pro-inflammatory mediators such as intratumoral TNF-α and systemic IFN-γ.³⁶

Calcium Overload and Immunomodulation

Calcium overload not only induces tumor cell death directly, but it can also promote immunogenic cell death (ICD) and act with Salmonella’s immunogenicity to better remodel the tumor immune microenvironment and reverse the immunosuppressive metastatic tumor microenvironment. In these systems, Salmonella‑triggered immunogenic cell death drives tumor cells to release damage-associated molecular patterns (such as calreticulin and HMGB1). Ca²⁺ acts as an immune signaling molecule, increasing the efficiency of antigen presentation by dendritic cells, advancing their maturation and secretion of TNF-α, along with activating specific activity of CD8⁺ T cells to kill tumor cells.³⁶ This synergistic effect promoted prominent intratumoral CD8⁺ T cell infiltration in B16F10 melanoma animal models, with a significant effect in reversing the “cold” tumor microenvironment.³⁶

Calcium overload stimulates NLRP3 inflammasome activation via lysosomal damage. It facilitates the release of IL-1β, and amplifies the body’s innate immune response.¹¹⁷ Ca²⁺ also stimulates macrophage phagocytic abilities and promotes the polarization of M1 macrophages.¹¹⁷ Such calcium‑mediated immunomodulatory events may potentiate antitumor therapeutic efficacy.

Combination of Salmonella with Ferroptosis Mechanism

Ferroptosis is a regulated cell death triggered by iron-dependent lipid peroxide accumulation, and its core mechanism lies in the collapse of intracellular antioxidant defense (especially the glutathione-GPX4 axis). Although it has broad prospects, ferroptosis therapy faces challenges such as poor targeting of inducers, insufficient intratumoral iron/ROS supply, and difficulty in activating systemic immune responses. The intervention of attenuated S. Typhimurium is expected to serve as a “one-stop” platform for solving these bottlenecks. Nanobiotechnology enables the efficient combination of the tumor-targeting ability of S. Typhimurium and the killing mechanism of ferroptosis by constructing iron-based nanocarriers and realizing nanoscale delivery of ferroptosis inducers. Its tumor hypoxia-targeting ability facilitates the efficient delivery of iron-based materials and inducers, while its inherent adjuvant activity can amplify the immunogenic effect caused by ferroptosis.

Bacteria-Mediated Targeted Delivery of Iron-Based Carriers and Ferroptosis Inducers

The attenuated S. Typhimurium VNP20009 can serve as a living delivery platform for iron-based carriers and ferroptosis inducers due to its selective colonization characteristics in tumor hypoxic areas. Researchers have realized the stable combination of iron-based nanomaterials and S. Typhimurium through nanoscale assembly methods such as host–guest interaction and cell membrane encapsulation with the help of nanobiotechnology, achieving controllable release of iron ions in combination with the acidic characteristics of the tumor microenvironment and simultaneously enhancing the lipid peroxidation effect.

Xie’s team developed a nanoparticle carrier system that carried a biomimetic nanoparticle FeAM via host-guest chemistry, namely tumor-tropic attenuated strain VNP20009 acting as a carrier.¹¹⁸ FeAM is composed of an Fe₃O₄ nano-core loaded with the ferroptosis inducer sulfasalazine (SAS) and cloaked with 4T1 tumor‑cell‑derived membranes.¹¹⁸ Following intravenous injection, VNP20009 can function as a “navigator” to precisely deliver FeAM to 4T1 breast tumor tissue.¹¹⁸ One of SAS’s mechanisms of action is to suppress the System Xc⁻ transporter to block cystine import, which causes decreased intracellular glutathione synthesis, resulting in the reduction of critical antioxidant GPX4 activity.¹¹⁸ The generated Fe²⁺ catalyzes the Fenton reaction to produce abundant hydroxyl radicals, which attack polyunsaturated fatty acids. This large amount of hydroxyl radicals, coupled with exhausted antioxidant machinery, causes dramatic lipid peroxidation and eventually leads to ferroptosis in tumor cells.¹¹⁸ This platform exerted potent antitumor efficacy in the 4T1 mouse tumor model and showed negligible damage to liver or kidney function.¹¹⁸

Another innovative work uses the ability of S. Typhimurium to reshape the tumor microenvironment combined with nano-self-assembly technology to “utilize local materials” for tumor therapy. Xu et al revealed that the intravenous infusion of VNP20009 could trigger flagellin‑driven secretion of TNF‑α, which in turn disrupts immature tumor neovasculature.¹¹⁹ This provoked significant extravasation of huge amounts of erythrocytes and their infiltration into CT26 colorectal tumor tissue.¹¹⁹ When the erythrocytes were then phagocytosed by tumor cells, heme was catabolized into Fe²⁺ by the oxidative enzyme heme oxygenase-1 (HMOX-1). Upon administration of zoledronic acid (ZOL), Fe²⁺ subsequently coordinated with ZOL inside tumor cells to form nanoparticles.¹¹⁹ These nanoparticles exhibited peroxidase mimicking activity and facilitated a continuous catalytic reaction of intratumoral hydrogen peroxide by generating free hydroxyl radicals to induce oxidative stress.¹¹⁹ Experiments revealed that the combination approach significantly elevated tumor cell apoptosis in CT26 and enhanced expression level of γ-H2AX (which is a marker of DNA damage), and exhibited negligible systemic toxicity.¹¹⁹

Synergistic Enhancement of Immunomodulation and Ferroptosis

The combination of S. Typhimurium and ferroptosis goes beyond simple direct killing, and its strength lies in triggering profound immune synergy. The composite system constructed by nanobiotechnology makes this synergistic effect more precise and durable. On the one hand, ferroptotic cells release damage-associated molecular patterns such as calreticulin (CALR) and high‑mobility group box 1 (HMGB1), initiating immunogenic signals. On the other hand, Salmonella’s pathogen-associated molecular patterns, the LPS, highly stimulate innate immunity via receptors such as TLR4.

For the FeAMV system, this combination effect appeared as follows: ferroptosis-related signaling and bacterial immune stimulation synergistically promoted the polarization of tumor-associated macrophages from the immunosuppressive M2 type toward the antitumor M1 type and elevated pro‑inflammatory cytokines.¹¹⁸ Such immune activation may further activate the NLRP3 inflammasome, promote the expression of pro‑inflammatory factor IL‑1β, and increase intratumoral CD8⁺ T‑cell infiltration as well as IFN‑γ secretion, effectively reversing the immunosuppression state.

Tumor cell lysis caused by a combination of VNP20009 and ZOL can trigger release of antigens from tumor cells. These tumor antigens, together with the immunoadjuvant effect of VNP20009, could promote dendritic cell uptake and presentation of tumor antigens. TUNEL assays demonstrated that tumor cell apoptosis in the combination group was elevated relative to the ZOL alone group.¹¹⁹ CD8⁺ T cell proliferative activity in tumor-draining lymph nodes was enhanced, and mice developed immune memory, showing prolonged growth delay upon rechallenge with homologous tumor cells. Additionally, the local inflammatory response triggered by Salmonella colonization within tumors may increase the concentration of hydrogen peroxide in the TME, providing ample substrate for Fe²⁺-mediated Fenton reactions to amplify oxidative‑stress‑driven cytotoxicity and establish a positive feedback cycle.¹¹⁹

Synergistic Combination of Salmonella OMVs with Sonodynamic Therapy

Sonodynamic therapy (SDT) has the advantages of deep tissue penetration and minimal invasiveness and achieves tumor killing by exciting sonosensitizers to generate reactive oxygen species through ultrasound. However, its single-agent application has problems such as insufficient targeting of sonosensitizers and weak immune activation effect. Salmonella outer membrane vesicles (OMVs) are natural biological nanocarriers with both tumor-targeting and adjuvant activities. Nanobiotechnology builds an efficient bridge for the combination of OMVs and SDT. By integrating sonosensitizers with OMVs through nanocoating, hybrid membrane construction, and other methods, it can form efficient synergy with SDT, not only improving the enrichment of sonosensitizers and ROS production but also amplifying the immunogenic cell death effect, providing an effective path for breaking through the bottleneck of SDT efficacy.

Zhang et al designed the composite nanosystem TMRC@VPm based on hybrid membranes (VPm) constructed by the fusion of OMVs derived from attenuated S. Typhimurium VNP20009 with platelet membranes at a 2:1 protein mass ratio.¹²⁰ The mesoporous organosilicon nanoparticles possess sonosensitizer TCPP covalently hybridized within the framework and are loaded with siSLC4A4.¹²⁰ OMVs confer immune-activating functions to the system, while the platelet membranes provide tumor‑targeting, prolonged circulation stability and circulating tumor cell (CTC) capture ability. Intravenously injected, the system can easily be enriched in pancreatic cancer tissue through CD44-mediated targeting and exhibits superior tumor accumulation compared with the group without VPm hybrid‑membrane modification.¹²⁰ Under ultrasound excitation, TCPP produces abundant ROS. Together with siSLC4A4 alleviating TME acidosis, TMRC@VPm + US elicits robust tumor‑cell apoptosis in vitro.¹²⁰ OMV-loaded PAMPs synergize with SDT-induced DAMPs to promote dendritic cell maturation; M2-to-M1 macrophage polarization is induced, accompanied by marked expansion of splenic CD8⁺ T‑cell populations.¹²⁰ This system ultimately achieves prominent tumor regression, effectively suppressing lung metastasis and greatly prolonging the survival of tumor‑bearing mice, and shows no obvious pathological damage to major organs including liver and kidney.¹²⁰

Multimodal Combination Strategies

The multifaceted antitumor activity observed with combination therapies mentioned above has greatly improved the complementary functional effect to provide effective support in clinical treatment. In contrast, under the complex challenges of physical barriers and immunosuppressive microenvironment, and distant metastasis, the system still experienced limitations such as partial clearance of some lesions, residual drug resistance, as well as inadequate deep penetration. Moreover, combination therapies involving three or more therapies to further integrate the core functions such as targeted delivery, local killing, immune stimulation, and microenvironment modulation can give more holistic synergistic effects, which has now become the supplementary direction of breaking through the aforementioned restrictions.

The philosophical principle behind multimodal combination is “functional superposition and weakness compensation”. Using Salmonella as a core carrier, various means including nanotechnology, chemo/radiotherapy, and immune modulation can be integrated into it to still retain this bacterial tumor-targeting colonization and immune activation advantages while combining with other therapies to strengthen local tumor-killing effectiveness and enhance the effect of delivery.

Similarly, based on combining bacteria with radiotherapy and immune checkpoint inhibitors, introducing nanozyme modification and low‑dose X-ray pretreatment could be utilized to enhance radio-immuno synergistic effects of nanozymes relieving tumor hypoxia and X-rays reinforcement bacterial enrichment, while addressing at the same time the clearing of metastatic foci.³⁹ By integrating bacterial biomimetic delivery systems, the combination of photothermal therapy and chemotherapy could benefit the penetrability advantages of biomimetic carriers to compensate for the tough problems of the depth of chemo.¹²¹

It can be inferred from these multimodal combinations to systematically integrate the core advantages of different therapies and achieve targeting resolutions of the foregoing restrictions to accomplish not only better primary tumor inhibition but also reinforcing immune memory and inhibiting distant metastasis to provide the treatment plan.

Challenges and Future Perspectives

Challenges

Despite the remarkable antitumor potential of engineered S. Typhimurium validated in preclinical studies via synthetic biology and nanobiotechnology modifications, its translation from laboratory research to widespread clinical application remains confronted with substantial challenges.

Underlying Limitations of Engineered Strain Optimization

Previous sections have systematically summarized Salmonella modification strategies from interdisciplinary engineering perspectives including synthetic biology and nanobiotechnology, as well as combinatorial therapeutic regimens. Nevertheless, the intrinsic drawbacks of bacterial strains have not been fully resolved, and the core contradiction between therapeutic efficacy and biosafety remains prominent. Fundamentally, current engineering strategies suffer from fragmented design frameworks: these approaches only conduct partial optimization targeting individual functions, while integrated and synergistic design logic for multi-modular systems remains absent.

At the molecular level, the vast majority of therapeutic payloads and lysis-controllable circuits are expressed via plasmid vectors to date. Under the dual selective pressure of rapid intratumoral proliferation and host immune surveillance, plasmid loss and spontaneous mutations occur frequently.¹²² Current regulatory elements merely respond to single stimuli such as hypoxia or acidic pH, lacking multi-signal cascade regulation that enables dynamic modulation of bacterial proliferation and drug release in accordance with tumor regression extent and systemic inflammatory status.⁸⁸ Simultaneous co-expression of diverse functional components for attenuation, tumor targeting and tumor cytotoxicity triggers metabolic competition and signal crosstalk, which prevents all functional modules from reaching optimal performance synchronously.¹²³

From the in vivo functional perspective, existing engineered strains only passively adapt to static tumor microenvironmental features including hypoxia and acidity, yet fail to actively penetrate multiple physical and nutritional barriers constituted by dense extracellular matrix, neutrophil extracellular traps (NETs), and intratumoral microbiota.⁷⁰ Furthermore, substantial interpatient tumor heterogeneity leads to dramatic individual variability in bacterial tumor colonization efficiency.⁶⁸ Even when effective intratumoral enrichment is achieved, continuous secretion of PAMPs by engineered bacteria persistently drives massive infiltration of Tregs and MDSCs, inducing T‑cell exhaustion and establishing a negative immunosuppressive feedback loop that impairs durable antitumor immune responses.⁴⁷

In terms of multimodal combinatorial therapies, current regimens mostly rely on simple physical combination without synergistic design at the molecular signaling pathway level. Inflammation triggered by bacteria may synergize with radiotherapy/chemotherapy and accumulated nanomaterials, exacerbating systemic organ toxicities.³⁷

The core bottleneck lies in the long-term segregated advancement of distinct research branches including targeted engineering, lysis regulation, immunotherapeutic payload delivery, nanocoating modification, and combinatorial treatment. Few systematic studies have verified potential functional antagonism or offset effects after multi-module assembly. Critical scientific questions, such as whether stacked multi-layer modifications result in metabolic overload, impaired colonization capacity, and amplified toxic side effects, still lack comprehensive validation frameworks. Even with multi-layer synthetic biology and nanocoating modification, these interventions only partially alleviate rather than fully eliminate the intrinsic risks of inflammatory toxicity, intratumoral bacterial overgrowth, and incomplete bacterial clearance, which remain unresolvable core barriers for clinical translation.

Core Translational Bottleneck

Disparities Between Murine Preclinical Models and Human Patients

Genetically attenuated Salmonella strains consistently achieve tumor-to-normal tissue CFU ratios exceeding 1000:1 in immunodeficient xenograft/orthotopic mouse models, yet this robust tumor tropism cannot be recapitulated in human clinical trials. This translational gap arises from fundamental interspecies differences in tumor physical architecture and host innate/adaptive immunity. Murine xenografts feature loose stroma and highly permeable vasculature, whereas human solid tumors develop dense fibrotic matrices that block bacterial extravasation. In addition, human hepatic and splenic reticuloendothelial systems, complement cascades, and neutrophil populations exert far stronger bacterial clearance activity compared to athymic nude mice lacking functional T-cell immunity. Consequently, intravenously administered Salmonella is rapidly eliminated from human peripheral circulation, limiting the quantity of bacteria that successfully infiltrate and persist within tumor parenchyma.

The landmark phase I trial of the backbone strain VNP20009 clearly demonstrates this translational failure: only 12% of enrolled patients presented detectable intratumoral bacterial colonies post-infusion, and no objective tumor regression was observed across all subjects.⁶⁸ A systematic summary of human clinical data for leading engineered Salmonella therapeutics is provided in Table 5 to quantitatively illustrate preclinical-to-clinical efficacy divergence. Such robust immune clearance in human patients also leads to uncontrollable bacterial loss, making precise systemic dose adjustment impossible to date.

Table 5.

Clinical Studies of Salmonella-Based Cancer Therapeutics

Strain	Genetic Modification	Administration Route	Trial Phase	Indications	MTD	DLT & Safety Profile	Tumor Colonization & Clinical Outcomes	References
VNP20009 (parental vector)	ΔpurI, ΔmsbB, empty backbone without effector gene	IV	Phase I	Metastatic melanoma, renal cell carcinoma	3 × 108 CFU/m2; 1×109 CFU/m2 triggers DLT	High-dose events: persistent bacteremia, thrombocytopenia, anemia, hyperbilirubinemia, severe gastrointestinal adverse events, hypotension	Only 3 out of 25 patients (12%) of patients had detectable intratumoral bacteria; ORR = 0, no tumor shrinkage observed	[68]
Salmonella-IL2 (Saltikva)	Δcya/Δcrp/Δasd, plasmid encoding truncated human IL-2	PO	Phase I (monotherapy) Phase II (combined with FFX)	Phase I: metastatic gastrointestinal cancers Phase II: stage IV pancreatic adenocarcinoma	MTD not reached (tested up to 1010 CFU in Phase I; 109 CFU used in Phase II)	No strain-related severe adverse events; all 41 serious adverse events attributed to FFX chemotherapy	No biopsy-confirmed intratumoral colonization data; single-agent ORR = 0; ≥5 doses + FFX yielded ORR = 70%, mPFS = 15 months, mOS = 20.3 months	[124,125]
SGN1	VNP20009 backbone overexpressing L-methioninase (METase)	IV / intratumoral injection	Phase I/II ongoing (NCT05103345, NCT05038150)	Broad advanced solid tumors		In pre‑clinical murine models: no severe hematological or organ‑related toxicities observed	Selective tumor enrichment and methionine depletion in murine xenograft and human PDO models; human full-scale ORR and intratumoral CFU data unpublished	[126]

Abbreviations: MTD, maximum tolerated dose; DLT, dose-limiting toxicity; ORR, objective response rate; CFU, colony-forming unit; IV, intravenous; PO, oral; OS, overall survival; PFS, progression-free survival; FFX, FOLFIRINOX; PDO, patient-derived organoid.

Restricted Applicability of Administration Routes

The three dominant administration routes for engineered Salmonella are all hindered by intractable physiological delivery barriers, and no delivery regimen is available to simultaneously target systemic metastatic lesions and deep-seated tumors while maintaining low systemic toxicity.¹²⁷

Intravenous injection enables systemic delivery, yet circulating bacteria are rapidly cleared by the liver and spleen, resulting in extremely low intratumoral accumulation efficiency. Continuous stimulation of TLR inflammatory signaling induced by bacterial lipopolysaccharide and flagellin means high doses readily trigger hyperthermia, cytokine storm, or even bacteremia.⁹ Although oral administration exhibits superior patient compliance, most bacteria are inactivated by gastric acid, bile acids, and intestinal commensal microbiota; this route only acts on primary gastrointestinal tumors and fails to target distal lesions in organs such as the liver and lung. Intratumoral injection achieves the highest bacterial colonization efficiency with minimal systemic adverse effects, but this procedure is only applicable to superficial, isolated solid tumors. It cannot be deployed for deep visceral primary tumors, diffuse peritoneal metastases, or disseminated microlesions throughout the body and merely serves as a local palliative intervention. All administration routes are accompanied by unavoidable off-target colonization risks and poor controllability of in vivo bacterial loads. To a certain extent, administration routes limit the spectrum of cancer types and eligible clinical populations suitable for engineered Salmonella therapy.

Challenges in Standardizing Pharmacokinetics of Live Bacterial Therapeutics

Engineered Salmonella Typhimurium represents self-replicating live biotherapeutic products (LBPs), which differ fundamentally from static agents including small-molecule drugs and monoclonal antibodies (mAbs). Conventional pharmacokinetic (PK) analytical models are entirely incompatible with their dynamic in vivo behaviors.¹²⁸ Administration dosage presents a prominent safety-efficacy trade-off: high bacterial doses tend to cause ectopic colonization in the liver and spleen, and massive release of pathogen-associated molecular patterns drives systemic inflammation, with drastically elevated risks in immunocompromised patients. By contrast, low-dose bacteria are swiftly eliminated by host immunity, leaving intratumoral viable bacterial loads below the therapeutic threshold.⁶⁸ For intravenous delivery, higher initial bacterial concentrations are required to offset hepatic and splenic clearance losses, which further narrows the therapeutic window; no linear correlation exists between administered dosage and tumor-suppressive efficacy.

In parallel, patient baseline immune status, intratumoral hypoxic and lactic acid microenvironment, as well as genetically engineered modules integrated into bacterial strains profoundly alter the colonization and proliferation kinetics of Salmonella. Standardization will require quantitative assays for viable bacteria, serial biodistribution and shedding measurements, and prespecified rules for dose adjustment and antimicrobial rescue. No universal evaluation metrics for blood bacterial concentration or half-life have been established. At present, clinically translatable real-time tracking techniques for intratumoral viable bacteria remain absent, and mature protocols for individualized dosage adjustment are lacking. Collectively, the narrow therapeutic window and non-uniform live-bacteria pharmacokinetic characteristics impede the standardized clinical translation of this therapeutic modality.¹²⁹ This extremely narrow therapeutic window creates persistent conflicts between anti-tumor efficacy and systemic toxic reactions without standardized solving strategies.

Challenges in Industrial Manufacturing

The core bottleneck in chemistry, manufacturing and controls (CMC) quality assurance lies in the genetic stability of engineered plasmids, which represents the most prominent obstacle to large-scale production of live bacterial therapeutics. Under antibiotic-free fermentation procedures as well as dual selective pressures imposed by intratumoral and intestinal microenvironments in vivo, engineered Salmonella strains are highly susceptible to the loss of therapeutic plasmids encoding oncolytic proteins and immunomodulatory cytokines, which directly abrogates their antitumor functions.¹³⁰ Furthermore, horizontal gene transfer can occur between engineered strains and indigenous human gut commensal bacteria. The dissemination of antibiotic resistance and virulence genes introduces dual biosafety hazards, including invasive human infections and environmental biocontamination.

In addition, formulation storage and transportation constitute major barriers to commercial translation. Current cryopreservation and cold-chain logistics strategies fail to sustain long-term retention of bacterial viability and tumor colonization capacity, leading to pronounced attenuation of therapeutic potency after prolonged storage. Modification via calcium carbonate or manganese dioxide nano-biomineralization suffers from poor process controllability and severe inter-batch variability of finished formulations. At present, the industry lacks unified CMC quality control specifications tailored to live bacteria and bacteria-nano hybrid formulations, hindering consistent large-scale industrial production.³⁷

Existing in vitro evaluation platforms exhibit limited predictive capacity. Conventional two-dimensional (2D) cell culture models fail to recapitulate the intact tumor microenvironment.¹³¹ Patient-derived organoids and microfluidic chips are only applicable to small-scale laboratory screening, while positron emission tomography (PET) and near-infrared (NIR) tracing techniques cannot achieve quantitative in vivo measurement of intratumoral bacterial colonization in human subjects.¹²⁹ Moreover, globally unified specialized regulatory frameworks and long-term toxicological assessment criteria for genetically engineered live bacterial agents remain absent. Without standardized workflows to assess diverse biological risks, the clinical translation of this therapeutic modality is substantially delayed. Long-term biosafety risks including horizontal gene transfer cannot be fully excluded under current production systems.

Future Perspectives

Future research on engineered S. Typhimurium tumor therapeutics will advance along four interconnected technical and translational routes.

First, integrated multiplex genetic circuits will replace scattered plasmid modules. Chromosomal fixed integration, toxin-antitoxin, and redundant dual-inducible promoters (hypoxia+lactic acid) will mitigate plasmid loss and in vivo functional drift, enabling synchronized lysis and sequential payload release to balance killing efficacy and biosafety.¹³⁰ Orthogonal quorum-sensing systems will be constructed to realize multi-strain synthetic microbial consortia that jointly remodel immunosuppressive tumor microenvironments.⁸⁸ Nevertheless, such integrated circuits cannot completely avoid inflammatory toxicity and bacterial clearance issues in human bodies.

Second, nanobiology combined with physical exogenous control will become a core precision regulation tool. Biomineralized CaCO₃/MnO₂ coatings and macrophage biomimetic cloaks will prolong blood circulation while responding to acidic tumors for on-demand deprotection; near-infrared/ultrasound optogenetic switches will achieve non-invasive spatiotemporal activation of cytotoxic and immune effector genes, avoiding uncontrolled bacterial overproliferation.⁴³ But these nanocloaking and optogenetic tools only mitigate partial safety risks rather than fundamentally resolve dose control limitations.

Third, patient-derived organoids coupled with microfluidic chips will serve as standardized pre-screening platforms, narrowing the translational gap between mouse models and human patients, and rapidly screen personalized engineered strains to overcome interindividual tumor heterogeneity.¹³¹ Multimodal synergistic regimens combining bacterial vectors with chemo-, radio-, photothermal, and immune checkpoint blockade will be systematically optimized to eliminate deep and metastatic lesions.⁹² Even personalized screening platforms cannot bridge the interspecies gap and guarantee stable intratumoral bacterial colonization in patients.

Fourth, standardized CMC production pipelines and exclusive regulatory evaluation criteria for live biotherapeutics need to be established. Long-term, traceable in vivo PET/NIR imaging technology will be developed to dynamically monitor intratumoral bacterial colonization and predict therapeutic response, requiring massive clinical verification before any clinical popularization.¹²⁹ Ultimately, modular programmable S. Typhimurium may serve as potential preclinical research platforms into a personalized precision therapy complementary to traditional tumor treatments. But standardized production and tracing technology alone cannot address unresolved toxicity and immune clearance barriers to establish reliable clinical efficacy.

Most engineering and combination strategies discussed above remain at the in vitro or animal-study stage, whereas human evidence is limited to a small number of early-phase studies and trial records. Consistent, durable anti-tumor efficacy of engineered S. Typhimurium has not been validated in human clinical trials, and unresolved toxicity, bacterial clearance, dose regulation, and regulatory hurdles will continue to restrict its clinical transformation in the long term.

Funding Statement

This work was supported by the National Natural Science Foundation of China (No. 82472515); the Basic Research Program of Jiangsu Province (No. BK20250121); and the Suzhou Youth Program for Applied Basic Research (No. SYW2024171).

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Disclosure

The authors declare that they have no competing interests.