Leveraging the bacteria for enhanced cancer immunotherapy: from a perspective of synthetic biology.

Introduction

Cancer remains a major global health challenge, with malignancies such as lung, gastric, and hepatocellular carcinoma imposing significant clinical burdens. 1 Although conventional treatments, including surgery, radiotherapy, and cytotoxic chemotherapy, have demonstrated efficacy, their severe side effects often compromise patients’ quality of life. 2 Therefore, the development of safer and more targeted therapeutic strategies is urgently needed. Among emerging approaches, cancer immunotherapy—particularly chimeric antigen receptor T-cell (CAR-T) therapy—has shown transformative potential by achieving remarkable efficacy in refractory malignancies. 3 However, tumor immune evasion mechanisms—particularly programmed death-ligand 1 (PD-L1) expression—continue to limit treatment efficacy, underscoring the need for innovative therapeutic strategies. 4 Recent advances suggest that cytokine modulation and immune checkpoint blockade represent promising approaches to overcome these challenges. 5

Notably, a deeper understanding of the tumor microenvironment (TME) has reinforced bacterial immunotherapy as a promising therapeutic strategy. Compared with conventional treatments, this approach offers superior tumor specificity and reduced systemic toxicity. Its conceptual foundation originates from early observations and has since been refined through advances in microbiology and genetic engineering. Over time, bacterial anticancer strategies have progressed from direct bacterial administration (e.g., Coley’s toxins and the therapeutic use of Bacillus Calmette-Guérin (BCG)) to the precise modulation of intratumoral microbiota using synthetic biology (exemplified by engineered strains such as Salmonella VNP20009 and Clostridium novyi -NT). 6-11 Indeed, synthetic biology revolutionizes anticancer therapeutics through the engineering of bacterial vectors to augment specific functions, including targeted payload delivery and immune activation. 12-15 Engineered bacterial systems have been shown to produce antineoplastic agents in situ, act as biosensors, and serve as targeted delivery platforms. 16 To illustrate this evolutionary path, Figure 1 provides a chronological overview of pivotal milestones, from empirical beginnings to contemporary engineered approaches, in the development of bacterial immunotherapy.

Specifically, this review elucidates: (i) synthetic biological strategies to enhance bacterial targeting and immunomodulation within the TME; (ii) mechanistic insights into innate and adaptive immune activation including antigen presentation pathways; (iii) therapeutic applications of bacteria-nanomaterial conjugates for precision therapy; and (iv) synergistic potential with photodynamic therapy (PDT), photothermal therapy (PTT), and radiotherapy. Collectively, the integration of synthetic biology with bacterial immunotherapy represents a paradigm-shifting strategy in oncological treatment, potentially transforming the therapeutic landscape against this pervasive disease.

Effects of bacterial therapeutic agents on immune system

This section focuses on the fundamental immunological mechanisms by which bacterial agents activate host antitumor immunity, providing the mechanistic rationale for the therapeutic strategies detailed in Chapter 4. We first dissect the innate immune pathways triggered by bacterial pathogen-associated molecular patterns and then analyze the subsequent engagement of adaptive immunity, laying the groundwork for understanding their translational applications.

Bacterial PAMPs as activators of antitumor innate immunity

Bacterial immunotherapy exerts its antitumor effects primarily by activating the innate immune system through pathogen-associated molecular patterns (PAMPs). These microbial structures, known as PAMPs, are recognized by specific pattern-recognition receptors (PRRs) on or in immune cells. This recognition initiates downstream signaling pathways that trigger innate immune activation and shape adaptive immunity. Key bacterial PAMPs and their receptors include: lipopolysaccharide (LPS), which is recognized by Toll-like receptor 4 (TLR4) 17 ; flagellin, detected by TLR5 18 ; unmethylated CpG-rich bacterial DNA, a ligand for TLR9 19 ; and peptidoglycan fragments, which activate intracellular nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD1/2) receptors. 20 Initial PAMP recognition by PRRs, including TLRs, triggers an immunostimulatory cascade that activates immune cells and induces inflammatory mediator production. 21 Subsequent studies have shown that PRR engagement recruits adapter proteins such as myeloid differentiation primary response 88 (MyD88) or TIR-domain-containing adapter-inducing interferon- β (TRIF) via TIR domain interactions, forming signaling complexes that initiate downstream pathways. MyD88-dependent signaling activates the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) cascades, driving the expression of pro-inflammatory cytokines, including tumor necrosis factor- α (TNF- α ), interleukin-6 (IL-6), and IL-12. These cytokines activate innate effector cells such as natural killer (NK) cells and macrophages, regulate immune cell recruitment, and amplify inflammatory responses. In contrast, TRIF-dependent signaling induces type I interferon (IFN) production, which directly enhances innate antitumor immunity by augmenting NK cell cytotoxicity and promoting dendritic cell maturation. 22 Bacterial immunotherapeutic agents serve as potent PAMP delivery systems; a notable example is Mycobacterium bovis BCG, a strong TLR2/4/9 agonist widely used in bladder cancer therapy. 23 A key outcome of PRR activation is the maturation and migration of dendritic cells (DCs), which significantly improves their ability to secrete immunostimulatory cytokines and activate innate lymphocytes such as NK cells, thereby potentiating early innate antitumor responses. 24 , 25 Collectively, these mechanisms not only amplify innate immune responses but also provide essential signals for initiating adaptive immunity.

Harnessing adaptive immunity for antitumor responses

This section aims to delineate the mechanisms by which bacterial immunotherapy mobilizes the adaptive immune system to achieve targeted and durable anticancer effects. The discussion is structured around three key processes: antigen-specific T cell activation, immune memory formation, and the strategic use of combination therapies to amplify these effects. These mechanistic principles are later manifested in specific therapeutic designs, such as engineering bacteria to deliver tumor-associated antigens or immunomodulatory fusion proteins, as will be explored in Chapter 4.

Beyond activating innate immunity, bacterial immunotherapeutic agents also engage adaptive responses to achieve tumor-specific effects. A key event in this process is the activation of DCs, which bridge innate and adaptive immunity. Upon encountering bacteria, DCs present antigens on major histocompatibility complex (MHC) molecules to T cells. These antigens include tumor-associated or tumor-specific antigens expressed by malignant cells, as well as bacterial antigens. Potent bacterial PAMPs such as flagellin (FlaB) serve as potent immune stimulants. For instance, flagellin signaling through TLR5 promotes dendritic cell maturation and shapes the adaptive immune response, with the Th polarization direction being context-dependent and influenced by the local cytokine milieu. This activation of antigen-presenting cells (APCs) leads to the upregulation of co-stimulatory molecules and enhanced antigen presentation. Consequently, this elicits robust T-cell responses directed against the bacterial vector. Through mechanisms such as epitope spreading or bystander effects, this activated immune state can be redirected against the tumor, linking bacterial immune stimulation to antitumor immunity. 24 To exploit this, engineered bacteria are often designed to deliver defined tumor antigens. A notable example is attenuated Listeria monocytogenes expressing the human papillomavirus (HPV) E7 protein for cervical cancer therapy. 26 This strategy activates cytotoxic T lymphocytes (CTLs), enabling tumor cell recognition and elimination. 27 Consequently, the degree of CTL infiltration within tumors is considered a strong prognostic indicator.

Bacterial agents also modulate helper T-cell subsets, notably Th1 and T helper 17 (Th17) cells, inducing the secretion of cytokines such as IFN- γ and IL-17, which enhance inflammatory responses and immune surveillance. 28 Among these, IFN- γ plays a pivotal role by promoting antigen presentation and upregulating MHC expression on tumor cells, thereby exposing additional tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs) for CTL recognition. 29 Furthermore, bacterial immunotherapy drives the formation of immune memory, providing rapid and durable protection against tumor recurrence. To amplify these adaptive immune responses, combination strategies are actively explored. These include synthetic TLR agonizts (e.g., monophosphoryl lipid A [TLR4], CpG oligodeoxynucleotides [TLR9]) and stimulator of interferon genes (STING) agonizts (e.g., cGAMP derivatives, DMXAA analogs), which synergize with bacterial PAMPs to amplify DC activation, cytokine production, and T-cell priming. 30 , 31 For instance, certain intracellular bacteria such as Listeria monocytogenes naturally produce cyclic di-AMP (c-di-AMP), a potent STING agonist that cooperates with bacterial PAMPs to enhance type I interferon production and antitumor T-cell responses. 32 Beyond these direct PAMP-receptor interactions, bacterial components can also reshape the immunological set point of the TME to potentiate adaptive immunity. For example, gut-derived LPS remodels the pancreatic tumor microenvironment by stimulating T cell infiltration via the TLR4/MyD88/AKT/NF-κB signaling cascade. This LPS-driven pathway transcriptionally upregulates PD-L1 expression on tumor cells, thereby creating a condition in which PD-L1 checkpoint blockade synergizes with the remodeled immune context to suppress tumor growth. 33

In summary, bacterial immunotherapy activates multiple adaptive immune components through a multi-tiered mechanism ( Figure 2 ). This strategy leverages both the intrinsic immunogenicity of bacteria and the synergistic potential of immunotherapeutic agents and small-molecule modulators, forming a robust conceptual foundation for next-generation cancer immunotherapy.

The production of bacteria-based therapeutic agents

The development of contemporary bacterial immunotherapeutics from discovery to clinical application follows a defined translational pipeline, as illustrated in Figure 3 . This multistage workflow encompasses the rational selection and engineering of safe, tumor-targeting bacterial chassis, rigorous preclinical and clinical validation, and ultimately, scalable current Good Manufacturing Practice (cGMP) production.

The ideal bacterial chassis must exhibit low pathogenicity, a favorable safety profile, and intrinsic tumor tropism. Common candidates include attenuated strains of Salmonella spp. , Escherichia coli , and Listeria monocytogenes. 24 , 34 , 35 Their genetic engineering relies on versatile vector systems. Beyond synthetic plasmids, the strategic engineering of a bacterium’s native (cryptic) plasmids offers advantages in host compatibility and stable, antibiotic-free maintenance. For stable genomic integration, plasmid-based homologous recombination remains a fundamental strategy.

Precise genetic modifications employ both traditional and advanced genome-editing technologies. While the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is widely used for targeted gene knockout, the CRISPR/Cas12a platform has emerged as a potent alternative for multiplexed editing. Cas12a’s ability to process its own guide RNA arrays facilitates simultaneous regulation of multiple targets. For instance, the dCas12a-based “MultiduBE” system enables multiplexed base editing to drive complex metabolic reprogramming in bacteria. 36 Concurrently, optimizing transgene expression is critical. Codon optimization has evolved into sophisticated strategies that balance codon preference with translation kinetics and protein folding. Advanced deep learning models now intelligently design coding sequences to maximize heterologous protein expression in bacterial chassis like E. coli , significantly improving therapeutic protein yield. 37

Engineered strains undergo purification to confirm genotypic stability and phenotypic consistency. 38 Concurrently, genotypic verification is performed using PCR or Sanger sequencing. 39 Phenotypic attributes—including antibiotic susceptibility, growth kinetics, and therapeutic protein expression—are characterized via disc diffusion assays, ELIZA, or immunoblotting. To improve bacterial stability and functionality, nanoparticle encapsulation strategies have been explored. For instance, liposomal coatings enhance resistance to gastric acids, while gold nanoparticle conjugation improves photoacoustic imaging and bactericidal properties. 40

Upon successful preclinical validation, therapeutic batches are produced to meet clinical demand. This bioprocessing pipeline includes bioreactor cultivation, biomass harvesting, purification, and final formulation. Techniques such as hollow-fiber bioreactors (HFBs), continuous centrifugation, tangential flow filtration, and affinity chromatography are implemented under Good Manufacturing Practice (GMP) standards. 41 Subsequently, phased clinical trials—from preclinical studies through phase III—are conducted to assess pharmacokinetics, safety, and efficacy, representing a critical step toward clinical translation. 42 After regulatory approval, manufacturing processes are iteratively refined to increase yield, reduce production costs, enhance product quality, and explore novel therapeutic applications.

Advances in synthetic biology and nanotechnology have transformed the landscape of scalable bacterial production, enabling innovative strategies for next-generation immunotherapies.

Strategies of bacteria-based cancer immunotherapy

Building upon the fundamental immunological mechanisms described in Chapter 2, this chapter explores their translational application. We categorize the diverse bacteria-based anticancer strategies into distinct therapeutic paradigms and detail their design, delivery, and clinical potential.

Bacteria-based anticancer strategies can be broadly categorized into two distinct paradigms based on their mode of application: direct and indirect use. Direct strategies employ live bacteria—either wild-type (WT) or engineered—as intratumoral agents that locally deliver therapeutics, induce oncolysis, or modulate the TME. Indirect strategies utilize bacterial components (e.g., outer membrane vesicles, attenuated strains) or bioengineered bacteria as platforms for producing vaccines, viral vectors, or immunostimulatory molecules ex vivo, which are then administered separately. The following sections delineate these strategies, with their key features summarized in Figure 4 .

Engineered tumor tropism via synthetic biology

As a direct application strategy, bacterial tumor tropism can be classified into two categories: non-engineered (native) tropism and engineered tropism. Non-engineered tropism relies on the inherent biological properties of certain bacterial species—such as the facultative anaerobic metabolism of Escherichia coli and Salmonella spp. that drives colonization of hypoxic tumor cores, or native chemotaxis toward tumor-derived metabolites. In contrast, synthetically engineered bacteria exhibit enhanced or entirely novel tumor tropism achieved through genetic modification, enabling targeted delivery of immunotherapeutic agents in response to programmed chemotactic cues within the TME. This selective targeting reduces off-tumor cytotoxicity. Below, we first discuss native tropism mechanisms shared by many bacterial chassis, then detail how synthetic biology further augments these capabilities.

Bacterial accumulation in tumors occurs via both passive and active mechanisms. Passive targeting is mediated by the enhanced permeability and retention (EPR) effect in leaky tumor vasculature, whereas active tropism is driven by engineered or native responses to hypoxia, necrosis, and metabolic gradients, as well as engineered sensing circuits. 43 Importantly, the role of native tumor-associated bacteria (TAB) within tumors is complex and context-dependent. Certain species, such as Fusobacterium nucleatum in colorectal cancer and Pseudomonas aeruginosa in lung cancer, have been shown to actively promote tumor progression. They achieve this through mechanisms like forming protective biofilms, secreting metabolites (e.g., iron-scavenging pyoverdine) that shield tumor cells from ferroptosis, and inducing epithelial-mesenchymal transition. 44 This dual nature—being both a component of the TME and a potential driver of malignancy—highlights the significance of understanding native bacteriotropism.

While the above mechanisms represent inherent bacterial properties, synthetic biology enables the rational design and augmentation of tumor tropism. Synthetic biology enables the design of bacteria that respond to TME-specific signals, including acidic pH and tumor-derived metabolites. Hypoxia, a hallmark of the TME, drives metabolic reprogramming, invasion, metastasis, radioresistance, and immune evasion by reducing tumor immunogenicity. 45 While facultative anaerobes such as Escherichia coli Nissle 1917 (EcN) and Salmonella spp. naturally colonize hypoxic tumor regions via their inherent metabolic versatility, synthetic engineering efforts have primarily focused on rewiring bacteria to sense and respond to other TME cues that are not naturally exploited. Furthermore, engineering often co-opts native TAB strategies. For instance, bacteria can be designed to sense and respond to the acidic, lactate-rich TME, restricting therapeutic gene expression to tumors for enhanced specificity. 46 Bacterial translocation into tumor tissue is further facilitated by motility systems, including flagella and type IV pili, as well as chemoreceptor-mediated taxis.

Genetic engineering can further enhance tumor tropism by endowing bacteria with synthetic adhesion properties. Piñero-Lambea et al. developed a modular synthetic adhesin (SA) platform in which the immunoglobulin-like domains of the native Intimin adhesin from enteropathogenic Escherichia coli were replaced with single-domain nanobodies (VHHs). By selecting nanobodies that recognize tumor-associated antigens such as HER2, the engineered bacteria acquired de novo tumor-specific adhesion and colonized solid tumors expressing the cognate antigen with significantly greater efficiency than their non-adherent counterparts. 47 Complementing adhesion-based targeting, bacteria can also be equipped with engineered sensing systems to detect metabolic gradients within solid tumors. Panteli and Forbes demonstrated that E. coli engineered to express a glucose-sensing chemotaxis receptor (Trz1) can detect spatial profiles in glucose concentration within solid tumor cell masses, enabling the bacteria to actively track sugar gradients generated by metabolically heterogeneous tumor regions—a capability that passive diffusion alone cannot achieve. 48 Additionally, tumor cells undergo aerobic glycolysis, producing lactate that acidifies the TME. This acidic environment, potentiated by histone lysine lactylation (Kla), promotes tumor progression. 49 Acid-responsive bacterial systems have therefore been engineered to secrete enzyme inhibitors or lysins, modulating TME biochemistry and impairing tumor viability. 50

Beyond environmental responsiveness, bacterial swarming behavior (BSB), exemplified by Pseudomonas aeruginosa , provides a motility mechanism for deep tumor penetration. During swarming, P. aeruginosa forms multicellular rafts that migrate rapidly across surfaces via flagellar propulsion, generating complex vortical patterns. 51 In cancer applications, this native swarming capability has been harnessed by engineered Salmonella flagellar systems to enhance intratumoral infiltration and replication, creating novel therapeutic opportunities. 52 Clinical translation, however, requires a detailed understanding of swarming dynamics, including fission–fusion kinetics during collective migration. To this end, chemotactic behaviors are being reprogrammed to optimize tumor-directed migration, while small-molecule inhibitors of flagellin biosynthesis or c-di-GMP signaling are employed to spatially constrain bacterial dissemination. 53

Overall, the interplay between native bacterial tropism and synthetic reengineering—from exploiting inherent anaerobic metabolism to programming novel sensing circuits and swarming mechanisms—enables precise therapeutic delivery while minimizing collateral tissue damage.

Targeted cancer therapies: bacterial conjugates and EVs

An alternative strategy for tumor-specific delivery employs bacteria–nanoparticle conjugates and bacterial extracellular vesicles (BEVs) as engineered platforms. These systems synergize with tropic bacteria to enhance targeting precision and primarily represent an indirect application paradigm, wherein bacterial components or engineered vectors are produced ex vivo and administered, rather than relying on intratumoral bacterial colonization.

Bacteria–nanoparticle conjugates integrate principles of synthetic biology and nanomedicine, leveraging bacterial tropism for site-specific nanocarrier deposition. Nanoparticle (NP) properties—including size, morphology, surface charge, and material composition—can be tuned to optimize drug loading, release kinetics, and biocompatibility. For example, colorectal cancer membrane-coated mesoporous silica NPs (Mel-SiO₂@CCM) exploit homotypic adhesion for tumor targeting and Gal-GalNAc-Fap2 interactions for selective Fusobacterium nucleatum elimination, achieving 91% tumor suppression via dual bacteriolytic and immunomodulatory effects. 54 Functionalized NPs enable spatiotemporal delivery of diverse payloads, including chemotherapeutics and siRNAs. Flagella-drug nanoconjugates (FDNCs), for instance, utilize flagellar immunogenicity to enhance cellular uptake and acid-labile linkers for triggered release. 55 NP selection depends on therapeutic goals: magnetic NPs facilitate magnetothermal therapy, gold NPs enable photothermal therapy through efficient photoconversion, and silica NPs serve as robust delivery vehicles. 56-58 Mechanistically, these conjugates mediate direct tumoricidal effects, immune activation, angiogenesis inhibition, and enhanced intratumoral drug accumulation. Despite these advantages, translational challenges remain. Key obstacles include biosafety concerns, immune clearance, optimization of NP pharmacokinetics, and scalable manufacturing.

Concurrently, bacterial extracellular vesicles (BEVs) have emerged as promising nanocarriers due to their inherent biocompatibility, stability, and tumor-homing capabilities. These lipid-bilayer vesicles mediate intercellular communication by transporting proteins, nucleic acids, and metabolites. 59 BEVs can be internalized by tumor cells through endocytosis or membrane fusion, enabling efficient payload delivery. 60 For example, Bifidobacterium bifidum BEVs intrinsically inhibit tumor growth while delivering therapeutic agents against triple-negative breast cancer. 61 Similarly, Lactobacillus -derived BEVs act as immunotherapeutic vectors by transporting immunostimulants and repolarizing macrophages toward antitumor phenotypes. 62 To further enhance the performance of conjugates and BEVs, surface functionalization strategies—including covalent conjugation or physical adsorption—attach targeting ligands such as peptides or antibodies to nanoparticle or BEV membranes, thereby improving tumor specificity and therapeutic payload delivery. 63

In summary, although bacteria–NP conjugates and BEVs exhibit considerable therapeutic potential, their clinical translation requires addressing biosafety concerns, manufacturing complexity, and pharmacokinetic optimization to achieve reproducible, tumor-specific efficacy.

Specific applications of synthetic biology: from gene editing to immune regulation

This direct application strategy employs live, engineered bacterial vectors capable of colonizing tumors and performing localized genome editing or immune modulation within the TME. Bacterial genome editing is a key tool for programming bacteria to deliver tumor-specific antigens or immunomodulators. Among available technologies, the CRISPR–Cas system stands out for its high efficiency and multiplexed targeting; however, its application is constrained by the requirement for protospacer-adjacent motifs (PAMs) and the persistent risk of off-target effects. To improve CRISPR–Cas9 specificity, strategies such as guide RNA (gRNA) design optimization, Cas9 protein engineering, and delivery system innovations are actively pursued. 64 Complementing these genomic modifications, synthetic biology circuits increasingly incorporate tunable gene-expression modules. Gene silencing tools—such as CRISPR interference (CRISPRi), transcription activator-like effector nucleases (TALENs), and RNA interference (RNAi)—allow precise knockdown of target genes, though they may produce transient effects and off-target transcript degradation. Conversely, gene overexpression systems enhance production of immunostimulatory cytokines but face challenges in achieving precise temporal control within the TME. For instance, Mansurov et al. demonstrated that engineered bacteria can be programmed to overexpress cytokines such as interleukin-12 (IL-12) locally, maximizing antitumor effects while minimizing systemic toxicity. 65

Beyond genome manipulation, live bacterial vectors can modulate immune responses within the TME in a context-dependent manner. These constructs induce secretion of cytokines, including IL-2, TNF- α , and IFN- γ , activating immune effectors and potentiating tumor eradication. For example, IL-12-expressing Salmonella typhimurium stimulates Th1-polarized immunity and enhances CTL activity. 21 Similarly, attenuated Salmonella downregulates epithelial cell adhesion molecule (EpCAM) via AKT/mTOR signaling to suppress metastasis, while poly-arginine-coated antigen delivery establishes antigen-specific CD8+ T cell immunity, synergistically augmented by type I interferon adjuvants. 66 Engineered E. coli Nissle 1917 also colonizes tumors, converts ammonia to L-arginine, delivers PD-L1-blocking antibodies, and enhances immune-mediated clearance. 67

Critically, synthetic gene circuits enable spatiotemporal control of bacterial viability and therapeutic output, ensuring localized cytokine production and subsequent clearance. These feedback-regulated systems respond to multiple TME cues. Despite these advances, suboptimal antigen presentation efficiency remains a challenge, motivating the development of dynamic regulatory circuits to enhance CD4⁺/CD8⁺ T cell cooperation in tumor elimination. Notably, sialic acid-binding immunoglobulin-type lectin G (Siglec-G) modulates dendritic cell-mediated CD8⁺ T cell priming, and strategic manipulation of this pathway could amplify antigen presentation and improve vaccine efficacy. 68 Concurrently, bacterially delivered immune checkpoint inhibitors (ICIs) targeting programmed cell death protein 1 (PD-1), PD-L1, and CTLA-4 have demonstrated therapeutic potential. 69 To reduce immune-related adverse events (irAEs) associated with systemic monoclonal antibodies, engineered bacteria can serve as localized ICI delivery platforms. For example, Shapiro et al. developed a thermally responsive bacterial strain that produces anti-PD-L1 and anti-CTLA-4 antibodies exclusively within tumors. 70

Extending beyond local delivery, engineered bacteria have been developed as systemic anticancer vaccines. By leveraging their intrinsic PAMPs as built-in adjuvants, bacterial vectors deliver tumor antigens to APCs and elicit durable T cell immunity—addressing a key limitation of conventional peptide vaccines that often lack sufficient immunogenicity. 71 Clinically, the most advanced example is ADXS11-001, a live-attenuated Listeria monocytogenes secreting an HPV E7-LLO fusion protein, which achieved objective tumor responses in a randomized Phase II cervical cancer trial. 72 More recently, Redenti et al. engineered the probiotic Escherichia coli Nissle 1917 to deliver arrays of patient-specific neoantigens, eliciting potent CD4⁺ and CD8⁺ T cell responses that controlled established primary and metastatic tumors and synergized with anti-PD-L1 blockade in preclinical models. 73

Collectively, synthetic biology advances have revolutionized both genome editing and immunomodulatory strategies, enabling safer, more effective anticancer therapies.

Bacterial oncolysis and virotherapy

Tumor-selective oncolytic bacteria and viruses represent emerging therapeutic modalities that exploit pathogen tropism to selectively destroy malignant cells. This section is organized into two complementary strategies: direct bacterial oncolysis, and the integration of bacteria with oncolytic virotherapy, including bacteria-mediated viral delivery and direct viral oncolysis.

Direct bacterial oncolysis

Oncolytic bacteria employ multiple mechanisms for tumor-specific translocation, including vascular extravasation, interstitial migration, and direct cellular invasion. Synthetically engineered Salmonella YB1, an oxygen-sensitive attenuated strain, suppresses metastasis through an IFN- γ - and NK cell-dependent immune mechanism, in which NK cell-derived IFN- γ promotes the accumulation, activation, and cytotoxicity of NK cells against metastatic cancer cells, while Clostridium spp. lyse tumor cells via exotoxin release, recruiting immune effectors and enhancing antitumor immunity. 74 , 75 Mechanistically, C. difficile exotoxins A and B mediate distinct effects: toxin A increases vascular permeability to facilitate immune cell infiltration, whereas toxin B binds cell surface receptors, inducing apoptosis. 76 Additionally, Salmonella and E. coli engineered to synthesize and locally release cytotoxic payloads (e.g., small molecules, immunotoxins) profoundly reshape the TME. Notably, Wang et al. paraformaldehyde-fixed Salmonella coated with manganese dioxide to activate innate immune pathways, achieving abscopal antitumor effects. 77 Consistent with the ability of Clostridium to thrive in hypoxic tumor cores, the attenuated strain C. novyi-NT has demonstrated promising clinical activity when combined with immune checkpoint blockade; a recent Phase Ib trial of intratumoral C. novyi-NT plus pembrolizumab in patients with treatment-refractory solid tumors reported a confirmed objective response rate of 25% with a median duration of response of 10.93 months and stable disease in 69% of patients. 78 Collectively, these direct oncolytic strategies leverage the intrinsic tumor-homing and tumor-lytic capabilities of bacteria to achieve localized antitumor effects, and they provide the foundational principles for more sophisticated combination approaches discussed below.

Bacteria-mediated viral delivery and direct virotherapy

Beyond direct bacterial oncolysis, a rapidly evolving strategy harnesses bacterial tumor tropism to overcome the pharmacokinetic limitations that hamper conventional virotherapy, such as rapid systemic clearance and poor tumor penetration. By functioning as delivery vehicles for oncolytic viruses or their genetic material, bacteria combine their intrinsic tumor-targeting capabilities with the potent oncolytic and immunostimulatory properties of viruses.

The covalently attached programmable polysaccharide-based encapsulation of Salmonella for intracellular delivery (CAPPSID) platform exemplifies innovative delivery strategies, using engineered Salmonella typhimurium to transport oncolytic Senecavirus A RNA directly into tumor cells, where bacterial lysis releases the viral genome, launching a potent oncolytic viral infection that can bypass circulating antiviral antibodies. 79 Similarly, Recent advances include “Virus-Delivering Salmonella ” (VDS) strains designed to protect and deliver the genome of oncolytic viruses such as the minute virus of mice (MVMp) into cancer cells, leading to successful intratumoral viral propagation and potent bystander effects. 80

Furthermore, oncolytic virotherapy exploits viral tropism to selectively infect and lyse malignant cells. Contemporary research demonstrates that oncolytic viruses not only induce direct oncolysis but also elicit systemic antitumor immunity—an immunostimulatory effect that is further potentiated when combined with immune checkpoint inhibitors or tumor antigen vaccines. 81 Building upon these intrinsic properties, oncolytic viruses can be directly employed or genetically engineered for tumor-selective destruction, representing a complementary strategy within the virotherapy paradigm. Viral therapy primarily utilizes the natural properties of viruses or genetic engineering for targeted infection and lysis of tumor cells. Modified adenovirus and herpes simplex virus (HSV) strains have shown significant efficacy in tumor therapy by replicating within tumor cells, inducing cell lysis, and stimulating the body's immune memory. 82 , 83 Moreover, synthetic biology tools have been employed to design genetic circuits capable of sensing and responding to the TME. 84 This capability enables on-demand control of viral activity and autonomous payload release. Furthermore, viral therapies also enhance tumor killing by delivering therapeutic genes or immune-stimulating molecules that activate or enhance the host's immune system and strengthen the immune response to the tumor. 85

In summary, the integration of bacterial delivery platforms with oncolytic virotherapy—whether through bacteria-mediated viral genome delivery or complementary direct viral oncolysis—represents a powerful convergence of two therapeutic modalities. This combined approach exploits the tumor-homing precision of engineered bacteria and the potent lytic and immunogenic properties of oncolytic viruses, offering synergistic potential for next-generation cancer immunotherapy.

Modular and containment systems in bacterial therapies

Implementation of modular design and biocontainment systems is critical for developing safe and effective bacterial immunotherapies. These foundational technologies provide essential safety and control mechanisms for both direct and indirect therapeutic strategies, ensuring precise spatial and temporal regulation of therapeutic activity regardless of the delivery mode. Biocontainment restricts bacterial proliferation and dissemination to prevent off-tumor toxicity. For example, leucine-auxotrophic Salmonella typhimurium A1-R requires exogenous leucine for viability, limiting its expansion in vivo. 86 Suicide gene systems offer density-dependent control, triggering autolysis at threshold populations to maintain ecological equilibrium. Din et al. ’s synchronized lysis circuit (SLC) exemplifies this approach: bacterial quorum sensing induces lysis factor production, releasing therapeutics while regulating population dynamics. 87

The modular design strategy enhances the flexibility and controllability of therapeutics by incorporating various functional modules, including targeting, release, and safety. This design paradigm enables the customization of bacterial behavior to meet specific therapeutic needs, thereby improving the precision and safety of treatment strategies. Targeting modules primarily modify bacterial surface proteins or ligands to enable selective binding to tumor cells. For instance, Massa et al. enhanced bacterial aggregation at tumor sites and improved invasiveness against CD20+ lymphomas by conjugating an anti-CD20 antibody to the surface of Salmonella. 88 Similarly, Park et al. endowed bacteria with the ability to specifically bind overexpressed αvβ3 integrins by incorporating an arginine-glycine-aspartate (RGD) peptide into the bacterial outer membrane protein A, resulting in significant targeted anti-tumor effects in xenograft models of melanoma and breast cancer. 89 The release module controls the timing and dosage of the therapeutic agent delivered by the bacteria, utilizing the bacteria's natural secretory pathways, such as the type III secretion system (T3SS), to efficiently target the host cell membrane or cytoplasm and activate specific immune responses. The safety module, on the other hand, consists of suicide circuits or externally controlled kill switches to ensure the safety of the treatment process. Two main types of kill switches have been identified: “dead man” kill switches and “codon” kill switches. The “dead man” lethal switch maintains cell viability through unbalanced transcriptional repression in combination with specific input signals, whereas the “codon” lethal switch employs a two-tiered transcriptional structure that combines a set of hybrid LacI-GalR transcription factors to regulate bacterial survival through a triad of different signals. 90

Integration of these modules ensures precise, contained therapeutic action.

Personalized and smart bacterial therapies

Microbiome-informed personalized therapies and environmentally responsive “smart” bacteria represent advanced translational paradigms. These approaches strategically leverage both direct intratumoral mechanisms and indirect systemic effects to achieve precision oncology goals.Tumor–microbiome interactions significantly influence carcinogenesis, treatment response, and immunotherapy efficacy. Rare commensal strains may serve as novel biotherapeutics, and patient-specific microbiota profiling can guide the selection of therapeutic bacteria. For example, genetically modified gut commensals have been engineered to release anticancer agents within the TME. Tanoue et al. demonstrated that defined Listeria consortia enhance host resistance and potentiate ICI efficacy. 91 Intratumoral microbiota also modulate chemoresistance; for instance, Fusobacterium spp. confer gemcitabine resistance. 92 Currently, personalized therapies aim to improve immune responses against specific tumors by leveraging the patient’s microbiome, such as using probiotic strains engineered to deliver antitumor drugs.

Smart bacterial therapies utilize engineered bacteria capable of sensing specific TME signals to selectively release therapeutic agents—including chemotherapeutics, immunostimulatory factors, or lysogenic viruses. This on-demand release minimizes systemic toxicity and enhances therapeutic efficacy. Therapeutic precision is further improved when these bacteria are combined with nanocarriers, enabling higher drug concentrations at tumor sites. 93

Although clinically nascent, these strategies offer transformative potential for precision oncology.

Development of bacteria-based combination treatment

Multi-mechanism immunotherapy has emerged as a superior strategy for tumor. Although conventional modalities—including chemotherapy, radiotherapy, and immunotherapy—remain therapeutic cornerstones, clinical evidence demonstrates suboptimal efficacy of monotherapies. 94 Similarly, standalone bacterial immunotherapy rarely achieves complete tumor clearance. Consequently, bacterially-mediated synergistic regimens are being actively explored. Herein, we elucidate prominent combination strategies, as summarized in Table 1 and illustrated in Figure 5 .

Combination with photodynamic therapy

Photodynamic therapy (PDT) employs photosensitizers to generate cytotoxic reactive oxygen species (ROS) upon oxygen-mediated photoactivation.Despite its tissue-sparing advantage, PDT efficacy is constrained by suboptimal photosensitizer biodistribution, limited light penetration, and tumor hypoxia. Bacterially-mediated delivery systems precisely overcome these limitations. Notably, Guo et al. utilized Salmonella expressing fluorogen-activating protein (FAP) for tumoral targeting; FAP complexation with fluorogens generated ROS, inducing oxidative stress-mediated apoptosis while stimulating pro-inflammatory cytokine production. 100 Furthermore, Porphyromonas gingivalis (Pg) functioned as a potent endogenous photosensitizer in oral squamous cell carcinoma (OSCC), activating innate immunity and promoting antitumor responses. 101 Conversely, Zhang et al. demonstrated enhanced therapeutic outcomes using HER-2-specific nanobody-photosensitizer conjugates with catalyst-binding complexes. 102

Combination with photothermal therapy

Photothermal therapy (PTT) selectively ablates tumors via photothermal agents (PTAs) that convert near-infrared (NIR) light into localized hyperthermia. While valued for noninvasiveness and precision, PTT efficacy is hampered by inadequate PTA accumulation, tissue attenuation, and oxygen dependency. Bacterially-derived nanoplatforms offer innovative solutions.

For instance, Shuang et al. engineered calcium phosphate-coated E. coli outer membrane vesicles (OMVs) loaded with indocyanine green (ICG). Under NIR irradiation, these constructs elevated intratumoral temperatures and activated immune cells, significantly amplifying PTT effects. 103 Similarly, Chen et al. developed Shewanella MR-1-based phototropic bacteria ( PTBs ) encapsulating methylene blue (MB), which inhibited mitochondrial function and suppressed heat shock protein (HSP) expression to potentiate PTT. 104 To address thermal resistance, Wang et al. designed thermally activated biohybrids (TAB@Au) that accumulate at tumor sites, producing localized heat to induce cytolysin A (ClyA)-mediated lysis. 105 Notably, VNP20009 —a hypoxia-tropic Salmonella strain—was combined with polydopamine-mediated PTT, enhancing malignant melanoma eradication. 106

Collectively, bacterially-enhanced phototherapy demonstrates potential to significantly enhance therapeutic indices through tumor-specific targeting and spatiotemporal control.

Combination with radiation therapy

Radiotherapy (RT) eradicates tumors by inducing DNA damage via ionizing radiation. However, off-target exposure risks damaging adjacent healthy tissues, causing acute/chronic toxicities. Moreover, radiotherapy-induced TME alterations—including vascular rarefaction and hypoxia—exacerbate radioresistance, thereby diminishing therapeutic efficacy.

Consequently, bacterially-enhanced radiotherapy represents a promising strategy to circumvent these limitations. Engineered bacteria can deliver radiosensitizing payloads or potentiate immune-mediated tumor clearance. Notably, Listeria monocytogenes has been repurposed as a vaccine vector for antigen delivery and immune stimulation. In seminal work, Hannan et al. demonstrated complete tumor regression in prostate cancer models using ADXS-31-142 (a Listeria -based vaccine) combined with RT. 107 Subsequently, Qi et al. pioneered radioimmunotherapy by immobilizing ¹³¹I-labeled anti-PD-L1 antibodies on bacterial cellulose, which amplified immunogenic cell death (ICD) and activated diverse immune populations. 97 Parallel innovations include radiolabeled inactivated vectors ( ¹²⁵I-VNP/¹³¹I-VNP ) that achieve sustained intratumoral retention and target tumor-associated macrophages (TAMs). 108 Furthermore, gut microbiota modulation may enhance colorectal cancer immunogenicity, radiosensitivity, and mitigate radiotoxicity—a frontier demanding exploration. 109

Combination with chemotherapy drugs

Despite therapeutic advances, chemotherapy remains an oncological cornerstone, yet confronts dose-limiting toxicity, tumor heterogeneity, and multidrug resistance. Bacterially-mediated drug delivery leverages tumor tropism and hypoxic penetration to enhance precision and safety. 110

Specifically, Salmonella typhimurium colonizes necrotic/hypoxic tumoral niches inaccessible to conventional therapies. 111 Against multidrug-resistant tumors, engineered Salmonella delivered siRNAs targeting MDR1, effectively suppressing tumor growth. 112 Similarly, Staphylococcus aureus cell wall-derived liposomal biomimetics (Bio-Bac) encapsulating doxorubicin (DOX@Bio-Bac) demonstrated synergistic antitumor activity. 113 These platforms not only improve drug targeting but also remodel the immunologically suppressive TME.

Bacterially-derived minicells constitute ideal vehicles for chemotherapeutic encapsulation; their physicochemical properties enable versatile payload delivery. 114 siRNA-loaded minicells enhance chemosensitivity, permitting dose reduction and treatment abbreviation. 115 To ensure payload stability, covalent conjugation (e.g., amide bonding) facilitates controlled drug release at tumor sites. 116 Future designs should incorporate TME-responsive linkers to optimize spatiotemporal drug release, thereby maximizing therapeutic indices.

Combination with immune checkpoint inhibitors

The human gut microbiota, a complex ecosystem residing in the gastrointestinal tract, is increasingly recognized as a pivotal modulator of host immunity and a determinant of therapeutic responses in cancer. 117 Beyond its local effects, the gut microbiome exerts systemic influence, shaping the tumor immune microenvironment and thereby affecting the efficacy of anticancer therapies, particularly immune checkpoint blockade (ICB). 118 Specific commensal bacteria and their metabolites (e.g., short-chain fatty acids) have been mechanistically linked to enhanced antitumor T cell responses and improved outcomes with ICB. 119 This foundational understanding positions the gut microbiota not merely as a bystander but as a viable therapeutic target. Strategies to modulate it, ranging from dietary interventions and probiotics to more precise engineered microbial therapeutics, represent a highly studied frontier for overcoming limitations of current immunotherapies. 120

Building on this foundation, the gut microbiota has emerged as a critical determinant of response to ICB. ICB targeting CTLA-4 and PD-1/PD-L1 restores antitumor T cell function. Despite improved tolerability, its clinical utility is limited by response heterogeneity, frequent dosing requirements, and immune-related adverse events (irAEs). Compelling evidence implicates the gut microbiota in modulating ICB efficacy. 121 For example, Park et al. identified commensal suppression of PD-L2/RGMb signaling as a mechanism for enhanced antitumor immunity. 122 Furthermore, Enterococcus spp. potentiated ICB responses; Joachim et al. demonstrated that deaminotyrosine (DAT) amplifies type I interferon signaling and remodels the microbiota to overcome resistance. 123 Synthetic biology enables engineered probiotics to locally deliver checkpoint inhibitors within the TME, minimizing systemic exposure while maximizing intratumoral concentrations. Danino's group pioneered probiotic-guided CAR-T cell therapy: E. coli Nissle 1917 secreting synthetic antigens and CXCL11 recruits and activates systemically administered CAR-T cells within tumors, overcoming poor infiltration and immunosuppression characteristic of solid tumors. 124

Moreover, photo-activatable bacterial vectors can achieve spatiotemporally controlled release of checkpoint inhibitors. Bacteria modified by internal and external modifications can produce a localized thermal effect under light irradiation and simultaneously release immune checkpoint inhibitors to trigger immune activation. 125 This precision targeting elevates the therapeutic index of ICB and provides a rational platform for combination regimens.

Combination with oncolytic virotherapy

As described in Section 4.4 , oncolytic viruses selectively infect and lyse tumor cells while eliciting systemic antitumor immunity. This section focuses on the synergistic co-administration of oncolytic viruses with tumor-targeting bacteria as a distinct combination paradigm. Herpesviridae have become a predominant focus in OVT development due to their inherent oncotropism. Specific strains exhibit intrinsic tumor selectivity, while others have been genetically engineered to enhance tumor-specific targeting. 126 In this combinatorial approach, bacteria and viruses function as co-therapeutics. For instance, the immunomodulatory properties of bacteria can be harnessed to reshape the tumor microenvironment, thereby potentially overcoming residual antiviral defenses and creating a more permissive niche for viral replication. For example, Xie et al. demonstrated that herpesvirus-mediated delivery of the E3 ubiquitin ligase IpaH9.8 effectively degraded GBP1, resulting in significant antitumor efficacy in vivo. 127 When such viral strategies are combined with tumor-colonizing bacteria, the local immune activation and enhanced intratumoral distribution mediated by bacteria can synergistically amplify the overall therapeutic outcome.

Nevertheless, bacterial–OV synergistic therapy faces translational challenges. Precise spatial–temporal control of both bacterial and viral dissemination is essential to prevent collateral damage to healthy tissues. Modulation of the compounded immune responses is also required to mitigate adverse events, and maintaining the stability and predictable interaction of both vectors in vivo remains a critical technical barrier to therapeutic efficacy.

Combination with other cancer therapies

The convergence of bacterial immunotherapy and ferroptosis induction represents an emerging anticancer paradigm. Ferroptosis—characterized by iron-dependent phospholipid peroxidation—potentiates antitumor immunity through dendritic cell activation by damage-associated molecular patterns (DAMPs) released during ferroptotic cell death. This cascade promotes CD8⁺ T cell cross-priming and IFN- γ production, establishing a proimmunogenic milieu. Cai et al. elucidated that ferroptosis induction overcomes resistance to immunotherapeutics. 128 Furthermore, photodynamic therapy synergizes with ferroptosis inducers by exploiting Fenton reaction-derived reactive oxygen species to amplify cytotoxic effects. 129 Bacterial vectors can be engineered to deliver ferroptosis triggers with tumor specificity, enhancing tumoricidal activity while stimulating robust systemic immunity.

Notably, engineered Salmonella SL7207 displaying the HPV E7 antigen (9RE7) on its outer membrane—designated Sal-9RE7 —significantly enhanced immunogenicity and antitumor efficacy when co-administered with the albumin-IFNβ (Alb-IFNβ) adjuvant. 130 Therefore, adjuvants are posited to critically augment anticancer immunotherapy, particularly in in situ tumor vaccine formulations designed to optimize immune activation efficiency. 131 These findings collectively demonstrate that bacterial immunotherapy combined with adjuvant approaches may enhance therapeutic indices, mitigate adverse effects, and potentiate adaptive immune responses, offering novel translational avenues for oncology.

The clinical trials of bacteria-based cancer immunotherapy

Bacteria-based cancer immunotherapies are undergoing extensive clinical evaluation, primarily leveraging four microbial vectors ( Table 2 ). Notably, intravesical Mycobacterium bovis BCG remains the first-line therapy for non-muscle-invasive bladder cancer (NMIBC), supported by its established tolerability and safety profile. Its genetically attenuated derivative, VPM1002BC, has shown improved therapeutic responses in clinical trials. Combination regimens pairing BCG with agents such as the IL-15 superagonist N -803, as well as novel approaches like nadofaragene firadenovec and the nano-immunotherapy OncoTherad (MRB-CFI-1), have demonstrated encouraging efficacy in BCG-unresponsive NMIBC cohorts. 132

Significant progress has also been achieved with engineered Escherichia coli vectors, particularly in HPV vaccinology. An E. coli –produced bivalent HPV-16/18 vaccine achieved 100% efficacy against HPV-16/18-associated high-grade genital lesions in Phase III trials, demonstrating robust immunogenicity, favorable safety, and sustained clinical protection despite minor schedule deviations. 136 This platform offers substantial potential to enhance global vaccine accessibility.

Attenuated Listeria monocytogenes strains have emerged as promising candidates for treating pancreatic adenocarcinoma and metastatic castration-resistant prostate cancer (mCRPC). 138 , 139 In parallel, obligate anaerobes such as the non-toxic Clostridium novyi -NT spores have shown promise in trials by selectively germinating in hypoxic tumor cores, inducing oncolysis and local immune responses in advanced solid tumors. 78 , 143 Finally, probiotic organisms exert antitumor effects through gut microbiome modulation and function as vaccine delivery vehicles. Clinical evidence suggests that strains such as Clostridium butyricum CBM588 and Bifidobacterium lactis BLS-M07 can potentiate responses to immune checkpoint inhibitors and CAR T-cell therapies, underscoring their value as adjuvants in multimodal oncology regimens. 144

Collectively, these clinical advances highlight the transformative potential of bacterial immunotherapies, though broader clinical implementation faces challenges including precise control of bacterial viability in vivo, managing potential systemic toxicity, heterogeneous patient responses, and complex manufacturing and regulatory pathways for live biotherapeutic products. Ongoing trials continue to refine safety, dosing, and efficacy across diverse malignancies, positioning these strategies as an emerging pillar in the oncologic treatment paradigm.

The translational challenges and opportunities of bacteria-based cancer immunotherapy

Synthetic biology breakthroughs over the past decade have transformed the landscape of bacterial cancer immunotherapy, enabling engineered vectors to selectively proliferate within the TME and suppress neoplastic growth. 148 However, this rapid progress is tempered by translational hurdles, including innate bacterial cytotoxicity, manufacturing complexities, and unresolved clinical safety-efficacy paradigms.

The primary challenge lies in mitigating inherent pathogenicity during bacterial vector engineering. Attenuation strategies often employ CRISPR-Cas9–mediated inactivation of virulence determinants, as exemplified in the development of Listeria monocytogenes and Salmonella enterica serovar Typhimurium vaccines. These approaches reduce pathogenic potential while preserving or enhancing antitumor efficacy. 149 Furthermore, synthetic biology enables the engineering of bacteria resilient to physiological stressors, thereby improving therapeutic durability. Raman et al. exemplified this strategy by systematically integrating bacterial chassis, therapeutic payloads, delivery mechanisms, immunomodulatory elements, and genetic circuitry, establishing novel anticancer platforms. 150

Concurrently, manufacturing scalability and quality assurance remain significant translational challenges. To enhance production efficiency and product quality, researchers have implemented advanced bioreactor technologies, multi-step purification processes, and stringent quality control measures, including comprehensive safety assessments and molecular dynamics simulations. These strategies aim to ensure both safety and efficacy throughout the manufacturing pipeline. Developing an efficient production system requires the careful selection of bacterial strains, balancing attenuation with therapeutic potency, and leveraging bioreactor platforms to achieve high-density cultures. Recent advances, such as microfluidic chemotaxis-based devices, have further improved the screening of bacterial candidates with strong tumor-targeting capabilities. 151 Quality control is equally critical. Robust safety evaluations—including toxicology, immunogenicity, and sensitization assays—are essential to confirm compliance with established criteria for bacterial enumeration, purity, and activity. In addition, molecular dynamics simulations and 3D structural prediction tools facilitate the assessment of vaccine constructs for structural stability and immunogenicity. 152 From a cost perspective, process optimization and technological innovation remain key to reducing manufacturing expenses without compromising quality. A notable example is the STING-targeting bacterial immunotherapeutic developed by Daniel S. Leventhal et al., which was designed to meet regulatory standards while minimizing synthesis costs. 153

Notably, clinical implementation faces multifaceted challenges. Preclinical safety evaluation must address both immunogenicity profiling and ecotoxicological risk assessment. 154 Although strategies such as type III secretion system–mediated effector delivery can enhance antitumor potency, 155 clinical outcomes often fail to replicate preclinical efficacy. A well-documented example is Salmonella VNP20009 , which exhibited an acceptable safety profile but demonstrated limited therapeutic benefit due to suboptimal tumor colonization and rapid immune clearance. 156 Administration route optimization adds another layer of complexity. Intravenous injection carries the risk of systemic dissemination, intratumoral delivery is constrained by anatomical accessibility, and oral administration must overcome gastrointestinal degradation. Nanotechnology-based encapsulation offers a promising solution by improving bacterial stability and tumor-targeting specificity, particularly in pulmonary malignancies. 157 Similarly, dose-limiting toxicities—such as systemic inflammatory responses observed in BCG-treated bladder cancer—may be mitigated by incorporating bioactive adjuncts, 158 underscoring the need for precise dose and frequency optimization to maximize therapeutic indices.

Despite these hurdles, interdisciplinary collaboration continues to drive innovation. Advances in synthetic biology, biomaterials, and immunoengineering, combined with rigorous clinical evaluation, position bacterial immunotherapy as a potential cornerstone of future cancer treatment, offering the prospect of highly specific and durable tumor eradication.

Outlooks

The convergence of synthetic biology and bacterial immunotherapy represents a paradigm shift in oncology, offering substantial therapeutic promise. Advances in bioengineering and nanotechnology have driven the development of tumor-targeted engineered bacteria capable of disrupting metabolic pathways and eliciting robust antitumor immune responses. This emerging field introduces innovative translational strategies, including microbiota-based interventions, multimodal combination therapies, and utilization of bacterial components as therapeutic agents.Microbiota engineering—empowered by synthetic biology—enables precise genetic modification of microbial vectors, transforming them into localized biofactories for in situ delivery of immunotherapeutic payloads such as nanobodies, PAMPs, STING agonizts, and cytokines. However, clinical translation of STING agonizts remains limited by metabolic instability and off-target immune activation. To address these challenges, nano-agonist formulations and engineered probiotics have been developed to enhance targeting specificity and improve safety, thereby facilitating clinical application. Despite encouraging preclinical data, successful clinical implementation requires rigorous safety validation, efficacy confirmation within the TME, and establishment of GMP-compliant manufacturing pipelines for large-scale production. 42 Importantly, environmentally responsive bacterial vectors enable tumor-selective drug delivery through sensing of TME cues. Combination strategies integrating bacterial immunotherapy with photothermal, photodynamic, radiotherapeutic, or chemotherapeutic adjuvants can overcome resistance mechanisms and enhance overall efficacy. Collectively, these integrated approaches offer solutions to the challenges of tumor heterogeneity and acquired resistance, advancing next-generation multimodal cancer treatment paradigms.

Ultimately, synthetic biology-driven innovations pave the way for highly targeted, efficient, and personalized cancer therapeutics. Continued interdisciplinary collaboration will be essential to overcome translational hurdles. With sustained innovation and clinical validation, bacterial immunotherapy is poised to become a cornerstone in the oncologic armamentarium, delivering transformative therapeutic options for cancer patients.

Acknowledgments

All authors have made substantial contributions to this work, including but not limited to conception, study design, execution, data acquisition, analysis, and interpretation. They have participated in drafting, revising, or critically reviewing the manuscript, provided final approval for the version to be published, consented to the journal submission, and take full responsibility for all aspects of the work.

Disclosure statement

All authors declare that they have no financial or non-financial conflicts of interest related to the subject matter or materials discussed in this manuscript, nor any affiliations with organizations or entities that could influence the content of this work.

Funding

No funding or sponsorship was received for this study or publication of this article.

Data availability statement

Data sharing is not applicable to this article as no data were created or analyzed in this research.

Consent for publication

All authors have seen and approved the manuscript and consent publication.

References

1. Siegel RL , Giaquinto AN , Jemal A . Cancer statistics, 2024 . CA Cancer J Clin . 2024 ; 74 ( 1 ): 12 – 49 . doi: 10.3322/caac.21820 . 38230766

2. Kaur R , Bhardwaj A , Gupta S . Cancer treatment therapies: traditional to modern approaches to combat cancers . Mol Biol Rep . 2023 ; 50 ( 11 ): 9663 – 9676 . doi: 10.1007/s11033-023-08809-3 . 37828275

3. Li X , Sun S , Zhang W , Liang Z , Fang Y , Sun T , Wan Y , Ma X , Xu Y , Tian R . Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening . J Exp Clin Cancer Res . 2024 ; 43 ( 1 ): 95 . doi: 10.1186/s13046-024-03027-6 . 38561797 PMC10986136

4. Xie QH , Zheng JQ , Ding JY , Wu YF , Liu L , Yu ZL , Chen G . Exosome-mediated immunosuppression in tumor microenvironments . Cells . 2022 ; 11 ( 12 ): 1946 . doi: 10.3390/cells11121946 . 35741075 PMC9221707

5. Wang Y , Li Y , Jing Y , Yang Y , Wang H , Ismtula D , Guo C . Tubulin alpha-1b chain was identified as a prognosis and immune biomarker in pan-cancer combing with experimental validation in breast cancer . Sci Rep . 2024 ; 14 ( 1 ): 8201 . doi: 10.1038/s41598-024-58982-z . 38589634 PMC11001892

6. Hoption Cann SA , van Netten JP , van Netten C . Dr william coley and tumour regression: a place in history or in the future . Postgrad Med J . 2003 ; 79 ( 938 ): 672 – 680 . doi: 10.1093/postgradmedj/79.938.672 . 14707241 PMC1742910

7. Coley WBII . Contribution to the knowledge of sarcoma . Ann Surg . 1891 ; 14 ( 3 ): 199 – 220 . doi: 10.1097/00000658-189112000-00015 . PMC1428624 17859590

8. Livingston-wheeler therapy . CA Cancer J Clin . 1990 ; 40 ( 2 ): 103 – 108 . doi: 10.3322/canjclin.40.2.103 . 2106368

9. Starnes CO . Coley's toxins in perspective . Natur . 1992 ; 357 ( 6373 ): 11 – 12 . doi: 10.1038/357011a0 . 1574121

10. Huang X , Pan J , Xu F , Shao B , Wang Y , Guo X , Zhou S . Bacteria-based cancer immunotherapy . Adv Sci (Weinh) . 2021 ; 8 ( 7 ): 2003572 . doi: 10.1002/advs.202003572 . 33854892 PMC8025040

11. Sepich-Poore GD , Zitvogel L , Straussman R , Hasty J , Wargo JA , Knight R . The microbiome and human cancer . Science (New York, NY) . 2021 ; 371 ( 6536 ): eabc4552 . doi: 10.1126/science.abc4552 . PMC8767999 33766858

12. Mi Z , Guo L , Liu P , Qi Y , Feng Z , Liu J , He Z , Yang X , Jiang S , Wu J , et al. Trojan Horse" salmonella enabling tumor homing of silver nanoparticles via neutrophil infiltration for synergistic tumor therapy and enhanced biosafety . Nano Lett . 2021 ; 21 ( 1 ): 414 – 423 . doi: 10.1021/acs.nanolett.0c03811 . 33356313

13. Li X , Liu J , Zhang W , Wu Y , Li J , Foda MF , Han H . Biogenic hybrid nanosheets activated photothermal therapy and promoted Anti-PD-L1 efficacy for synergetic antitumor strategy . ACS Appl Mater Interfaces . 2020 ; 12 ( 26 ): 29122 – 29132 . doi: 10.1021/acsami.0c09111 . 32501679

14. Cheng K , Zhao R , Li Y , Qi Y , Wang Y , Zhang Y , Qin H , Chen L , Liang J , Xu J , et al. Bioengineered bacteria-derived outer membrane vesicles as a versatile antigen display platform for tumor vaccination via plug-and-display technology . Nat Commun . 2021 ; 12 ( 1 ): 2041 . doi: 10.1038/s41467-021-22308-8 . 33824314 PMC8024398

15. Cameron DE , Bashor CJ , Collins JJ . A brief history of synthetic biology . Nat Rev Microbiol . 2014 ; 12 ( 5 ): 381 – 390 . doi: 10.1038/nrmicro3239 . 24686414

16. Zhu C , Ji Z , Ma J , Ding Z , Shen J , Wang Q . Recent advances of nanotechnology-facilitated bacteria-based drug and gene delivery systems for cancer treatment . Pharmaceutics . 2021 ; 13 ( 7 ): 940 . doi: 10.3390/pharmaceutics13070940 . 34202452 PMC8308943

17. Mahla RS , Reddy MC , Prasad DV , Kumar H . Sweeten PAMPs: role of sugar complexed PAMPs in innate immunity and vaccine biology . Front Immunol . 2013 ; 4 : 248 . 24032031 10.3389/fimmu.2013.00248 PMC3759294

18. Hajam IA , Dar PA , Shahnawaz I , Jaume JC , Lee JH . Bacterial flagellin—a potent immunomodulatory agent . Experimental & Molecular Medicine . 2017 ; 49 ( 9 ): e373 – e373 . doi: 10.1038/emm.2017.172 . 28860663 PMC5628280

19. Mogensen TH . Pathogen recognition and inflammatory signaling in innate immune defenses . Clin Microbiol Rev . 2009 ; 22 ( 2 ): 240 – 273 Table of Contents. doi: 10.1128/CMR.00046-08 . 19366914 PMC2668232

20. Keestra-Gounder AM , Tsolis RM . NOD1 and NOD2: beyond peptidoglycan sensing . Trends Immunol . 2017 ; 38 ( 10 ): 758 – 767 . doi: 10.1016/j.it.2017.07.004 . 28823510 PMC5624830

21. Gurbatri CR , Arpaia N , Danino T . Engineering bacteria as interactive cancer therapies . Science (New York, NY) . 2022 ; 378 ( 6622 ): 858 – 864 . doi: 10.1126/science.add9667 . PMC10584033 36423303

22. Kawasaki T , Kawai T . Toll-like receptor signaling pathways . Front Immunol . 2014 ; 5 : 461 . doi: 10.3389/fimmu.2014.00461 . 25309543 PMC4174766

23. Adams S . Toll-like receptor agonists in cancer therapy . Immunotherapy . 2009 ; 1 ( 6 ): 949 – 964 . doi: 10.2217/imt.09.70 . 20563267 PMC2886992

24. Zheng JH , Nguyen VH , Jiang SN , Park SH , Tan W , Hong SH , Shin MG , Chung I , Bom H , Choy HE , et al. Two-step enhanced cancer immunotherapy with engineered salmonella typhimurium secreting heterologous flagellin . Sci Transl Med . 2017 ; 9 ( 376 ). doi: 10.1126/scitranslmed.aak9537 . 28179508

25. Moretta A . Natural killer cells and dendritic cells: rendezvous in abused tissues . Nat Rev Immunol . 2002 ; 2 ( 12 ): 957 – 964 . doi: 10.1038/nri956 . 12461568

26. Shahabi V , Maciag PC , Rivera S , Wallecha A . Live, attenuated strains of listeria and salmonella as vaccine vectors in cancer treatment . Bioeng Bugs . 2010 ; 1 ( 4 ): 235 – 243 . doi: 10.4161/bbug.1.4.11243 . 21327055 PMC3026462

27. Jhunjhunwala S , Hammer C , Delamarre L . Antigen presentation in cancer: insights into tumour immunogenicity and immune evasion . Nat Rev Cancer . 2021 ; 21 ( 5 ): 298 – 312 . doi: 10.1038/s41568-021-00339-z . 33750922

28. Dong C . Seventeen on inflammation . Nat Immunol . 2020 ; 21 ( 8 ): 821 – 822 . doi: 10.1038/s41590-020-0649-x . 32616860

29. Muranski P , Boni A , Antony PA , Cassard L , Irvine KR , Kaiser A , Paulos CM , Palmer DC , Touloukian CE , Ptak K , et al. Tumor-specific Th17-polarized cells eradicate large established melanoma . Blood . 2008 ; 112 ( 2 ): 362 – 373 . doi: 10.1182/blood-2007-11-120998 . 18354038 PMC2442746

30. Wu Y , Yang Z , Cheng K , Bi H , Chen J . Small molecule-based immunomodulators for cancer therapy . Acta Pharm Sin B . 2022 ; 12 ( 12 ): 4287 – 4308 . doi: 10.1016/j.apsb.2022.11.007 . 36562003 PMC9764074

31. Falahat R , Berglund A , Perez-Villarroel P , Putney RM , Hamaidi I , Kim S , Pilon-Thomas S , Barber GN , Mulé JJ . Epigenetic state determines the in vivo efficacy of STING agonist therapy . Nat Commun . 2023 ; 14 ( 1 ): 1573 . doi: 10.1038/s41467-023-37217-1 . 36949064 PMC10033671

32. Woodward JJ , Iavarone AT , Portnoy DA . c-di-AMP secreted by intracellular listeria monocytogenes activates a host type I interferon response . Science (New York, NY) . 2010 ; 328 ( 5986 ): 1703 – 1705 . doi: 10.1126/science.1189801 . PMC3156580 20508090

33. Yin H , Pu N , Chen Q , Zhang J , Zhao G , Xu X , Wang D , Kuang T , Jin D , Lou W , et al. Gut-derived lipopolysaccharide remodels tumoral microenvironment and synergizes with PD-L1 checkpoint blockade via TLR4/MyD88/AKT/NF-κB pathway in pancreatic cancer . Cell Death Dis . 2021 ; 12 ( 11 ): 1033 . doi: 10.1038/s41419-021-04293-4 . 34718325 PMC8557215

34. Song D , Yang X , Chen Y , Hu P , Zhang Y , Zhang Y , Liang N , Xie J , Qiao L , Deng G . Advances in anti-tumor based on various anaerobic bacteria and their derivatives as drug vehicles . Front Bioeng Biotechnol . 2023 ; 11 : 1286502 . doi: 10.3389/fbioe.2023.1286502 . 37854883 PMC10579911

35. Mayer RL , Verbeke R , Asselman C , Aernout I , Gul A , Eggermont D , Boucher K , Thery F , Maia TM , Demol H , et al. Immunopeptidomics-based design of mRNA vaccine formulations against listeria monocytogenes . Nat Commun . 2022 ; 13 ( 1 ): 6075 . doi: 10.1038/s41467-022-33721-y . 36241641 PMC9562072

36. Wu Y , Li Y , Liu Y , Xiu X , Liu J , Zhang L , Du G , Lv X , Chen J , Ledesma-Amaro R . Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor . Nucleic Acids Res . 2024 ; 52 ( 8 ): 4739 – 4755 . doi: 10.1093/nar/gkae228 . 38567723 PMC11077070

37. Fu H , Liang Y , Zhong X , Pan Z , Huang L , Zhang H , Xu Y , Zhou W , Liu Z . Codon optimization with deep learning to enhance protein expression . Sci Rep . 2020 ; 10 ( 1 ): 17617 . doi: 10.1038/s41598-020-74091-z . 33077783 PMC7572362

38. Yu X , Han X , Li Y , Sun Z , Dong C . Isolation, identification and prokaryotic expression of a bacteriocin-like substance from bacillus licheniformis . Sheng Wu Gong Cheng Xue Bao . 2021 ; 37 ( 7 ): 2453 – 2462 . 34327910 10.13345/j.cjb.210181

39. Cui Y , Luo L , Wang X , Lu Y , Yi Y , Shan Y , Liu B , Zhou Y , Lü X . Mining, heterologous expression, purification, antibactericidal mechanism, and application of bacteriocins: a review . Compr Rev Food Sci Food Saf . 2021 ; 20 ( 1 ): 863 – 899 . doi: 10.1111/1541-4337.12658 . 33443793

40. Cao Z , Cheng S , Wang X , Pang Y , Liu J . Camouflaging bacteria by wrapping with cell membranes . Nat Commun . 2019 ; 10 ( 1 ): 3452 . doi: 10.1038/s41467-019-11390-8 . 31388002 PMC6684626

41. Won S , Lee C , Bae S , Lee J , Choi D , Kim MG , Song S , Shin H , Basukala A , Gho YS . Mass-produced gram-negative bacterial outer membrane vesicles activate cancer antigen-specific stem-like CD8(+) T cells which enables an effective combination immunotherapy with anti-PD-1 . J Extracell Vesicles . 2023 ; 12 ( 8 ): e12357 . doi: 10.1002/jev2.12357 . 37563797 PMC10415594

42. St-Denis-Bissonnette F , Cummings SE , Qiu S , Stalker A , Muradia G , Mehic J , St‐Denis‐Bissonnette F , Mediratta K , Kaczmarek S , Burger D , et al. A clinically relevant large-scale biomanufacturing workflow to produce natural killer cells and natural killer cell-derived extracellular vesicles for cancer immunotherapy . J Extracell Vesicles . 2023 ; 12 ( 12 ): e12387 . doi: 10.1002/jev2.12387 . 38054534 PMC10698709

43. Chien T , Harimoto T , Kepecs B , Gray K , Coker C , Hou N , Pu K , Azad T , Nolasco A , Pavlicova M , et al. Enhancing the tropism of bacteria via genetically programmed biosensors . Nat Biomed Eng . 2022 ; 6 ( 1 ): 94 – 104 . doi: 10.1038/s41551-021-00772-3 . 34326488 PMC8956018

44. Yeung YWS , Ma Y , Deng Y , Khoo BL , Chua SL . Bacterial iron siderophore drives tumor survival and ferroptosis resistance in a biofilm-tumor spheroid coculture model . Adv Sci (Weinh) . 2024 ; 11 ( 39 ): e2404467 . doi: 10.1002/advs.202404467 . 39135304 PMC11496991

45. Wu Q , You L , Nepovimova E , Heger Z , Wu W , Kuca K , Adam V . Hypoxia-inducible factors: master regulators of hypoxic tumor immune escape . J Hematol Oncol . 2022 ; 15 ( 1 ): 77 . doi: 10.1186/s13045-022-01292-6 . 35659268 PMC9166526

46. Zou ZP , Wang XG , Shi XR , Sun ST , Mi J , Zhang XP , Yin B , Zhou Y , Ye B . Self-adjusting engineered probiotic for targeted tumor colonization and local therapeutics delivery . Adv Sci (Weinh) . 2025 ; 12 ( 31 ): e06486 . doi: 10.1002/advs.202406486 . 40432606 PMC12376540

47. Piñero-Lambea C , Bodelón G , Fernández-Periáñez R , Cuesta AM , Álvarez-Vallina L , Fernández L . Programming controlled adhesion of E. Coli to target surfaces, cells, and tumors with synthetic adhesins . ACS Synth Biol . 2015 ; 4 ( 4 ): 463 – 473 . doi: 10.1021/sb500252a . 25045780 PMC4410913

48. Panteli JT , Forbes NS . Engineered bacteria detect spatial profiles in glucose concentration within solid tumor cell masses . Biotechnol Bioeng . 2016 ; 113 ( 11 ): 2474 – 2484 . doi: 10.1002/bit.26006 . 27159665 PMC5037000

49. Chen L , Huang L , Gu Y , Cang W , Sun P , Xiang Y . Lactate-lactylation hands between metabolic reprogramming and immunosuppression . Int J Mol Sci . 2022 ; 23 ( 19 ): 11943 . doi: 10.3390/ijms231911943 . 36233246 PMC9569569

50. Kim S , Kim Y , Lee S , Kim Y , Jeon B , Kim H , Park H . Live biotherapeutic lactococcus lactis GEN3013 enhances antitumor efficacy of cancer treatment via modulation of cancer progression and immune system . Cancers (Basel) . 2022 ; 14 ( 17 ): 4083 . doi: 10.3390/cancers14174083 . 36077619 PMC9455052

51. Choi JK , Naffouje SA , Goto M , Wang J , Christov K , Rademacher DJ , Green A , Stecenko AA , Chakrabarty AM , Das Gupta TK , et al. Cross-talk between cancer and pseudomonas aeruginosa mediates tumor suppression . Commun Biol . 2023 ; 6 ( 1 ): 16 . doi: 10.1038/s42003-022-04395-5 . 36609683 PMC9823004

52. Manieri FZ , Moreira CG . Salmonella typhimurium O-antigen and VisP play an important role in swarming and osmotic stress response during intracellular conditions . Braz J Microbiol . 2022 ; 53 ( 2 ): 557 – 564 . doi: 10.1007/s42770-022-00701-9 . 35303296 PMC9151935

53. Rütschlin S , Böttcher T . Inhibitors of bacterial swarming behavior . Chemistry . 2020 ; 26 ( 5 ): 964 – 979 . doi: 10.1002/chem.201901961 . 31268192 PMC7027876

54. Wu W , Guo F , Wang C , Qu X , Wang Q , Yang Y , Yang Y , Ma Y . Biomimetic "Trojan Horse" nanoparticles with biotactic behavior toward tumor-associated bacteria for targeted therapy of colorectal cancer . Cell Biomaterials . 2025 ; 1 ( 6 ): 100092 . doi: 10.1016/j.celbio.2025.100092 .

55. Fu Z , Wang L , Guo H , Lin S , Huang W , Pang Y . Bacterial flagellum-drug nanoconjugates for carrier-free immunochemotherapy . Small . 2024 ; 20 ( 10 ): e2306303 . doi: 10.1002/smll.202306303 . 37919854

56. Kuzajewska D , Wszołek A , Żwierełło W , Kirczuk L , Maruszewska A . Magnetotactic bacteria and magnetosomes as smart drug delivery systems: a new weapon on the battlefield with cancer? Biology (Basel) . 2020 ; 9 ( 5 ): 102 . doi: 10.3390/biology9050102 . 32438567 PMC7284773

57. Dheyab MA , Aziz AA , Khaniabadi PM , Jameel MS , Oladzadabbasabadi N , Rahman AA , Braim FS , Mehrdel B . Gold nanoparticles-based photothermal therapy for breast cancer . Photodiagnosis Photodyn Ther . 2023 ; 42 : 103312 . doi: 10.1016/j.pdpdt.2023.103312 . 36731732

58. Kuang Y , Zhai J , Xiao Q , Zhao S , Li C . Polysaccharide/mesoporous silica nanoparticle-based drug delivery systems: a review . Int J Biol Macromol . 2021 ; 193 ( Pt A ): 457 – 473 . doi: 10.1016/j.ijbiomac.2021.10.142 . 34710474

59. Elsharkasy OM , Nordin JZ , Hagey DW , de Jong OG , Schiffelers RM , Andaloussi SE , Vader P . Extracellular vesicles as drug delivery systems: why and how? Adv Drug Deliv Rev . 2020 ; 159 : 332 – 343 . doi: 10.1016/j.addr.2020.04.004 . 32305351

60. Ho MY , Liu S , Xing B . Bacteria extracellular vesicle as nanopharmaceuticals for versatile biomedical potential . Nano Converg . 2024 ; 11 ( 1 ): 28 . doi: 10.1186/s40580-024-00434-5 . 38990415 PMC11239649

61. Jiang Y , Wang L , Yang B , Ma G , Chen Z , Ma J , Chang X , Fang L . Bifidobacterium-derived membrane vesicles inhibit triple-negative breast cancer growth by inducing tumor cell apoptosis . Mol Biol Rep . 2023 ; 50 ( 9 ): 7547 – 7556 . doi: 10.1007/s11033-023-08702-z . 37498438

62. Kim W , Lee EJ , Bae IH , Myoung K , Kim ST , Park PJ , Pham AVQ , Ko J , Oh SH , Cho E . Lactobacillus plantarum-derived extracellular vesicles induce anti-inflammatory M2 macrophage polarization in vitro . J Extracell Vesicles . 2020 ; 9 ( 1 ): 1793514 . doi: 10.1080/20013078.2020.1793514 . 32944181 PMC7480564

63. Harimoto T , Hahn J , Chen YY , Im J , Zhang J , Hou N , Li F , Coker C , Gray K , Harr N , et al. A programmable encapsulation system improves delivery of therapeutic bacteria in mice . Nat Biotechnol . 2022 ; 40 ( 8 ): 1259 – 1269 . doi: 10.1038/s41587-022-01244-y . 35301496 PMC9371971

64. Bravo JPK , Liu MS , Hibshman GN , Dangerfield TL , Jung K , McCool RS , Johnson KA , Taylor DW . Structural basis for mismatch surveillance by CRISPR-Cas9 . Natur . 2022 ; 603 ( 7900 ): 343 – 347 . doi: 10.1038/s41586-022-04470-1 . PMC8907077 35236982

65. Mansurov A , Hosseinchi P , Chang K , Lauterbach AL , Gray LT , Alpar AT , Budina E , Slezak AJ , Kang S , Cao S , et al. Masking the immunotoxicity of interleukin-12 by fusing it with a domain of its receptor via a tumour-protease-cleavable linker . Nat Biomed Eng . 2022 ; 6 ( 7 ): 819 – 829 . doi: 10.1038/s41551-022-00888-0 . 35534574 PMC11155269

66. Yen WC , Li QZ , Wu LH , Lee WY , Chang WW , Chien PJ , Lee CH . Salmonella inhibits tumor metastasis by downregulating epithelial cell adhesion molecules through the protein kinase-b/mammalian target of rapamycin signaling pathway . Int J Biol Macromol . 2023 ; 253 ( Pt 3 ): 126913 . doi: 10.1016/j.ijbiomac.2023.126913 . 37716656

67. Canale FP , Basso C , Antonini G , Perotti M , Li N , Sokolovska A , Neumann J , James MJ , Geiger S , Jin W , et al. Metabolic modulation of tumours with engineered bacteria for immunotherapy . Natur . 2021 ; 598 ( 7882 ): 662 – 666 . doi: 10.1038/s41586-021-04003-2 . 34616044

68. Ding Y , Guo Z , Liu Y , Li X , Zhang Q , Xu X , Gu Y , Zhao D , Cao X . The lectin Siglec-G inhibits dendritic cell cross-presentation by impairing MHC class I-peptide complex formation . Nat Immunol . 2016 ; 17 ( 10 ): 1167 – 1175 . doi: 10.1038/ni.3535 . 27548433

69. Ni JJ , Zhang ZZ , Ge MJ , Chen JY , Zhuo W . Immune-based combination therapy to convert immunologically cold tumors into hot tumors: an update and new insights. 2023 . (1745-7254 (Electronic)). doi: 10.1038/s41401-022-00953-z PMC9889774 35927312

70. Abedi MH , Yao MS , Mittelstein DR , Bar-Zion A , Swift MB , Lee-Gosselin A , Barturen-Larrea P , Buss MT , Shapiro MG . Ultrasound-controllable engineered bacteria for cancer immunotherapy . Nat Commun . 2022 ; 13 ( 1 ): 1585 . doi: 10.1038/s41467-022-29065-2 . 35332124 PMC8948203

71. Bolhassani A , Zahedifard F . Therapeutic live vaccines as a potential anticancer strategy . Int J Cancer . 2012 ; 131 ( 8 ): 1733 – 1743 . doi: 10.1002/ijc.27640 . 22610886

72. Basu P , Mehta A , Jain M , Gupta S , Nagarkar RV , John S , Petit R . A randomized phase 2 study of ADXS11-001 listeria monocytogenes-listeriolysin O immunotherapy with or without cisplatin in treatment of advanced cervical cancer . Int J Gynecol Cancer . 2018 ; 28 ( 4 ): 764 – 772 . doi: 10.1097/IGC.0000000000001235 . 29538258 PMC5929492

73. Redenti A , Im J , Redenti B , Li F , Rouanne M , Sheng Z , Sun W , Gurbatri CR , Huang S , Komaranchath M , et al. Probiotic neoantigen delivery vectors for precision cancer immunotherapy . Natur . 2024 ; 635 ( 8038 ): 453 – 461 . doi: 10.1038/s41586-024-08033-4 . PMC11560847 39415001

74. Yu B , Yang M , Shi L , Yao Y , Jiang Q , Li X , Tang L , Zheng B , Yuen K , Smith DK , et al. Explicit hypoxia targeting with tumor suppression by creating an "obligate" anaerobic salmonella typhimurium strain . Sci Rep . 2012 ; 2 : 436 . doi: 10.1038/srep00436 . 22666539 PMC3365283

75. Weerakkody LR , Witharana C . The role of bacterial toxins and spores in cancer therapy . Life Sci . 2019 ; 235 : 116839 . doi: 10.1016/j.lfs.2019.116839 . 31499068

76. Ciesielski-Treska J , Ulrich G , Rihn B , Aunis D . Mechanism of action of clostridium difficile toxin B: role of external medium and cytoskeletal organization in intoxicated cells . Eur J Cell Biol . 1989 ; 48 ( 2 ): 191 – 202 . 2545447

77. Wang C , Zhong L , Xu J , Zhuang Q , Gong F , Chen X , Tao H , Hu C , Huang F , Yang N , et al. Oncolytic mineralized bacteria as potent locally administered immunotherapeutics . Nat Biomed Eng . 2024 ; 8 ( 5 ): 561 – 578 . doi: 10.1038/s41551-024-01191-w . 38514774

78. Nelson BE , Janku F , Fu S , Dumbrava EE , Hong DS , Karp DD , Naing A , Rodon J , Tsimberidou AM , Murthy R , et al. Phase ib study of pembrolizumab in combination with intratumoral injection of clostridium novyi-NT in patients with advanced solid tumors . Clin Cancer Res . 2025 ; 31 ( 18 ): 3864 – 3875 . doi: 10.1158/1078-0432.CCR-24-3952 . 40643985 PMC12434394

79. Singer ZS , Pabón J , Huang H , Sun W , Luo H , Grant KR , Obi I , Coker C , Rice CM , Danino T . Engineered bacteria launch and control an oncolytic virus . Nat Biomed Eng . 2025 ; 10 : 490 – 500 . doi: 10.1038/s41551-025-01476-8 . 40817284 PMC12696630

80. Khanduja S , Bloom SMK , Raman V , Deshpande CP , Hall CL , Forbes NS . Intracellular delivery of oncolytic viruses with engineered salmonella causes viral replication and cell death . iSci . 2024 ; 27 ( 6 ): 109813 . doi: 10.1016/j.isci.2024.109813 . PMC11126981 38799578

81. Arab A , Behravan N , Razazn A , Barati N , Mosaffa F , Nicastro J , Slavcev R . The viral approach to breast cancer immunotherapy . J Cell Physiol . 2019 ; 234 ( 2 ): 1257 – 1267 . doi: 10.1002/jcp.27150 . 30146692

82. Nemunaitis J , Cunningham C , Buchanan A , Blackburn A , Edelman G , Maples PA . Intravenous infusion of a replication-selective adenovirus (ONYX-015) in cancer patients: safety, feasibility and biological activity . Gene Ther . 2001 ; 8 : 746 – 759 . doi: 10.1038/sj.gt.3301424 . 11420638

83. Markert JM , Medlock MD , Rabkin SD , Gillespie GY , Todo T , Hunter WD , Palmer CA , Feigenbaum F , Tornatore C , Tufaro F , et al. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial . Gene Ther . 2000 ; 7 ( 10 ): 867 – 874 . doi: 10.1038/sj.gt.3301205 . 10845725

84. Kitada T , DiAndreth B , Teague B , Weiss R . Programming gene and engineered-cell therapies with synthetic biology . Science (New York, NY) . 2018 ; 359 ( 6376 ). doi: 10.1126/science.aad1067 . PMC7643872 29439214

85. Zhu B , Yin H , Zhang D , Zhang M , Chao X , Scimeca L , Wu M . Synthetic biology approaches for improving the specificity and efficacy of cancer immunotherapy . Cell Mol Immunol . 2024 ; 21 ( 5 ): 436 – 447 . doi: 10.1038/s41423-024-01153-x . 38605087 PMC11061174

86. Hoffman RM . Tumor-targeting amino acid auxotrophic Salmonella typhimurium. (1438-2199 (Electronic)). 2009 . doi: 10.1007/s00726-009-0261-8 19291366

87. Din MO , Danino T , Prindle A , Skalak M , Selimkhanov J , Allen K , Julio E , Atolia E , Tsimring LS , Bhatia SN , et al. Synchronized cycles of bacterial lysis for in vivo delivery . Natur . 2016 ; 536 ( 7614 ): 81 – 85 . doi: 10.1038/nature18930 . PMC5048415 27437587

88. Massa PE , Paniccia A , Monegal A , de Marco A , Rescigno M . Salmonella engineered to express CD20-targeting antibodies and a drug-converting enzyme can eradicate human lymphomas . Blood . 2013 ; 122 ( 5 ): 705 – 714 . doi: 10.1182/blood-2012-12-474098 . 23736700

89. Park SH , Zheng JH , Nguyen VH , Jiang SN , Kim DY , Szardenings M , Min JH , Hong Y , Choy HE . RGD peptide cell-surface display enhances the targeting and therapeutic efficacy of attenuated salmonella-mediated cancer therapy . Theranostics . 2016 ; 6 ( 10 ): 1672 – 1682 . doi: 10.7150/thno.16135 . 27446500 PMC4955065

90. Chan CT , Lee JW , Cameron DE , Bashor CJ , Collins JJ . Deadman' and 'Passcode' microbial kill switches for bacterial containment . Nat Chem Biol . 2016 ; 12 ( 2 ): 82 – 86 . doi: 10.1038/nchembio.1979 . 26641934 PMC4718764

91. Tanoue T , Morita S , Plichta DR , Skelly AN , Suda W , Sugiura Y , Narushima S , Vlamakis H , Motoo I , Sugita K , et al. A defined commensal consortium elicits CD8 T cells and anti-cancer immunity . Natur . 2019 ; 565 ( 7741 ): 600 – 605 . doi: 10.1038/s41586-019-0878-z . 30675064

92. Battaglia TW , Mimpen IL , Traets JJH , van Hoeck A , Zeverijn LJ , Geurts BS , de Wit GF , Noë M , Hofland I , Vos JL , et al. A pan-cancer analysis of the microbiome in metastatic cancer . Cell . 2024 ; 187 ( 9 ): 2324 – 35.e19 . doi: 10.1016/j.cell.2024.03.021 . 38599211

93. Mi P . Stimuli-responsive nanocarriers for drug delivery, tumor imaging, therapy and theranostics . Theranostics . 2020 ; 10 ( 10 ): 4557 – 4588 . doi: 10.7150/thno.38069 . 32292515 PMC7150471

94. Lou X , Chen Z , He Z , Sun M , Sun J . Bacteria-mediated synergistic cancer therapy: small microbiome has a big hope . Nanomicro Lett . 2021 ; 13 ( 1 ): 37 . doi: 10.1007/s40820-020-00560-9 . 34138211 PMC8187705

95. Yang Z , Zhu Y , Dong Z , Hao Y , Wang C , Li Q , Wu Y , Feng L , Liu Z . Engineering bioluminescent bacteria to boost photodynamic therapy and systemic anti-tumor immunity for synergistic cancer treatment . Biomaterials . 2022 ; 281 : 121332 . doi: 10.1016/j.biomaterials.2021.121332 . 35066286

96. Jiang N , Jiang M , Chen J , Mohsin A , Mu Y , Yi X , Zhuang Y , Qian J , Huang J . Living hybrid material based on probiotic with photothermal properties inhibits PD-L1 expression after tumouricidal photothermal therapy . Biomater Transl . 2025 ; 6 ( 1 ): 73 – 84 . doi: 10.12336/biomatertransl.2025.01.006 . 40313568 PMC12041808

97. Qi Z , Pei P , Zhang Y , Chen H , Yang S , Liu T , Zhang Y , Yang K . (131)I-αPD-L1 immobilized by bacterial cellulose for enhanced radio-immunotherapy of cancer . J Control Release . 2022 ; 346 : 240 – 249 . doi: 10.1016/j.jconrel.2022.04.029 . 35469982

98. Gurbatri CR , Lia I , Vincent R , Coker C , Castro S , Treuting PM , Hinchliffe TE , Arpaia N , Danino T . Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies . Sci Transl Med . 2020 ; 12 ( 530 ): eaax0876 . doi: 10.1126/scitranslmed.aax0876 . 32051224 PMC7685004

99. Haralambieva I , Iankov I , Hasegawa K , Harvey M , Russell SJ , Peng KW . Engineering oncolytic measles virus to circumvent the intracellular innate immune response . Mol Ther . 2007 ; 15 ( 3 ): 588 – 597 . doi: 10.1038/sj.mt.6300076 . 17245355 PMC3833616

100. Guo Y , Song M , Liu X , Chen Y , Xun Z , Sun Y , Tan W , He J , Zheng JH . Photodynamic therapy-improved oncolytic bacterial immunotherapy with FAP-encoding S. Typhimurium . J Control Release . 2022 ; 351 : 860 – 871 . doi: 10.1016/j.jconrel.2022.09.050 . 36181917

101. Shi E , Shan T , Wang H , Mao L , Liang Y , Cao M , Wu Q , Li C . A bacterial nanomedicine combines photodynamic-immunotherapy and chemotherapy for enhanced treatment of oral squamous cell carcinoma . Small . 2023 ; 19 ( 52 ): e2304014 . doi: 10.1002/smll.202304014 . 37653616

102. Zhang Q , Wu L , Liu S , Chen Q , Zeng L , Chen X , Zhang Q . Moderating hypoxia and promoting immunogenic photodynamic therapy by HER-2 nanobody conjugate nanoparticles for ovarian cancer treatment . Nanot . 2021 ; 32 ( 42 ): 425101 . doi: 10.1088/1361-6528/ac07d1 . 34319255

103. Qing S , Lyu C , Zhu L , Pan C , Wang S , Li F , Yue H , Gao X , Jia R , Wei W , et al. Biomineralized bacterial outer membrane vesicles potentiate safe and efficient tumor microenvironment reprogramming for anticancer therapy . Adv Mater . 2020 ; 32 ( 47 ): e2002085 . doi: 10.1002/adma.202002085 . 33015871

104. Chen QW , Liu XH , Fan JX , Peng SY , Wang JW , Wang XN , Zhang C , Liu CJ , Zhang XZ . Self‐mineralized photothermal bacteria hybridizing with mitochondria‐targeted Metal–Organic frameworks for augmenting photothermal tumor therapy . Adv Funct Mater . 2020 ; 30 ( 14 ): 1909806 . doi: 10.1002/adfm.201909806 .

105. Wang L , Qin W , Xu W , Huang F , Xie X , Wang F , Ma L , Zhang C . Bacteria-mediated tumor therapy via photothermally-programmed cytolysin A expression . Small . 2021 ; 17 ( 40 ): e2102932 . doi: 10.1002/smll.202102932 . 34472212

106. Chen W , Wang Y , Qin M , Zhang X , Zhang Z , Sun X , Gu Z . Bacteria-driven hypoxia targeting for combined biotherapy and photothermal therapy . ACS Nano . 2018 ; 12 ( 6 ): 5995 – 6005 . doi: 10.1021/acsnano.8b02235 . 29786420

107. Hannan R , Zhang H , Wallecha A , Singh R , Liu L , Cohen P , Alfieri A , Rothman J , Guha C . Combined immunotherapy with listeria monocytogenes-based PSA vaccine and radiation therapy leads to a therapeutic response in a murine model of prostate cancer . Cancer Immunol Immunother . 2012 ; 61 ( 12 ): 2227 – 2238 . doi: 10.1007/s00262-012-1257-x . 22644735 PMC11028516

108. Pei P , Zhang Y , Jiang Y , Shen W , Chen H , Yang S , Yi X . Pleiotropic immunomodulatory functions of radioactive inactivated bacterial vectors for enhanced cancer radio-immunotherapy . ACS Nano . 2022 ; 16 ( 7 ): 11325 – 11337 . doi: 10.1021/acsnano.2c04982 . 35819107

109. Yuan H , Gui R , Wang Z , Fang F , Zhao H . Gut microbiota: a novel and potential target for radioimmunotherapy in colorectal cancer . Front Immunol . 2023 ; 14 : 1128774 . doi: 10.3389/fimmu.2023.1128774 . 36798129 PMC9927011

110. Kang SR , Nguyen DH , Yoo SW , Min JJ . Bacteria and bacterial derivatives as delivery carriers for immunotherapy . Adv Drug Deliv Rev . 2022 ; 181 : 114085 . doi: 10.1016/j.addr.2021.114085 . 34933064

111. Badie F , Ghandali M , Tabatabaei SA , Safari M , Khorshidi A , Shayestehpour M , Mahjoubin-Tehran M , Morshedi K , Jalili A , Tajiknia V , et al. Use of salmonella bacteria in cancer therapy: direct, drug delivery and combination approaches . Front Oncol . 2021 ; 11 : 624759 . doi: 10.3389/fonc.2021.624759 . 33738260 PMC7960920

112. Deng J , Guo Y , Jiang Z , Yang M , Li H , Wang J . Enhancement of ovarian cancer chemotherapy by delivery of multidrug-resistance gene small interfering RNA using tumor targeting salmonella . J Obstet Gynaecol Res . 2015 ; 41 ( 4 ): 615 – 622 . doi: 10.1111/jog.12598 . 25370788

113. Meng J , Li L , Xu Y , Wei X , Chen D , Chen C , Liu S , Wang Z , Shi G , Zhou X , et al. A simple liposome-based bionic bacterium for tumor treatment by re-education of tumor-associated microphages in combination with chemotherapy . Colloids Surf B Biointerfaces . 2023 ; 222 : 113069 . doi: 10.1016/j.colsurfb.2022.113069 . 36508889

114. de Boer PA , Crossley RE , Rothfield LI . A division inhibitor and a topological specificity factor coded for by the minicell locus determine proper placement of the division septum in E. Coli . Cell . 1989 ; 56 ( 4 ): 641 – 649 . doi: 10.1016/0092-8674(89)90586-2 . 2645057

115. MacDiarmid JA , Amaro-Mugridge NB , Madrid-Weiss J , Sedliarou I , Wetzel S , Kochar K , Brahmbhatt VN , Phillips L , Pattison ST , Petti C , et al. Sequential treatment of drug-resistant tumors with targeted minicells containing siRNA or a cytotoxic drug . Nat Biotechnol . 2009 ; 27 ( 7 ): 643 – 651 . doi: 10.1038/nbt.1547 . 19561595

116. Yu C , Han Z , Sun H , Tong J , Hu Z , Wang Y , Fang X , Yue W , Qian S , Nie G . Balancing mechanical property and swelling behavior of bacterial cellulose film by in-situ adding chitosan oligosaccharide and covalent crosslinking with γ-PGA . Int J Biol Macromol . 2024 ; 267 ( Pt 1 ): 131280 . doi: 10.1016/j.ijbiomac.2024.131280 . 38640644

117. Hajjar R , Mars RAT , Kashyap PC . Harnessing the microbiome for cancer therapy . Nat Rev Microbiol . 2026 ; 24 : 392 – 407 . doi: 10.1038/s41579-025-01268-6 . 41486395 PMC12914569

118. Dohlman AB , Pan X , Zitvogel L , Iliev ID . The multi-kingdom cancer microbiome . Nat Microbiol . 2025 ; 10 ( 10 ): 2369 – 2383 . doi: 10.1038/s41564-025-02103-7 . 40926099 PMC12716404

119. Chen L , Li B , Zu M , Reis RL , Kundu SC , Xiao B . Advances and mechanisms of gut microbiota modulation in enhancing immune checkpoint inhibitor efficacy . Semin Cancer Biol . 2025 ; 114 : 150 – 172 . doi: 10.1016/j.semcancer.2025.06.012 . 40617533

120. Elkrief A , Pidgeon R , Maleki Vareki S , Messaoudene M , Castagner B , Routy B . The gut microbiome as a target in cancer immunotherapy: opportunities and challenges for drug development . Nat Rev Drug Discov . 2025 ; 24 ( 9 ): 685 – 704 . doi: 10.1038/s41573-025-01211-7 . 40457025

121. Vétizou M , Pitt JM , Daillère R , Lepage P , Waldschmitt N , Flament C , Rusakiewicz S , Routy B , Roberti MP , Duong CPM , et al. Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota . Science (New York, NY) . 2015 ; 350 ( 6264 ): 1079 – 1084 . doi: 10.1126/science.aad1329 . PMC4721659 26541610

122. Park JS , Gazzaniga FS , Wu M , Luthens AK , Gillis J , Zheng W , LaFleur MW , Johnson SB , Morad G , Zhou Y , et al. Targeting PD-L2-RGMb overcomes microbiome-related immunotherapy resistance . Natur . 2023 ; 617 ( 7960 ): 377 – 385 . doi: 10.1038/s41586-023-06026-3 . PMC10219577 37138075

123. Joachim L , Göttert S , Sax A , Steiger K , Neuhaus K , Heinrich P , Fan K , Orberg ET , Kleigrewe K , Ruland J , et al. The microbial metabolite desaminotyrosine enhances T-cell priming and cancer immunotherapy with immune checkpoint inhibitors . EBioMedicine . 2023 ; 97 : 104834 . doi: 10.1016/j.ebiom.2023.104834 . 37865045 PMC10597767

124. Vincent RL , Gurbatri CR , Li F , Vardoshvili A , Coker C , Im J , Ballister ER , Rouanne M , Savage T , de los Santos-Alexis K , et al. Probiotic-guided CAR-T cells for solid tumor targeting . Science (New York, NY) . 2023 ; 382 ( 6667 ): 211 – 218 . doi: 10.1126/science.add7034 . PMC10915968 37824640

125. Wang L , Cao Z , Zhang M , Lin S , Liu J . Spatiotemporally controllable distribution of combination therapeutics in solid tumors by dually modified bacteria . Adv Mater . 2022 ; 34 ( 1 ): e2106669 . doi: 10.1002/adma.202106669 . 34687102

126. Liu BL , Robinson M , Han ZQ , Branston RH , English C , Reay P , McGrath Y , Thomas SK , Thornton M , Bullock P , et al. ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties . Gene Ther . 2003 ; 10 ( 4 ): 292 – 303 . doi: 10.1038/sj.gt.3301885 . 12595888

127. Xie J , Wang S , Zhong Y , Gao M , Tian X , Zhang L , Pan D , Qin Q , Wu B , Lan K , et al. Oncolytic herpes simplex virus armed with a bacterial GBP1 degrader improves antitumor activity . Mol Ther Oncolytics . 2023 ; 29 : 61 – 76 . doi: 10.1016/j.omto.2023.04.006 . 37223114 PMC10200819

128. Cai H , Ren Y , Chen S , Wang Y , Chu L . Ferroptosis and tumor immunotherapy: a promising combination therapy for tumors . Front Oncol . 2023 ; 13 : 1119369 . doi: 10.3389/fonc.2023.1119369 . 36845720 PMC9945274

129. Chang Q , Wang P , Zeng Q , Wang X . A review on ferroptosis and photodynamic therapy synergism: enhancing anticancer treatment . Heliyon . 2024 ; 10 ( 7 ): e28942 . doi: 10.1016/j.heliyon.2024.e28942 . 38601678 PMC11004815

130. Wang S , Cheng M , Chen CC , Chang CY , Tsai YC , Yang JM , Wu T , Huang C , Hung C . Salmonella immunotherapy engineered with highly efficient tumor antigen coating establishes antigen-specific CD8+ T cell immunity and increases in antitumor efficacy with type I interferon combination therapy . Oncoimmunology . 2024 ; 13 ( 1 ): 2298444 . doi: 10.1080/2162402X.2023.2298444 . 38170154 PMC10761047

131. Duszenko N , van Willigen DM , Bunschoten A , Velders AH , Roestenberg M , van Leeuwen FWB . Chemically enhanced immunogenicity of bacteria by supramolecular functionalization with an adjuvant . ChemBioChem . 2022 ; 23 ( 23 ): e202200434 . doi: 10.1002/cbic.202200434 . 36177993 PMC10098600

132. Alonso JCC , de Souza BR , Reis IB , de Arruda Camargo GC , de Oliveira G , de Barros Frazão Salmazo MI , Gonçalves JM , de Castro Roston JR , Caria PHF , da Silva Santos A , et al. OncoTherad(®) (MRB-CFI-1) nanoimmunotherapy: a promising strategy to treat bacillus Calmette-Guérin-Unresponsive non-muscle-invasive bladder cancer: crosstalk among T-Cell CX3CR1, immune checkpoints, and the toll-like receptor 4 signaling pathway . Int J Mol Sci . 2023 ; 24 ( 24 ): 17535 . doi: 10.3390/ijms242417535 . 38139364 PMC10743608

133. Hahn NM , O'Donnell MA , Efstathiou JA , Zahurak M , Rosner GL , Smith J , Kates MR , Bivalacqua TJ , Tran PT , Song DY , et al. A phase 1 trial of durvalumab in combination with bacillus calmette-guerin (BCG) or external beam radiation therapy in patients with BCG-unresponsive non-muscle-invasive bladder cancer: the hoosier cancer research network GU16-243 ADAPT-BLADDER study . Eur Urol . 2023 ; 83 ( 6 ): 486 – 494 . doi: 10.1016/j.eururo.2023.01.017 . 36717286 PMC10192088

134. Rentsch CA , Thalmann GN , Lucca I , Kwiatkowski M , Wirth GJ , Strebel RT , Engeler D , Pedrazzini A , Hüttenbrink C , Schultze-Seemann W , et al. A phase 1/2 single-arm clinical trial of recombinant bacillus Calmette-Guérin (BCG) VPM1002BC immunotherapy in non-muscle-invasive bladder cancer recurrence after conventional BCG therapy: SAKK 06/14 . Eur Urol Oncol . 2022 ; 5 ( 2 ): 195 – 202 . doi: 10.1016/j.euo.2021.12.006 . 35012889

135. Inman BA , Hahn NM , Stratton K , Kopp R , Sankin A , Skinner E , Pohar K , Gartrell BA , Pham S , Rishipathak D , et al. A phase 1b/2 study of atezolizumab with or without bacille Calmette-Guérin in patients with high-risk non-muscle-invasive bladder cancer . Eur Urol Oncol . 2023 ; 6 ( 3 ): 313 – 320 . doi: 10.1016/j.euo.2023.01.013 . 36803840

136. Qiao YL , Wu T , Li RC , Hu YM , Wei LH , Li CG , Chen W , Huang S , Zhao F , Pan Q , et al. Efficacy, safety, and immunogenicity of an escherichia coli-produced bivalent human papillomavirus vaccine: an interim analysis of a randomized clinical trial . J Natl Cancer Inst . 2020 ; 112 ( 2 ): 145 – 153 . doi: 10.1093/jnci/djz074 . 31086947 PMC7019098

137. Yu XJ , Li J , Lin ZJ , Zhao H , Lin BZ , Qiao YL , Hu Y , Wei L , Huang W , Wu T , et al. Immunogenicity of an escherichia coli-produced bivalent human papillomavirus vaccine under different vaccination intervals . Hum Vaccin Immunother . 2020 ; 16 ( 7 ): 1630 – 1635 . doi: 10.1080/21645515.2020.1761202 . 32544361 PMC7482734

138. Stein MN , Fong L , Tutrone R , Mega A , Lam ET , Parsi M , Vangala S , Gutierrez AA , Haas NB . ADXS31142 immunotherapy ± pembrolizumab treatment for metastatic castration-resistant prostate cancer: open-label phase I/II KEYNOTE-046 study . Oncologist . 2022 ; 27 ( 6 ): 453 – 461 . doi: 10.1093/oncolo/oyac048 . 35373299 PMC9177110

139. Tsujikawa T , Crocenzi T , Durham JN , Sugar EA , Wu AA , Onners B , Nauroth JM , Anders RA , Fertig EJ , Laheru DA , et al. Evaluation of Cyclophosphamide/GVAX pancreas followed by listeria-mesothelin (CRS-207) with or without nivolumab in patients with pancreatic cancer . Clin Cancer Res . 2020 ; 26 ( 14 ): 3578 – 3588 . doi: 10.1158/1078-0432.CCR-19-3978 . 32273276 PMC7727397

140. Huh WK , Brady WE , Fracasso PM , Dizon DS , Powell MA , Monk BJ , Leath CA , Landrum LM , Tanner EJ , Crane EK , et al. Phase II study of axalimogene filolisbac (ADXS-HPV) for platinum-refractory cervical carcinoma: an NRG oncology/gynecologic oncology group study . Gynecol Oncol . 2020 ; 158 ( 3 ): 562 – 569 . doi: 10.1016/j.ygyno.2020.06.493 . 32641240 PMC7487015

141. Nair N , Chen SY , Lemmens E , Chang S , Le DT , Jaffee EM , Murphy A , Whiting C , Müller T , Brockstedt DG . Single-cell immune competency signatures associate with survival in phase II GVAX and CRS-207 randomized studies in patients with metastatic pancreatic cancer . Cancer Immunol Res . 2020 ; 8 ( 5 ): 609 – 617 . doi: 10.1158/2326-6066.CIR-19-0650 . 32132105 PMC7832081

142. Gniadek TJ , Augustin L , Schottel J , Leonard A , Saltzman D , Greeno E , Batist G . A phase I, dose escalation, single dose trial of oral attenuated salmonella typhimurium containing human IL-2 in patients with metastatic gastrointestinal cancers . J Immunother . 2020 ; 43 ( 7 ): 217 – 221 . doi: 10.1097/CJI.0000000000000325 . 32554977 PMC7458080

143. Janku F , Zhang HH , Pezeshki A , Goel S , Murthy R , Wang-Gillam A , Shepard DR , Helgason T , Masters T , Hong DS , et al. Intratumoral injection of clostridium novyi-NT spores in patients with treatment-refractory advanced solid tumors . Clin Cancer Res . 2021 ; 27 ( 1 ): 96 – 106 . doi: 10.1158/1078-0432.CCR-20-2065 . 33046513

144. Dizman N , Meza L , Bergerot P , Alcantara M , Dorff T , Lyou Y , Frankel P , Cui Y , Mira V , Llamas M , et al. Nivolumab plus ipilimumab with or without live bacterial supplementation in metastatic renal cell carcinoma: a randomized phase 1 trial . Nat Med . 2022 ; 28 ( 4 ): 704 – 712 . doi: 10.1038/s41591-022-01694-6 . 35228755 PMC9018425

145. Park YC , Ouh YT , Sung MH , Park HG , Kim TJ , Cho CH , Lee J . A phase 1/2a, dose-escalation, safety and preliminary efficacy study of oral therapeutic vaccine in subjects with cervical intraepithelial neoplasia 3 . J Gynecol Oncol . 2019 ; 30 ( 6 ): e88 . doi: 10.3802/jgo.2019.30.e88 . 31576684 PMC6779607

146. Kawana K , Kobayashi O , Ikeda Y , Yahata H , Iwata T , Satoh T , Akiyama A , Maeda D , Hori-Hirose Y , Uemura Y , et al. Phase I and II randomized clinical trial of an oral therapeutic vaccine targeting human papillomavirus for treatment of cervical intraepithelial neoplasia 2 and 3 . JNCI Cancer Spectr . 2023 ; 7 ( 6 ): pkad101 . doi: 10.1093/jncics/pkad101 . 38001029 PMC10748578

147. Hu Y , Li J , Ni F , Yang Z , Gui X , Bao Z , Zhao H , Wei G , Wang Y , Zhang M , et al. CAR-T cell therapy-related cytokine release syndrome and therapeutic response is modulated by the gut microbiome in hematologic malignancies . Nat Commun . 2022 ; 13 ( 1 ): 5313 . doi: 10.1038/s41467-022-32960-3 . 36085303 PMC9461447

148. Yaghoubi A , Khazaei M , Jalili S , Hasanian SM , Avan A , Soleimanpour S , Cho WC . Bacteria as a double-action sword in cancer . Biochim Biophys Acta Rev Cancer . 2020 ; 1874 ( 1 ): 188388 . doi: 10.1016/j.bbcan.2020.188388 . 32589907

149. Liu CY , Jin M , Guo H , Zhao HZ , Hou LN , Yang Y , Wen Y , Wang F . Concurrent gene insertion, deletion, and inversion during the construction of a novel attenuated BoHV-1 using CRISPR/Cas9 genome editing . Vet Sci . 2022 ; 9 ( 4 ): 166 . doi: 10.3390/vetsci9040166 . 35448664 PMC9029512

150. Raman V , Deshpande CP , Khanduja S , Howell LM , Van Dessel N , Forbes NS . Build-a-bug workshop: using microbial-host interactions and synthetic biology tools to create cancer therapies . Cell Host Microbe . 2023 ; 31 ( 10 ): 1574 – 1592 . doi: 10.1016/j.chom.2023.09.006 . 37827116

151. Stehnach MR , Henshaw RJ , Floge SA , Guasto JS . Multiplexed microfluidic screening of bacterial chemotaxis . eLife . 2023 ; 12 : e85348 . doi: 10.7554/eLife.85348 . 37486823 PMC10365836

152. Shafaghi M , Bahadori Z , Madanchi H , Ranjbar MM , Shabani AA , Mousavi SF . Immunoinformatics-aided design of a new multi-epitope vaccine adjuvanted with domain 4 of pneumolysin against streptococcus pneumoniae strains . BMC Bioinf . 2023 ; 24 ( 1 ): 67 . doi: 10.1186/s12859-023-05175-6 . PMC9951839 36829109

153. Leventhal DS , Sokolovska A , Li N , Plescia C , Kolodziej SA , Gallant CW , Christmas R , Gao J , James MJ , Abin-Fuentes A , et al. Immunotherapy with engineered bacteria by targeting the STING pathway for anti-tumor immunity . Nat Commun . 2020 ; 11 ( 1 ): 2739 . doi: 10.1038/s41467-020-16602-0 . 32483165 PMC7264239

154. Zhang S , Chen Y , Wang J , Tang F , Li M . Role of nontoxigenic clostridium novyi in solid tumor therapy . Reviews in Medical Microbiology . 2014 ; 25 ( 3 ): 71 – 76 Publish Ahead of Print(3):1. doi: 10.1097/MRM.0000000000000005 .

155. Ruano-Gallego D , Álvarez B , Fernández L . Engineering the controlled assembly of filamentous injectisomes in E. Coli K-12 for protein translocation into mammalian cells . ACS Synth Biol . 2015 ; 4 ( 9 ): 1030 – 1041 . doi: 10.1021/acssynbio.5b00080 . 26017572 PMC4603727

156. Chorobik P , Marcinkiewicz J . Therapeutic vaccines based on genetically modified salmonella: a novel strategy in cancer immunotherapy . Pol Arch Med Wewn . 2011 ; 121 ( 12 ): 461 – 466 . doi: 10.20452/pamw.1117 . 22129784

157. Han AR , Wang Z , Zhu T , Zhou M , Sun M . X. Pulmonary delivery of nano-particles for lung cancer diagnosis and therapy: recent advances and future prospects . Wiley Interdiscip Rev Nanomed Nanobiotechnol . 2024 ; 16 ( 1 ): e1933 . doi: 10.1002/wnan.1933 . 37857568

158. Sylvester RJ , van der Meijden AP , Oosterlinck W , Hoeltl W , Bono AV . The side effects of bacillus calmette-guerin in the treatment of ta T1 bladder cancer do not predict its efficacy: results from a european organisation for research and treatment of cancer genito-urinary group phase III trial . Eur Urol . 2003 ; 44 ( 4 ): 423 – 428 . doi: 10.1016/S0302-2838(03)00371-3 . 14499675