Probiotic Carriers for Tumor-Targeted Therapy: Applications and Challenges

Abstract

Cancer poses an escalating threat to global public health, characterized by continuous rises in both incidence and mortality. While conventional tumor interventions including surgery, chemotherapy and radiotherapy have made considerable progress, their clinical efficacy is severely constrained by systemic toxic side effects, inadequate targeting precision and frequent severe postoperative and treatment-related sequelae. Nanodrug delivery systems have evolved as a viable therapeutic alternative to optimize tumor targeting and minimize systemic toxicity. However, conventional nanocarriers suffer from rapid clearance by the mononuclear phagocyte system, and the intricate, heterogeneous tumor microenvironment (TME) further impairs effective intratumoral drug penetration and uniform distribution. As an emerging class of living delivery vectors, probiotics have recently gained extensive research attention by virtue of their intrinsic tumor tropism, unique capacity to adapt to and remodel the TME, and excellent synergistic compatibility with multiple therapeutic regimens. This review systematically elaborates the biological mechanisms governing the tumor-targeted accumulation and TME-responsive properties of probiotics, summarizes core strategies for probiotic genetic modification and hybrid system construction, and compares the strengths and applicable scenarios of diverse probiotic chassis strains. We further discuss the synergistic potential of probiotic-based platforms combined with immunotherapy, chemotherapy, radiotherapy and physical therapy. Finally, this article underscores the key bottlenecks and future research directions for the clinical translation of probiotic-based antitumor therapeutics, aiming to provide rigorous theoretical support for their broader clinical application in cancer treatment.

Graphical Abstract

Introduction

Cancer, which is one of the most important challenges in global public health. Its incidence and mortality continue to increase, and the number of deaths is projected to reach 29.4 million by 2040.¹ The prevention and treatment of high-incidence malignancies such as colorectal cancer, gastric cancer, and breast cancer remain especially daunting.² Current clinical treatments mainly include surgery, radiotherapy, and chemotherapy, yet all have significant limitations.^3–5 Although surgery can be curative at an early stage, more than 70% of patients are diagnosed at middle or late stages, thereby missing the optimal window for surgical intervention.⁶ Accordingly, there is an urgent need to develop novel anti-tumor strategies that are more effective and less toxic.

Nanodrug delivery systems, as an important area of tumor therapy research, can achieve tumor targeting to a certain extent while reducing systemic adverse effects. Based on the enhanced permeability and retention (EPR) effect, nanomedicines can selectively accumulate at tumor sites. Carriers such as poly (lactic-co-glycolic acid) nanoparticles, liposomes, and iron nanoparticles have been widely used in cancer therapy.^7,8 However, because drug carriers are rapidly cleared by the mononuclear phagocyte system in the liver and spleen, the efficiency of nanodrug delivery to solid tumors is typically below 1% (approximately 0.7%).⁹ Moreover, the complex tumor microenvironment, including dense extracellular matrix and elevated interstitial fluid pressure, further impedes drug penetration and homogeneous distribution, thereby weakening therapeutic efficacy.

Against this background, bacteria and their derivatives have attracted widespread attention as biological delivery carriers. Their intrinsic tumor tropism and colonization capacity may partially overcome stromal barriers and reduce nonspecific damage to healthy tissues. Among them, probiotics are regarded as particularly promising delivery “chassis”. Probiotics can colonize the gastrointestinal tract, oral cavity, and reproductive system; when administered in sufficient quantities, they can alter the microenvironment of specific host sites through their own activity and secreted metabolites.¹⁰

The principal characteristics of probiotics include acid resistance, bile resistance, immune-stimulatory activity, antagonism against pathogens, and antimutagenic effects.^11,12 Studies have shown that probiotics can intervene in cancer progression by modulating the tumor microenvironment through multiple dimensions. This review aims to clarify the biological mechanisms underlying probiotic tumor accumulation and microenvironmental responsiveness; summarize key modules in engineering modification and hybrid system design; compare the advantages, disadvantages, and suitability of different genera as chassis carriers; explore their integration with existing therapeutic modalities; and finally analyze the challenges and future frontiers of clinical translation, thereby providing a feasible reference for related research and translational development (Figure 1).

Figure 1.

Targeted tumor therapy using engineered probiotics. Systemically administered probiotic carriers accumulate in the tumor microenvironment (TME) and hypoxic core regions, enhance the efficacy of immunotherapy through T cells and dendritic cells, and simultaneously mediate drug release. When combined with chemotherapy and radiotherapy, this approach can generate synergistic antitumor effects.

Key Superiorities of Probiotic Carriers

The term “probiotic” derived from the Greek meaning “for life”, was introduced by Lilley and Stillwell. It generally refers to nonpathogenic live microorganisms that confer health benefits on the host when administered in adequate amounts.¹³ Owing to their distinctive biological properties, probiotics are gradually emerging as highly promising drug-delivery platforms for tumor therapy.

The Natural Homing Mechanism and Accumulation Mechanism of Tumor

During the expansion of cancerous tissue, excessive cell proliferation, abnormal angiogenesis, and insufficient perfusion jointly create hypoxic regions within the tumor microenvironment. This feature is particularly advantageous for certain obligate or facultative anaerobes, such as Escherichia coli and Clostridium, which exhibit tropism toward hypoxic niches and can therefore selectively accumulate and proliferate within tumors.^14–16 In contrast to conventional nanocarriers, which are often limited by inadequate tumor vascular perfusion and interception by the mononuclear phagocyte system in the liver and spleen,⁹ probiotics are better able to traverse abnormal vascular regions, stably accumulate locally within tumors, and reduce drug leakage,¹⁷ thereby enabling more sustained and efficient local therapy. Nevertheless, it must be emphasized that while this strong enrichment capacity offers therapeutic advantages, it may also entail biosafety risks.

Microenvironmental Responsiveness: Achieving Localised Tumour-Specific Triggering

TME is a dynamic ecosystem surrounding tumor cells and is composed mainly of blood vessels, fibroblasts, immune cells, extracellular matrix (ECM), and a variety of signaling molecules. Together, these components support tumor cell proliferation, growth, metastasis, and therapeutic resistance.¹⁸ Compared with normal tissues, the extracellular pH of most tumors is acidic, mainly due to enhanced glycolysis and the resulting production of lactate and H⁺, which establish an acidic milieu.¹⁹

Lactate and lactic acid generated by tumor cells can directly activate cancer-associated fibroblasts (CAFs).²⁰ In turn, activated CAFs exacerbate immunosuppression and tissue acidosis via the secretion of lactate and other bioactive mediators.²¹

Dual Functional Roles: Delivery Vehicle and Immunomodulator

Current research is increasingly focused on constructing multifunctional probiotic delivery systems capable not only of transporting therapeutic agents but also of participating in immune regulation. During the process of surface functionalization and the loading or delivery of therapeutic substances, probiotics can stimulate dendritic cells, lymphocytes, and macrophages to produce cytokines and chemokines, promote IgA-related immune responses, and induce IgA secretion.^22–25 Accordingly, probiotics may reshape tumor immune status by influencing antigen presentation, immune-cell infiltration, and inflammatory cytokine networks. Moreover, some probiotics, such as Clostridium butyricum, have been shown to enhance the efficacy of immune checkpoint inhibitor.²⁶ This suggests that the rational use of probiotic immunomodulatory properties may help transform “cold tumors” into “hot tumors”, although the risk of excessive immune activation and systemic inflammation must also be considered.

From Natural Bacteria to Controllable Drug-Loading Systems: Engineering and Hybrid Platforms

Selection of Chassis Strains

The construction of engineered probiotic delivery systems begins with the selection of an appropriate chassis strain. An ideal chassis should possess a good safety record and low pathogenicity, reliable in vivo tumor-colonization capacity, feasible routes of administration (such as oral delivery), and sufficient stability for industrial-scale fermentation and manufacturing. Therefore, evaluation criteria should be multidimensional rather than focused solely on targeting ability.

Engineering Strategies for Key Functional Modules

Engineering design typically centers on key modules including targeting, expression, and release.

Targeting Module

Targeting can be divided into two stages: “pre-targeting” and “post-targeting”. Pre-targeting emphasizes maintaining probiotic viability and survival before arrival at the target site. In the context of oral delivery, for example, probiotics must survive adverse gastric and intestinal conditions such as enzymatic degradation, low pH, and bile salts; adhere effectively to intestinal epithelial surfaces to establish colonization; maintain metabolic activity; compete with resident microorganisms; and ultimately release sufficient numbers at the target site in a controlled fashion to exert therapeutic effect.²⁷ Microbial encapsulation technology is a common strategy for protecting probiotics and improving targeting efficiency in oral delivery.²⁸ The most widely used encapsulation or microencapsulation techniques embed probiotics in protective matrices before administration, thereby improving survival during delivery and storage.^29,30 However, conventional microencapsulation still faces problems such as bacterial leakage and low in vivo delivery efficiency. Emerging single-cell nanocoating technologies offer new solutions to these limitations.³¹

Once viability and activity are preserved, the post-targeting stage focuses on improving local tumor enrichment, retention, and tissue penetration. Enhancing colonization and tissue penetration is critical for enabling system performance and for transforming natural bacteria into controllable platforms. Strategies based on chemotaxis, adhesion, and surface ligands can further strengthen accumulation and persistence at lesion sites. For example, Song used bioorthogonal reactions (azide and DBCO) to modify probiotic surfaces and regulate their adhesion to resident intestinal microbes, thereby enhancing colonization capacity.³²

Expression and Release Modules

Tumor-specific promoters and logic gating can markedly reduce background leakage, bringing the system closer to a “living drug factory” capable of “site-specific, on-demand” delivery. For example, a dual-responsive gated design in Escherichia coli Nissle 1917 (EcN) enables spatiotemporal control of induced expression while simultaneously triggering bacterial self-destruction, thereby achieving, to some extent, a closed-loop system of “on-demand” function and “clearability”.³³ Even after solving the questions of “when and where to express” and “how much to release”, further improvements are still needed in release precision and rate control. The former can exploit the tumor-targeting colonization ability and programmability of probiotics to achieve high-level local release of tumor neoantigens through genetic circuit design, thereby guiding in situ targeting of tumor neoantigens.³⁴ The latter can combine microbial cell-surface charge properties with self-assembly technologies; dual-coating designs or co-encapsulation strategies can further enable sustained and controlled release.³⁵

Design of Hybrid Systems

Based on safety and controllability, hybrid systems have gradually become a research hotspot for further improving robustness and therapeutic efficacy.

The basic concept is that “bacteria are responsible for homing and retention, while materials are responsible for loading and gating”, thereby increasing drug-loading capacity and reducing leakage risk. For example, a hybrid system was constructed by coupling Lactobacillus acidophilus (LA) with two-dimensional copper-molybdenum layered double hydroxide nanosheets (CoCuMo-LDH). Under exogenous near-infrared light stimulation, the CoCuMo-LDH gating device responds, effectively inducing apoptosis and promoting tumor eradication.³⁶

Innovative Advances and Design Strategies of Common Probiotic Carriers

EcN: The Most Mature General-Purpose Chassis for Engineering

Because of its well-developed genetic toolbox and its amenability to the construction of complex regulatory circuits, EcN has become one of the most widely used chassis strains in engineered probiotic delivery systems. Its advantages in drug delivery lie not only in tumor enrichment, but also in its capacity to control expression, release, and post-therapy bacterial clearance, thereby forming a closed-loop system in which “expression occurs only after reaching the tumor, release occurs only after reaching a threshold, and the bacteria can self-destruct after release”. For instance, one study constructed a dual-responsive engineered EcN strain by introducing operators associated with both lactate-responsive and temperature-responsive systems and decorating the bacterial surface with gold nanoparticles.³³ Through synergistic action, the system achieved spatiotemporally controlled release of cytolysin while simultaneously inducing bacterial release of lysozyme and self-destruction, thereby improving the precision and efficacy of antitumor therapy and illustrating the evolution of engineered bacteria from “expressible” systems to “precisely controllable” systems.

Furthermore, EcN can be engineered as bioactive factories that continuously produce immunomodulatory molecules in tumors to complement immune checkpoint therapy. The anti-PD-1/IL-12-engineered probiotic INP-EcN (IL-12 nanoparticle-loaded EcN) biosynthesizes anti-PD-1 and releases IL-12, eliciting systemic antitumor immunity through cascaded T-cell regulation (Figure 2).³⁷ At the same time, formulation strategies can improve oral delivery and the reversal of immunosuppression. For example, an oral microgel delivery system based on electrostatic interactions between chitosan (CS) and sodium alginate (SA), termed EcN@(CS-SA)₂, not only preserved the beneficial features of therapeutic E. coli but also synergistically enhanced the antitumor efficacy of the TGF-β inhibitor galunisertib (Gal).³⁸

Figure 2.

Liao developed anti-PD-1/IL-12 engineered probiotics (INP-EcN) as a living drug factory, using anti-PD-1 biosynthesis and IL-12 nanoengineering to regulate T-cell cascade responses. Cascade regulation based on CD8+ T cell immune checkpoint blockade (ICB) and cytokine-driven proinflammatory signaling can shift T cells from an exhausted to an activated state, offering new hope for clinical cancer immunotherapy. Solid arrows indicate process progression, functional activation, or signal transduction; upward arrows (↑) indicate increased response rate or numbers of activated T cells; downward arrows (↓) indicate reduced immune suppression or numbers of exhausted T cells. Reprinted with permission from Ref.37

Overall, EcN-engineered bacteria have evolved from single-function delivery carriers into programmable therapeutic platforms featuring integrated “localization–gating–output–clearance” cascades. With unique tumor-penetrating colonization capability, favorable intestinal stability, precise tumor-targeting performance, and the ability to modulate multiple signaling pathways (eg, Bax/Bcl-xL and AKT/PTEN), EcN stands out as a preferred chassis strain for biosafety-oriented therapeutic design. Nevertheless, its clinical translation remains constrained by unresolved challenges, including the demand for redundant safety engineering, guaranteed batch-to-batch consistency, and reliable in vivo clearance controllability. Accordingly, lactic acid bacteria are frequently prioritized for immune-dominant therapeutic scenarios due to their superior biosafety and excellent adaptability to oral administration. The comprehensive characteristics, advantages, and limitations of EcN and other probiotic chassis are systematically summarized in Table 1.

Table 1.

A Classification-Based Comparison of Different Probiotic Carriers by Chassis Type

Strain Type	Key Biological Characteristics	Key Engineering & Design Strategies	Therapeutic Mechanisms & Mechanism of Action	Advantages & Limitations	References
Escherichia coli Nissle 1917 (EcN)	Facultative anaerobe with robust tumor colonization capacity; well-characterized genetic background and mature synthetic biology toolkit	Intelligent logic gating: quorum-sensing-based “colonization–sensing–synchronized lysis” circuit; Living drug factory: in situ biosynthesis of nanobodies (eg, anti-CD47/PD-L1) or cytokines (IL-12); Surface modifications: biomineralized coatings or polymeric functionalization	Pulsatile release: quorum-triggered lysis for high-concentration payload delivery; Immune remodeling: T cell recruitment to drive cold-to-hot tumor transition	Advantages: Highly programmable with diverse functional modules; rapid clinical translation progress. Limitations: LPS-related inflammatory risks require management; safety kill switches may be needed	[33–38]
Lactic Acid Bacteria (LAB)	GRAS-certified (Generally Recognized as Safe); suitable for mucosal and localized immune applications	In situ vaccination: surface display of tumor antigens and immune adjuvants (eg, OX40, R848); Continuous secretion: chemokines (eg, Flt3L) or fusion proteins	Mucosal immune activation: local DC/NK cell stimulation; Antigen presentation: enhanced tumor antigen uptake and T-cell priming	Advantages: Extremely safe with high public acceptance; ideal for oral delivery. Limitations: Heterologous expression lower than E. coli; weaker plasmid stability	[39–43]
Bifidobacterium spp./Bifidobacterium longum	Obligate anaerobe with high specificity for hypoxic tumor cores; key regulator of intestinal homeostasis	Enzyme–prodrug therapy (DEPT): CD expression to convert 5-FC to 5-FU; Nano-hybrid systems: conjugation with therapeutics (eg, HMME@BiL)	Hypoxia-specific cytotoxicity: selective activity in hypoxic tumor cores; Microenvironment remodeling: Treg reduction, preserved CD8+ T-cell activity	Advantages: Exceptional tumor-targeting specificity; minimal systemic chemotoxicity. Limitations: Slow growth; highly oxygen-sensitive, strict storage requirements	[44–49]
Clostridium butyricum	Strong butyrate-producing ability; natural HDAC inhibitor; sporulation potential	Immune adjuvant: synergistic enhancer for ICI therapy; Metabolic intervention: pathway modulation via bioactive metabolites; Epigenetic regulation: HDAC inhibition for tumor apoptosis; Signaling blockade: Wnt suppression for proliferation inhibition	Epigenetic regulation: HDAC inhibition by butyrate for tumor apoptosis; Signaling blockade: Wnt pathway suppression for proliferation inhibition	Advantages: Spore-form gastric acid tolerance; natural synergy with immunotherapy. Limitations: Fewer genetic tools than EcN; potential colitis risk in specific contexts	[26,50–52]
Bacillus spores	Dormant form; extreme resilience; hydrophobic surface with large specific surface area and negative charge	Particulate carrier: cation-treated spores adsorb DNA for gene gun delivery; genetic engineering for heterologous antigen delivery to induce immunity	Barrier crossing: cargo protection against gastric degradation, gut-specific germination; Oral delivery: improved oral bioavailability for antigens and drugs	Advantages: Easy formulation and storage; effective oral delivery. Limitations: Therapeutic efficacy depends on germination efficiency induced by host environment	[53–60]

Abbreviations: CD, Cytosine Deaminase; DCs, Dendritic Cells; DEPT, Enzyme-Directed Prodrug Therapy; EcN, Escherichia coli Nissle 1917; 5-FC, 5-Fluorocytosine; 5-FU, 5-Fluorouracil; GRAS, Generally Recognized as Safe; HDAC, Histone Deacetylase; ICI, Immune Checkpoint Inhibitor; IL-12, Interleukin-12; LAB, Lactic Acid Bacteria; LPS, Lipopolysaccharide; NK cells, Natural killer cells; Treg, Regulatory T cells.

Engineered Lactic Acid Bacteria as in situ Vaccines: Local Immune Costimulatory Strategies

Lactic acid bacteria (LAB), such as Lactobacillus and Lactococcus lactis, are generally recognized as safe (GRAS) and are well suited for oral administration. They have long been used in fermented foods and can naturally colonize the human gastrointestinal tract and vaginal cavity.^61–64 In addition, their amenability to genetic engineering and adaptability to human-associated microenvironments have made LAB highly promising chassis strains for live bacterial drug delivery. Compared with conventional tumor vaccines that rely on complex ex vivo antigen preparation, engineered LAB-based in situ vaccination (ISV) strategies can achieve spatiotemporally coordinated antigen release, adjuvant exposure, and immune cell activation through local intratumoral delivery, thereby initiating antitumor immune responses directly within the tumor microenvironment. This process is typically accompanied by dendritic cell (DC) maturation, enhanced antigen cross-presentation, activation of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), and increased expression of effector molecules such as IFN-γ and TNF-α, ultimately promoting the conversion of immunosuppressive “cold” tumors into immune-active “hot” tumors.³⁹ Zhu⁴⁰ constructed an engineered Lactococcus lactis strain (FOLactis) secreting a Flt3L–OX40L fusion protein. Following intratumoral administration, the engineered L. lactis remodeled the immune microenvironment in both the injected tumors and tumor-draining lymph nodes, promoted DC-mediated cross-presentation, and enhanced NK-cell and CTL responses. FOLactis activated NK cells, cytotoxic T lymphocytes, and conventional type 1 dendritic cells (cDC1s) in tumors and tumor-draining lymph nodes, enhanced tumor antigen-specific immune responses, and drove the canonical cold-to-hot tumor transition (Figure 3).

Figure 3.

Schematic illustration of an engineered lactic acid bacteria-based in situ vaccination platform for local tumor immunotherapy. Intratumoral administration of engineered Lactococcus lactis remodels the immune microenvironment in the tumor and tumor-draining lymph nodes, enhances dendritic cell-mediated antigen cross-presentation, and activates NK cells and cytotoxic T lymphocytes. Solid arrows indicate cell migration, cytokine secretion, signalling transduction, or functional activation; dashed circular arrows indicate plasmid replication or gene circuit expression; red bold text indicates key biological outcomes; and red laser beams indicate primary tumour ablation leading to tumour cell death. Reprinted with permission from Ref.40

On this basis, another study simultaneously anchored α-OX40 costimulatory signals and the TLR7/8 agonist resiquimod (R848) on the LAB surface, achieving localized “costimulation plus innate immune activation” within the tumor microenvironment and thereby reinforcing ISV efficacy.⁴¹ This immunobacteria-based living (IBL) platform efficiently promotes dendritic cell (DC) maturation and rescues the functional exhaustion of CTLs. As a potent small-molecule immunoadjuvant, R848 specifically targets TLR7/8 and triggers the secretion of multiple proinflammatory cytokines, including IL-6 and TNF-α, which further activates antigen-presenting cells and initiates downstream immune signaling cascades.^42,43 The dual integration of costimulatory signals and innate adjuvants enables coordinated activation of both innate and adaptive immunity, exhibiting great potential for synergistic antitumor immunotherapy. In this design, intratumorally delivered OR@Lac efficiently enhances DC-mediated antigen cross-presentation in both the TME and tumor-draining lymph nodes (TDLNs). Serving as a versatile living delivery chassis, L. lactis distributes immunotherapeutic payloads throughout tumor tissues to maximize local therapeutic synergy.

In parallel with surface engineering strategies, a secretion-type LAB delivery system has also been developed, in which genetically modified L. lactis continuously secretes Flt3L–OX40L fusion proteins to sustain immune stimulation.⁴⁰ This technical advancement highlights a critical conceptual transition in ISV design, shifting from passive carrier-based payload delivery to the localized and sustained provision of immunostimulatory signals. Collectively, such LAB-based immunotherapeutic strategies markedly improve the efficiency of local tumor immune activation while minimizing systemic immune toxicity. Nevertheless, the clinical application of these approaches still requires careful control of inflammatory intensity and duration, as well as comprehensive consideration of interpatient immune heterogeneity. The overall strengths, mechanisms, and limitations of LAB chassis compared with other probiotic platforms are systematically summarized in Table 1.

Bifidobacterium Spp

Bifidobacterium plays important roles through colonization of the small and large intestine and participation in host metabolism. Its mechanisms include the production of beneficial factors, regulation of intestinal homeostasis, and adaptation to anaerobic environments.⁶⁵ Bifidobacterium exhibits numerous beneficial properties, including the inhibition of pathogenic bacteria, regulation of inflammatory responses, maintenance of intestinal barrier function, synthesis of vitamins and amino acids, enhancement of calcium bioavailability, and certain antitumor effects.^44,45

Both natural and genetically modified Bifidobacterium strains have demonstrated promise in targeted drug delivery for a variety of cancers. For example, in a dimethylbenz[a]anthracene-induced rat mammary tumor model, genetically engineered Bifidobacterium longum was shown to be a feasible tumor-specific gene-delivery carrier for breast cancer.⁴⁶ Fujimori et al combined Bifidobacterium with gene therapy by introducing the cytosine deaminase (CD) gene into B. longum, thereby developing Bifidobacterial Selective Targeting–Cytosine Deaminase (BEST-CD) therapy, which converts the prodrug 5-fluorocytosine (5-FC) into active 5-fluorouracil (5-FU) within tumors for Enzyme-directed prodrug therapy (DEPT) of hypoxic tumors.⁴⁷

Further, a genetically modified CD-expressing Bifidobacterium strain (APS001F) has been used in combination therapy. In a CT26 colorectal cancer mouse model, Shioya et al combined APS001F with 5-FC and anti-PD-1 monoclonal antibody. Compared with monotherapy, this regimen showed stronger antitumor efficacy and significantly prolonged survival, while reducing the number of CD4⁺ T cells and maintaining a stable proportion of CD8⁺ T cells, suggesting both enhanced antitumor activity and a degree of immune modulation.⁴⁸

In addition, nanoparticle-based chemotherapeutic formulations have attracted considerable interest due to their capacity for sustained release, improved stability, and targeted delivery. One study developed a biohybrid delivery system based on Bifidobacterium infantis for delivering drug-loaded nanoparticles to hypoxic breast tumors. This system, termed Bif@BDC-NPs, consists of B. infantis conjugated with doxorubicin-loaded albumin nanoparticles coated with chitosan. In an in vivo breast cancer model, Bif@BDC-NPs achieved efficient targeting and a tumor inhibition rate of 94%, while significantly reducing the typical side effects of doxorubicin (DOX) therapy.⁴⁹

Beyond immunotherapy combinations and nanomaterial modification, Lu grafted the sonosensitizer hematoporphyrin monomethyl ether (HMME) onto a Bifidobacterium longum strain to construct sonosensitized bifidobacteria (HMME@BiL). Owing to its hypoxia-directed chemotactic properties, this system achieved strong tumor-targeted sonodynamic therapeutic effects both in vitro and in vivo.⁶⁶ Overall, Bifidobacterium exhibits outstanding hypoxic tumor targeting specificity and versatile combinatorial therapeutic potential. A detailed overview of its unique strengths and inherent drawbacks relative to other probiotic carriers is presented in Table 1.

Clostridium butyricum

Within the genus Clostridium, Clostridium butyricum has strong butyrate-producing capacity and therefore considerable potential in tumor prevention and treatment, especially in studies of intestinal tumors. Many recent investigations have focused on its capacity to enhance the efficacy of immune checkpoint inhibitors (ICIs) in combination therapies. For example, Xie found that C. butyricum enhanced the antitumor efficacy of anti-programmed cell death protein-1 (PD-1) immunotherapy in colorectal cancer and proposed its use as an adjuvant to immune checkpoint blockade. Mechanistically, the surface protein secD of C. butyricum binds to the GRP78 receptor on colorectal cancer cells, thereby abrogating IL-6-mediated immunosuppression of CD8⁺ T cell (Figure 4).⁵⁰

Figure 4.

Schematic representation of the binding of Clostridium butyricum surface protein secD to the GRP78 receptor on colorectal cancer cells, thereby eliminating IL-6-mediated immunosuppression of CD8⁺ T cells. Humanized mouse models and patient-derived organoid/tumor-infiltrating lymphocyte (TIL) coculture models confirmed that C. butyricum suppresses colorectal tumorigenesis by modulating tumor-cell signaling and the immune microenvironment, promoting the conversion of tumors from “cold” to “hot” and increasing responsiveness to anti-PD-1 immunotherapy. Solid black arrows indicate pathway activation, molecular secretion, or functional regulation; blue downward arrows (↓) indicate reduced tumor burden; brown upward arrows (↑) indicate enhanced CD8⁺ CTL activation and upregulated LCK expression; brown downward arrows (↓) indicate downregulated IL-6 expression, reduced M2 TAM numbers, and suppressed PI3K-AKT/NF-κB signaling; red upward arrows (↑) indicate improved therapeutic efficacy or enhanced cytotoxicity; and gradient red-blue horizontal arrows indicate conversion from anti-PD-1 resistance to response. Reprinted with permission from Ref.50

However, another study evaluating the combined effects of levofloxacin (LVFX) and Clostridium butyricum MIYAIRI 588 (CBM588) on immune checkpoint inhibitor efficacy found that although the combination enhanced PD-1 therapy, it could also aggravate dextran (Dex) sulfate sodium (DSS)-induced colitis, indicating that immune activation may be accompanied by inflammatory risk.²⁶ In addition, studies have shown that C. butyricum significantly suppresses high-fat diet-induced intestinal tumor progression in Apc (min/+) mice. After treatment, tumor-cell proliferation decreased and apoptosis increased; these effects may be related to regulation of the Wnt signaling pathway and the gut microbiota, further highlighting the potential value of C. butyricum in colorectal cancer prevention and treatment.⁵¹ In short, Clostridium butyricum effectively reshapes intestinal tumor microenvironments and improves the responsiveness of immune checkpoint therapy, despite potential inflammatory risks. The key features of this gut-targeted probiotic chassis are summarized in Table 1.

Bacillus Spores

Spores of Bacillus species, including Bacillus subtilis, Bacillus pumilus, and Bacillus coagulans, have been widely used as oral probiotic formulations to improve the gut microbiota and enhance immune function.⁵² Bacillus spores are dormant life forms that can withstand harsh conditions, including extreme temperatures, acidic and alkaline environments, desiccation, and toxic chemicals.⁵³ Therefore, after oral administration, Bacillus spores can safely transit through the stomach and survive to germinate in the intestine. Owing to their hydrophobicity and large specific surface area, Bacillus spores can carry substantial amounts of hydrophobic drugs through adsorption or covalent conjugation. Zhen⁵⁴ exploited the hydrophobic components of the Bacillus coagulans spore shell (SS) to design a biomimetic spore nanoplatform (SS@Lipo/Hb/GOx/JQ1), in which the SS was associated with liposomes co-loaded with hemoglobin, glucose oxidase, and JQ1. By integrating glucose consumption and Fenton reaction-mediated oxidative damage with JQ1-enhanced immunomodulation, this platform achieved synergistic anticancer effects (Figure 5).

Figure 5.

(A) A spore-shell biomimetic nanoparticle platform developed from hydrophobic components in the spore shell (SS) of Bacillus coagulans to enhance BMAT, CDT, and antitumor immunity for synergistic therapy. Solid black arrows indicate catalytic reactions, signal transduction, or molecular release; red cross symbols indicate blockade of the PD-L1/PD-1 interaction to restore T-cell activation; and the dashed box in (B) magnifies the cascade reaction mechanism (glucose oxidation, Fenton reaction, and NLRP3 inflammasome activation) triggered by the nanocarrier in the tumor microenvironment. Reprinted with permission from Ref.54

When Bacillus spores germinate in the intestine, their outer layers rupture and degrade. Based on these characteristics, Bacillus spores are being explored as oral carriers for delivering a variety of beneficial substances to the gut.⁵⁵ Among them, Bacillus subtilis spores possess negatively charged surfaces under physiological conditions and can serve as ideal carriers for positively charged molecules, including proteins,⁵⁶ enzymes,⁵⁷ and viral particles.⁵⁸ It has also been shown that, after treatment with cationic molecules, purified spore surfaces can adsorb DNA. DNA-coated Bacillus subtilis spores have been used as microparticle carriers to replace gold microparticles for the biolistic delivery of plasmid DNA in mice, and spore-adsorbed DNA vaccines were efficiently delivered in vivo, inducing specific cellular and humoral immune responses.⁵⁹ In brief, most recombinant Bacillus spores have been used as carriers for delivering heterologous antigens to the gastrointestinal tract to induce immune responses, whereas nonrecombinant spores have served as oral carriers for heterologous molecules through covalent conjugation and adsorption.⁵⁷ In brief, Bacillus spores act as robust, orally adaptable delivery vehicles with excellent environmental tolerance for gut immune regulation and vaccine delivery. The comparative attributes of spore-based and other probiotic platforms are listed in.

Combination Therapies

As heterologous bacteria to humans, probiotics elicit diverse beneficial responses and exert multiple positive biological functions, as detailed in Figure 6.⁶⁰ Benefiting from advances in biosynthetic technology and their inherent structural and functional plasticity, probiotics can act as safe carriers or active components in combination with various therapeutic strategies. Such combinatorial regimens yield prominent synergistic effects, including enhanced local drug exposure and attenuated systemic toxicity, while overcoming the inherent drawbacks of monotherapy including immunosuppression, excessive toxicity, inadequate tissue penetration, and poor targeting.

Figure 6.

Schematic diagram of probiotic-based combination therapies. Reprinted with permission from Ref.60

Synergy with Immunotherapy: Local Expression and Systemic Immune Regulation

Immunotherapy is a powerful anticancer strategy but is hindered by low response rates, autoimmune toxicity, and nonspecific inflammation. Probiotics adapt to the gastrointestinal environment, mitigate microbial dysbiosis to reduce cancer-related disorders, enhance antigen presentation and immune cell infiltration through local expression, and alleviate systemic immune-related toxicity. In particular, anaerobic probiotics naturally target hypoxic tumor regions, driving the development of probiotic–immunotherapy combinations.⁶⁷

Li et al engineered tumor-resident living immunotherapeutic agents by modifying EcN with a triple immune nano-activator.⁶⁸ Tumor-specific ovalbumin (OVA) antigen and anti-PD-1 (α-PD-1) were conjugated to polydopamine (PDA) nanoparticles and anchored onto bacteria via in situ polymerization. The engineered probiotics enabled tumor-specific retention and proliferation-dependent drug release. The photothermal effect of PDA repolarized tumor-associated macrophages (TAMs) from M2 to proinflammatory M1 phenotype. OVA stimulated dendritic cells (DCs) to initiate antitumor immunity, while surface-anchored α-PD-1 blocked immune checkpoints and activated cytotoxic T lymphocytes, establishing a promising probiotic–immunotherapy platform.

Liu et al constructed the EcN-aPD1-S1 complex by coating EcN with a hybrid nano-surface carrying checkpoint inhibitors and viral antigens. Benefiting from EcN’s natural hypoxia tropism, the engineered strain achieved sustained release and prolonged immune stimulation of aPD1 and S1.⁶⁹ Lactic acid bacteria further enhance host immunity mainly by promoting the function of DCs, macrophages, and other myeloid cells, triggering their activation and cytokine secretion to strengthen antigen presentation and direct T-cell activation.^70–75

In addition, probiotics improve T-cell responses to checkpoint inhibitors by regulating immune-cell trafficking in the TME, including TAMs and myeloid cells.^76,77 Patients enriched in Bifidobacterium, Pseudomonas aeruginosa, and Enterococcus faecalis show better responses to checkpoint therapy, with improved T-cell activity and anti-PD-L1 efficacy.⁷⁸ In oral squamous cell carcinoma, anaerobic P. aeruginosa combined with nanoparticle-embedded hydrogels synergizes with PD-L1 therapy to boost antitumor immunity.⁷⁹ Notably, some probiotics strongly potentiate immunotherapy by activating CD8⁺ T cells and M1 macrophages.⁸⁰ For example, Akkermansia muciniphila combined with FOLFOX chemotherapy exerted superior antitumor effects in colon cancer models, inhibiting tumor growth, increasing infiltrating CD4⁺/CD8⁺ T cells and NK cells, reducing regulatory T cells (Tregs), and enhancing immune responses.⁸¹

With ongoing research, probiotics act as natural carriers to activate innate and adaptive immunity and serve as immune adjuvants to amplify therapeutic effects. Through synthetic biology, engineered probiotics overcome limitations of traditional carriers and provide efficient delivery platforms for cancer immunotherapy. Probiotic-based tumor vaccines also show great promise.³⁷

Synergy with Conventional Therapies

Combination of Probiotics and Chemotherapy

Chemotherapy remains a mainstay anticancer treatment, but its efficacy is limited by off-target toxicity and poor intratumoral drug delivery. Only a small fraction of intravenously administered drugs reaches tumor tissue, due to high solid stress and interstitial fluid pressure caused by rapid tumor proliferation, abnormal stromal accumulation, and impaired lymphatic drainage. Bacteria with tumor-colonizing and hypoxia-penetrating abilities can overcome these barriers.⁶⁰

Drug-loaded bacteria preferentially migrate to tumor stroma and release drugs in response to the TME. For instance, EcN was used as a carrier to conjugate DOX via an acid-sensitive linker, generating EcN-ca-Dox that enables tumor accumulation and pH-responsive drug release.⁸² As a well-studied, non-toxigenic probiotic, EcN remains viable after drug loading, proliferates in solid tumors but not in normal organs, and survives in both necrotic and oxygenated tumor regions. Importantly, EcN can be rapidly eliminated in vivo using antibiotics if adverse events occur.⁸³

Beyond direct antitumor effects, probiotics alleviate chemotherapy-induced toxicity, especially in mucosal and gastrointestinal tissues. In a nasopharyngeal carcinoma model, Lactobacillus rhamnosus-containing next-generation probiotics combined with chemoradiotherapy reduced severe oral mucositis from 14.7% to 2.8% and restored CD4⁺ T-cell proportions.⁸⁴ Probiotics also improve intestinal barrier function and microbial balance, reducing intestinal inflammation and immunosuppression. Faecalibacterium prausnitzii enhances barrier integrity and secretes short-chain fatty acids (SCFAs) to suppress tumors and mitigate chemotherapy toxicity.⁸⁵ Similarly, Lactobacillus plantarum and Bacteroides fragilis improve immune responses and reduce chemotherapy-related adverse effects.⁸⁶

Combination of Probiotics and Radiotherapy

Radiotherapy is a cornerstone of cancer treatment, but ionizing radiation injures rapidly dividing normal cells, especially gastrointestinal (GI) epithelial cells, causing acute enteropathy with nausea, vomiting, and diarrhea.⁸⁷ It also generates reactive oxygen species (ROS) that induce DNA damage and chronic inflammatory tissue injury, while tumor radioresistance further limits efficacy.^88,89 Given their ability to target the TME, regulate immunity, and exert radiosensitization, probiotics have been widely combined with radiotherapy.⁹⁰

The primary mechanism involves immunomodulation. Bioactive components from probiotics, including lipopolysaccharide (LPS), outer membrane vesicles (OMVs), peptidoglycans, and RNA, activate pattern recognition receptors (PRRs) through Toll-like receptors (TLRs), stimulating DC, macrophage, and neutrophil activation and interfering with the cancer-immunity cycle.^91,92 Clostridium species produce SCFAs (butyric, acetic, propionic acids) that modulate DC and macrophage functions.⁹³ Butyric and propionic acids downregulate LPS-induced IL-6 and IL-12 expression, enhance leukocyte migration, and inhibit proinflammatory chemokines.⁹⁴ Butyric acid also regulates DC costimulatory molecules and antigen presentation without directly affecting T-cell IFN-γ production.⁹⁵ SCFAs modulate Treg levels and enhance radio-chemotherapy efficacy; butyric acid inhibits histone deacetylases (HDACs) involved in cell-cycle regulation,⁹⁶ while indole metabolites also improve radiotherapy outcomes.⁹⁷ Butyric acid was shown to enhance radiosensitivity in colorectal cancer patient-derived organoids (PDOs) by activating FOXO3A and inducing cell-cycle arrest.⁹⁸

A second major mechanism is probiotic-mediated remodeling of the tumor microenvironment to enhance radiosensitivity. The TME differs markedly from normal tissue environments and greatly restricts clinical treatment. Some facultative anaerobes, including EcN, Bifidobacterium, and Clostridium, can proliferate extensively in hypoxic tumor regions.⁹⁹ Current strategies mainly aim to increase oxygen content in tumor tissue, for example by delivering oxygen-releasing therapeutics, destroying hypoxic tumor cells, or enhancing tumor blood circulation. Based on these principles, many groups have used bacteria and their extracellular vesicles to deliver radiosensitizing agents, turning them into innovative anticancer tools.¹⁰⁰ Multiple strains can serve as efficient carriers for transporting specific drugs to tumor sites and targeting hypoxic cells, thereby improving radiotherapy outcome.¹⁰¹ Fu demonstrated that combining Bifidobacterium infantis with a specific monoclonal antibody effectively disrupted hypoxic tumor regions, enhanced radiation-induced DNA double-strand breaks, and induced tumor-cell death in a Lewis lung carcinoma xenograft model.¹⁰²

Another promising approach is suicide gene therapy, in which bacteria-derived prodrug-sensitive genes are expressed in tumor tissue, converting nontoxic prodrugs into cancer-killing metabolites. In this context, bacterial toxins and the E. coli CD gene are of particular interes.¹⁰³ The CD gene encodes an enzyme that catalyzes the conversion of cytosine to uracil; when expressed in tumors, it can convert the prodrug 5-fluorocytosine into 5-fluorouracil, thereby producing a radiosensitizing effect.¹⁰⁴

Combination of Probiotics and Pharmacotherapy

Disorganized tumor vasculature and high interstitial pressure prevent most nanomedicines from penetrating the stroma, resulting in low tumor targeting efficiency.^105–108 Probiotics with inherent TME tropism provide a feasible strategy for precise drug delivery. Conjugating drug-loaded nanocarriers to bacterial surfaces enhances antitumor activity.^109,110

He covalently conjugated DOX-loaded CaP/SiO2 nanoparticles (DNPs) to Bi to construct DNPs@Bi for tumor-targeted therapy. On the one hand, the pH-responsive release of doxorubicin (DOX) could induce chemotherapeutic effects and immunogenic cell death within the immunosuppressive tumor microenvironment (ITME). On the other hand, the tumor-targeting biotherapeutic Bi significantly enhanced the presentation of tumor-associated antigens derived from B16F10 cells to dendritic cells (DCs).¹¹¹ However, achieving controlled drug release at target sites remains challenging in dynamic microenvironments such as tumors and inflamed tissues. In this context, probiotic spores (PS) have emerged as attractive carriers for controlled drug delivery. PS are dormant life forms with low water content and exhibit high resistance to harsh conditions, including heat, acidic or alkaline environments, ultraviolet irradiation, and various chemical agent.¹¹² Notably, PS can withstand the highly hostile gastric environment. After reaching the intestine, PS absorb water, shed their hydrophobic spore coat proteins, germinate into probiotic bacteria, and subsequently colonize the gut.^113,114 Following colonization, Bacillus can exert multiple biological effects, including modulation of the gut microbiota, secretion of therapeutic metabolites such as short-chain fatty acids, and enhancement of both innate and adaptive immunity.¹¹⁵

Inspired by these properties, researchers have exploited hydrophobic interactions, electrostatic adsorption, and hydrogen bonding to integrate PS with free drugs or drug-loaded nanoparticles. This strategy takes advantage of the natural controlled-release properties of spores while improving drug bioavailability. Song developed an oral nanoparticle generator in which chemotherapeutic agents, namely doxorubicin and sorafenib (DOX/SOR), were loaded into spores and further modified with deoxycholic acid (DA).¹¹⁶ This nanogenerator not only protected the payloads from the harsh intestinal environment, but also enabled the in situ generation of DOX/SOR/spore-DA nanoparticles, which effectively penetrated epithelial cells and thereby improved basolateral drug release. Both in vitro and in vivo studies demonstrated that this system autonomously generated large amounts of nanoparticles in the intestine, providing a new strategy for cancer therapy. Zheng grafted chemically modified prebiotic Dex onto commercially available Clostridium butyricum spores through host–guest chemistry to obtain Spores-Dex.¹¹⁶ The results showed that, after oral administration, Spores-Dex specifically accumulated at tumor sites. Dex can be fermented by bacteria such as C. butyricum to produce short-chain fatty acids, thereby significantly increasing the overall richness of the microbiota. This study highlighted the potential of modulating the gut microbiota through a highly safe strategy and suggested a promising approach for the treatment of various gastrointestinal diseases. In addition, probiotics/spores and free drugs or drug-loaded nanoparticles can also be encapsulated within hydrogels or microspheres to promote probiotic colonization and regulate drug release through stimulus-responsive release mechanisms.^117,118

Synergy with Physical Therapies

Combination with Photodynamic Therapy

Photodynamic therapy (PDT) uses photosensitizers to generate ROS under light irradiation, killing tumor cells and enhancing antitumor immunity. However, PDT is limited by poor tumor penetration, rapid photodynamic attenuation, and low photosensitizer retention.^119–121 Probiotics’ natural accumulation in hypoxic TMEs supports targeted PDT systems.

Because probiotics readily accumulate in hypoxic and ITME, researchers have developed probiotic-based tumor-targeted therapeutic systems. Guo et al constructed a photoacoustic–photothermal ternary system in BL21 cells.¹²² Intratumoral injection combined with irradiation significantly promoted tumor regression and survival in colon and breast cancer models. Yang et al coupled Lactobacillus acidophilus with 2D CoCuMo-LDH nanosheets for NIR-II PDT.³⁶ Low pH and high GSH in the TME induced nanosheet amorphization, enhancing photodynamic activity and tumor-cell apoptosis. Probiotic–photosensitizer combinations improve PDT efficacy and targeting without increasing systemic toxicity.

Combination with Photothermal Therapy

Photothermal therapy (PTT) employs photosensitizers with specific near-infrared absorption properties to convert light energy into heat and kill target cells. In bacteria-mediated PTT, photosensitizers are attached to bacterial surfaces to construct bacteria-driven photosensitizer delivery systems for precise tumor treatment. After efficiently binding photosensitizers, bacteria can remain viable and, by virtue of their self-propelled properties, penetrate deeply into solid tumors. Under near-infrared irradiation, photosensitizer-coated bacteria exhibit outstanding tumor-killing capacity and can completely eradicate solid tumors. This combined therapy has significant value in solid tumor treatment because of its high efficacy and low toxicity.¹²³

Chen’s group conjugated indocyanine green nanoparticles to attenuated Salmonella YB1, forming YB1-INP.¹²⁴ Hypoxia-targeted colonization enabled efficient tumor ablation and fluorescence imaging under NIR light. The safe strain VNP20009 was coated with PDA; after injection, it accumulated in tumors and generated hyperthermia to kill cancer cells with minimal organ damage.^125,126

Bacteria can be employed to in situ load functional nanomaterials through biomineralization, establishing naturally targeted bacterial photothermal platforms for precise cancer therapy.^127,128 Nevertheless, most bacteria-based photothermal systems rely solely on exogenously loaded photothermal agents, leading to monotonous therapeutic modes, incomplete tumor ablation, and a lack of controllable in situ photothermal conversion. Meanwhile, conventional engineered bacterial systems commonly adopt constitutive gene expression, which suffers from persistent off-target toxicity and poor spatiotemporal controllability, greatly hindering their clinical translation. Hu et al introduced upconversion nanoparticles (UCNPs) that efficiently convert near-infrared light into blue light, and genetically engineered EcN as a living vector to specifically express and secrete α-hemolysin (αHL), a pore-forming protein, under blue light stimulation, thereby constructing a near-infrared-responsive optogenetic bacterial system (EcNαHL-UCNPs). This system induces extravasation of blood components and in situ thrombosis within tumor tissue, which acts as an endogenous photothermal agent to achieve precise and efficient photothermal tumor therapy (Figure 7).¹²⁹

Figure 7.

Schematic illustration of the construction of EcNαHL-UCNPs and its antitumor immune response, enabling synergistic photothermal therapy via NIR-optogenetic release of αHL and subsequent thrombosis formation. Reprinted with permission from Ref.129

Combination with Sonodynamic Therapy

Sonodynamic therapy (SDT) combines low-frequency ultrasound with sonosensitizers to activate therapeutic agents within tumors, leading to the release of cytotoxic species. Hematoporphyrin monomethyl ether (HMME) has already been approved for tumor treatment. However, its low molecular weight, limited tumor accumulation, and poor retention severely restrict its utility in SDT. Moreover, the immunosuppressive nature of tumor tissue greatly limits the immunogenic antitumor effects of SDT.¹³⁰ In this regard, probiotics, owing to their accumulation and retention within hypoxic tumor cores, have become effective carriers for sonosensitizer.¹³¹

Lu constructed an HMME@BiL complex using Bifidobacterium longum (Figure 8).⁶⁶ This complex exploited the anaerobic properties of BiL to achieve tumor-targeted delivery of HMME, significantly improving tumor-site accumulation and producing effective SDT. When further combined with the STING agonist SR717, which promoted infiltration of cytotoxic T cells and NK cells into tumor tissue and stimulated cytokine release, the system effectively activated antitumor immunity and opened a new avenue for sonoimmunotherapy. In a CT26 colorectal cancer metastasis model, the combined treatment of HMME@BiL + ultrasound + SR717 effectively inhibited primary tumors and shortened disease progression.

Figure 8.

Schematic illustration of probiotic microorganism-based sono-immunotherapy combined with a STING agonist for cancer treatment. Solid black arrows indicate molecular transport, signalling activation, or therapeutic delivery; red upward arrows (↑) indicate increased expression of pro-inflammatory cytokines (IL-6, TNF-α, IFN-γ) and enhanced sonoimmunotherapy efficacy; blue downward arrows (↓) indicate exacerbated tumor hypoxia (decreased intratumoral O₂ level); and dashed boxes highlight key immune cell populations. Reprinted with permission from Ref.66

To address the problem of payload release outside tumors and the resulting risk to healthy tissues, Abedi used focused ultrasound to enable directed delivery of therapeutic microorganisms.¹³² They developed a genetically engineered EcN strain expressing a temperature-sensitive genetic switch and tumor-suppressive nanobodies. When focused ultrasound heated the tumor site, the engineered EcN expressed nanobodies that killed tumor cells. By introducing a temperature-dependent gene switch and a nanobody-encoding gene, the researchers created strains capable of synthesizing anticancer nanobodies at trigger temperatures of 42–43°C. These bacteria do not induce antitumor nanobody production at normal body temperature (37°C), but rather grow covertly within the tumor until externally applied heat raises them to the activation threshold. This technology provides a key tool for achieving spatiotemporally targeted probiotic therapy in a range of biological and clinical contexts.⁶⁰

Combination with Magnetodynamic Approaches

Although engineered probiotics exhibit adaptive tropism, their in vivo localization remains somewhat unpredictable and imprecise. Similarly, while magnetic therapy can penetrate biological tissues, it is limited in controlling deep-tissue deposition. Combining engineered probiotics with magnetic strategies therefore represents an attractive solution. By decorating probiotic surfaces with magnetic particles and applying external magnetic fields to guide their in vivo movement, probiotics can be delivered to diseased sites with improved precision.

Buss developed a cell localization strategy assisted by magnetic particles, in which oral administration was combined with an external magnetic field to enable the site-directed deposition of composite biomagnetic materials in vivo.¹³³ By combining probiotics (EcN BL21 and EcN 1917) with magnetic microparticles (carboxyl-functionalized superparamagnetic iron oxide microparticles), the probiotics could be captured and retained within the mouse gastrointestinal tract. This technology enhanced probiotic enrichment and colonization at target sites and provides a new concept for externally controlled microbial therapy.

Other Combinations in Bioimaging and Diagnosis

Probiotics are capable of colonizing diverse in vivo biointerfaces, particularly the oral mucosa, reproductive tract, and gastrointestinal tract, and can also preferentially accumulate at pathological sites including tumors. Owing to their intrinsic properties, including motility, genetic programmability, and targeting capability, probiotics integrated with imageable agents have emerged as promising living probes for bioimaging and disease diagnosis.^134,135 As important constituents of the mammalian microbiota, both Gram-positive and Gram-negative bacteria can colonize multiple biological interfaces in vivo, thereby influencing host homeostasis and disease progression.¹³⁵

Jiang engineered the probiotic strain EcN 1917 to coexpress firefly luciferase and luciferin-regenerating enzyme (LRE), enabling robust, continuous, and red-shifted bioluminescence for bacterial tracking in both in vitro and in vivo settings (Figure 9A).¹³⁶ This strategy established an optical in vivo tumor-targeting platform for monitoring bacterial behavior and provided a useful tool for investigating bacteria-mediated cancer therapy.

Figure 9.

Probiotic-based bioimaging and diagnosis. (A) EcN was engineered to contain the core module Fluc-LRE. Reprinted with the permission from Ref.136 (B) Schematic diagram of the components and sensory pathways of i- ROBOT. Upward arrows (↑) indicate increased expression of the anti-inflammatory cytokine IL-10; and downward arrows (↓) indicate decreased expression of pro-inflammatory cytokines (IL-6, TNF-α, MCP-1) and inflammatory T cells. Reprinted with the permission from Ref.137

Meanwhile, intelligent whole-cell biosensors engineered via synthetic biology have emerged as powerful tools for disease diagnosis and targeted therapy. Zou et al developed an intelligent probiotic platform named i-ROBOT for the detection, in situ recording, and therapeutic intervention of inflammatory bowel disease (IBD).¹³⁷ This platform is constructed based on the probiotic chassis EcN 1917, which is programmed to sense gradient levels of the IBD inflammatory biomarker thiosulfate. Thiosulfate stimulation activates the intracellular base-editing system, enabling heritable genomic recording and concomitant colorimetric signal output (Figure 9B). Furthermore, thiosulfate fluctuation serves as a regulatory switch to control the release of the immunomodulatory protein Acanthocheilonema viteae cystatin. In vivo experiments in colitis mice demonstrated that orally administered i-ROBOT successfully recorded intestinal molecular signatures in fecal and colon tissues and markedly alleviated IBD pathological symptoms. Collectively, this engineered living system presents great potential for the precise management of gastrointestinal disorders and other metabolic diseases.

Despite the growing evidence supporting the application of engineered probiotics in combination with other therapeutic strategies for diverse diseases, as well as their utility in bioimaging and diagnosis, biosafety remains a critical issue, particularly for modified probiotics. Genetic or functional modification may alter the native behavior of probiotic strains, potentially enhancing epithelial adhesion or reducing susceptibility to physiological clearance, thereby increasing the risk of unintended colonization and even pathogenicity. Moreover, some engineered probiotic strains may transfer exogenous genes to resident gut microorganisms, which could contribute to the dissemination of antibiotic resistance or other adverse health outcomes.^138,139

Combination of Chemotherapy and Physical Therapies with Probiotics

Through non-covalent conjugation, lipid-based nanoparticles have been widely employed to functionalize probiotics with therapeutic cargos, thereby enabling combinational strategies integrating chemotherapy with physical modalities such as PTT and magnetic guidance. Akolpoglu combined magnetic nanoparticles (mNPs) with nanoliposomes (NLs) co-loaded with a photothermal agent and a chemotherapeutic drug, and subsequently engineered ICG–DOX NL-decorated EcN with an assembly efficiency of approximately 90%.¹⁴⁰ As shown in Figure 10, this biohybrid microrobot was constructed by immobilizing synthetic components, streptavidin-coated mNPs, and biotinylated NLs onto the surface of EcN through non-covalent interactions.

Figure 10.

The construction of bacterial biohybrids carrying mNPs and NLs and the cancer cell killing efficiency. Reprinted with the permission from Ref.140

When applied to cancer cells in a three-dimensional tumor spheroid model, these bacterial biohybrids induced substantially higher levels of cell death than the control groups, and this therapeutic effect was further enhanced under near-infrared (NIR) irradiation. Notably, these bacterial biohybrids outperformed several previously reported EcN-based microrobotic systems because they preserved the intrinsic motility of the bacteria, enabling active migration through biological matrices and magnetic field-guided accumulation within tumor spheroids. In addition, they allowed on-demand release of therapeutic payloads in response to NIR stimulation. Collectively, these bacterial biohybrid constructs provide a multifunctional microrobotic platform for guided locomotion within three-dimensional biological networks and represent a promising stimulus-responsive therapeutic strategy for diverse biomedical applications.

Frontier Directions and Challenges

Frontier Directions

Genetically Engineered Probiotics for Tumor-Targeted Drug/Nucleic Acid Delivery

A central goal of current research is to use synthetic biology to engineer probiotics capable of specifically colonizing and proliferating within the tumor microenvironment and of precisely releasing therapeutic molecules on demand. Some groups have engineered anti-PD-1-producing probiotics, effectively transforming them into “living drug factories” capable of in situ anti-PD-1 generation. Such systems can continuously interfere with the PD-1/PD-L1 pathway, alleviate T-cell immunosuppression, and reduce immune-related adverse events.¹⁴¹

Liao’s team genetically and nanoengineered EcN to generate IL-12 nanoparticle-loaded EcN (INP-EcN), enabling intratumoral biosynthesis of anti-PD-1 antibodies and TME-responsive release of IL-12 agonist.³⁷ After systemic administration, INP-EcN selectively colonized the TME and delivered anti-PD-1 antibodies together with inducible IL-12 agonists in situ. Locally produced anti-PD-1 strongly and continuously interfered with PD-1/PD-L1-mediated immunosuppression, significantly restoring T-cell antitumor activity. Meanwhile, the TME-responsive release of IL-12 agonists effectively enhanced T-cell activation, proliferation, and infiltration.¹⁴² Conversely, anti-PD-1 could also compensate for immunosuppression induced by PD-L1 upregulation. Taken together, this anti-PD-1/IL-12-engineered living drug factory initiated relevant cascade regulation in vivo and promoted systemic integrated antitumor immunity, offering new possibilities for future cancer bioimmunotherapy (Figure 11A).

Figure 11.

(A) Western blot showing that engineered EcN can produce anti-PD-1 antibodies recognizing PD-1 protein. (B and C) Confocal imaging and flow cytometry showing the blocking efficacy of EcN-derived anti-PD-1 antibodies in a T cell–melanoma coculture system. (D) Growth comparison between EcN and INP-EcN. (E) ELISA monitoring anti-PD-1 production by EcN and INP-EcN from 0 to 72 h in vitro. (F) Release kinetics of IL-12 from INP and INP-EcN under simulated tumor (pH 6.5) and physiological (pH 7.4) environments. (G–I) T-cell proliferation, IFN-γ secretion, and cytotoxicity against B16F10 melanoma cells under INP-EcN treatment. Significance was calculated using one-way ANOVA with Dunnett’s post-test, *p < 0.05, 0.05 < **p < 0.01, and ***p < 0.001. Reprinted with permission from Ref.37

Western blot analysis first confirmed that the transfected bacteria could produce functional anti-PD-1 antibodies. The biosynthesized anti-PD-1 was detected as a 28 kDa protein, consistent with the expected molecular weight of the murine PD-1 inhibitor. Competition assays with commercial anti-PD-1 antibodies confirmed the antagonist capacity of the biosynthesized antibody. Functional assays, including coculture of mouse T cells with B16F10 melanoma cells (Figure 11B and C) combined with confocal immunofluorescence imaging and flow cytometry, demonstrated that the biosynthesized anti-PD-1 effectively blocked PD-1 surface exposure and inhibited PD-1/PD-L1 interaction. Importantly, no significant difference in bacterial bioactivity was observed before and after chemical conjugation (Figure 11D). ELISA further verified that INP-EcN could rapidly initiate and stably produce anti-PD-1, indicating that EcN’s proliferation and protein-production capacity were retained after engineering (Figure 11E). Because of the severe toxicity associated with systemic IL-12 administration, Liao also designed a TME-responsive polymer-encapsulated IL-12 nanoagonist, which showed responsive release, high cytotoxicity, and strong immune activation under tumor-like conditions (Figure 11F–I).³⁷

Spatiotemporally Controlled Release

Engineered probiotics can be programmed to initiate the expression and secretion of therapeutic molecules in response to distinct tumor microenvironmental cues, including hypoxia and acidic pH. For instance, one study employed the nonpathogenic Gram-negative probiotic EcN to construct a pH-responsive bacteria-powered microrobot for the targeted therapy of hypoxic intestinal tumors, thereby improving intratumoral drug penetration.¹⁴³ In another investigation, genetically engineered EcN was combined with azo-modified hyaluronic acid hydrogels and near-infrared irradiation to realize spatiotemporally controlled therapy against solid tumors. This strategy effectively restricted bacterial spread and off-target therapeutic release in healthy tissues, consequently enhancing the biosafety of the treatment.¹⁴⁴

Diverse Therapeutic Payloads

Engineered probiotics can deliver various therapeutic molecules to enhance tumor treatment. Common payloads include oncolytic toxins; for example, EcN has been engineered to produce the pore-forming theta toxin (Nis-Theta), which showed tumor-selective effects in pancreatic ductal adenocarcinoma models by overcoming drug-delivery barriers and the immunosuppressive microenvironment.¹⁴⁵ Engineered probiotics can also secrete chemokines or cytokines to remodel the TME and activate antitumor immunity. For instance, engineered Lactococcus lactis secreting soluble CD80 reduced tumor growth by 64%, while engineered EcN producing a PD-L1 nanobody achieved complete tumor regression in 50% of mice.¹⁴⁶ Other studies have delivered tumor-specific neoantigen epitopes through engineered probiotics,¹⁴⁷ including EcN-based delivery systems that stimulated specific CD4⁺ and CD8⁺ T-cell responses and thereby enhanced antitumor immunity.¹⁴⁸ Enzymes and gene-editing tools are also being incorporated into these delivery systems. One example is EcN engineered to continuously convert ammonia into L-arginine and further modified with PDA to form argEcN@P, which enhanced colorectal tumor clearance and demonstrated the potential of metabolic remodeling to boost tumor photoimmunotherapy.¹⁴⁹ Another is the use of CRISPR-Cas systems to precisely edit EcN, endowing it with nonnatural functions and paving the way for future innovations in probiotic engineering.¹⁵⁰

Dual Nanobody Delivery

Some groups have combined the above strategies to develop externally triggered dual-nanobody delivery systems. In such systems, bacteria lyse in response to near-infrared irradiation, releasing anti-PD-L1 and anti-CD9 nanobodies at tumor sites to target tumor-derived exosomes carrying PD-L1, thereby reversing tumor immunosuppression and effectively inhibiting tumor proliferation and metastasis.¹⁵¹

Probiotic-Mediated Immunoregulation and Synergistic Therapy

Probiotics, as beneficial members of the human microbiota, have become an important focus for enhancing tumor immunotherapy through their regulation of the gut microbiota and host immune responses.^64,152

Probiotic-Mediated Immunoregulation and Immune Checkpoint Blockade

The key role of the gut microbiota in tumorigenesis and in treatment with ICIs has become increasingly evident. Probiotics can regulate the gut microbiota and influence the tumor microenvironment, thereby improving responsiveness to immune checkpoint blockade. For example, a randomized Phase I study showed that renal cancer patients receiving Clostridium butyricum probiotic supplementation responded better to immunotherapy.¹⁵³ Probiotics can not only modulate the immune system but also improve tumor immune evasion by altering the TME, thereby offering new directions for cancer immunotherapy.

Probiotics and Multimodal Combination Therapy

The combination of probiotics with other therapeutic modalities is becoming a powerful tool for strengthening antitumor effects. By integrating probiotics with PTT, sonodynamic therapy, chemotherapy, and oncolytic virotherapy, synergistic antitumor effects can be achieved. For example, HMME combined with Bifidobacterium longum enabled tumor-targeted sonodynamic therapy, and further combination with the STING agonist SR717 enhanced antitumor immunity.⁶⁶ In another case, anaerobic Bifidobacterium infantis delivered bovine serum albumin nanoparticles loaded with doxorubicin to hypoxic breast tumors, overcoming poor chemotherapy efficacy and improving therapeutic outcomes.¹⁵⁴ In addition, OMVs from EcN have been used to encapsulate the oncolytic virus OH2, enhancing its tumor accumulation, remodeling tumor-associated macrophages, and killing prostate cancer cells.¹⁵⁵ OMVs, as naturally released nanocarriers, can activate immune responses through pathogen-associated molecular patterns and thus combine delivery and immunomodulatory functions.¹⁷ Microneedle delivery systems have also attracted considerable attention; dissolvable microneedle patches combining engineered EcN with L-arginine and indocyanine green composite nanoparticles can enable synergistic photothermal–immune therapy while enhancing local bacterial delivery and immune activation.¹⁵⁶

Probiotic–Nanomaterial Hybrid Carriers

The combination of probiotics with nanomaterials to form biohybrid systems can overcome the limitations of either probiotics or nanomaterials alone and significantly improve delivery efficiency and safety. In recent years, a variety of innovative nanohybrid carriers have been developed for tumor therapy.

Bacterial OMVs, as nanocarriers, offer multiple advantages including safety, stability, and ease of modification. They can target therapeutic molecules to tumors and improve efficacy through immune activation. Studies have shown that OMVs activate the immune system through pathogen-associated molecular patterns and possess good biocompatibility and therapeutic potential.^155,157 OMV-based carriers have also attracted interest in combination with PTT; for example, encapsulating oncolytic adenovirus within engineered exosomal microcapsules significantly enhanced viral enrichment within tumor tissue and improved immunotherapeutic efficacy.¹⁵³

Researchers have also exploited nonreplicative bacterial forms, including bacterial minicells and bacterial ghosts. These nonreplicative forms can serve as drug carriers while reducing potential biosafety risks. Because they do not proliferate, they can deliver drugs stably with fewer adverse effects on the host.¹⁷ Other material-based combinations are also highly active research areas. For instance, carbon-dot-labeled Lactiplantibacillus plantarum has been used as a multifunctional biocarrier capable of both anticancer drug delivery and bioimaging, thereby supporting targeted delivery and real-time monitoring.¹⁵⁸ Hydrogels loaded with probiotics, particularly protein/polysaccharide-based hydrogels, can effectively protect probiotic viability and achieve controlled release, thereby improving therapeutic durability and efficacy.¹⁵⁹ Ultimately, single-cell encapsulation techniques afford personalized protection to probiotics, thereby surmounting the inherent limitations of conventional encapsulation strategies.¹⁶⁰ Such approaches enhance tolerance to oxidative stress and harsh gastrointestinal conditions, facilitate site-specific controlled release, and prolong product shelf life.¹⁶¹

Risks and Challenges

Biosafety Challenges

Biosafety is a core issue in the use of engineered probiotics for cancer therapy. It is essential to strictly control the pathogenic potential of engineered strains, the risk of horizontal gene transfer, and the stability of in vivo colonization. Although many probiotics are considered nonpathogenic, potential risks remain in immunocompromised patients and in individuals receiving long-term treatment.¹⁶² To address this, researchers are actively developing safe delivery platforms incorporating “kill switches”, such as auxotrophic or temperature-sensitive self-destruction switches. These mechanisms can ensure timely elimination of probiotics after they have achieved the intended therapeutic effect, thereby reducing adverse effects and misuse risk.

In vivo Colonization Kinetics and Quantitative Imaging

More precise technologies are needed to track probiotic distribution, colonization, and activity in vivo.¹²⁴ At present, the colonization kinetics of probiotics within the body remain incompletely understood, and quantifying their distribution across tissues—especially within the tumor microenvironment—remains a major challenge. Further development of quantitative imaging technologies will help clarify the mechanisms and therapeutic value of probiotics in cancer therapy and thereby optimize treatment efficacy.

Large-Scale GMP Manufacturing and Clinical Translation

Translating probiotic formulations from the laboratory into clinically compliant products remains highly challenging. Key issues include manufacturing cost, formulation stability and purity, and batch-to-batch consistency. GMP-grade production requires standardized workflows and stringent quality control. This involves not only control over strain stability, viability, and purity, but also the production environment, production scale, and consistency of the final product. Ensuring quality and safety during large-scale production is crucial for advancing clinical translation.

Drug Delivery Efficiency

Before reaching tumor sites, probiotics may encounter physiological barriers such as gastric acid, bile salts, and digestive enzymes, which can reduce viability or even completely inactivate them, thereby affecting targeting efficiency and therapeutic efficacy. In response, researchers are actively developing more effective delivery systems to protect probiotic viability and ensure successful arrival at tumor sites. Technologies such as microencapsulation, nanocoating, emulsions, and hydrogels are being used to protect probiotics and improve gastrointestinal survival and targeting efficiency. Single-cell encapsulation provides individualized protection and better addresses environmental challenges during production, storage, and in vivo transport.¹⁶³ In addition, hybrid systems formed by combining probiotics with nanomaterials are receiving increasing attention, as such combinations can further enhance drug-loading capacity and targeting while protecting probiotics from degradation.¹⁶⁴

Complexity of Host–Microbiome Interactions

Interindividual differences in gut microbiota and their complex interactions with the host immune system may lead to substantial variability in probiotic-related therapeutic outcomes.¹⁶⁵ Differences in microbial composition and function among patients, as well as variation in physiological environments, can influence probiotic colonization, proliferation, and immune modulation, ultimately producing different therapeutic effects. Therefore, an important future direction for safer clinical translation will be the development of personalized probiotic therapeutic strategies and the use of precise microbiome analyses to predict patient responsiveness.¹⁶⁶

Conclusion

In summary, probiotics, as “living drugs”, hold enormous promise for precision tumor therapy. However, their broad clinical application will require overcoming numerous risks and challenges. By addressing issues such as delivery efficiency, and interindividual variation in the host microbiome, probiotics may come to play a unique and important role in future cancer treatment.

Funding Statement

This work was supported by Natural Science Foundation of Jilin Province (YDZJ202501ZYTS821).

Data Sharing Statement

Data sharing is not applicable to this article, as no datasets were generated or analysed during the current study.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no competing interests in this work.