Engineered oncolytic bacteria for malignant solid tumor treatment

Bacteria have been explored for their potential in fighting against cancer for decades. Due to their outstanding tumor‐targeting capacity and high biocompatibility, live bacteria can serve as microrobots delivering and producing anti‐tumor agents. In addition, live bacteria have intrinsic immune‐activating functions that aid in the generation of anti‐tumor immunity both systemically and locally in the tumor microenvironment. While bacteria‐based cancer therapy is still facing great challenges, progress in this platform combined with nanobiotechnologies has shown promise in terms of safety and effectiveness. Here, basic development strategies of bacteria‐based delivery systems armed with nanotechnologies, virulence attenuation, and genetic manipulation are summarized and the design of a spatiotemporal selectivity is particularly emphasized. In conclusion, the engineered bacteria platform has a high potentiality in the development of novel cancer therapeutics and holds prospects for future investigation and clinical use.

Malignant tumor poses a huge global health threat to human life. 1 Chemotherapeutics have traditionally and widely been used for cancer therapy through direct killing.However, serious side effects occur frequently post chemotherapy due to the concomitant cytotoxicity on normal cells. 2In addition, drug resistance and undesirable solubility/stability of chemical drugs under physiological conditions are major concerns. 3Compared to chemotherapy, targeted therapy can benefit genetically defined patient. 4However, toxicity still remains in this context, which is largely caused by the off-target effects on normal cells. 5Moreover, drug resistance and insufficient delivery efficacy are also the stumbling block in the clinical implementation of targeted therapy. 6Immunotherapy has emerged as a novel and promising therapeutic strategy that deploys the immune system to fight against cancer. 7uring the past decades, immune-checkpoint inhibitors targeting PD-1, PD-L1, CTLA-4 have successfully been developed and proven to be effective in cancer treatment. 8owever, clinical outcomes of immune-checkpoint inhibitors differ in different types of tumors. 9For example, the so-called "cold tumor" characterized by a low-level T cell infiltration and a highly immunosuppressive microenvironment normally demonstrated a low response rate to treatment. 10o tackle these challenges, nanotechnology has been extensively implemented in the development of novel cancer therapeutics on the basis that solid tumor tissues are normally highly vascularized. 11Due to the "enhanced permeability and retention (EPR) effect", nanodrug delivery systems (NDDS) have been designed to control drug release precisely. 12Nanoparticles in bloodstream can be easily leaked from the gaps in the endothelial lining of the vessels and finally accumulate in the solid tumor interstitium. 13Even so, solid tumors are normally characterized by hypoxia that is formed when oxygen and nutrients are supplied insufficiently as a result of heterogeneous blood perfusion. 14Tumor cells in hypoxic areas stay in a dormancy state with stem cell-like properties. 15Therefore, the existence of hypoxic areas is considered as one of the major hurdles to NDDS-based treatments.As such, it also results in chemoresistance and radio-resistance which may lead to the clinical relapse. 16On the other hand, these hypoxic and necrotic areas in solid tumor sites are suitable niches for anaerobic bacteria to colonize and therefore demonstrate high potentiality for anaerobic bacteria-directed tumor therapy. 17naerobic facultative species were historically deployed in fighting against cancer and have recently gained attention due to their advantages in breaking drug resistance caused by immunosuppressive environment in tumor. 18In addition, anaerobic bacteria contribute to the restriction of tumor growth and can also compete with tumor cells in consuming nutrients, accompanied by secretion of proteins, toxins and other stimuli activating the immune system locally.These organisms could therefore serve as appropriate therapeutic payloads for tumor treatment. 19However, the potential risk of infection remains a major concern on live bacteria-based therapy, especially when administered systemically. 20ow the live bacteria can be further manipulated to be safe and versatile at producing various therapeutic proteins awaits further investigation. 21In this review, we summarized the recent progress of engineered bacteria used in tumor targeted therapy (Figure 1).

| MATERIAL-BASED ENGINEERED BACTERIAL THERAPEUTICS
When designing a bacterial-based delivery system, a high spatiotemporal selectivity should be taken into consideration for precision, safety and controllability. 22ompared to biological engineering, material-based strategies are applicable to some unique scenarios, especially in stimuli-responsive systems.In response to external/internal stimuli, nanomaterials exhibit multiple functions, for example, can enhance bacteria motility and regulate a precise drug release. 23Here, we emphasized different types of therapeutic strategies that have deployed various materials to achieve an efficient stimuliresponse in solid tumor (Table 1).

| Photothermal therapy-based engineered bacterial delivery system
Photothermal agents (PTAs) normally have a high lightto-heat conversion capacity and therefore have been widely used in different contexts. 24Near-infrared (NIR) light irritates PTAs to generate a photothermal effect leading to an elevation of tumor temperature while causing limited damage to circumambient normal tissues.Engineered bacteria armed with PTAs are therefore proposed to be a promising therapeutic tool. 25t has been proved that photothermal therapy (PTT) can induce an immune-stimulatory tumor microenvironment (TME) favoring the generation of antitumor immunity. 26PTT has also been utilized in combination with immunotherapy to achieve a synergistic anti-tumor effect and has demonstrated promising efficacy in tumor elimination and metastasis inhibition. 27

F I G U R E 1 Development of engineered bacterial therapeutics.
T A B L E 1 Material-based engineered bacterial therapeutics.

Types Strategies References
Photothermal therapy PTT combined with immunotherapy 28,30 N-V-J. 28NHS-N782, a synthesized photosensitizer, can elevate the temperature in tumor to destroy malignant cells under an NIR laser.Meanwhile, JQ-1 carboxylic acid can be released upon NIR irradiation and reduce PD-L1 expression in tumors (Figure 2).This provides an effective combinatorial strategy that turns a "cold tumor" into a "hot tumor", breaks the immune tolerance and ultimately exerts a strong anti-tumor effect. 29  Different from the traditional approaches of attaching nano-agents onto the bacterial surface, the incorporation of nano-agents into bacteria has become a new tactic aiming to prevent early releasing of payload and improve stability.For example, a bacterial delivery system loaded with glucose polymer (GP) and photosensitive indocyanine green (ICG) silicon-nanoparticles (GP-ICG-SiNPs) has been tested in glioblastoma photothermal immunotherapy.Facultatively anaerobic bacteria internalized the GP-ICG-SiNPs through bacteria-specific ATPbinding cassette (ABC) transporter and formed a "Trojan horse" that could penetrate tumor tissue.Upon 808 nm irradiation, ICG molecules were triggered to produce photothermal effects. 13Similarly, Chu et al. have constructed a "Trojan" nanobacteria system consisting of Escherichia coli (E.coli) DH5α conjugated with maltodextrin and ICG internally.In this context, ICG could exert a photothermal effect to destroy the tumor tissue under 808 nm irradiation. 31ther attempts were made to overcome the shortages of photosensitizers such as unitarity, instability, limited tumor penetration and retention through combination of different types of photosensitizers.E. coli BL21 integrated with a monochromatic irradiation-mediated ternary combination of photosensitizers including natural melanin, ICG and polydopamine has been reported to demonstrate photoacoustic imaging-guided synergistic photothermal effect. 32All the three photosensitizers could be irritated at 808 nm, therefore generating sufficient thermal energy and stable imaging signals.This work conferred engineered bacteria a stable triple photoacoustic and photothermal effect under a monochromatic irradiation.

| Photodynamic therapy-based engineered bacterial delivery system
As a newly arising therapeutic strategy, photodynamic therapy (PDT) has attracted much attention due to its unique properties such as spatiotemporal targeting, easy manipulability and non-invasive intervention. 33Distinguished from photothermal effects, photochemical reactions rely on the generation of endogenous anti-tumor reactive oxygen species produced upon photosensitization. 34Moreover, it is easy to select the irritated tumor sites specifically to obtain more precise targeting and killing. 35ypoxic and necrotic tumor sites are major obstacles in photodynamic therapy, which is largely due to the fact that the photosensitizer functions only at the sites with a higher O 2 concentration.To this end, anaerobic bacteria can offer an ideal complementation to photodynamic therapy.Our group has recently tested this strategy and showed that the combinatorial use of anaerobic oncolytic bacteria and photodynamic therapy could ablate malignant melanoma efficiently. 36Clostridium belongs to obligate anaerobes that only propagate in anoxic or necrotic areas of solid tumors, exhibiting significant oncolytic effect and its side effect on normal tissues is very low.D-alanine (D-Ala), a metabolic substrate of Clostridium butyricum, was conjugated with a photosensitizer (TPApy) that exhibited aggregation-induced emission (AIE).The metabolic substrate of D-Ala-TPAPy could be metabolically incorporated into bacterial peptidoglycan.In this case, the engineered C. butyricum could colonize and ablate the hypoxia region of tumor, where the photosensitizer demonstrated a photodynamic effect in the oxygen-rich region and destroyed the residual tumor under light irradiation (Figure 3).It has also been proposed that endogenous oxygen generated inside tumor may enhance PDT. 37Photosynthetic cyanobacteria (Cyanobacteria Synechococcus elongatus PCC 7942) modified with inorganic two-dimensional black phosphorus nanosheets (BPNSs) can produce oxygen continuously under light irradiation, which elevated oxygen concentration at tumor sites to boost the photodynamic effect of BPNSs.
Although photodynamic therapy-based engineered bacterial therapy has made a great progress, the uneven illumination of external light irradiation in deep-seated solid tumors has limited further development of this strategy. 38To tackle this, several approaches have been developed to improve the illumination mode. 39Yang et al. reported a kind of engineered bioluminescent bacteria (Salmonella typhimurium) for the purpose of illuminating the entire tumor evenly. 40Attenuated Salmonella typhimurium was engineered with the plasmid encoding firefly luciferase and photosensitizer chlorin e6 (Ce6), which forged an internal light source in the engineered bacteria.Under the internal illumination, the photosensitizer Ce6 could be activated to function in photodynamic therapy subsequently.In addition, to overcome limited depth of laser penetration on treating deep tumors inside abdominal cavity, Zhu et al. developed a hypoxia-targeting E. colibased AIE luminogen (TBP-2) system aiming at eliminating orthotopic colon tumors via an interventional method. 41They used an optical fiber and an endoscope to visualize the orthotopic tumor and irradiated it with a laser in the abdominal cavity.This strategy largely minimized the requirements for deep laser penetration.

| Magnetically steerable biohybrid microrobots
To enhance the intrinsic propulsion of these bacteria, different types of micro-robotic platforms have been developed recently.Driven by external signals, the biohybrid microrobots armed with artificial components (e.g., micro/nanocarriers) have shown outstanding performance for their self-propelling, sensing and targeting mechanisms. 42So far, various driven modes have been unlocked, including magnetism, light, acoustics, etc. 43 Here, researchers constructed a magnetically steerable biohybrid microrobot with enhanced high-throughput fabrication, efficient motility, tissue penetration capability, multifunctional operation, and stimuli-responsive external control.Akolpoglu et al. engineered E. coli MG1655 with two synthetic material arms, streptavidin magnetic nanoparticles (mNPs) and nanoliposomes (NLs). 44Externally applied magnetic fields could stimulate mNPs to achieve dynamic navigation, and therefore drive the bacterial biohybrids through 3D porous microenvironments for targeted localization.On the other hand, researchers encapsulated drug molecules [doxorubicin (DOX)] and PTA ICG in a delicate prepared liposomal formulation, which was photothermally active WANG ET AL. upon NIR light irradiation, resulting in the release of chemotherapeutic molecules spatiotemporally (Figure 4).Chen et al. designed a biohybrid microrobot with a magnetothermal switch. 45Conjugated on Escherichia coli Nissle1917 (EcN), the Zn-doped Fe 3 O 4 mNPs (Zn 0.4 Fe 2.6 O 4 ) could respond to external alternating magnetic field (AMF) and provide magnetotaxis, which also served as a magnetothermal switch and heated the biohybrid microrobot.Meanwhile, a thermally sensitive promoter was encoded in EcN in response to high temperature and subsequently induced NDH-2 enzyme (respiratory chain enzyme II) and mCherry expression by AMF, which could promote the anti-tumor effect and imaging-guided tracking and actuation.
Aiming at enhancing the propulsive forces of biohybrid microrobots to infiltrate across robust tissue barriers, Gwisai et al. constructed a scalable magnetic torque-driven control strategy. 46Compared to scalable magnetic field gradients or employed directing magnetic field-based control strategies, researchers deployed magnetic torque driven motion to promote the autonomous taxis-based locomotion of Magnetospirillum magneticum AMB-1 in deep tissue, which acted as a carrier for covalently-coupled liposomes (MTBLP).

| TME stimuli-responsive bacterial delivery system
Most normal tissues maintain a stable state with pH from 7.3 to 7.4. 47Tumor sites are usually characterized by a condition with deregulated energy metabolism, insufficient perfusion, and uncontrolled proliferation.In this case, acidic metabolic waste products are abnormally produced in the TME, where the pH values were reported between 6.4 and 7.0 as a result of anaerobic glycolysis and the accumulation of lactic acid. 48Due to this, a variety of acid-labile linkers have been developed and used in the engineering of bacterial delivery systems.Xie et al. engineered bacteria with acid-labile linkers of cis-aconitic anhydride to induce the release of therapeutical agents precisely when entering into TME. 49EcN was constructed as a carrier, which was conjugated with Doxorubicin (DOX) on the surface of EcN with cis-aconitic anhydride, forming EcN-ca-Dox.EcN-ca-Dox could stay at a stable state in blood circulation, while DOX could be released readily in the acid TME to inhibit the tumor growth.In addition, Xie et al. used the acid-labile linker 2-propionic-3-methylmaleic anhydride (CDM) to achieve an acid-labile release in TME. 50α-tocopheryl succinate F I G U R E 3 Photodynamic therapy-based engineered live bacteria.D-Ala-TPApylabeled C. butyricum colonize in and ablate the hypoxia region of malignant melanoma.TPApy exerts a photodynamic effect in the left oxygen-rich region.Reproduced under terms of the CC-BY license. 36Copyright 2022, The Authors, published by John Wiley and Sons.
(TOS) and DOX were both conjugated with poly (ethylene glycol) and formed as amphiphilic promicelle polymers (PM TOS and PM DOX ).Then, PM DOX (H2N-PEG-ss-DOX) and PM TOS (H2N-PEG-ss-TOS) polymers were attached to bacteria through CDM and tetrazine derivatives.Therefore, when PM DOX and PM TOS co-polymers separated from the surface of bacterial microrobots in acid TME, they self-assembled into hybrid micelles (M D/T ) and entered tumor cells through endocytosis.Further, in response to cytosolic glutathione (GSH), DOX and TOS were released from micelles achieving an intracellular drug release (Figure 5).
In summary, researchers designed a dual internal stimuli-responsive bacteria delivery system.Biophysicochemical characteristics of the TME have been exploited as internal stimulation to activate or enhance the functions of nanoparticles, which can be coordinated with the bacterial delivery system.

ENGINEERED BACTERIAL THERAPEUTICS
The main reason that genetic engineering technology was first applied was to make tumor-targeted microorganisms less virulent and immunogenic in vivo. 51Bacteria are a flexible platform for delivering therapeutic payloads because they may be manipulated using a range of bioengineering techniques including genetic engineering to decrease their pathogenicity and toxicity and further increase their anticancer activity and safety.There are increasing opportunities for scientists to create genetically modified bacteria that can produce immunotherapeutic drugs as a result of the rapid development of genetic engineering technology and synthetic biology.Bioengineered bacteria can effectively express immunotherapeutic medicines in local tumors, evoking a potent immune response against the tumor while minimizing the off-target and pharmacological side effects (Table 2).

| Virulence attenuation
Several studies have demonstrated that bacteria can be both pathogenic and carcinogenic.Yet, when their pathogenicity is attenuated by the knock-down of specific virulent genes, their ability to target and kill tumors can be retained or even increased. 52Safety issues of using live bacteria for cancer therapy have been extensively questioned, and how to reduce the risk is growing to be of paramount importance, particularly in the context when bacteria were administered intravenously. 53However, it should be noted that some bacterial virulence factors may be responsible for their intrinsic antitumor activity; therefore, attenuation must be achieved without ablation of their antitumor activity.Deletion of key virulent genes has successfully transformed fatally hazardous strains into essentially safe variants. 54For example, lipopolysaccharide (LPS) in the outer membrane of Salmonella can activate toll-like receptor 4 (TLR4) to induce TNF-α production, which has antitumor effects and is an important driving-factor in severe inflammation or sepsis. 55Deletion of the msbB gene can result in the loss of lipid A myristoylated to improve the safety of Salmonella. 56Salmonella VNP20009, a pathogenic Salmonella typhimurium-based attenuated strain, demonstrated excellent safety profiles due to the deletion of purI and msbB genes. 57It has been shown that VNP20009 can induce apoptosis of various leukemia cell types in vitro and in vivo (Figure 6A). 58Moreover, VNP20009 can significantly inhibit the proliferation of MLL-af9-induced acute myeloid leukemia cells and promote the production of TNF-α, interferon-γ (IFN-γ) and leukemia suppressor (LIF).Moreover, frequencies of multiple immune cell subsets were elevated in VNP20009-treated AML mice, suggesting that VNP20009 can deploy the immune system to combat tumors.However, it should be noted that some bacterial virulent genes are indispensable for their inherent anti-tumor activity, so they should be attenuated for subsequent tumor diagnosis and treatment.
Bacteria attenuation can also be achieved by downregulating or inhibiting the expression of related genes and their functions.S. Typhimurium with a deficiency in guanosine 5 0 -diphosphate 3 0 -diphosphate synthesis (ΔppGpp) is an attenuated strain with excellent antitumor activity and good biosafety, 59 which can activate inflammasome (NLRP3, IPAF) and induce proinflammatory cytokine production (IL-1β, IL-18, and TNF-α). 60nother approach is to introduce specific nutrient deficiencies in bacteria that allow them to survive under specific conditions to achieve selective tumor colonization and growth at the sites of target and reduce toxic effects on normal tissues.For example, S. Typhimurium A1-R(A1-R), a leucine-arginine auxotrophic variant, cannot sustain infectivity in normal tissues but can survive and proliferate in tumor sites. 61The tumor-targeting capacity of A1-R has been tested in a variety of malignant models including melanoma, 62 mammary cancer, 63 sarcoma, 64 pancreas, 65 cervical carcinoma, 66 gastrointestinal stromal tumor. 67Moreover, the mutated strain demonstrated robust anti-tumor ability or increased tumor sensitivity to chemotherapy. 68In another example, a F I G U R E 6 (A) VNP20009 can significantly promote cytokine production.Reproduced under terms of the CC-BY license. 58Copyright 2020, The Authors, published by Elsevier.(B) Therapeutic efficacy of the tryptophan auxotrophic ST in an aggressive metastatic cancer mode.Reproduced under terms of the CC-BY license. 69Copyright 2022, The Authors, published by Cell Press.
tryptophan auxotroph bacterium ST2514P3 was created by removing the gene encoding tryptophan synthase (trpA) and anthro phthalate synthase (trpE) in S. Typhimurium, 69 the bacterial motility decreased in normal tissues but was not altered in tumor sites (Figure 6B).This strain showed a strong anti-tumor capacity in a highly aggressive metastatic 4T1 breast cancer model, which ultimately inhibited the growth of both primary and metastatic tumors.Of note, deletion of certain genes can also lead to increased virulence of bacteria, which may favor the anti-tumor ability.Deletion of aroA has been shown to generate pronounced alterations in metabolism and cellular physiology, and increased the immunogenicity and adjuvant effect of Salmonella, leading to an improved anti-tumor efficacy. 70

| Enhancement of tumor targeting
Bacteria can target tumors actively or passively.Some bacteria, such as Salmonella and Bifidobacterium, have been shown to preferentially accumulate in tumors, invade the hypoxic area, 71 and proliferate in tumor cells to promote local immune-therapeutic effect. 72Increasing the targeting precision and efficiency of bacteria can further improve the anti-tumor efficacy and meanwhile ensure the safety by reducing or avoiding unnecessary damage to normal tissues.One way is to genetically manipulate the expression of tumor-specific ligands on the bacterial surface.For example, arginine-glycine-aspartate (RGD) peptide is a tumor homing peptide which can bind to α v β 3 integrin that is over-expressed on tumor cells and blood vessels during angiogenesis. 73ttenuated S. Typhimurium strain ΔppGpp expressing RGD peptide on the outer membrane protein A (ompA) showed a highly targeting specificity and strong tumor inhibition in human melanoma MDA-MB-231 (Figure 7). 74ing et al. constructed an "obligate" anaerobic Salmonella strain YB1 in which the key gene asd has been controlled by the hypoxia promoter through genetic engineering.Asd is responsible for the synthesis of diaminopimelic acid (DAP), an important part of the cell wall of gram-negative bacteria.Lack of DAP could lead to bacterial lysis.In the absence of DAP, YB1 can only survive under hypoxic conditions.In aerobic normal tissues, YB1 is rapidly cleared due to the lack of expression of asd.Experimentally proved, YB1 can specifically targets and colonizes tumors in nude mice and suppress tumor growth, while it is rapidly eliminated in normal tissues. 75umor antigens can also be used as targets for bacterial therapy.Artificially engineered bacteria secreting tumor antigens can induce specific anti-tumor immunity as vaccines.CRS-207, a strain of Listeria monocytogenes DactA/DinlB, was genetically engineered to express human mesothelin, which is a typical tumor-specific antigens (TSAs) expressed by a variety of tumors.CRS-207 elicited a robust mesothelin-specific T cell response when tested in a mesothelin-expressing tumor mouse model F I G U R E 7 Surface display of RGD peptide enhances the targeting and therapeutic efficacy of attenuated Salmonella.Reproduced under terms of the CC-BY license. 74Copyright 2016, The Authors, published by Ivyspring International Publisher.
and was also reported to be well-tolerated in patients with refractory mesothelioma, and ovarian, lung cancer, and pancreatic. 76Similarly, an aroA/aroD mutant of SL1344, S. enterica serovar typhimurium BRD509 expressing Human papillomavirus (HPV)-related antigens, SipB160/HPV16 E7 fusion protein, showed an effective anti-tumor effect in a cervical cancer mouse model. 77

| Introduction of an inducible expression system
Some anaerobic microorganisms capable of colonizing the TME tend to express heterologous genes in a sustained manner, which may inevitably induce undesired toxicity.The promoters of gene expression systems can be triggered by tumor-derived internal signals or external signals such as chemicals, radiation, etc. 21 Therefore, transforming bacteria into an inducible expression system can provide spatiotemporal control to maximize their therapeutic efficacy while minimizing toxicity.In a recent study, researchers developed a genetically encoded microbial packaging system-inducible capsular polysaccharide (iCAP), 78 which dynamically expressed CAP to improve systemic delivery efficiency and reduce unwanted non-specific toxicity.Based on a small RNA screening of the capsular biosynthetic pathway, researchers "hijacked" the CAP system in EcN and constructed an induced synthetic gene circuit that regulates the bacterial envelope to control bacterial survival by altering IPTG levels.This dynamic dosing strategy not only increased the maximum tolerated dose of bacterial therapy by 10-fold, but also increased the proportion of bacterial translocations in tumors and improved killing efficacy in distal tumors.Therefore, it can be further expected that the inducible, programmable encapsulation system would improve the therapeutic applications of engineered live bacteria in malignancies (Figure 8).EcN was also engineered to acquire the ability to release flagellin B (flaB) that binds to lanthanide upconversion nanoparticles (UCNPs) after sensing blue light (Figure 9). 79Under 808 nm irradiation, EcN is activated to efficiently secrete flaB and re-polarize tumorassociated macrophages after binding to TLR5 receptors.Subsequently, the activated macrophages produce pro-inflammatory cytokines favoring the activation of CD8þ T cells to kill tumors.In various tumor models and The biosynthetic pathway of bacterial CAP for tunable and dynamic surface modulation of EcN with synthetic gene circuits.(B) Programmable CAP system enhances the systemic delivery of bacteria by instantaneously expressing CAP.The iCAP (blue) system enables transient encapsulation of bacteria and allows for in situ activation of CAP at local tumor sites, leading to the translocation of inducible bacteria to distant uninitiated tumors.Reproduced under terms of the CC-BY license. 78Copyright 2022, The Authors, published by Springer Nature.
WANG ET AL. metastatic tumors, this synergistic effect resulted in significant tumor regression with negligible side effects.This justifies the use of optogenetic tools in combination with the natural propensity of certain bacteria for cancer immunotherapy based on the NIR nanophotogenetic platform.
The use of ultrasound to control gene expression spatiotemporally can also be used in caner immunotherapy. 80For example, researchers engineered an ultrasound-responsive bacterium (URB) to control gene expression through focused-ultrasound-induced hypnotherapy (Figure 10).80a The insertion of IFN-γ allowed the bacteria to accumulate at multiple sites of colonization under the PL and PR promoter, and further transformed into E. coli MG1655 in the hypoxic and necrotic regions of the tumor in this system.Focused ultrasound irradiation and heating could trigger the expression of IFN-γ to activate anti-tumor immunity during bacterial invasion.This combinatorial strategy has greatly inhibited the growth of distal metastatic tumors and prolonged the survival time of metastatic tumor mice, which provided a novel therapeutic strategy for the treatment of deep tumors.
Wang et al. modified bacteria using synthetic biology and interfacial chemistry to express photothermal melanin internally and immune checkpoint inhibitors on the surface. 81Melanin can repeatedly induce moderate and uniform tumor heating under spatiotemporally controlled light irradiation and checkpoint blockade can reverse the immune-suppressive milieu to immune-stimulatory status, which synergistically achieved improved antitumor effects.

| Expression of therapeutic proteins
In general, the cytotoxicity of natural bacteria is caused by the sensitization of the immune system and the competition for nutrients. 81Attenuated bacteria alone often fail to eradicate solid tumors. 78It was first proposed in the mid-1990s that therapeutic payloads through bacterial targeting enhanced their efficacy. 82Drug delivery systems based on genetically engineered bacteria have properties that cannot be achieved with conventional therapeutic interventions. 83As a result, attenuated bacteria have drawn attention in cancer therapy, as they can specifically invade tumor tissues and play a significant role in anti-tumor therapy as "factories" for a variety of therapeutic agents.Transformation of various plasmids expressing therapeutic proteins or tumor antigens into bacterial cells can promote anti-tumor activity and reduce systemic toxicity. 84acteria can also be used as an adjunct therapy to PTT to precisely inhibit tumor growth. 85Combining PTT and immunotherapy to achieve deep tumor targeting provides a new paradigm for tumor immunotherapy. 86Abedi et al. utilized the ability of engineered bacteria to infiltrate the tumor while activating them by using ultrasound to release effective drugs within the tumor.This team engineered cellular agents by adapting temperaturesensitive repressors to EcN, and designing gene circuits in which they control an integrase-based state switch, resulting in long-term therapy production.Two new sets of genes were transferred into the EcN.One acts as a heat switch that is expressed when the bacteria reach a certain temperature range.The other express immune checkpoint inhibitors targeting CTLA-4 and PDL1 that elicit anti-tumor immune responses to attack the tumor.Using focused ultrasound technology, ultrasound waves are heated to 42-43°C to activate the engineered bacteria to express nanoantibodies and inhibit tumor proliferation (Figure 11). 87umor cells can also be killed directly by toxic proteins produced by bacteria.Cytolysin A (ClyA), a poreforming toxin in which ClyA at high concentrations induces cell lysis while at low concentrations affects intracellular signaling, is a bacterial virulence factor with potential applications in tumor therapy. 88It is widely known that pancreatic cancer has a high mortality rate and is resistant to conventional therapeutic interventions due to the presence of stromal cells causing poor immune cell infiltration. 89A novel targeted therapy approach based on attenuated S. Typhimurium was recently reported, which allows modified S. Typhimurium expressing ClyA to destroy cancer stromal cells (Figure 12). 90pon intravenous administration, the attenuated S. Typhimurium accumulated and proliferated inside tumors releasing ClyA into TME.A single dose administration was shown to inhibit pancreatic tumor growth potently in subcutaneous xenografts and orthotopic nude tumor-bearing mice models.Further histological analysis revealed that expression of stromal cell markers significantly decreased after colonization with ClyA-expressing bacteria, while at the same time infiltration of immune cells (neutrophils and macrophages) into the tumor was remarkably enhanced.
Therapeutic ability of ClyA-expressing bacteria was also evaluated in a mouse model of human pancreatic cancer.Jiang et al. modified E. coli strain K-12 to produce ClyA and assessed its tumor-killing effect in primary and metastatic tumor models. 91A single-dose treatment with ClyA-expressing E. coli significantly reduced the growth rate of tumors during a short period of observation but showed recurrence of tumor growth thereafter.Radiation therapy alone (RT; 21 Gy) inhibited the growth rate of the tumors but not their size.However, combined use of ClyA-expressing E. coli and irradiation resulted in a significant tumor reduction or even a complete eradication in a colon cancer tumor-bearing mouse model.In addition, treatment with ClyA-expressing E. coli significantly inhibited the growth of metastatic tumors and prolonged the survival time of mice.These results suggest that treatment with genetically engineered E. coli can significantly improve the outcome of RT and significantly inhibit the development of lung metastases.Likewise, the Nissle (ΔECHy)-based bacterial system was shown to distribute the fusion peptide ClyA-hyaluronidase (Hy) through outer membrane vesicles (OMV).ΔECHy, when in combination with immune checkpoint inhibitor and tyrosine kinase inhibitors (TKIs), improved the efficacy of immunotherapy by remodeling the tumor stroma. 92n addition, vector strains can be generated to carry a synchronous lysis loop in the bacteria to release therapeutic payloads once the bacteria colonize the solid tumor. 93Chowdhury et al. used bacteria as a chassis for immune checkpoint-blocking therapy. 94In this context, they carried a synchronized lysis circuit (SLC) gene line in non-pathogenic E. coli encoded for anti-CD47 nanobody (Figure 13A).They showed that the release of anti-CD47 nanobodies in the tumor enhanced the activity of tumor-infiltrating T cells and therefore significantly inhibited tumor growth.
Gurbatri et al. further applied the SLC to probiotics to release nanobodies against PD-L1 and CTLA-4. 95They established EcN expressing PD-L1 and CTLA-4, which significantly limited tumor growth upon intratumor injection accompanied by a clear reverse of immune-suppressive state to immune-stimulatory milieu (Figure 13B).Mice studies showed that intratumoral release of anti-PD-L1 and anti-CTLA-4 nanobodies induced strong T cell activation and promoted the generation of T cell memory responses.Moreover, such effects were further promoted when combined with granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. 95

| Transgenesis
Mammalian proteins normally require proper folding and post-translational modifications to be functional.However, this is difficult to achieve in bacteria, so DNA or RNA can be introduced into target cells by gene transfer to improve therapeutic efficacy compared to directly expressed proteins. 21or example, RNA interference (RNAi) therapy enabled by genetically engineered bacteria is through the transport of therapeutic RNAi molecules (short hairpin RNA, shRNA) into target cells to silence tumorassociated mRNA.Phan et al. modified the attenuated S. Typhimurium strain to allow them to express shRNA F I G U R E 1 3 (A) E. coli with synchronized lysis circuit induces the phagelysis protein ϕX174E, leading to bacterial lysis and release of anti-CD47 blocking nanobody binding to CD47 on the tumor cell surface.Reproduced with permission.Copyright 2019, Springer Nature.(B) The mechanism of controlled release of engineered bacteria is to produce PD-L1 and CTLA-4 blocking nanobodies within tumors.Reproduced with permission. 95Copyright 2020, The American Association for the Advancement of Science.specific to indoleamine 2,3-dioxygenase 1 (IDO), which was shown to increase neutrophil infiltration into tumor sites causing tumor cell death when tested in CT26 and MC38 colorectal tumor-bearing mouse models. 96Howsince the spread of pre-bacterial bacteria is not limited to tumors, it is critical to control the precise expression of RNAi molecules spatially-temporally to avoid unnecessary damage to normal tissues.

| CONCLUSION AND OUTLOOK
Malignant tumors pose a serious threat to human health, and the effect of current strategies to treat tumors is not very optimistic, mainly because the targeting and deep penetration of drugs into tumors are not ideal, resulting in severe side effects on normal tissues, poor prognosis, and high costs. 97The blood vessels of tumors usually exhibit morphological abnormalities that lead to the formation of hypoxic and/or necrotic regions in solid tumors. 98These hypoxic and/or necrotic regions provide a critical niche for bacterial colonization.With the development of the microbial discipline, bacteria have shown great targeting ability and antitumor activity in many animal models of antitumor experiments due to their unique biological properties. 99Furthermore, bacteria have great gene packaging capabilities, making them optimal candidates for providing therapeutic loading systems. 98Currently, several bacterial strains have been used to target tumors, including E. coli, Clostridium, Listeria monocytogenes, magnetotactic bacteria, and S. typhimurium, but their safety is of concern, and the development of live bacterial anticancer drugs still faces significant challenges. 99Various strategies have been developed to achieve virulence attenuation of bacteria, including deleting key virulent genes and down-regulating or inhibiting the expression of related virulent genes.Moreover, it has been proved that introducing specific nutrient deficiencies in bacteria can lead to a reduction of toxic effects on normal tissues.To enhance the targeting precision and efficiency of bacteria, some studies manipulated the expression of tumor-specific ligands genetically on the bacterial surface so that engineered bacteria can bind to tumors specifically.
The convenience of bacterial gene manipulation allows the integration of complex gene circuits into bacteria, such as species-specific and inducible promoters, to express therapeutic proteins in specific environments.In addition, it is also a promising strategy to construct engineered bacteria secreting tumor antigens which can induce specific anti-tumor immunity as vaccines.
Meanwhile, materials engineering has developed different strategies to improve the antitumor efficiency of bacteria while reducing the insecurity of gene transfer and uncertainty of appropriate dosage to allow bacteria to play complex roles in tumor therapy.Bacteria can be engineered to exert PTT or PDT in tumors, which leads to high spatiotemporal selectivity and limited damage to circumambient normal tissues.To improve the intrinsic propulsion of these bacteria, different types of magnetically steerable micro-robotic platforms have been developed, which can achieve dynamic navigation for targeted localization.Besides, various TME stimuli-responsive linkers have been developed and used in the engineering of bacterial delivery systems.
Bacteria have demonstrated their great potential as immunotherapeutic drug carriers in many aspects of biocompatibility and biological function in vivo, but it is difficult to combine all the properties of ideal drug delivery systems in the same bacterium. 100Through genetic engineering and surface modification, the development of a biological hybrid "microrobot" in combination with other treatment methods offers a new direction and new strategies for the field of bacterial therapy. 101lthough the use of genetically engineered bacteria in mouse tumor models enables tumor regression and prolonged mouse survival, there are still many factors that limit the use of genetically engineered bacteria in the clinic.First, in view of tumor heterogeneity and dynamicity and complexity of bacteria, the targeted effect of bacteria is inestimable, so efficacy may vary in different tumor patients in the treatment process.Second, the expressed levels of immunotherapeutic factors in bacteria are variable, lower with inefficiency and higher with toxicity.It is very important to ensure the long-term and stable production of bacteria in the host body.Third, although therapeutic agents and surface-modifying materials do not multiply with live bacteria, there is a risk that multiplication of artificial bacteria and some uncontrollable mutations during bacterial proliferation may cause undesirable harm to the patient.
Currently, the treatment of malignant tumors still focuses on surgery, chemotherapy, radiotherapy, and immunotherapy.Bacillus Calmette-Guerin (BCG) is the only bacterial strain approved for clinical use in the treatment of bladder cancer, which provides a benchmark and example for subsequent development of bacteria cancer therapy.Besides, different engineered oncolytic bacteria are being evaluated clinically and the results can reveal a new direction of development.With the continuous development of scientific research, the deepening of synthetic biology, and the exploration of various mechanisms, the engineering of live bacteria could be a perfect modulator to be combined with the traditional methods of antitumor treatment.Furthermore, the combination of bacteria therapies and other anti-tumor cellular therapies external modalities also shows great potential to launch the treatment of malignant tumors.

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Photothermal therapy using engineered live bacteria.(A) N-V-J is formed by a VNP20009 loaded with NHS-N782 and a JQ-1.(B) Schematic drawing of N-V-J inducing an inflamed "hot" tumor under a near-infrared laser irradiation.Reproduced with permission.28Copyright 2022, Elsevier.CuS, which can facilitate tumor-specific PTT under local NIR laser irradiation.Concomitantly, Cu þ ions dissolve from Cu 2 O into the low-pH TME and then catalyze the Fenton-like reaction to decompose endogenous H 2 O 2 into cytotoxic hydroxyl radicals (•OH).Bacterial metabolism serves as a biological-switch in this scenario and induces a massive release of tumor antigens and DAPMS, which favors the PTT-enhanced nanocatalytic treatment leading to a high sensitivity of tumors to immune checkpoint blockade (ICB) therapy.

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TME stimuli-responsive engineered live bacteria.(A) Synthetic scheme of norbornene-terminated and CDM-linked PMDOX and (B) PMTOS copolymers.(C) The acid-labile linker CDM and norbornene terminals are linked to PM DOX and PM TOS copolymers, then EcN load PM DOX /PM TOS copolymers via tetrazine derivatives.When PM DOX /PM TOS copolymers released from EcN in acid TME, they form M D/T hybrid micelles and enter into tumor cells by endocytosis.In response to cytosolic GSH, DOX and TOS were released from micelles achieving an intracellular drug release.Reproduced with permission.50Copyright 2018, Elsevier.

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Schematic illustration of the anti-tumor therapy of EcN flaB -UCNPs.EcN flaB -UCNPs activate the TLR5 signaling pathway by releasing flabs and inducing innate immune responses to produce cytokines.Reproduced with permission.79Copyright 2022, John Wiley and Sons.

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I G U R E 1 0 IFN-γ produced and secreted by URB induced macrophage polarization, promoted apoptosis of cancer cells, and activated CD4þ and CD8þ T cells (right).High levels of IFN-γ activated T cells and macrophages in the spleen, inhibiting distant tumor growth and lung metastasis through immune memory response (left).Reproduced under terms of the CC-BY license.80a Copyright 2022, The Authors, published by Springer Nature.

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I G U R E 1 1 (A) Illustration of the genetic circuit of temperature sensitive repressors in EcN.(B) Illustration of the genetic circuit to establish a temperature-responsive state switch.TetR is the tetracycline resistance cassette.(C) Illustration of modification of the temperature-responsive state switch to release αCTLA-4 or αPD-L1 nanobodies.The circuit includes an Axe-Txe stability cassette.Reproduced under terms of the CC-BY license. 87Copyright 2022, The Authors, published by Springer Nature.F I G U R E 1 2 Modified attenuated S. Typhimurium accumulate and proliferate in tumor sites after intravenous injection secreting ClyA into the TME by the infiltration of immune cells and disrupted cancerous stromal cells and cancer cells in a mouse model of human pancreatic cancer.Reproduced with permission. 90Copyright 2021, Elsevier.
Biotechnology-based engineered bacterial therapeutics.
T A B L E 2Vector strains generated to carry synchronous lysis loop in bacteria 94, 95TransgenesisIntroducing DNA or RNA introduced into target cells by gene transfer 96