Nanomaterial‐assisted oncolytic bacteria in solid tumor diagnosis and therapeutics

Abstract Cancer presents a formidable challenge in modern medicine due to the intratumoral heterogeneity and the dynamic microenvironmental niche. Natural or genetically engineered oncolytic bacteria have always been hailed by scientists for their intrinsic tumor‐targeting and oncolytic capacities. However, the immunogenicity and low toxicity inevitably constrain their application in clinical practice. When nanomaterials, characterized by distinctive physicochemical properties, are integrated with oncolytic bacteria, they achieve mutually complementary advantages and construct efficient and safe nanobiohybrids. In this review, we initially analyze the merits and drawbacks of conventional tumor therapeutic approaches, followed by a detailed examination of the precise oncolysis mechanisms employed by oncolytic bacteria. Subsequently, we focus on harnessing nanomaterial‐assisted oncolytic bacteria (NAOB) to augment the effectiveness of tumor therapy and utilizing them as nanotheranostic agents for imaging‐guided tumor treatment. Finally, by summarizing and analyzing the current deficiencies of NAOB, this review provides some innovative directions for developing nanobiohybrids, intending to infuse novel research concepts into the realm of solid tumor therapy.


| INTRODUCTION
Oncolytic bacteria are genetically engineered or naturally occurring bacterial species with the remarkable ability to selectively target, colonize, and ultimately eradicate cancer cells.2][3][4][5] Numerous types and strains of bacteria, such as Salmonella, Escherichia coli (E.7][8][9] Notably, these promising candidates for advanced cancer treatment strategies show preferential colonization of hypoxic regions within tumors, 10 excellent intratumoral penetration, 11 and the ability to deliver drugs via convenient genetic manipulation. 12Despite exhibiting unique characteristics, oncolytic bacteria often pose inevitable side effects and dose-dependent toxicity due to their high pathogenicity and immunogenicity, even in instances using live attenuated strains. 13Additionally, the limitations imposed by the single route of administration and the body's inherent clearance mechanisms lead to insufficient intratumoral colonization and accumulation. 14,15Low clinical efficacy severely restricted the advancement and practical application of oncolytic bacteria, necessitating the urgent development of strategies that enhance effectiveness while minimizing risks. Nanomaterials are materials with at least one dimension within the 1-100 nanometer scale, exhibiting exceptional optical, electrical, magnetic, and thermodynamic properties. 16[19] These effects enable nanomaterials to assume distinctive benefits in tumor diagnosis and treatment, such as ideal biocompatibility, drugtargeted delivery, controlled release, and multimode bioimaging. 20,21tegrating customized functional materials with oncolytic bacteria holds immense potential and practical value in tumor therapy.As targeted carriers, nanomaterials surface-modified with different molecules like chemotherapeutic drugs and genes present an ideal solution for minimizing damage to normal cells and mitigating undesired toxicity. 22,235][26] As therapeutic agents, multifunctional nanomaterials bestow oncolytic bacteria with characteristics that surpass their inherent capabilities.[29][30][31] These nanomaterials also serve as imaging probes, generating specific signals for tumor therapy visualization. 32Nanomaterial-assisted oncolytic bacteria (NAOB) further broaden the means and modes of oncolytic bacterial therapy, leading to precision tumor therapy.
This review comprehensively summarizes the various applications of NAOB in solid tumor diagnosis and treatment.Specifically, we focus on leveraging them to augment the tumor's therapeutic efficacy and act as nanotheranostic agents for visualizing treatment pathways.
Besides, to fully exploit the potential of NAOB in clinical practice, we undertake a rigorous assessment of the current research in this field and provide recommendations for further investigations.Overall, this review provides an essential reference for researchers and clinicians to expand the indications for bacterial-mediated tumor therapy and develop innovative and effective treatment strategies for solid tumors.

| ASSESSMENT OF TUMOR THERAPY: MERITS AND DEMERITS OF CONVENTIONAL TREATMENT METHODS
Cancer is a severe global public health problem with extremely high morbidity and mortality.According to the American Cancer Society, in 2024, there will be an estimated 2,001,140 new cases and 611,720 deaths from cancer in the United States. 33Tumor development, characterized by aberrant cell differentiation and hyper-proliferation, is a complex process. 34Composed of these aberrant cells, the tumor microenvironment (TME) is hypoxic, acidic, and immunosuppressive, which makes solid tumors challenging to treat.The hypoxic TME triggers anaerobic glycolysis in tumor cells and stimulates the production of neovascularization factors, like vascular endothelial growth factor (VEGF). 35Anaerobic glycolysis exacerbates excess lactic acid production and activates specific proteases, which may be responsible for turning off some chemotherapy drugs. 36VEGF induces vascular hyper-permeability and promotes abnormal neovascularization, 37 contributing to generating the immunosuppressive TME. 38,39Encouragingly, these current challenges in cancer treatment have vigorously promoted the continuous development of novel therapeutic approaches.Surgery, radiotherapy, and chemotherapy are orthodox treatment methods for solid tumors, and each has unique virtues and drawbacks.Surgical extirpation is the preferred choice for most patients with early or intermediate-stage solid tumors, which is highly effective and easy to perform.Tumors and corresponding organs can be removed directly without considering cell proliferation or treatment sensitivity.
However, surgery impairs the immune system and may not guarantee complete cancer cell eradication, bringing about recurrence and metastasis of some malignant tumors. 40A recent study demonstrated that neutrophils were activated and subsequently underwent NETosis in the TME and nearby surgical wound, which may be a vital postoperative risk factor for postoperative tumor relapse and metastasis. 41ile exerting therapeutic effects, radiotherapy can also induce DNA damage by causing double-strand breaks, leading to cell apoptosis or necrosis. 42Additionally, radiation contributes to genetic mutations, mainly characterized by small fragment deletions closely associated with tumor radiotherapy resistance. 43Another feature of radiotherapy is that the therapeutic effect often depends on cell susceptibility to radiation.Scientists showed that the imbalanced cytokinesis of normal esophageal cells might make it difficult for radiation to damage all oesophageal squamous cell carcinoma cells. 44With the higher fraction of dividing daughter cells, an aberrant rise in cell proliferation rates and squeezing of surrounding normal esophageal epitheliums resulted in tumor recurrence.However, high-precision radiotherapy is capable of reducing local recurrence, 45 downsizing the primary tumor, 46 and improving overall survival.For patients who cannot undergo surgery, radiotherapy is considered a life-prolonging alternative. 47The above traits make palliative and curative radiotherapy recommended for nasopharyngeal carcinoma and prostate cancer. 48,49fferent from surgery and radiotherapy, chemotherapy is usually a systemic therapy.Chemotherapeutic agents damage tumor cells by interfering with cell division, dysregulating cellular metabolism, and inhibiting nucleic acid or protein biosynthesis. 50Multidrug resistance is a significant factor in chemotherapy failure, and it may involve protein-protein interactions, oxidative stress, and genomic mutations.
The WNT16B released by fibroblasts after chemotherapy damage can weaken the cytotoxicity of chemotherapy drugs in vivo and promote tumor cell survival. 51High-level reactive oxygen species (ROS) are critical in maintaining cellular redox balance.However, the interaction between the inhibitor of apoptosis-stimulating protein of p53 (iASPP) and the antioxidant core factor Nrf2 could suppress their production.
The iASPP/Nrf2/ROS signaling pathway may be pivotal in renal carcinoma resistance to 5-fluorouracil (5-FU). 52During neoadjuvant chemotherapy, tumor cells changed their genome and phenotypic evolution in triple-negative breast cancer. 53The experimental results indicate that resistant genotypes are pre-existing and adaptively selected by cancer cells.With the increasing emphasis on individualization and low side effects in cancer therapy, molecular-targeted therapy has emerged.Although the efficacy of molecular-targeted therapy varies widely among individuals, it is possible to achieve long-term disease control by combining it with selective drug combinations for different tumor resistance mechanisms. 54spite the plethora of available tumor treatment methods, it has become increasingly apparent in clinical practice that each approach needs to be revised.In 2019, an article in Nature reported that an engineered E. coli strain could induce sustained tumor regression. 31is study first substantiated an abscopal effect caused by oncolytic bacteria, showcasing their ability to generate potent, tumor-specific adaptive immune responses with systemic efficacy in clearing distant tumor lesions.Subsequently, mounting evidence indicates the substantial promise of oncolytic bacteria in tumor therapy.

| The history of oncolytic bacterial therapy
The origin of oncolytic bacterial therapy dates back to the late nineteenth century when William Coley creatively injected a combination of heat-killed Streptococcus pyogenes and Serratia marcescens (known as Coley's toxin) into patients with osteosarcoma. 55However, the high degree of individual variability, poor reproducibility, and unknown mechanisms severely hampered this approach as a routine oncology treatment.Progress in this field met a turning point in 2012 when scientists, such as Karbach, reported that the mechanism of Coley's toxin was to reawaken the host's immune system to suppress tumor cells. 56This new finding renewed researchers' interest in Coley's toxin, with Teoh following Coley's work and validating that heat-inactivated Clostridium sporogenes reduced the proliferation of CT26 and HCT116 colorectal cancer cells to 20% and 26.2%, respectively. 57These works suggest that bacterial therapeutics is a promising area for further investigation by researchers as a novel approach to dealing with solid tumors.Autophagy is a catabolic process widespread in eukaryotes and essentially a lysosome-dependent protein degradation pathway. 58Lucas et al.

| Multifaceted oncolytic mechanisms
observed high mRNA expression levels of specific autophagy-related genes when the colibactin-producing E. coli (CoPEC) strain colonized the intestinal mucosa.It suggested that CoPEC infection triggered autophagy in colonic mucosal epithelial cells, potentially preventing DNA double-strand breaks in healthy cells and the occurrence of colorectal cancer. 1 The caspase family of cysteine proteases directly leads to apoptotic and is central to the network of apoptotic mechanisms.
Research has shown that engineered Salmonella Typhimurium (S.Typhimurium) SL7207 transfected with a luminescent gene cluster induced caspase-dependent apoptosis in HepG2 hepatocellular carcinoma cells by activating Caspase-3. 2Intracellular pro-apoptotic signaling molecules include oxidative stress (ROS, glutathione, etc.), cytochrome C, calcium ions (Ca 2+ ), and endoplasmic reticulum stress.Among these, ROS are crucial to caspase-independent apoptosis. 59Lactobacillus can enhance multifarious ROS-dependent apoptosis-inducing signals, 60 while Listeria monocytogenes can increase intracellular levels of Ca 2+ and ROS by activating nicotinamide adenine dinucleotide phosphate (NADP + ) oxidase. 61Both of these mechanisms result in high levels of ROS production and directly induce apoptosis of tumor cells through intracellular oxidative damage.Utilizing oncolytic bacteria to produce cytotoxic proteins at the tumor site enables targeted destruction of non-programmed cancer cell necrosis.Alpha-toxin secreted by Clostridium novyi-NT (C.novyi-NT), 62 the Exotoxin T produced by Pseudomonas aeruginosa, 63 and the pore-forming toxin produced by Clostridium perfringens 64 65 Simultaneously, the release of vasodilator inflammatory factors further exacerbated the formation of hemorrhagic inflammation, prompting blood outflow and the construction of thrombi.Qin and colleagues conducted a comparable study and found that infection with E. coli MG1655, expressing cytolysin A (ClyA), resulted in hemoconcentration at tumor sites with a substantial increase in hemoglobin levels.Eventually, it facilitated thrombosis, preventing tumor growth by disrupting the nutrient supply. 4A principal target of current anti-angiogenic agents is the vascular endothelium cells (VECs), a pivotal participant in angiogenesis.Tumstatin and HM-3 serve as two distinct tumor-specific angiogenesis inhibitors.Tumstatin suppresses the proliferation of VECs by downregulating VEGF-A, while HM-3 inhibits the migration of VECs by binding to integrin αvβ3.Two independent studies have shown a noticeable decrease in the expression of platelet endothelial cell adhesion molecule-1 (CD31, a marker of vascular endothelial differentiation) when utilizing S. Typhimurium VNP20009 in combination with Tumstatin and HM-3.The investigation suggests the competence of oncolytic bacteria in effectively targeting tumor vessels and impeding the angiogenesis process. 66,67e innate immune system employs pattern-recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) in oncolytic bacteria, with Toll-like receptors (TLRs) playing a crucial role in this initial recognition. 68Salmonella flagella can activate TLR5 in antigen-presenting cells (APCs like macrophages). 69TLR5 further initiates the activation of the Nuclear factor kappa-B (NF-κB) pathway, which promotes the enormous production of interferon (IFN), interleukin-17 (IL-17), and macrophage chemokines. 70Simultaneously, major histocompatibility complex class II molecules situated on APCs engage with the tyrosine kinase Btk through the costimulatory molecule cluster of differentiation (CD) 40, facilitating the synthesis of tumor necrosis factor (TNF). 71 Both pathways elicit robust responses from CD4 + helper T (Th) cells and CD8 + cytotoxic T cells (CTLs), ultimately leading to the potent antitumor effect through the secretion of inflammatory cytokines and cytotoxic molecules. 72,73 concert with TNF signaling, lipopolysaccharide (LPS) activates downstream TLR4/NF-κB signaling pathway, augmenting intestinal B cell survival and proliferation. 74TLR2 recognizes lipoprotein, 75 and TLR9 identifies bacterial DNA containing cytosine-phosphate-guanine (CpG) dideoxynucleotides. 76The rapid progress in synthetic biology has facilitated the innovative development of genetically engineered bacteria capable of producing and expressing various immunotherapeutics.IFN-γ is a widely recognized cytokine immunomodulator known for its antitumor effects and stimulation of immune cells. 77In a recent study, researchers conducted attenuated S. Typhimurium with the ability to synthesize IL-18, a factor known for inducing IFN-γ production and immune cell activation. 78The stimulator of interferon genes (STING) is a cell-intrinsic metabolic checkpoint in innate immunity. 79Daniel et al. genetically modified a strain of E. coli called SYNB1891 to express STING-agonist cyclic diAMP.Upon intratumoral injection of SYNB1891 into murine tumors, a substantial upregulation of IFN-γ was observed. 80Another fundamental mode of oncolytic bacteria-mediated immunotherapy is the reactivation of the TME.Engineered E. coli, designed to deliver immune checkpoint inhibitors targeting programmed cell death protein-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4), recruits and activates Th cells while reducing the proportion of regulatory T (Treg) cells. 81The injection of E. coli outer membrane vesicles (OMVs) as exogenous antigens into tumors similarly induced a durable therapeutic response mediated by the adaptive immune system. 82

| Advantages and disadvantages
Natural oncolytic bacteria can penetrate the peripheral bloodstream, accumulate and proliferate within hypoxic regions, and present the F I G U R E 1 A comprehensive overview of the mechanisms underlying tumor eradication through oncolytic bacteria.
tumor-suppressive effect. 10In contrast to inactivated bacteria, live bacteria have the fabulous talent to target tumors through intratumoral colonization.For example, C. novyi localized precisely in glioblastomas, whereas no colonization was observed in normal brain tissues. 83Bacterial motility can overcome the diffusion limitation of chemotherapeutic drugs, allowing them to penetrate tumor tissue and treat drug-resistant areas away from the vasculature. 84Motility also influences the degree of colonization and spatial distribution of oncolytic bacteria. 85Unlike live cell-dependent oncolytic viruses, oncolytic bacteria are safer and easier to control with medication. 86Despite numerous studies reporting the remarkable efficacy of oncolytic bacteria against tumors, their application as anticancer agents can be challenging.First, some strains remain toxic even at therapeutic doses, while reducing the dosage can compromise treatment efficacy. 14,87ditionally, probiotic bacteria, like Lactobacillus, may be associated with potential side effects such as systemic infections, adverse metabolic activities, and excessive immune stimulation in susceptible individuals. 88Engineered bacteria maintain wild bacteria's kinetic activity and reduce toxicity.Within the appropriate dosage range, engineered bacteria are harmless to the organism and target and colonize tumors more precisely.Nonetheless, engineered bacteria exhibit inherent instability, and the potential for genetic mutations over time introduces the risk of functional escape. 89,90Therefore, utilizing oncolytic bacterial therapy as a monotherapy cannot provide an efficient and safe approach to antitumor treatment.

| ADVANCING SOLID TUMOR THERAPEUTICS: APPLICATIONS OF NANOMATERIAL-ASSISTED ONCOLYTIC BACTERIA
The first section focuses on boosting therapeutic effectiveness through nanomaterial-assisted strategies, which involve encapsulating oncolytic bacteria during tumor-targeted transportation, collaboratively synthesizing antitumor agents, and modulating the TME to trigger immune responses.In the second section, we illustrate and list some representative bacterial-nanotheranostic agents that integrate tumor imaging and treatment (Table 1).Generally, our review provides an analysis of the specific applications of NAOB in tumor treatment and visualization while offering insights into the bright prospects of this approach (Figure 2).

| Bacterial encapsulation
2][93] Bacterial encapsulation presents appreciable potential for clinical applications of targeted bacterial delivery based on incorporating nanoencapsulation technology with oncolytic bacteria. 94colytic bacteria may exhibit their well-tried therapeutic effects by transient colonization of the host's GI tract mucosal surface.Thus, selecting nanomaterials with mucoadhesive properties is a staple consideration in prolonging the temporary retention of oncolytic bacteria in the GI tract. 95Silk fibroin is an anti-inflammatory protein that targets ulcers and damaged areas of the intestine while generating protective nanoshells by self-assembling on the surface of nanoparticles (from a random coil to β-sheet conformation). 96With these ascendant traits, silk fibroin nanocoated E. coli Nissle 1917 (EcN) outperformed uncoated germs in a 5.8-fold higher intestinal colonization and improved oral bioavailability.By tannic acid (TA) and Fe III chelation, Luo et al. synthesized a polyphenol-metal nanocoated EcN (TA@EcN) capable of encoding bacterial colonization and therapeutic modalities (Figure 3a). 25 The TA coating resulted in an astounding 41.3-fold, 39.6-fold, and 30.1-fold increase of EcN to adherently colonize the jejunum, cecum, and colon (Figure 3b).In the in vivo assessment, the oral bioavailability of TA@EcN was 32.7 times higher than native EcN, suggesting that nanocoatings can endow robust bacterial localization to enhance therapeutic effects synergistically.
From bacterial culture to GI tract colonization, the viability of oncolytic bacteria is paramount during manufacturing.The carboxymethyl cellulose-chitosan (CMC-Cht) hybrid micro-and macroparticles are potent, heat-resistant, and insensitive to the potential of hydrogen (pH) changes.Singh et al. successfully encapsulated Lactobacillus rhamnosus GG in CMC-Cht particles and validated the acceptable viability in all CMC-Cht systems. 97The average entrapment/encapsulation efficiencies were 64% for physical encapsulation and 56% for chemically cross-linking.Traditional oral biotherapeutics encountered inevitable manufacturing obstacles to bacterial viability, such as oxygen exposure and cellular stress reactions. 92,98Fan et al. successfully generated metal-phenolic networks (MPNs) on E. coli and Bacteroides thetaiotaomicron using Fe(III) ions and polyphenols. 99This biocompatible coating with better self-assembly can protect bacteria from production stresses like oxygen exposure and lyophilization and can be quickly disassembled under acidic conditions.Likewise, scientists evidenced a correlation between the viability of Lactobacillus plantarum (L.plantarum) and the nanocellulose proportion in cryoprotective agents. 95L. plantarum showed the best resistance and viability after freeze-drying at concentrations of skim milk 13.75%, trehalose 20.5%, and nanocellulose 13.75%.
Given the challenges rendered by environmental factors such as gastric acids, proteolytic enzymes, and bile salts, the NAOB hybrid should be competent to maintain metabolic activity in the harsh GI tract. 93Microencapsulation creates a shielding barrier between bacteria and the surrounding environment by being embedded inside or coated with nanomaterials. 100 Pediococcus pentosaceus Li05 encapsulated in the microgel doped with magnesium oxide nanoparticles (MgO NPs) demonstrated better stability than in the microgel alone.It might be attributed to the filling ability of MgO NPs, which partially isolated oxygen and impeded the diffusion of hydrogen ions, bile salts, or digestive enzymes into the alginate-gelatin microgel. 101Hydrogenbonded silicene (H-silicene) nanosheets show excellent stability in acidic conditions, making them ideal for encapsulation. 102Zhu et al.
reported a copolymer-modified two-dimensional H-silicene nanomaterial (SiH@TPGS-PEI) that shielded EcN from the strongly acidic environment and enzymatic lysis.Si-H bonds are relatively active in alkaline environments but stable under acidic conditions, which made SiH@TPGS-PEI present high stability in the simulated gastric fluid (pH 1.5) while rapidly degrading when suspended in the simulated intestinal fluid (pH 7.8) (Figure 3c,d).As expected, EcN@SiH exhibited more than 100-fold higher viability than naked EcN after exposure to simulated gastric fluid for 15, 30, and 60 min. 103Similarly, the relative survival of Salmonella at pH 3.0 improved from 10% to 58% by encapsulation in nanoparticles composed of cationic polymers. 104vertheless, it is noteworthy that nanoparticles used as static bacterial modifications lack the capability for in situ regulation. 105Surface bacterial nanocoatings may also disrupt biological activities, such as membrane protein expression and flagellar rotation. 25Therefore, further investigation is required to determine the clinical application of encapsulation.

| Synthesis of bioactive antitumor metabolites
Bacterial-directed enzyme prodrug therapy is an emerging cancer treatment whereby oncolytic bacteria employ enzymes to convert inactive prodrugs into antitumor substances. 106As a bioactive antitumor molecule, the dual relationship between nitric oxide (NO) and tumors is that an insufficient concentration of NO fosters tumor growth, while a relatively high level of NO exerts antitumor effects. 107eng  As living organisms, the ability of oncolytic bacteria to genetically construct the expression of virulence proteins makes them a potent tumor-killing agent.ClyA is an immunogenic poreforming toxin that forms the transmembrane channel either by assembling a growing pore or by creating a soluble pre-pore in the plasma membrane. 113Wang et al. made a thermally-activated biohybrid through the integration of a plasmid containing a thermally sensitive promoter and gene of ClyA into the nonpathogenic E. coli, leading to the controllable ClyA expression. 114 45 C, the thermally sensitive promoter enabled ClyA expression, leading to pore formation in the cancer cell membranes.
Shewanella algae (S. algae) is a proficient natural tetrodotoxin producer, but wild strains typically contain limited quantities of tetrodotoxin.Henceforth, researchers obtained the optically controlled material-assisted microbial system (called Bac@Au) by biosynthesizing gold nanoparticles (AuNPs) on the surface of hypoxia-targeted S. algae. 115Under light irradiation, photoelectrons from AuNPs were deposited on the bacterial surface and transferred to the bacterial cytoplasm.The transference finally facilitated the in situ synthesis of the antitumor agent tetrodotoxin with a 40% increase.
Substrate limitation and metabolite toxicity pose challenges in synthesizing anti-neoplastic drugs.High doses of pre-drug substrates may cause undesirable off-target toxicity despite enhancing therapy effectiveness. 116Besides, antitumor metabolites like tetrodotoxin often take toxicity risks.Further discussions on tetrodotoxin accumulation in normal tissues and its health implications are warranted.
Overall, creating NAOB hybrids to generate bioactive antitumor metabolites presents an optimistic therapeutic tactic after ensuring safety and sustainability.Another factor that influences the efficiency of PTT is the intrinsic tumor thermal tolerance.High-temperature treatment (above 50 C) may harm normal tissues, while low temperature (42-46 C) may yield poor therapeutic effects due to the expression of heat shock proteins (Hsps). 127  by increasing susceptibility to cell death and promoting apoptosis. 132 addition, excessive ROS can interact with biomolecules (DNA, proteins, lipids, etc.), destroy their structures, affect their functions, and ultimately cause cancer cell death. 131Inspired by ROS's acute tumor eradication capacity, chemodynamic therapy (CDT) and PDT propose revolutionary approaches to generate high ROS in a tumor-specific manner. 133,134T capitalizes Fenton or Fenton-like reactions to stimulate internal ROS production from endogenous sources, such as a particular concentration of mitochondrial oxygen or the continuous generation of H 2 O 2 . 135,136However, the hypoxic microenviron- The overexpression of GSH in the TME, which can provoke ROS depletion, is another determinant factor impacting the effectiveness of CDT. 140To improve ROS generation, researchers decorated Au@Pt compounds on the surface of E. coli, constituting a bacterium-based nanozyme (Bac-Au@Pt). 141Bac-Au@Pt has been validated to yield ROS at pH 6. PDT is an emerging method for tumor treatment that offers low toxicity, minimal invasiveness, high selectivity, and easy synergism. 133ere are two types of PDT based on ROS generation mechanisms.
The type I pathway necessitates the photosensitizer absorbing light energy and generating cytotoxic ROS through electron transfer.Upon absorption of light energy, the type II photosensitizer undergoes an electron spin exchange with ground-state oxygen ( 3 O 2 ), converting 3 O 2 to 1 O 2 .The 1 O 2 subsequently oxidizes biomacromolecules and stimulates an oxidative stress response in the target cells. 133,143e type I pathway is inherently oxygen-independent and can efficiently produce considerable ROS even under severe hypoxia conditions (2% oxygen), but the penetrating depth of conventional lasers is often restricted. 144Researchers deposited aggregationinduced emission photosensitizers on the outer membrane of E. coli (termed AE). 145 heavily on oxygen concentration, and the rapid oxygen consumption leads to deficient 1 O 2 production, making it challenging to attain the desirable therapeutic effect. 147A promising approach involves modifying cyanobacteria with the photosensitizer chlorin e6 (Ce6) to form the special Ce6-integrated photosensitive cells. 148Under 660 nm laser irradiation, cyanobacteria's intense photosynthetic oxygenation properties continuously produced oxygen, which was immediately applied for photosensitizer integration to facilitate 1 O 2 synthesis.Likewise, the following two independent tests validated that the self-oxygenating PDT system, co-constructed with cyanobacteria and photosensitizers, required no long-term external excitation for constant oxygenation and 1 O 2 supply. 149,150

| Immunomodulation
Cancer immunotherapy leverages the patient's immune system to initiate and maintain the inherent defensive mechanisms intended to control and eliminate tumors. 151,152There are three dominant strategies for NAOB-based cancer immunotherapy.One entails making immune-related cells redistribution and reactivation to recognize and attack tumor cells.The second approach is to express and secrete cytokines that target specific parts of the immune system.Additionally, oncolytic bacteria can serve as immunological adjuvants to collaborate combination therapy with immune checkpoint blockade (ICB).
Numerous immune cells, such as macrophages and dendritic cells (DCs), are immunosuppressed or hypo-immunoreactive in the tumor immunosuppressive microenvironment. 153,154Researchers biosynthesized copper sulfide nanomaterials (CuS NMs) within S. Typhimurium VNP20009 to create the nanosystem CuS VNP20009 NB , aiming to target and repolarize tumor-associated macrophages (TAMs). 155TAM constitutes a pivotal factor correlated with immune resistance, while LPS in VNP20009 is a well-accepted agent for TAM repolarization. 156perimental findings indicated that CuS VNP20009 NB could accumulate in tumor tissues and reprogram immunosuppressive M2-type macrophages into immunostimulatory M1-type macrophages to boost the immune response.The tumor cell lysate-coated polydopamine nanoparticles (PDA@CL) were designed to transfer tumor-associated antigens (TAAs) precisely, alleviating the restricted activation of CD8 T cells in the TME due to insufficient antigen presentation by DCs. 157en, engineered Salmonella (EnS) was encapsulated in PDA@CL (EnS@PDA@CL). 158In animal experiments, the quantities of DCs and CD8 T cells in the EnS@PDA@CL group were almost three times higher than in the PBS group, verifying that EnS@PDA@CL intensively promoted antigen cross-presentation and subsequent CD8 T cell stimulation by DCs.
Cytokines play a paramount regulatory role in cell development, differentiation, growth, and survival. 159Scientists constructed a thermosensitive drug delivery system in which programmable E. coli MG1655, expressing TNF-α, was decorated with bio-mineralized AuNPs. 160en exposed to NIR light, the AuNPs induced E. coli to precisely regulate the expression of TNF-α through controlled heat generation (Figure 4a-c).Cytotoxic protein presentation led to apoptotic cell death, underscoring the potency of bacteria-based antitumor cytokines delivery.Wang and colleagues devised a nano-STING agonist-decorated microrobot incorporating S. Typhimurium VNP20009 and mitochondriatargeted NPs. 161The sophisticated microrobot enabled the concurrent delivery of the STING agonist 2 0 3 0 -cyclic guanosine monophosphate- Over the past decade, there have been significant advances in cancer immunotherapies, with immune checkpoint inhibitors as a prospective tool for activating self-immunity against tumors. 162The nanobiohybrid synergized with prevalent ICBs, including the antiprogrammed death protein 1 (PD-1) and PD-L1 proteins, have been validated to potentiate the immune response by blocking the T-cell inhibitory pathway and promoting effector T-cell activation. 163,16447 is expressed in human cells but significantly upregulated in nearly all tumor cells.Through interacting with macrophages, CD47 transmits the "don't eat me" signal to inhibit macrophage phagocytosis and facilitate tumor immune evasion. 165CD47 has emerged as a compelling immunological target alongside PD-1/PD-L1.Tal Danino's team constructed a synchronized lysis circuit (eSLC) in E. coli that colonized tumors to cleave and release the encoded nanobody antagonist of CD47 (CD47nb). 31In BALB/c mice with tumors on both sides, intratumoral administration of eSLC-CD47nb resulted in rapid and sustained clearance of tumor cells within approximately 10 days of treatment initiation.Furthermore, researchers utilized alternating magnetic fields (AMFs) to manipulate tumor-homing E. coli.Fe 3 O 4 @lipid nanocomposites enabled controlled motion via the magnetic field, which enhanced tumor targeting and facilitated the release of CD47nb pre-expressed within the bacteria. 23This treatment exhibited remarkable therapeutic efficacy in both in situ and distal colon tumor models in mice, leading to rapid and long-lasting tumor clearance (Figure 4d-g).

| Others
Various studies under the International Microbiome Consortium have linked dysbiosis to metabolic syndromes, autoimmune diseases, autism, and tumors. 166Nanomaterials can regulate flora signaling and metabolites in the TME.As stated, intratumoral bacteria incurred gemcitabine resistance. 111Zhang et al. designed a nanoformulation responsive to both pH and enzymes to counteract bacterial-induced resistance, encapsulating gemcitabine modified with HA and the antibiotic ciprofloxacin. 167This nanoformulation targeted and controlled drug release in the acidic and hyaluronidase-rich TME and killed intratumoral bacteria to overcome chemotherapy resistance.It also promoted antigen-presenting dendritic cell maturation and depleted immunosuppressive myeloid-derived suppressor cells in bacterially infected tumors to activate T cell-mediated immune responses.
Resembly, mucin-crosslinked antibiotics and chemotherapeutic drugs have produced outstanding outcomes in eliminating microbially induced tumor resistance. 168This trial showcases MTB to act as drug carriers and drugs in the meantime, providing an innovative and multifunctional strategy for cancer therapy.
Magnetic-responsive bacteria are strains that undergo magnetization through the binding of magnetic materials.Evidence has shown that modification of Spirulina microalgae (Sp.) with nano-contrast agents, such as superparamagnetic magnetite suspensions, enabled the tracing of bacterial behavior in vivo by MRI. 185Researchers designed a biohybrid magnetic microrobot consisting of photothermal conversion agent Pd@Au NPs, the chemotherapeutics doxorubicin, and Fe 3 O 4 together with Sp. 186 Under a rotating magnetic field, this microrobot achieved highly targeted and synergistic chemo-PTT with optical imaging mediation.E. coli, with its high sensitivity, has been fond of many scientists in making non-magnetic bacteria magnetic.A micro-robotic system is composed after loading magnetic nanoparticles and an internal fluorescent protein DiR on the probiotic E. coli, which encodes the NDH-2 enzyme. 187The system triggered magne- negative bacteria in mouse tumor models. 189Targeted radionuclide therapy (TRT) is a rapidly growing field that utilizes substances like radiolabeled molecules, radioisotopes, nanoparticles, or microparticles to target cancer cells with cytotoxic α and β particles. 190An integrated microbe-based pretargeting approach used a bacteria-specific radiopharmaceutical to target solid tumors, employing E. coli Nissle 1917 as a delivery vehicle. 191This approach utilizes the siderophore-mediated metal uptake pathway to selectively concentrate 64 Cu and 67 Cu in complex with yersiniabactin (YbT) within transgenic bacteria.PET imaging with 64 Cu-YbT enabled visualization of bacteria within the tumor, while 67 Cu-YbT delivered a cytotoxic dose to the surrounding cancer cells.Cancerous mice carrying MC38 and 4T1 tumors showed impressive tumor reduction and prolonged survival with 67 Cu-YbT.
Computed tomography (CT) is a commonly utilized technique in contemporary clinical imaging due to its cost-effectiveness, quick examination durations, and user-friendly operation. 192Researchers developed a nano-bio emulsion that collaborated X-ray PDT with oncolytic bacterial therapy.The emulsion contained photosensitizercoated nanoscintillators (NaGdF 4 /Tb/Ce@NaGdF 4 ) and C. novyi-NT spores. 193Results indicated that image-guided X-ray PDT expressed elevated levels of apoptotic cell death in cancerous tissue observed under CT imaging.An alternative method in tumor therapy involves applying branched gold nanoparticles (BGNPs) coating on C. novyi-NT spores for CT-guided precise tumor therapy. 194Leveraging the anaerobic targeting specificity and tumor oncolytic capabilities of spores, guided by CT imaging, the deployment of this composite spore manifested robust antitumor effects in the PC3 prostatetumor-bearing mouse model.
Other alternative forms of modalities find application in the realm of cancer treatment.Zhang and colleagues have detailed an integrated nanosystem (Bac@BNP) comprising E. coli and bismuth sulfide nanoparticles (BNPs), demonstrating its capacity to augment radiotherapy sensitivity. 30When exposed to X-ray irradiation, the synergistic effect Compared with antibody-mediated AuNP surface modification on C. difficile spores, the non-specific modification of AuNPs on the Bifidobacterium breve led to lower tumor enrichment.This decrease can be attributed to the dilution resulting from bacterial cell growth and division. 196Bacterial motility may induce artifacts or blur images, affecting their localization and morphology accuracy. 197Moreover, various imaging modalities encounter challenges when utilizing NAOB.Optical imaging is limited by its penetration depth (1 cm) and usually receives small molecule interferences such as hemoglobin, water, and cytochromes. 198PET imaging tracers applied in preclinical and clinical studies may accumulate non-specifically in normal organs or off-target lesions. 195crucial therapeutic consideration is the safety and off-target possibility of NAOB.Many studies are now taking endogenous stimulants to detect and manipulate the nanobiohybrid.The acidity in the TME is often applied as an endogenous indicator.However, it is crucial to consider the similarly low lysosomal pH in normal cells, which may pose a risk of off-target damage from the hybrids.134,199 Additionally, nanoparticles, like gold and silver, are complex to degrade and can have adverse health effects if internally deposited.Green biomaterials are a novel category of biodegradability, biocompatibility, nontoxicity, and swift removal characteristics.200 Studies have shown that multifunctional and nontoxic green graphene emerges as a promising candidate in cancer nanodiagnostics, and microbial-mediated synthesis of eco-friendly metal nanoparticles exhibits remarkable efficacy in cancer therapy.201,202 Therefore, it is necessary to optimize the performance of oncolytic bacteria and green biomaterials by operating different tactics in various nanomedicine applications.
Based on the preceding, it is imperative to conduct further research in the cooperation of oncolytic bacteria and nanomaterials.
Ensuring the safety of nanomaterial-loaded bacteria is paramount,

ACKNOWLEDGMENTS
This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The graphic designs were created with BioRender software (biorender.com).Thanks to all the reviewers and editors for their support in the publication of this manuscript.
Oncolytic bacteria can impact tumor cells directly or indirectly, and the three main potential mechanisms are as follows: (1) induction of programmed cell death and unprogrammed necrosis in tumor cells,(2) modulation of tumor angiogenesis through vascular destruction or inhibition, and (3) activation of the immune system to suppress tumorigenesis (Figure1).The extensively investigated pathways of tumor cell death directly triggered by oncolytic bacteria encompass programmed cell death, including autophagy and apoptosis, as well as unprogrammed necrosis.
et al. constructed a nanobiosystem by linking carbon-dot doped carbon nitride (CCN) with E. coli carrying nitric oxide synthase (NOS). 108Upon reaching the tumor site, photoelectrons excited from CCN headed to NO synthases within E. coli.This mechanism facilitated the reduction of endogenous NO 3 À to cytotoxic NO, thereby instigating apoptosis in tumor cells.Chen et al. conducted a parallel investigation that combined E. coli with black phosphorus nanoparticles (BP NPs). 109When irradiated with a 635-nm laser, E. coli effectively captured photoelectrons generated by BP NPs and triggered the metabolism of nitrate reduction to NO.In addition to generating endogenous cytotoxic metabolites, the nanobiohybrid can also metabolize macronutrients in the TME, like amino acids and lactate.Researchers modified E. coli to express the l-methionine-γ-cleaving enzyme (MdeA) and loaded it with indocyanine green (ICG).In response to near-infrared laser irradiation, the complex released MdeA and depleted the essential amino acid methionine (Met) to disrupt the balance of the TME. 27Eubacterium hallii deposited by iron-polyphenol nanoparticles sustained the conversion of intratumoral lactate to butyrate, inhibiting the polarization of pro-tumor M2-like macrophages. 110Alongside catalyzing the generation of antitumor substances, biological enzymes could serve as an essential physical factor in tumor drug resistance.In 2017, According to Geller et al., intratumoral Gammaproteobacteria resulted in drug resistance in patients with pancreatic ductal adenocarcinoma, rendering them less responsive to the chemotherapy drug gemcitabine. 111It might attribute to the expression of a specific enzyme, cytidine deaminase (CDD), in the intratumoral bacteria.Consequently, selective inhibition of drug-resistant enzyme activity has become a prospective approach to overcome chemoresistance.Researchers bound nitrogen-doped carbon nanospheres (N-CSs) competitively to the active center of CDD, effectively blocking the metabolism of gemcitabine while catalyzing the production of hydroxyl radicals (•OH) to damage tumor cells. 112

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Conceptual diagram of nanomaterial-assisted oncolytic bacteria used in various imaging-guided solid tumor treatments.4.1.3| Nanocarriers for antitumor drug deliveryChemotherapeutic agents are the cornerstone of oncologic pharmacotherapy for their capacity to eradicate tumor cells.Scientists integrated nanomaterials with FuOXP (a prodrug conjugate of 5-FU and oxoplatin) to construct novel drug delivery nanoparticles.117,118When injected into mice, approximately 10% of the nanoparticles reached tumors, showing an immense leap over most other nanocarriers (with an average reach of 0.7%).However, their targeting still needs to catch up to the ideal level.Due to the innate anaerobic targeting ability and high load volume, NAOB can be harnessed as a safe and efficient system for chemotherapy.

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Photothermal agents (PTAs) convert light energy into heat to raise the temperature of the tumor site, triggering tumor cell death and tumor thermal ablation.123However, due to the poor tumortargeting properties of traditional PTAs, the outcome of PTT is partially affected.The leverage of oncolytic bacteria for delivering nano-PTAs that absorb near-infrared light is considered a prospective approach.124Sun's team created a nano-bacteria hybrid (pDA VNP) by coating S. Typhimurium VNP20009 with polydopamine via oxidation and self-polymerization.125With Salmonella's anaerobic targeting capacity and polydopamine's photothermal effect, tumors in pDA VNP-treated mice shrank from Day 4 and eventually disappeared without relapse or metastasis, and all mice survived for at least 90 days.YB1, a safe S. Typhimurium strain, has also emerged as a popular candidate for tumor treatment.Researchers conjugated ICG loaded with nanophotosensitizers onto YB1, successfully forming bacterial-material complexes known as YB1-INPs. 126After intravenous injection of YB1-INPs and irradiation with NIR laser, the fluorescent signal of YB1-INPs at the tumor site increased during the treatment course.At 28 days, the tumor suppression rate of YB1-INPs was 100%.It indicated that YB1-driven hypoxic targeting and photothermal-assisted bioaccumulation achieve low-dose and high-efficiency PTT.

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ment and endogenous H 2 O 2 are deficient in achieving optimal CDT efficacy, indicating the urgency for boosting intracellular H 2 O 2 levels.137,138E. coli was designed to overexpress NDHrespiratory streptokinase II), which accepted electrons from NADH (nicotinamide adenine dinucleotide) and transferred them to oxygen for H 2 O 2 production.139Subsequently, magnetic Fe 3 O 4 nanoparticles (MNPs) were surface-modified onto the bacteria to create a bioreactor as Ec-pE@MNP.With continuous respiration, Ec-pE@MNP consistently generated H 2 O 2 and converted them into cytotoxic •OH by the Fenton-like reaction, inducing severe tumor cell apoptosis and realizing a self-supplied CDT.
4 efficiently.Simultaneously, IFN-γ released by T cells disrupted the antioxidant defense of GSH, fostering ROS-induced plasma membrane oxidation and apoptosis in tumor cells.E. coli/MnOx-based nanospindles (EM NSs) were engineered to release DOX and Mn 2+ ions decomposed by GSH. 142DOX is a potent chemotherapeutic agent, whereas Mn 2+ ions catalyze the Fenton-like reaction to produce •OH.EM NSs presented high safety and efficiency in tumor eradication by GSH consumption and ROS generation in chemo-chemodynamic cancer therapy.
AE productively generated •OH via the type I photodynamic reaction while allowing successful targeting of hypoxic orthotopic colon tumor regions.This hybrid system guided by interventional light proficiently facilitated hypoxia-resistant PDT treatment by overcoming the limitations of light penetration depth.Moreover, engineering photosensitive bacteria through biosynthesis is another widely investigated direction to elevate the effectiveness of PDT.Guo et al. developed a genetically engineered strain of S. Typhimurium expressing fluorogen-activating proteins (FAP dL5**). 146When employing the fluorogen (MG-2I), the amalgamation of dL5**-MG-2I proved to be a robust photosensitizer, expediting the generation of phototoxic ROS to eliminate adjacent cancer cells and over-accumulating bacteria.Type II PDT relies

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adenosine monophosphate (cGAMP) and releasement of mtDNA via oxidative stress.Exogenous cGAMP and endogenous mtDNA demonstrated a synergistic augmentation of cyclic guanosine monophosphateadenosine monophosphate synthase (cGAS)/STING signaling, which dramatically bolstered the immunotherapeutic success of STING agonists.Nano-bacterial complex expressing tumor necrosis factorassociated apoptosis-inducing ligand (TRAIL) resembled assisting tumor apoptosis, activating T-lymphocytes, and releasing proinflammatory cytokines.I G U R E 4 Legend on next page.

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photothermal immunotherapy (Figure5a-c).32Focused ultrasound ablation surgery (FUAS) employs concentrated ultrasound to ablate target lesions through a combined thermal-mechanical effect.171Researchers designed E. coli carrying encoded gas vesicles (GVs) for ultrasound imaging, leveraging the genetically engineered bacterial vector to enhance ultrasound penetration.172This methodology allows the incorporation of GVs-E. coli to transport lipid nanoparticles containing the chemotherapeutic drug banoxantrone dihydrochloride (AQ4N), guiding multiple imaging and chemotherapy combined with FUAS ablation.Photoacoustic imaging (PAI) represents an emerging imaging modality aiming to detect mechanical waves from light absorption by endogenous or exogenous chromophores within the tissue.173Endogenous chromophores, such as melanin, exhibit light absorption at specific wavelengths.Erythrocyte membrane-nanocoated tothermal ablation and NDH-2-induced ROS damage, effectively rendering apoptotic responses in cancer cells under fluorescence imaging.4.2.3 | PET/CT imaging-guided nanotheranostic agentPositron emission tomography (PET) imaging, characterized by its high sensitivity, is the most prevalent tool in diagnosing and treating solid tumors.Live bacteria have proven to be successful carriers for delivering radionuclides.For instance, fluorescent dye Cy5.5 or 125 I labeled attenuated S. Typhimurium achieved fluorescent and nuclear dual-modality imaging of breast cancer and colon cancer.18818  Ffluorodeoxysorbitol (FDS) PET was capable of imaging E. coli and visualizing the colonization and proliferation of tumor-targeting Gram-

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of bacterial ClyA and BNPs, possessing high-Z element radiosensitivity, induced a substantial production of ROS and relevant DNA damage.Bacteria-specific molecular imaging is poised to expand the indications for bacterial-mediated tumor therapies and facilitate the clinical applicability of precision medicine.Optical/photoacoustic imaging-guided nanotheranostic agents mitigate pigment interference, enhancing signal aggregation and photoacoustic signal generation at specific sites.MRI, leveraged for high spatial resolution and absence of radiation, confronts limitations due to magnetic pores and contrast agents addressed effectively by MTB and magnetic-responsive strains. 195PET/CT achieves remarkable sensitivity (10 À10 to 10 À12 m), enabling bacteria-tumor co-localization, quantitative assessment, and accurate TRI diagnosis. 170Nevertheless, only a few studies have employed oncolytic bacteria as imaging and medical probes, and further experimental validation is warranted.POTENTIALITIES OF NAOB: CONSTRAINTS AND OPPORTUNITIES Several issues necessitate further investigation when utilizing the NAOB hybrid in solid tumor diagnosis.The metabolism and motility of nanobacteria can directly affect diagnostic results and imaging quality.
preferably with controlled release into tumor cells under external detection and regulation.Next, we advocate for extensive clinical research to explore alternative practical methods for promoting nanobiohybrid safety, efficacy, and metabolism.Optimizing imaging techniques, improving contrast agents, and reducing background disturbances are also essential parameters for reinforcing the stability of the nanobiological system.Although nanobiohybrids still have room for improvement, the combination of both properties and advantages has shown substantial potential for development and to become innovative and intelligent systems for future solid tumor treatment.6 | CONCLUSION Oncolytic bacteria-mediated tumor therapeutics offer superiority by leveraging their motility and penetration to directly or indirectly eliminate tumor cells while concurrently augmenting the anticancer immune response.Nanomaterials and nanocomposites attract global attention from researchers due to their transcendent performance in transmission, encapsulation, and conductivity.Combining these two approaches can complement the strengths and address their respective limitations.With the continuing development of biotechnology, nanomaterial-modified oncolytic bacteria can serve as potent tumor-targeting transmitters, drug carriers, and immunomodulators.While some shortcomings, like safety concerns and off-target possibility, require urgent attention, the future of utilizing NAOB in tumor therapy holds promise as a prospective methodology for clinicians and researchers aiming to broaden the applications of bacterial-mediated tumor therapy and advance precision medicine.AUTHOR CONTRIBUTIONS Xiangdi Zeng: Conceptualization; resources; software; writingoriginal draft; writingreview and editing.Qi Chen: Supervision; validation; visualization.Tingtao Chen: Conceptualization; resources; supervision; validation; visualization.
Summary of representative nanomaterial-assisted oncolytic bacteria utilized as nanotheranostic agents for tumor imaging and therapy.