Bacteria mediated tumor therapy recent advances challenges and future perspectives

Abstract

Bacteria, as simple unicellular microorganisms, can directly lyse tumor cells following intratumoral administration. Flagellin and cell wall components derived from bacteria can reshape the tumor microenvironment and activate host anti-tumor immune responses. Notably, certain anaerobic and facultative anaerobic bacteria exhibits preferential colonization in the hypoxic core of solid tumors, where they can serve as in situ “micro-bioreactors” and targeted delivery vectors for synergistic tumor therapy. Based on rapid advances in synthetic biology, diverse engineered bacteria and bacteria-based biohybrid systems have been developed for precision tumor therapy, including gene restoration, environment-responsive programmable expression, cytotoxic protein production, and multimodal combination therapy with nanomaterials, immune modulators, or photothermal agents. These living platforms enable targeted drug/gene delivery, sustained local therapeutic output, and durable anti-tumor immune activation. However, clinical translation remains hindered by insufficient biosafety control, off-target colonization, uncontrolled proliferation, immune clearance, and lack of standardized manufacturing and regulatory guidelines. This review systematically summarizes the historical development, inherent anti-tumor mechanisms, genetic engineering strategies, and multimodal biohybrid systems of bacteria-mediated tumor therapy. We comprehensively discuss the advantages, limitations, comparative characteristics, and core bottlenecks of different platforms, and propose rational design strategies for next-generation intelligent, controllable, and clinically translatable bacteria-derived living anti-tumor systems.

Introduction

Tumor represents a major disease threatening human life and health, causing enormous physical and psychological suffering worldwide. Characterized by difficulties in early diagnosis, poor prognosis in advanced stages, and high mortality, malignant tumors have long been regarded as the “king of diseases” [1, 2]. With the intensification of global population aging, tumor incidence and mortality rates continue to rise annually, increasing the clinical demand for more effective and accessible tumor therapies [3, 4]. Although surgery, chemotherapy, radiotherapy, targeted therapy, and emerging immunotherapies have improved patient prognosis, tumor heterogeneity and therapeutic resistance remain unresolved challenges [5, 6]. Once standard treatments fail, patients face limited options and poor outcomes. Therefore, innovative therapeutic strategies are urgently needed.

In contrast to conventional nanomedicines that are severely restricted by heterogeneous, unstable and clinically limited enhanced permeability and retention (EPR) effects, living engineered bacteria as natural bioactive delivery systems possess unique and irreplaceable therapeutic superiorities compared with passive inert nanocarriers and allogeneic cell therapies [7–9]. Different from passive accumulation relying on EPR effect, bacteria own intrinsic autonomous motility, strong hypoxia tropism and efficient extracellular matrix degradation ability, which endow them with superior deep tumor penetration capability to infiltrate dense and hypoxic tumor parenchyma that cannot be reached by traditional synthetic delivery vehicles [10, 11]. Obligate anaerobic and facultative anaerobic bacteria can preferentially colonize the immune-suppressed central area of solid tumors, and act as in situ micro-bioreactors to continuously release therapeutic payloads, thereby realizing sustained and efficient localized tumor suppression [12, 13].

Clinically, most malignant solid tumors are immunologically cold tumors with sparse intratumoral cytotoxic T cell infiltration, rich immunosuppressive cytokines, and weak therapeutic responses to single immune checkpoint blockade [14, 15]. To address this limitation, numerous recent studies have engineered biomaterials and nanoplatforms to remodel immunosuppressive tumor microenvironment (TME) and convert cold tumors into immunogenic hot lesions, a key strategy for boosting combinatorial immunotherapy efficacy [16]. Representative platforms include biomimetic nanoadjuvants, stimuli-responsive carriers and metal nanozymes that induce immunogenic cell death, recruit effector immune cells and relieve local immune suppression, as summarized in several landmark high-impact studies. Unlike synthetic nanovehicles restricted by poor deep tumor penetration, anaerobic and facultative anaerobic bacteria have intrinsic hypoxia tropism and innate immune adjuvant properties, serving as living micro-bioreactors to reshape suppressive TME and reverse cold tumor immune phenotypes [17]. Beyond targeted tumor delivery, these bacterial vectors also exert powerful immunomodulatory functions. The inherent components of bacteria including lipopolysaccharide and flagellin can strongly activate host innate and adaptive immune responses, comprehensively remodel the suppressive TME, reverse the cold immune state of tumors into hot phenotype, and initiate durable systemic antitumor T-cell immune responses [18, 19]. Furthermore, rationally optimized low-dose engineered bacterial formulations can serve as in situ tumor vaccine factories inside tumor tissues, continuously stimulating the body to establish stable long-term specific antitumor immune memory, which effectively inhibits tumor recurrence and distant metastasis [20, 21]. Nevertheless, the current research on bacterial-mediated tumor therapy still stays mostly at the preclinical research stage, and multiple formidable challenges greatly impede their large-scale clinical transformation and popularization [22, 23]. The main unresolved issues cover imperfect in vivo biosafety control strategies, uncontrollable off-target bacterial colonization in normal tissues, unclear long-term in vivo metabolic and toxicological risks, as well as the lack of unified industrialized preparation specifications and systematic clinical evaluation standards.

This review adopts a well-organized logical framework to systematically elaborate the research progress of bacteria-mediated tumor therapy. Firstly, we briefly retrospects the developmental history and elaborates the underlying biological mechanism of bacterial tumor therapy. Subsequently, we comprehensively illustrates mainstream genetic modification strategies for therapeutic bacteria, mainly focuses on tumor suppressor gene over-expression and tumor microenvironment-responsive genetic regulation modes. Furthermore, we summarizes the research progress of surface-functionalized engineered bacteria, and sort out diversified multimodal synergistic therapeutic regimens including reactive oxygen species (ROS) therapy, immunotherapy and photothermal therapy, meanwhile objectively comparing their respective superiorities and inherent limitations. Besides, we deeply analyzes the core obstacle restricting clinical transformation, and emphatically discuss in vivo uncontrollable bacterial proliferation, excessive host immune activation, undesired off-target tissue colonization as well as relevant pharmaceutical supervision and standardization difficulties. Finally, we puts forward feasible optimization directions and rational construction principles for next-generation intelligent living antitumor therapeutic platforms. Collectively, this review systematically consolidates the latest research advances of bacteria-based living drug delivery systems, profoundly clarifies their unique therapeutic strengths as well as currently unsolved scientific and technical bottlenecks, and provides targeted reference and novel insights for the further development and clinical translation of intelligent bacterial antitumor therapeutics.

Historical development and inherent anti-tumor mechanisms of bacteria

History of bacteria in tumor therapy

The clinical exploration of bacteria-mediated tumor therapy can be traced back to 1813, when Vautier and colleagues first observed spontaneous tumor regression in patients complicated with gas gangrene infection [24]. In 1891, William Coley pioneered the preparation of Coley’s toxins based on mixed bacterial strains to elicit potent antitumor immune responses, which marked the earliest prototype of tumor immunotherapy [25, 26]. Nevertheless, owing to unsatisfactory biosafety profiles, ambiguous therapeutic mechanisms and unstable clinical efficacy, this therapeutic strategy gradually fell into oblivion for decades after radiotherapy and chemotherapy became mainstream tumor treatment modalities. The rapid development of genetic engineering technology enabled the construction of various attenuated bacterial strains including Salmonella and Clostridium, which achieved safe and hypoxia-preferred tumor colonization and thus revived this once-abandoned research field [27, 28]. Furthermore, advances in modern synthetic biology have further endowed bacteria with programmable properties, turning them into versatile living drug factories that realize targeted cargo delivery, spatiotemporally controllable therapeutic factor expression and synergistic combination immunotherapy.

In general, the developmental course of bacterial tumor therapy can be divided into four distinct phases: initial clinical observation and preliminary exploration (late 19th to early 20th century), academic stagnation and clinical neglect (mid-20th century), field revival and rational strain design (late 20th century), as well as diversified engineering modification and multimodal combination therapy (21st century to the present) [29]. Reviewing this developmental history reveals that traditional empirical medical insights can be revitalized by cutting-edge modern technologies, providing promising solutions for intractable clinical medical dilemmas. Importantly, early clinical failures fully demonstrates that empirical bacterial application without genetic attenuation modification, precise in vivo behavior regulation and unified efficacy evaluation criteria inevitably causes severe safety hazards and poor experimental reproducibility. Historical experience fully proves that the successful clinical translation of bacterial tumor therapy relies on standardized and precise engineering transformation rather than simple empirical application. Collectively, the inherent biological properties of bacteria, such as natural hypoxia tropism, direct oncolytic capacity and endogenous immune-activating effects, lay the core mechanistic foundation for bacteria-based antitumor therapy.

Native anti-tumor capabilities of bacteria

Although utilizing bacteria as tumor-targeted carriers for drug delivery can enhance the targeted accumulation of antitumor agents and elevate local drug concentrations within tumor tissues to a certain degree, this strategy still has obvious limitations. Drug-loaded bacteria are prone to being eliminated by the host immune system, leading to gradual drug loss, and their in-situ proliferation fails to continuously augment effective therapeutic payloads [30]. Furthermore, genetic mutations are the fundamental pathogenesis of tumorigenesis [31]. Mutated oncogenes drive the malignant transformation of normal cells into tumor cells with unlimited proliferation and invasive metastatic capacities. These abnormally proliferative tumor cells seize nutrients from normal tissues, disrupt physiological organ functions, and eventually induce diverse clinical symptoms and even patient death [32, 33].

In view of the above facts, genetic-level therapeutic strategies possess great application potential for tumor treatment, which are expected to fundamentally tackle tumor pathogenesis. Accordingly, researchers adopt synthetic biology and genetic engineering technologies to rationally engineer bacteria to synthesize and secrete specific therapeutic molecules, such as cytotoxic proteins and immunomodulatory factors [34]. Through the introduction of exogenous inducible regulatory systems, the spatiotemporal expression and release of these therapeutic effects can be precisely manipulated [35]. Such engineered bacteria are capable of migrating into deep hypoxic tumor parenchyma, achieving sustained in-situ proliferation, and locally synthesizing therapeutic substances. This mode effectively elevates intratumoral drug abundance and remarkably improves overall antitumor therapeutic outcomes [36]. This emerging research direction that integrates synthetic biology with tumor therapy mainly takes full advantage of bacteria’s inherent tumor-homing ability and excellent genetic programmability, so as to realize precise and efficient targeted tumor suppression. Apart from exogenous gene modification, the inherent natural antitumor characteristics of bacteria also lay a solid biological foundation for their wide application in tumor therapy.

Tumor suppressor gene restoration

Apart from the construction of attenuated bacterial strains, tumor progression is commonly driven by the activation of oncogenes and the inactivation of tumor suppressor genes, which jointly lead to unrestrained malignant cell proliferation [36]. On this basis, the over-expression of tumor suppressor genes such as p53, PTEN and p16 can directly restore dysregulated signaling pathways in tumor cells, induce cell cycle arrest, cellular apoptosis or senescence, and thus inhibit tumor growth at the source [37, 38]. Accordingly, adopting genetically engineered bacteria as delivery vectors to colonize tumor tissues and achieve in-situ over-expression of tumor suppressor genes has emerged as a highly promising strategy for tumor treatment.

Tum-5 serves as an efficient targeting effector for precise tumor intervention, which specifically binds to integrin αvβ3 receptors on the surface of tumor neovascular endothelial cells [39]. Tum-5 further suppresses FAK, PI3K and PKB signaling cascades, reduces the phosphorylation level of mTOR kinase and the expression of eukaryotic initiation factor 4E, and ultimately blocks tumor angiogenesis [40]. He and colleagues successfully constructed engineered Escherichia coli Nissle 1917 (EcN) strains capable of over-expressing Tum-5, namely EcN (Tum-5), and verified their antitumor effects in a B16 melanoma xenograft mouse model (Fig. 1A) [41]. After intraperitoneal administration, EcN (Tum-5) was initially distributed throughout the whole body and gradually eliminated from normal visceral organs by the host immune system. Eventually, these engineered bacteria efficiently accumulated within hypoxic tumor regions with excellent tumor-targeting capability, and continuously secreted Tum-5 proteins locally. The released Tum-5 could restrain the proliferation of neovascular endothelial cells, block the formation of tumor vascular networks, upregulate the pro-apoptotic protein Bax and downregulate the anti-apoptotic protein Bcl-2, as well as activate Caspase-3 to trigger endothelial cell apoptosis, thereby exerting prominent tumor-inhibitory effects (Fig. 1B) [42]. Although mice exhibited slight body weight loss within the first two days after treatment, their body weight remained stable in the subsequent therapeutic period, confirmed the favorable biocompatibility of EcN (Tum-5) during in vivo tumor therapy. Despite that Tum-5 has not been approved as a clinical therapeutic agent, it can be efficiently expressed and secreted by engineered EcN strains. Such bacterial “Trojan horses” are able to sustainably produce functional Tum-5 inside tumor lesions, which can effectively suppress the progression of melanoma and other solid tumors without causing obvious systemic toxic side effects in vivo.

Fig. 1.

Schematic illustration of engineered EcN (Tum-5) for targeted anti‑angiogenesis tumor therapy. A Map of the recombinant plasmid encoding Tum-5 driven by the hypoxia‑responsive promoter Pvhb [41]. B Engineered EcN selectively colonizes hypoxic tumor tissues and secretes Tum-5 protein. Tum-5 targets integrin αvβ3 on vascular endothelial cells to induce apoptosis and suppress tumor angiogenesis, thereby inhibiting tumor growth [42]

Benefiting from the convenient genomic editing characteristics of bacteria, engineered strains can be rationally designed to express multiple therapeutic molecules simultaneously or sequentially. In accordance with the distinct tumor genetic mutation characteristics of individual patients such as p53 deficiency, targeted tumor suppressor genes can be selected to achieve precise genetic repair therapy [43]. As a core tumor suppressor gene and vital transcription factor, p53 is widely recognized as the guardian of genome stability [44]. More than half of human malignant tumors are accompanied by p53 mutation or functional inactivation, which represents one of the most prevalent genetic aberrations in tumorigenesis. p53 can be effectively activated when cells suffered from various stress stimuli including DNA damage, hypoxia, nutrient deficiency, oncogenic stimulation and telomere shortening. Once activated, p53 functions as a transcription factor to bind to specific DNA fragments and modulate the transcription of numerous downstream target genes, thereby determining cell survival or death fate. In short, p53 acts as an intracellular genome monitor and antitumor regulator. Upon DNA damage occurrence, activated p53 arrests the cell cycle to facilitate DNA repair, if DNA repair fails, it will further initiate cell apoptosis to block malignant transformation. On this basis, He et al. constructed fusion proteins with pro-apoptotic and anti-angiogenic dual functions by taking the matrix metalloproteinase (MMP) cleavage sequence PLGLWA as the fusion linker [41]. They further utilized EcN as a safe gene delivery chassis to establish tumor-homing EcN (Tum-5-p53) engineered bacteria, which could concurrently deliver p53 antitumor protein and Tum-5 anti-angiogenic factor into hypoxic tumor tissues for combined therapy. In vivo experimental results demonstrated that EcN (Tum-5-p53) could markedly inhibit the growth of human hepatocellular carcinoma SMMC-7721 cells in tumor-bearing BALB/c nude mice, possessing satisfactory antitumor efficacy and favorable biosafety without obvious systemic adverse reactions. Collectively, endogenous Tum-5 mainly suppresses tumor progression via cutting off tumor blood supply, while p53 exerts therapeutic effects by regulating intracellular malignant signaling pathways. The two molecules execute antitumor effects through two distinct strategies, namely intracellular malignant lesion elimination and peripheral nutrient supply blockade.

The combination of tumor suppressor gene overexpression strategy and engineered bacterial therapy has pushed tumor treatment into a novel programmable era, transforming the therapeutic paradigm from simple exogenous drug killing to intracellular pathological signal repair and microenvironment remodeling. This therapeutic pattern no longer merely relies on exogenous chemotherapeutic drugs to eliminate tumor cells, but employs engineered bacteria as living delivery vehicles to reconstruct the lost self-stabilization regulatory mechanisms inside tumor tissues, which provides a novel low-toxicity and high-efficiency intelligent therapeutic avenue for refractory solid tumor treatment. Nevertheless, this strategy still faces certain limitations. Its therapeutic efficacy is relatively limited against metastatic tumors and large-volume solid tumors. Besides, long-term ectopic gene expression mediated by engineered bacteria may bring potential risks such as host genomic disturbance and accelerated immune clearance, which have not yet been systematically explored and evaluated in relevant studies.

Environment-responsive and inducible systems

To minimize the damage exerted on normal tissues by cytotoxic proteins expressed by engineered bacteria, researchers rationally modify bacterial strains via synthetic biology strategies. Such engineered bacteria can actively sense alterations in the tumor microenvironment (TME), thereby realizing targeted colonization, controlled proliferation and localized release of therapeutic agents within tumor tissues [45]. With the continuous progress of related research, engineered bacteria has been confirmed that bacteria possess abundant endogenous bioactive molecules and functional proteins. Their intrinsic spontaneous redox reactions support in vivo bacterial biomineralization, which enables them to adsorb environmental metal ions and further synthesize metal nanoparticles inside bacterial cells [46]. Besides, bacteria can synthesize intracellular nanoparticles relying on their inherent active substances without altering their surface properties and core biological functions [47].

In view of this principle, Yao and colleagues supplemented sodium tellurite (Na₂TeO₃) into the culture medium of EcN. Taking EcN as a natural biological reactor, they successfully synthesized intracellular tellurium nanorods (TeNRs) and finally constructed an integrated Te@EcN therapeutic system with high drug loading capacity and favorable near-infrared (NIR) responsiveness (Fig. 2A) [48]. The obtained Te@EcN hybrids are capable of reprogramming tumor-associated macrophages to reshape the immunosuppressive TME. Under NIR irradiation, TeNRs generate abundant photothermal heat to trigger immunogenic cell death of tumor cells. Meanwhile, EcN acts as an effective immune adjuvant to facilitate dendritic cell (DC cells) maturation, which further promotes the activation and proliferation of cytotoxic T cells. Although Te@EcN can efficiently accumulate and achieve long-term retention in tumor sites, the synthesis of intracellular TeNRs inevitably impairs the intrinsic proliferative ability of EcN.

Fig. 2.

Construction and mechanism of bacteria‑based biomineralized therapeutic systems. A Preparation of Te@EcN via intracellular biosynthesis of tellurium nanorods (TeNRs) within EcN [50]. B Construction of thermo‑responsive gene‑engineered E. coli MG1655 carrying the ClyA gene. C Schematic of in situ biomineralization of gold nanoparticles on the bacterial surface through spontaneous reduction [53]

Given the unique biological characteristics of bacterial carriers, engineered bacteria can be further designed to secrete functional therapeutic proteins, so as to endow the therapeutic system with synergistic therapeutic effects beyond simple photothermal therapy (PTT) [49]. These effector proteins can exert antitumor effects via eliminating malignant cells, activating systemic antitumor immunity or inducing tumor cell apoptosis through regulating intracellular signaling cascades [50]. This multifunctional design fully reflects the concept of maximizing the utilization of biological resources, and has been widely recognized and applied in current biomedical research. Accordingly, Wang et al. transformed the recombinant plasmid pBV220 carrying thermosensitive promoters and cytolysin A (ClyA) coding sequences into Escherichia coli K-12 (MG1655) to construct engineered bacterial strain TBA, which enables in-situ intratumoral expression of cytotoxic ClyA protein (Fig. 2B) [51]. Subsequently, they adopted the biomineralization strategy to reduce chloroauric acid (HAuCl₄) adsorbed on the surface of TBA into gold nanoparticles (Fig. 2C). Under NIR irradiation, surface-deposited gold nanoparticles convert light energy into local hyperthermia, which further triggers the specific expression of pore-forming ClyA protein and ultimately achieves efficient tumor cell elimination.

Nevertheless, surface-modified nanomaterials inevitably interfere with the in vivo biological behaviors of host bacteria to varying degrees. To further reduce the off-target toxicity caused by cytotoxic proteins secreted by bacteria, researchers prefer to adopt TME-responsive gene expression vectors to restrict the synthesis of cytotoxic substances specifically within tumor lesions [52]. Furthermore, rapid malignant proliferation leads to structurally disordered and functionally defective tumor vascular networks, which results in severe oxygen and nutrient deprivation inside solid tumors. Such hypoxic TME is not merely a passive outcome of fast tumor growth, but also an active survival strategy adopted by tumors to enhance therapeutic resistance and accelerate distant metastasis [52]. Targeting this hypoxic characteristic, Leventhal et al. constructed recombinant plasmids containing hypoxia-inducible promoter PfnrS and cyclic dinucleotide synthase coding genes, and further delivered these plasmids into EcN strain [53]. The obtained engineered bacteria can initiate the synthesis of cyclic dinucleotides specifically under tumor hypoxic conditions. The secreted cyclic dinucleotides can effectively activate the STING signaling pathway in intratumoral antigen-presenting cells, upregulate the expression of type I interferons and pro-inflammatory cytokines, and ultimately promote the activation of tumor-specific cytotoxic T cells. Apart from hypoxia, tumor cells mainly rely on aerobic glycolysis for energy metabolism, which leads to massive accumulation of lactic acid and hydrogen ions in tumor interstitium and further forms an acidic TME. Based on this feature, Xie et al. conjugated doxorubicin (DOX) onto EcN via acid-labile cis-aconitic anhydride linkers, realizing pH-responsive targeted drug release under the low-pacid microenvironment inside tumors [54]. In vivo experimental results verified that the modified EcN still maintained 71% cell viability, could actively migrate toward hypoxic tumor regions and achieve precise local drug release, thereby efficiently inducing tumor cell apoptosis and prolonging the survival time of tumor-bearing mice.

In general, environment-inducible engineered bacteria can be classified into three major categories: exogenous signal-inducible systems with spatiotemporal precise regulation via external stimuli, TME-responsive systems automatically activated by intrinsic tumor characteristics such as hypoxia, acidosis and high lactate concentration to minimize off-target side effects, and bacterial self-activated systems that rely on quorum-sensing mechanisms to synchronously release therapeutic cargos when bacteria reach a certain in-situ density inside tumors. Collectively, benefiting from excellent inherent tumor tropism and intelligent environmental responsiveness, TME-inducible engineered bacteria greatly promote the development of precise and efficient tumor treatment strategies. With the in-depth interdisciplinary integration of synthetic biology, immunology and material science, such living bacterial drug platforms are expected to achieve groundbreaking progress in the treatment of refractory solid tumors. However, current inducible expression systems still face insufficient response sensitivity, serious background leakage expression and unstable working performance in complex in vivo microenvironments. In addition, undesired off-target activation in normal tissues remains a key biosafety bottleneck that severely restricts their further clinical transformation and practical application.

Comparative analysis and core limitations of engineered bacteria strategies

Engineered bacterial therapeutics constructed through tumor suppressor gene over-expression and microenvironment-inducible regulation possess respective superiorities and inherent bottlenecks in antitumor treatment. Strategies based on tumor suppressor gene over-expression can directly target the genetic pathogenesis of malignancies and exert precise therapeutic effects via regulating cell cycle progression and triggering tumor cell apoptosis. Nevertheless, they are hampered by low in vivo delivery efficiency, risk of host genomic disturbance, and unsatisfactory treatment outcomes against large-sized solid tumors and metastatic lesions [55]. In contrast, environment-responsive inducible systems can be specifically activated by intrinsic tumor characteristics, as well as exogenous external stimuli, which effectively minimize systemic off-target toxicities. Even so, such systems still face prominent defects such as low response sensitivity, severe basal leakage expression and poor functional stability within complex in vivo TME [56]. Importantly, both therapeutic strategies depend heavily on the natural hypoxia tropism of bacteria to realize intratumoral colonization, which greatly limits their popularization and application in non-hypoxic and highly vascularized tumor types. Furthermore, unregulated in vivo bacterial proliferation, rapid host immune-mediated clearance and ambiguous long-term biological safety hazards are regarded as common tough challenges restricting the clinical application of the above two engineering strategies [57]. In general, these two bacterial modification modes exhibit excellent genetic programmability and broad application prospects. Further systematic optimization in targeted colonization accuracy, spatiotemporal regulation precision and in vivo biosafety management is urgently needed to accelerate their clinical transformation and practical popularization.

Functional modification and combined therapeutic applications of genetically engineered bacteria

Externally functionalized bacteria-mediated combined tumor therapy

Nanocarriers including micelles, liposomes and inorganic nanoparticles fabricated via nanotechnology and biomedical material engineering are capable of optimizing in vivo pharmacokinetic properties of therapeutic agents, facilitating lysosomal escape, and improving drug stability and intratumoral retention duration. Such nanoplatforms can well compensate for multiple inherent deficiencies of genetically engineered bacteria in tumor treatment [58, 59]. Nevertheless, relevant early statistical data indicated that merely about 0.7% of administrated nanoformulations can successfully accumulate within solid tumor tissues. Although this classic evaluation value has been continuously questioned and revised in recent years owing to rapid progress in active targeting and deep tumor penetration strategies, the low tumor enrichment efficiency of passive nanomedicines still remains a prominent bottleneck [60].

Current research has verified that integrating nanomaterials onto bacterial surfaces through physical adsorption, biological affinity binding, chemical conjugation and immune recognition interaction can fully take advantage of the intrinsic hypoxia-tropic colonization ability of bacteria. This strategy not only remarkably elevates local drug accumulation and retention efficacy in tumor lesions, but also effectively lowers the practical administration dosage of engineered bacteria [61]. Bacterial chassis and surface-modified nanocomponents achieves perfect functional complementation to realize powerful synergistic antitumor outcomes. Importantly, exogenous nanomaterials can endow bacterial carriers with additional biological functions that original strains do not possess. The constructed bio-nano hybrid therapeutic systems can effectively break through the therapeutic bottlenecks of single bacterial monotherapy, thus greatly broadening the application scope of bacteria-based tumor treatment regimens.

Bacteria combined with ROS therapy

Aberrant cellular metabolism, dysregulated signaling cascades, heterogeneous immune cell infiltration and activated tumor-associated fibroblasts collectively lead to markedly higher intracellular ROS levels in tumor tissues than in normal tissues [62]. High concentrations of hydrogen peroxide (H₂O₂) can partially trigger the epithelial-mesenchymal transition process, endowing tumor cells with migratory potential to evade oxidative stress damage [63]. Nevertheless, excessive ROS will further elevate intracellular oxidative stress and ultimately induce tumor cell apoptosis or autophagic cell death.

Based on this biological feature, researchers propose two mainstream intervention strategies: one is to further sharply elevate intratumoral H₂O₂ content to overwhelm the saturated ROS scavenging capacity of tumor cells, and the other is to inhibit the endogenous antioxidant defense system of tumors and weaken their H₂O₂ elimination efficiency, so that intrinsically generated H₂O₂ can accumulate to cytotoxic concentrations [64]. Accordingly, abundant H₂O₂ within tumor lesions acts as a double-edged sword derived from rapid malignant metabolism, whcih facilitates tumor proliferation, invasion and distant metastasis under physiological conditions, while also serving as a promising and actionable therapeutic target for antitumor intervention. Nanozymes-mediated tumor chemodynamic therapy has emerged as a prevailing research direction, which relies on nanozymes to initiate intracellular Fenton-like reactions inside tumor cells [65]. Such reactions take endogenous high-level H₂O₂ as the substrate to catalyze the production of highly toxic hydroxyl radicals (•OH), which directly induce oxidative damage and irreversible tumor cell death. Besides, certain types of nanozymes are capable of depleting core intracellular antioxidants such as glutathione (GSH), further impairing the ROS clearance ability of tumor cells and enabling continuous accumulation of cytotoxic •OH [66]. Notably, nanozymes-triggered oxidative stress can also initiate immunogenic ferroptosis, a distinctive form of regulated cell death. This biological process releases multiple damage-associated molecular patterns to expose tumor-associated antigens, thereby recruiting and activating host immune cells effectively. Nanozymes-triggered oxidative stress therapeutic mode not only eliminates primary local tumor lesions, but also facilitates the establishment of long-term systemic antitumor immune memory, showing great potential for restraining tumor recurrence and metastasis.

In view of the above mechanisms, Li and colleagues firstly synthesized iron-based metal-organic framework (MOF, Materials of Institute Lavoisier, MIL) via self-assembly reaction between ferrous ions and terephthalic acid, and encapsulated chemotherapeutic drug DOX into the MOF structure [67]. Subsequently, calcium peroxide (CaO₂) nanoparticles were anchored onto the material surface by virtue of the strong adhesion property of polydopamine (PDA), and the final MCDP (MOF/DOX/CaO₂/PDA) composite nanosystem was successfully constructed (Fig. 3). To further improve tumor-targeted accumulation efficiency, the prepared MCDP was firmly modified on the surface of Bifidobacterium infantis (Bif) relying on the adhesion network of PDA, and the integrated therapeutic agent MCDP@Bif was obtained. Within the acidic TME, surface-modified CaO₂ nanoparticles undergo sustained decomposition to continuously supply exogenous MIL and calcium ions. The released calcium ions can block cellular membrane calcium channels, aggravate intracellular oxidative stress and accelerate tumor cell apoptosis. Meanwhile, ferrous ions released from collapsed MOFs participate in Fenton-like reactions with abundant local H₂O₂, which simultaneously destroys the MOF skeleton structure and promotes responsive DOX release. In vivo experimental results based on 4T1 breast tumor xenograft models confirmed that this tumor-targeted MCDP@Bif system could remarkably suppress tumor growth, prolong the survival time of tumor-bearing mice, elevate intratumoral DOX enrichment level and effectively reduce systemic adverse toxic effects.

Fig. 3.

Fabrication and antitumor mechanism of the biohybrid system MCDP@Bif. Schematic illustration of the MCDP nanocomposite composed of Fe‑based MOF, DOX, and CaO₂, coated on Bifidobacterium infantis via PDA. In the acidic TME, CaO₂ decomposes to generate H₂O₂ for chemodynamic therapy, accompanied by DOX release for synergistic chemotherapy [67]

Different from the strategy relying on CaO₂ to produce H₂O₂ under acidic microenvironments proposed by Li et al., Fan et al. adopted a genetically regulated microbial engineering strategy. They performed genetic modification on Escherichia coli MG1655 to achieve the overexpression of respiratory chain enzyme II (NDH-2), and successfully constructed the engineered strain Ec-pE (Fig. 4A) [68]. Afterwards, magnetic Fe₃O₄ nanoparticles were covalently anchored onto the surface of Ec-pE through amide bonds to fabricate the Ec-pE@MNP bio-nano composite system. When engineered bacteria proliferate inside tumor tissues, intracellular NDH-2 can capture electrons from nicotinamide adenine dinucleotide (NADH) and transfer these electrons to molecular oxygen, thus continuously driving endogenous H₂O₂ generation. This biosynthetic pathway effectively compensates for the shortage of intrinsic H₂O₂ within tumor lesions (Fig. 4B). In vivo experimental results verified that Ec-pE@MNP possessed excellent tumor-homing capability and could efficiently colonize hypoxic tumor regions. Meanwhile, surface-decorated Fe₃O₄ nanoparticles are able to trigger Fenton-like reactions with locally accumulated H₂O₂, further generating highly cytotoxic •OH. The produced ROS can markedly induce tumor cell apoptosis and ultimately exert prominent tumor growth inhibitory effects.

Fig. 4.

Engineered bacterial bioreactor for tumor chemodynamic therapy. A Expression of NDH‑2 in engineered E. coli MG1655 and its role in elevating endogenous H₂O₂ generation via the respiratory chain. B Schematic of the bacteria‑mediated Fenton‑like bioreactor: intracellular H₂O₂ reacts with Fe₃O₄ nanoparticles to produce cytotoxic •OH for tumor inhibition [68]

Taking full advantage of the inherent capability of bacteria to survive in anaerobic niches and actively migrate toward hypoxic tumor cores can efficiently resolve the problem of insufficient intratumoral diffusion of in-situ generated ROS. Despite the promising application potential of bacteria-assisted ROS-based tumor therapy, the universally hypoxic and acidic characteristics of TME directly restrict the catalytic efficiency of Fenton-like reactions [63]. Besides, massive accumulation of exogenous metal ions in vivo also raises serious concerns regarding systemic metal toxicity. In general, bacterial synergistic ROS therapy represents an innovative yet sophisticated therapeutic modality. Notably, Fenton-like catalytic reactions are highly susceptible to fluctuations in tumor microenvironmental pH and oxygen content, which easily result in unstable and inconsistent therapeutic outcomes. Furthermore, potential risks caused by long-term metal ion deposition and related biosafety hazards are frequently neglected in most current preclinical research studies.

Bacteria combined with immunotherapy

Different from conventional direct tumor cell killing strategies, tumor immunotherapy is centered on regulating host immune functions, reversing immunosuppressive TME and strengthening the specific recognition and elimination capacity of immune cells [69]. Immunotherapy realizes the therapeutic transformation from exogenous tumor elimination to activate endogenous antitumor immune defense systems, emerging as a revolutionary breakthrough in oncotherapy and bringing new opportunities for patients to achieve sustained therapeutic responses and long-term survival. Tumor immunotherapy has triggered a profound therapeutic paradigm shift, which mainly covers immune checkpoint blockade, adoptive cell therapy and tumor vaccines. All these strategies are designed to mobilize the host immune system to recognize and eradicate malignant cells [70]. In comparison with traditional treatment modalities, immunotherapy possesses prominent superiorities including high tumor specificity, low damage to normal tissues and the capability to establish long-term immune memory, and has established itself as the fourth mainstream tumor treatment modality alongside surgery, radiotherapy and chemotherapy [71].

In this research field, engineered bacteria have attracted widespread research attention by virtue of their excellent immune-modulating bioactivity [71]. Serving as inherent natural immune adjuvants, invasive bacteria can rapidly trigger robust innate immune responses, recruit and activate macrophages, natural killer cells and other immune effector cells, partially dismantle tumor-established immunosuppressive TME, and lay a solid foundation for subsequent systematic immune attack [51]. Nevertheless, bacterial immunotherapy still confronts enormous obstacles in clinical translation. Firstly, certain attenuated Salmonella strains, despite possessing favorable tumor-targeting ability, may impair intratumoral antitumor T-cell functions and hinder the formation of long-term immune memory [72]. Secondly, massive lactate secreted during bacterial metabolism is a core mediator sustaining immunosuppressive TME, which may counteract the intrinsic immune-activating effects of bacteria themselves [73]. Besides, solid tumors feature prominent heterogeneity and complicated immune evasion mechanisms, making single therapeutic strategies difficult to thoroughly reverse local immune suppression [11]. Accordingly, the combination of bacterial therapy and tumor immunotherapy has become an inevitable developmental trend in this field. The integration of engineered bacteria with immune checkpoint inhibitors and adoptive cell therapy enables synergistic regulation of multiple key biological processes, including DC cell maturation, cytotoxic T cell activation and TME remodeling [71]. Such multi-modal and multi-target combined strategies can produce powerful synergistic therapeutic effects, break through the bottlenecks of monotherapy, and induce stronger and more persistent systemic antitumor immune responses.

To further optimize the therapeutic efficacy of bacteria-based combined immunotherapy, researchers constructed a novel EcN@PPO living bacterial immunotherapeutic system. EcN@PPO platform adopts engineered EcN 1917 as biological carriers, and co-loads tumor-associated antigen OVA and anti-PD-1 antibody via PDA adhesion networks [74]. Benefiting from the inherent hypoxia tropism of EcN, the prepared system can deeply penetrate into hypoxic tumor regions and achieve sustained localized drug release under acidic TME. The released OVA antigens effectively enhance antigen presentation efficiency of DC cell, while released anti-PD-1 antibodies relieve T cell immune exhaustion, thereby synergistically activating systematic adaptive antitumor immunity. Meanwhile, PDA can generate mild photothermal effects under near-infrared irradiation, which further facilitates the phenotypic transformation of tumor-associated macrophages from immunosuppressive M2 phenotype to antitumor M1 phenotype and effectively remodels suppressive TME. In vivo experimental results confirmed that EcN@PPO could efficiently inhibit the growth and distant metastasis of 4T1 breast tumor xenografts and remarkably prolong the survival cycle of tumor-bearing mice via synergistically enhancing antigen presentation, blocking immune checkpoints and regulating macrophage polarization.

Although bacteria-mediated combined tumor immunotherapy still faces multiple challenges in terms of therapeutic efficacy, biosafety side effects and tumor heterogeneity regulation, which remains one of the most dynamic and promising research directions in current antitumor research. Clinically, the eligibility of patients for immunotherapy needs comprehensive assessment by professional oncologists according to specific tumor types, clinical stages, biomarker detection results and individual physical conditions. A major restrictive factor is that most relevant preclinical data are obtained from immunocompetent mouse models, while the actual therapeutic efficacy in immunodeficient hosts and advanced-stage tumor patients remains unpredictable. Moreover, excessive inflammatory responses triggered by bacterial intervention may even accelerate tumor progression under certain pathological conditions, which cannot be ignored in subsequent translational research.

Bacteria combined with photothermal therapy

Persistent metabolic reprogramming of tumor cells greatly weakens the therapeutic efficacy of conventional antitumor treatments [75]. As a novel minimally invasive therapeutic modality, photothermal therapy (PTT) enables photothermal agents to absorb specific-wavelength light and convert it into local hyperthermia, which further destroys cell membranes, induces protein denaturation and DNA damage, and achieves accurate tumor ablation [76]. In addition, PTT can trigger damaged tumor cells to release damage-associated molecular patterns, initiate immunogenic cell death (ICD) and activate systematic antitumor immune responses [77]. Accordingly, combined therapeutic strategies have gradually become the mainstream development trend of PTT, among which bacteria-assisted PTT shows outstanding application prospects. This strategy integrates the excellent tumor accumulation and deep penetration capacity of bacterial carriers with the precise tumor-killing property of PTT. At present, diverse loading strategies have been developed to modify photothermal agents onto bacterial surfaces.

For instance, Chen et al. covalently modified indocyanine green (ICG) onto the surface of hypoxia-targeted attenuated Salmonella YB1 via amide bonds to construct YB1-INPs hybrid system for large solid tumor treatment (Fig. 5A) [78]. The engineered YB1 strain carries a hypoxia-responsive promoter-controlled asd gene, which endows YB1 with specific targeting ability toward hypoxic tumor sites. Under near-infrared (NIR) irradiation, local tumor temperature can reach 63 °C to thoroughly ablate tumor tissues, and meanwhile eliminate residual intratumoral YB1, balancing antitumor efficacy and in vivo biosafety (Fig. 5B).

Fig. 5.

Schematic of YB1‑INPs for hypoxia‑targeted photothermal therapy of large solid tumors. A Preparation of YB1‑INPs via covalent conjugation of ICG‑based nanophotosensitizers to attenuated Salmonella YB1. B YB1‑INPs accumulate deeply in hypoxic tumor regions, under NIR irradiation, localized photothermal ablation achieves complete tumor elimination [78]

Nevertheless, the potential synergistic effect between bacterial infection-mediated tumor microenvironment remodeling and ICD-induced immune activation has not been fully clarified. Besides, most synthetic photothermal material involves complicated preparation procedures, which greatly restrict large-scale production and clinical transformation [79]. In view of this, modifying bacteria with endogenous biocompatible molecules such as dopamine, norepinephrine and serotonin has become a key optimization direction. In particular, dopamine can not only regulate tumor biological behaviors via receptor signaling pathways, but also undergo oxidative self-polymerization to form PDA. With favorable NIR responsiveness, biodegradability and abundant active groups, PDA serves as a universal interfacial modification material for conjugating various functional molecules [80]. Based on this property, Liu et al. constructed core-shell structured PDA@LAC delivery systems utilizing in-situ oxidative polymerization of dopamine on Lactobacillus surface [81]. The outer PDA shell endows the system with photothermal performance and efficient DOX loading capacity. After intravenous administration, Lactobacillus can actively home to hypoxic tumor regions and achieve targeted drug delivery. Upon NIR irradiation, PDA-mediated hyperthermia induces tumor cell ICD and releases tumor-associated antigens, reversing therapeutic resistance caused by tumor cell reprogramming. Moreover, acidic TME and high intracellular GSH levels can accelerate the degradation of PDA shells to realize sustained DOX release. Meanwhile, PTT treatment elevates tumor cell membrane permeability to facilitate intracellular drug uptake, thus achieving synergistic chemo-photothermal therapeutic effects.

Bacteria-based PTT possesses unique spatiotemporal controllability via adjustable light irradiation conditions, realizing programmable precise therapy. Local hyperthermia directly eradicates tumor lesions, and the subsequent ICD effect constructs an in-situ tumor vaccine. The combined effect of bacterial immunity and photothermal therapy can effectively transform immunosuppressive cold tumors into immunocompetent hot tumors, trigger systemic antitumor immunity and suppress tumor recurrence and metastasis [82]. Overall, PTT is a precise, minimally invasive and highly synergistic tumor treatment strategy. With continuous optimization of combined regimens and biocompatible materials, PTT will gain broader clinical application prospects. However, limited light penetration depth restricts its application in deep-seated tumors, and hyperthermia-induced thermal resistance also impairs long-term therapeutic outcomes.

Furthermore, the application scope of engineered bacterial synergistic therapy has been further expanded, including PTT, sonodynamic therapy, radiotherapy and CAR-T cell therapy. Bacterial carriers can enhance the enrichment and deep penetration of photosensitizers and sonosensitizers, reverse tumor hypoxia-induced treatment resistance, and improve the infiltration and persistence of CAR-T cells by remodeling immunosuppressive TME [83]. Such multi-modal combinations further broaden the application value of surface-functionalized bacteria and provide new ideas for solving clinical treatment dilemmas. Despite abundant research progress, current related reviews still have several research gaps. Firstly, the therapeutic potential of bacterial outer membrane vesicles as cell-free immunotherapeutic platform is rarely systematically summarized. Secondly, bacteria-assisted sonodynamic therapy with deep tissue penetration advantage lacks sufficient attention. Thirdly, bacterial membrane-camouflaged nanoparticles combining the merits of living bacteria and synthetic carriers are seldom discussed. Fourthly, systematic sorting of completed and ongoing clinical trials is insufficient. Future relevant studies need to supplement the above contents to build a more comprehensive research framework of bacteria-mediated tumor therapy.

Comparative analysis and core limitations of multimodal combination strategies

Although bacteria-mediated multimodal combination therapies can break through multiple drawbacks of single-modal tumor treatment, each therapeutic regimen still possesses inherent deficiencies. Bacteria-assisted ROS-based chemodynamic therapy can effectively trigger intracellular oxidative stress and tumor cell ferroptosis, while its therapeutic performance is highly correlated with endogenous intratumoral H₂O₂ content and greatly restricted by hypoxic and acidic TME [84]. Besides, cumulative deposition of exogenous metal ions also brings hidden risks to long-term in vivo biosafety. Bacteria-combined immunotherapy is capable of efficiently transforming immunosuppressive cold tumors into immunocompetent hot tumors, and evoking persistent systemic antitumor immune responses. Nevertheless, Bacteria-combined immunotherapy practical application is restricted by tumor heterogeneity, immunosuppressive metabolic metabolites secreted by proliferative bacteria, and unstable therapeutic outcomes in immunodeficient individuals. As for bacteria-based PTT that can realize spatiotemporally controllable precise treatment and readily induces immunogenic cell death [85]. However, its clinical popularization is greatly limited by insufficient light penetration depth, tumor thermal resistance effect and poor therapeutic effect against deep-seated solid tumors.

In general, these multimodal synergistic regimens can exert superior combined therapeutic effects, yet they share several universal translational bottlenecks, including complicated structural composition, difficulties in large-scale standardized preparation, undefined long-term biological safety risks, and excessive reliance on intrinsic TME features. Therefore, further rational structural simplification, targeted capability enhancement and intelligent regulatory system optimization are urgently required to promote the clinical transformation of such promising combined therapeutic strategies.

Conclusion and outlook

Bacterial tumor therapy combines microorganisms with modern precision medicine. After over a century of exploration, bacterial tumor therapy has evolved from fundamental research into a pivotal clinical translation stage [86]. Rather than replacing conventional tumor treatments, bacterial tumor therapy acts as a promising complementary strategy to address the bottlenecks of traditional cancer interventions (Fig. 6). Its core strength lies in utilizing inherent bacterial properties to compensate for the limitations of routine therapies, driving innovative progress in oncology. Given the distinct disparities of various strains in tumor targeting, biosafety, editability and therapeutic potency, it is essential to systematically summarize bacterial living therapeutics for rational design and clinical application. Herein, we overview mainstream preclinical and clinical strains, as well as their biological features, engineering tactics, applicable tumor models, anti-tumor effects and research advances in Table 1.

Fig. 6.

Overview of bacteria‑derived platforms for tumor therapy. Schematic illustration of diverse engineering strategies, including genetic modification, surface nanoengineering, intracellular biomineralization, environmental signal sensing, and autonomous targeting for multimodal synergistic tumor therapy

Table 1.

Comparative summary of representative bacterial strains for tumor therapy and corresponding clinical trials

Strain	Key characteristics	Common engineering modifications	Typical tumor models	Preclinical outcomes	Clinical trials & main outcomes
E. coli Nissle 1917 (EcN)	Probiotics, hypoxia‑targeting, high safety, easy genetic manipulation	Overexpress Tum‑5/p53/CD/STING agonists, hypoxia‑/acid‑responsive switches	Melanoma, HCC, breast cancer, CRC	Selective tumor colonization, local protein secretion, inhibited angiogenesis & tumor growth, good biocompatibility	SYNB1891 (EcN‑STING): Phase I (NCT04167137), intratumoral, manageable safety, activated IFN/T‑cell response
Salmonella VNP20009	Facultative anaerobic, strong tumor tropism, systemic delivery feasible	ΔpurI/ΔmsbB attenuated, express CD/CPG2/cytokines	Melanoma, CRC, pancreatic cancer	Tumor‑selective accumulation, suppressed tumor growth, prodrug conversion	Phase I (melanoma/renal cell carcinoma): MTD 3 × 108 CFU/m2, well tolerated, limited objective response, TAPET‑CD: Phase I, intratumoral, tumor colonization&5‑FU production observed
Clostridium (novyi‑NT, etc.)	Obligate anaerobic, strictly colonize hypoxic/necrotic tumor core, oncolytic	Non‑toxic spores, hypoxia‑dependent lysis	Soft tissue sarcoma, advanced solid tumors	Specific intratumoral germination, direct oncolysis, enhanced immune infiltration	C. novyi‑NT: Phase I (NCT01924689), combined with anti‑PD‑1: Phase Ib, ORR 25%, manageable safety, Clostridium spore: Phase I/II in China for advanced solid tumors
Listeria monocytogenes	Cytosolic delivery, strong immune adjuvanticity, vaccine platform	ΔprfA attenuated, express tumor antigens (E7, mesothelin)	Pancreatic cancer, cervical cancer, HCC	Induced robust CD8+ T‑cell response, anti‑tumor memory	ADXS11‑001: Phase III (cervical cancer), CRS‑207: Phase II (pancreatic cancer), safe, immune activation observed
Bifidobacterium	Probiotics, obligate anaerobic, high safety, oral/intravenous	Surface‑loaded MOF/nanoparticles, conjugate chemotherapeutics	Breast cancer, CRC, HCC	Hypoxia‑targeting, reduced systemic toxicity, synergistic chemo/chemodynamic therapy	Preclinical mainly, good safety profile supports clinical translation

Biosafety is the core barrier restricting clinical application of bacterial tumor therapy, with multiple unresolved challenges remaining [87]. In vivo proliferation of live bacteria may trigger systemic inflammation, infection risks and organ damage, while off-target colonization in normal tissues causes unexpected side effects. Additionally, bacterial components such as lipopolysaccharide and flagellin can induce excessive immune response and even cytokine storms. Long-term risks including gene insertion mutation, persistent exogenous gene expression and microbial leakage also require systematic safety assessment. To resolve these issues, various engineering approaches have been developed, including gene deletion-based attenuation, TME-responsive expression regulation, inducible suicide systems and quorum-sensing controlled proliferation. These modifications effectively reduce bacterial virulence, avoid non-specific effects and achieve controllable in vivo survival, laying a solid foundation for safe clinical transformation.

We further outline several cutting-edge research directions in this field. First, AI-assisted design accelerates engineered bacteria development via high-throughput screening, genetic circuit optimization, colonization prediction and therapeutic module regulation, greatly boosting translational progress [88]. Second, bacterial outer membrane vesicles (OMVs) serve as cell-free therapeutic platforms, retaining native tumor tropism and immunostimulatory effects while eliminating live bacteria-related proliferation and ectopic risks, showing superior clinical controllability [89]. Third, intratumoral bacteria-mediated in-situ vaccination remodels solid tumors into personalized antigen pools. Bacteria-induced immunogenic cell death releases tumor-associated antigens and damage-associated molecular patterns, which synergize with bacterial adjuvants to elicit potent and durable systemic anti-tumor immunity and immune memory. Fourth, combined application of oncolytic bacteria and gut microbiota modulation reshapes host immune homeostasis, enhances intratumoral infiltration of anti-tumor immune cells, and mitigates therapeutic heterogeneity caused by individual flora differences. These emerging strategies further broaden the research scope and application prospects of bacterial tumor therapy.

Systemically or locally administered therapeutic bacteria dynamically interact with host endogenous microbiota. Gut and intratumoral flora jointly regulate host immune status, metabolism and inflammation, thereby affecting the colonization efficiency, viability and anti-tumor activity of exogenous engineered bacteria, as well as in vivo biosafety and non-specific immune reactions. Elucidating such interaction mechanisms is critical to optimize therapeutic efficacy and safety. Despite unique advantages of different bacterial strains, their targeting accuracy, safety management and therapeutic stability still need further improvement. Ample preclinical achievements have been obtained, yet large-scale clinical promotion still faces great difficulties. The lack of unified technical standards stands as a major translational obstacle. Current preclinical studies lack consistent criteria for bacterial dosage, administration routes, detection methods and efficacy evaluation, leading to poor repeatability and comparability. For bacteria-based bio-nano hybrid systems, prominent chemistry, manufacturing, and controls (CMC) challenges exist in large-scale fermentation, activity maintenance, uniform cargo loading, storage stability, batch consistency and quality inspection. Different from conventional drugs and nanomedicines, replicable live bacterial agents require independent evaluation systems and stricter safety verification. At present, mainstream regulatory authorities have not established complete unified guidelines for live microbial drugs, and targeted supervision is needed for immunogenicity, ectopic proliferation and long-term biosafety risks. Hence, standardized CMC protocols, complete quality control systems and definite regulatory norms are urgently required to facilitate clinical translation and industrialization.

In the future, next-generation intelligent bacterial anti-tumor platforms will precisely recognize tumor hypoxia, acidity and specific protease signals to realize spatiotemporally controllable payload release. With the cross-integration of synthetic biology, materials science, immunology, clinical oncology and artificial intelligence, refined engineered bacterial therapeutics will become safer, smarter and more efficient. Nevertheless, current intelligent systems are still deficient in self-regulation, real-time monitoring and on-demand elimination, high costs and technical complexity also hinder clinical popularization. Thus, bacterial tumor therapy has entered an interdisciplinary development inflection point. Relying on inherent tumor targeting, immune activation and delivery capacity, it is capable of breaking through traditional treatment limitations. Multi-omics analysis helps clarify long-term immune mechanisms and screen optimal therapeutic regimens. Combined with genome editing, synthetic biology and AI design, bacterial therapy shows great potential to reverse tumor drug resistance. Ultimately, the establishment of predictable, quantifiable and controllable personalized treatment strategies will further accelerate clinical transformation. We believe that bacterial living therapeutics will become an indispensable part of precision oncology, bringing new therapeutic options and better clinical outcomes for cancer patients.

Author contributions

Weihong Mao: Conceptualization, Data curation, Formal analysis, Investigation, Software, Supervision, Validation, and Writing-original draft. Chunmin Deng: Funding acquisition, Methodology, Project administration, Resources, Visualization, and Writing-review & editing.

Funding

This research was supported by the “Scientific research project of Suzhou Municipal Health Commissio” (QNXM2024091) and “Medical Innovation Application Research of Suzhou Science and Technology Bureau” (SYWD2024238).

Data availability

No datasets were generated or analysed during the current study.

Declarations

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Not applicable.

Competing interests

The authors declare no competing interests.