Bacteria-based immunotherapy for cancer: a systematic review of preclinical studies

Immunotherapy has been emerging as a powerful strategy for cancer management. Recently, accumulating evidence has demonstrated that bacteria-based immunotherapy including naive bacteria, bacterial components, and bacterial derivatives, can modulate immune response via various cellular and molecular pathways. The key mechanisms of bacterial antitumor immunity include inducing immune cells to kill tumor cells directly or reverse the immunosuppressive microenvironment. Currently, bacterial antigens synthesized as vaccine candidates by bioengineering technology are novel antitumor immunotherapy. Especially the combination therapy of bacterial vaccine with conventional therapies may further achieve enhanced therapeutic benefits against cancers. However, the clinical translation of bacteria-based immunotherapy is limited for biosafety concerns and non-uniform production standards. In this review, we aim to summarize immunotherapy strategies based on advanced bacterial therapeutics and discuss their potential for cancer management, we will also propose approaches for optimizing bacteria-based immunotherapy for facilitating clinical translation.


Introduction
Traditional cancer therapies focus on inhibiting tumor growth while largely ignoring the role of the immune system in killing cancer cells (1). In the last ten years, numerous groundbreaking immunotherapies have been authorized for clinical practice (2). Especially cancer vaccines and immune checkpoint inhibitors (ICIs) (3). However, there is still no effective strategy to implement a cure since the hypoxia and immunosuppressive tumor microenvironment significantly restrict antitumor immunity (3). Additional research demonstrated that bacteria activate immune cells even in the immunosuppressive microenvironment of tumors. The rich pathogen-associated molecular patterns (PAMPs) of bacteria make them highly immunogenic, which further improve the specific immune recognition and elimination of cancer cells (4). Indeed, bacteriabased immunotherapy is of great potential to enhance the antitumor effect and is gradually being developed (5).
Bacteria act as natural immune antigens, stimulating the immune system to kill tumors by activating immune cells. In the 19th century, William B. Coley pioneered the use of bacteria for cancer immunotherapy by treating sarcomas patients with the injection of streptococcus pyogenes (6,7). Further discovery of a growing number of gut bacteria that can be used to fight immune elimination and immune escape in cancer (3,8). Recent clinical trials show Actinobacteria of Firmicutes and Lachnospiraceae/ Rumococcus provoke strong antitumor immunity (9). Further understanding of bacterial-host interaction in the immune environment promotes improved tumor immunotherapy (10). With in-depth research, bacterial antigens, and bacterial outer membrane vesicles (OMVs) (11,12), have been shaped into favorable immune activators to fight cancer (13). In addition, personalized engineered bacteria have been developed to enhance the antitumor immune response (14,15). For example, attenuated Listeria-based vaccines effectively activate antitumor immune responses and eliminate tumors (16). There have also been several effective strategies that combine bacterial immunotherapy with conventional therapies (17). However, clinical transition still exists issues, such as objective adverse immune events and biosafety concerns (18).
As mentioned above, naive bacteria and modified bacteria can both activate the antitumor immune response. Here, we focus on reviewing the recent progress on bacteria attenuated techniques, diverse use patterns for bacterial vaccines, and emerging combination therapy. The opportunities and challenges between the preclinical research on bacterial immunotherapy and the need for clinical application are discussed. To further consolidate the knowledge base used to improve immunotherapy for tumors by learning about new preclinical findings in cancer immunotherapy.

Bacteria-based natural antitumor immunity roles
Numerous studies have been conducted on the intricate immune response of bacteria in the tumor environment (14,19,20). In several cancer models, it has been discovered that bacteria specifically colonize tumors, multiply, have tumor-lysing effects, and stimulate the immune system (21,22). In addition, bacteria, components, and related derivatives contain a variety of pathogenassociated molecular patterns that trigger immune responses (23, 24) ( Figure 1). The key factor in the effectiveness of bacterial antitumor immunotherapy has been identified through a review and analysis of bacteria-based tumor immunomodulatory effects.

Naive bacteria
The discovery of tumor regression caused by bacterial infection opened up bacterial direct antitumor immunotherapy (25). The extensively researched agent in bacterial-mediated cancer therapy is Salmonella, which has a strong potential to induce direct tumor cell death and manipulate the immune elements of the tumor microenvironment (TEM) to assist tumor inhibition (26, 27). For example, tumor-programmed death-ligand 1 (PD-L1) expression can be downregulated by Salmonella, which alters tumor immune tolerance (28). Based on this feature, a recent research study increased the effectiveness of PD-L1 blockers in the treatment of Salmonella colorectal cancer (29). Salmonella may increase the therapeutic benefits of ICIs in the treatment of cancer. Besides, Salmonella is one of the strong candidates to overcome current obstacles to cancer immunotherapy. Three main methods direct antitumor, drug or antigen delivery, and combination immunotherapy can be used by Salmonella to treat cancer (30, 31).
Substantial reported clinical trials show a strong link between intestinal flora and anti-cancer immunity (32-35). Firstly, gut bacteria can act as immune adjuvants to improve the therapeutic coverage and potentially limit ICI toxicity (36, 37), such as the combination of Escherichia coli (E.coli) and TGF-beta blockade galunisertib (Gal) significantly boosts antitumor immunity (38). Probiotics, such as Lactobacillus, Bifidobacterium, Helicobacter pylori (H.pylori), and Lactobacillus rhamnosus probiotic-M9, have come into focus as the driving force behind this synergistic effect (39-41). Recently, a mixture of four Clostridium strains (CC4) has been used to successfully optimize anti-PD-1 therapy (41). By colonizing them separately, it was concluded that they might function as independent immunotherapeutic agents for melanoma and colorectal cancer (CRC) (42). There is hope for more strategies of bacterial synergistic immunotherapy for cancer. As akkermansia muciniphila induces immunoglobulin G1 (IgG1) antibodies and antigen-specific T cell responses in mice, mechanisms by which intestinal bacteria increase immune effects are being studied in greater depth (43). Emerging research validates that uropathogenic E. coli (UPEC) derived from prostatic patients can enhance the immunogenicity of drug-resistant cancer cells and restore immune response (44). Enough to understand the importance of gut microbiota in cancer immunotherapy.
The discovery of more natural bacteria for various immunomodulatory functions is ongoing. For example, colonization of Helicobacter hepaticus (Hhep) in CRC mice boosted tumor infiltration by T follicular helper (Tfh) cells and limited tumor growth (45). Besides, a retrospective clinical trial showed that the manipulation of the gut microbiota by C. butyricum MIYAIRI 588 (CBM588) greatly increased the efficacy of ICI in treating lung cancer (46). However, adverse immune responses remain unaddressed due to bacterial toxicity and individual variability (47). With the advancement of bioengineering technology, one trial evaluated transferrin-modified nano-thin polyelectrolyte shell-coated Subtilis cells in vitro, which induces leukemia cell death more strongly than controls (48). The modification mode weakens the immunogenicity of the host to bacteria while optimizing the targeting ability of bacteria in the complex TEM (47).

Bacterial components
Not only bacteria but also bacterial components behave as immune adjuvants. Recent research reviewed the essential mechanism by which bacterial flagellin acts as an immunomodulator (49, 50). On the one hand, it reveals that flagellin recognizes receptors specifically with the germ line-encoded pattern. Highlighting the physical connection of flagellin to antigens enhances the activation of immune cells. On the other hand, the interaction of flagellin with highly immunogenic tumors induces Th1 responses and suppresses Tregs, thereby inhibiting tumor growth independently (51). It is further convincing that Salmonella typhimurium (S. typhimurium) was modified to express flagellin B, which enhanced tumor immune control via a TLR-dependent mechanism (52).
The immunogenicity of bacterial wall components is well-used in antitumor therapy (53). For example, monophosphorylate lipid A (MPLA) was designed to eradicate toxic proinflammatory effects while maintaining sufficient immunogenicity (54). Emerging data show that beta-glucan and muramyl peptides from bacterial walls specifically recognize receptors to stimulate immune cells (55,56). Besides, lipopolysaccharide (LPS) increased the effectiveness of adoptive CD8(+) T-cell transfer in CRC patients while weakening tumor suppressive immunity (57,58). Additionally demonstrated to be outstanding immunomodulators in the TEM, modified LPS on bacteria offer significant potential for immunotherapy (59). Interestingly, bacterial ghosts (BGs) were found to be substantially more immunostimulatory than LPS (60). BGs are empty envelopes of gram-negative bacteria, which can effectively induce immune cells to produce long-term antitumor immune memory effects (61). Bacterial cyclic dinucleotides (CDNs) were also found to amplify immune effects in tumor regions by triggering the STING pathway (62). Overall, bacterial components have the potential to be used as anticancer immune adjuvants in therapeutic settings. Further needs to be combined with continually improving medical technology to develop into a mature therapeutic agent.

Bacterial derivatives
More naturally occurring active compounds released by bacteria have been identified as a result of contemporary bioprospecting approaches (63). The beneficial immune regulation of bacterial metabolites in the oncogenic mechanism is becoming clear (64,65). Monophosphorylate lipid A (MPL) was the first bacterial immune adjuvant approved for human use. But it does insufficiently elicit an effective response in weakly immunogenic tumors (66). There is considerable scientific evidence that bacterial toxins can activate antitumor adaptive immune as immunotherapeutic proteins (67). However, when colonized in patients, their innate immunogenicity renders them inert against malignancies. In addition, there is an antitumor mechanism of bacterial metabolites based on immunotherapy activation. Under the premise that T cells have been stimulated with ICIs to express adenosine A2A receptors, B. pseudolongum-derived inosine could enhance antitumor immunity by co-stimulation (68). Some bacterial secretions allow for indirect manipulation of the immune system in malignancies. For example, the transforming growth factor-b1 (TGF-b1) produced by certain strains promotes the secretion of interferon-g (IFN-g) to inhibit B16F10 tumor growth (69). Taken together, bacterial metabolites have the potential to be developed as antitumor immunotherapeutic.
Bacterial membrane vesicles (BMVs) gain widespread attention in the biomedical field, such as bacterial outer membrane vesicles (OMVs), inner membrane vesicles (IMVs), and double-membrane vesicles (DMVs) (70). BMV function as adjuvants by increasing antigen absorption and presentation to activate innate immunity (71). Further have found their ability to specifically target tumors, BMVs may be used as a drug delivery vehicle to increase the accumulation at the tumor site (72,73). For example, doxorubicin-loaded with OMVs or IMVs, can be transported more efficiently to non-small lung cancer A549 cells (74,75). More importantly, pathological antigens, PAMPs, and other immunostimulatory molecules are contained in BMVs (76), which is beneficial for the development of bacterial vaccines (77). For example, vaccinations based on P. aeruginosa-derived DMV successfully induce innate and adaptive immune responses in mouse models (78). In addition, the nano size of BMVs facilitates accumulation in tumor regions, further enhancing the permeation and retention (EPR) effect to trigger an antitumor immune response. Because of these qualities, BMV is expected to participate in antitumor treatment as an immunotherapeutic agent. For example, OMV of similar size to diseased antigens has been designed as an effective antitumor immune vaccine (79). Interestingly, a study artificially produced a non-toxic synthetic bacterial vesicle (SyBV). SyBV, when combined with other immunotherapeutic drugs, activates immune cells, producing melanoma regression, and better adjuvant effects increasing anti-PD-1 inhibitor therapeutic efficacy (80). Furthermore, bacteriaderived vesicles can be genetically and chemically manipulated to serve as vaccines or vaccine vectors, making them a promising cancer treatment option (73).

Effects of bacterial therapeutic agents on immune cells Regulating nonspecific immune cells
The non-specific immune function of phagocytes is the first line of defense against pathogenic invasion of the immune system. Nonpathogenic bacteria can aggressively target phagocytes even though they cannot actively enter host cells. This characteristic was used to create transgenic bacteria that target tumors and promote the "passive transfection" of phagocytes (81). Typical phagocytes that modulate the tumor immune microenvironment include macrophages, dendritic cells, and neutrophils. (Figure 1) Some engineered bacterial therapeutics, such as Salmonella coated with allylamine hydrochloride and engineered FlaB-secreting bacteria, are colonized in TEM to promote the infiltration of numerous macrophages and neutrophils (49, 82). Specifically, high levels of macrophage maturation factors were detected in NSI mice injected with live BCG, such as IL-6, IL-1b, IL-12P70, TNF superfamily member 11, tumor necrosis factor-a and monocyte chemotactic protein 1 (83). Besides, specific nanomaterials are selected to modify bacteria into nanocomposites, such as PLGA-R848, and polydopamine binds to the surface of E. coli, which can induce or promote the polarization of macrophages from M2 type to proinflammatory M1 type (84,85). Similarly, Akkermansia muciniphila-derived OMV polarizes macrophages as one of the immune mechanisms against pancreatic tumors (86).
Early induction of monocyte-derived dendritic cells is one of the indications of successful cancer immune response activation (87). Factors derived from bacteria are often used as immune adjuvants for DC-based antitumor therapy, such as the 50S ribosomal protein and w-chorismate mutase TBCM of Mycobacterium tuberculosis, FcgR secreted by Staphylococcus aureus (87)(88)(89). TBCM activates DC in a dose-dependent manner through TLR4-mediated signaling, including upregulating co-stimulatory molecules, increasing proinflammatory cytokine secretion, and migration, and triggering Th1 immune response (88). Besides, cross-presentation of DCs within the tumor region can also be initiated by direct colonization of Bifidobacteria via the STING pathway (90). Moreover, the codelivery of bacteria with other adjuvants aids in DC maturation (91). For example, the co-delivery of cysteine protease inhibitor U-Omp19 with Ag of Brucella can lengthen the half-life of Ag in DCs, which aids in the sustained expression of MHC class I/peptide complexes on the DC surface (92). As a result, when U-Omp19 increases Ag cross-presentation from DCs to CD8(+) T cells, a powerful antitumor adaptive immune response is triggered (92).
Natural killer cells (NKs) are members of the innate lymphocyte (ILC) family, which exhibit pleiotropic immune behavior in tumors (92). Bacteria, such as Salmonella and Mycobacterium, can inhibit the spread of many cancers in an NK cell-dependent way (93,94). Mechanistically, NK cells are stimulated by bacteria to produce IFN-g, which regulates their accumulation, activation, and cytotoxicity to inhibit tumor metastasis (95). More comprehensively, more cytokines, including TNF-g, perforin, IL-2, and IL-1, were validated in a preclinical study in which the Nocardia redis cell wall skeleton (Nr-CWS) inhibited melanoma-induced lung metastasis in mice (96). Besides, it was found that the expression of CD69, TRAIL, and FasL on NK cells increased, leading to the hypothesis that one of the anti-metastatic mechanisms is promoting the terminal differentiation of NK cells (96). Bacteria-derived immunogenic substances, such as Clostridium clostridia-spores (CVN-NT) and the E. coli fusion protein dsNKG2D-IL-15, can also support the continuation of NK cell toxic function on tumor cells (97, 98).

Regulating specific immune cells
Numerous preclinical evaluations have shown that bacteria rely on T cells to generate antitumor adaptive immune responses (99) (Figure 1), such as the CD3(+) T cell immune response activated by the Helicobacter pylori vaccine to inhibit GC cell growth (100). Through the analysis of Listeria, E. coli in common immune models, it was revealed that bacterial immune antigens activated CD8(+) T cells in the induction phase to eliminate tumors, and simultaneously promoted CD4(+) and CD8(+) T cells to eradicate tumors (19, 101,102). Further found that bacteria primarily induce specific CD4 (+) or CD8(+) T cell immune function enhancement through IFN-signaling in tumors, such as attenuated Listeria monocytogenes (LADD-Ag) and BCG (103, 104). Besides, adoptive metastasis specific to Bacteroides fragilis T cells can address the ineffectiveness of CTLA-4 blockade, which occurs in antibiotic-treated conditions or a sterile tumor setting (105). Interestingly, SVYRYYGL (SVY) immunity derived from the commensal bacterium Bifidobacterium breve, which refers to T cells targeting an epitope, such as SVY-specific T cells cross-reacting with the melanoma antigen SIYRYYGL (106). Furthermore, bacteria can be engineered to enhance tumor-infiltrating T cells by regulating chemokines or directly upregulating L-arginine concentration (107, 108), such as S.typhimurium A1-R and E.coli expressing CD47nb (109, 110). Overall, bacterial therapeutics have the potential to induce or enhance the immune response of T cells in favor of tumor eradication.

Bacteria-based preclinical treatment modes
Routine colonization of bacteria-based vaccine formulations in oncology is still limited by biological barriers (111). For example, Clostridium endophytic spores are poorly immunogenic, do not induce an immune response, and remain in multiple normal organs after intravenous injection, but reach the tumor region triggering a strong inflammatory and immune response that massively destroys the tumor (112). So, it is inferred that intratumoral injection may better reflect the medical application value of the antigen. Special emphasis is given to rational colonization of bacterial formulations which is beneficial for the immune response to penetrate the TEM (113) (Figure 2 and Table 1). We also reveal that practical animal models are the key to the clinical success of immunotherapy (155,156). Provide a reference for creating potential opportunities for clinical translation of bacterial vaccinology research.

Oral bacterial vaccine
Oral tumor vaccines have been shown to successfully cross the intestinal epithelial barrier, be taken up by DCs in the intestinal lamina propria, result in the production of draining lymph nodes and the presentation of tumor antigens, and ultimately induce a potent antitumor immune response (157). For example, autologous vascular endothelial growth factor receptor 2 (VEGFR2) was successfully delivered into tumor blood vessels by an attenuated Salmonella-based oral-engineered vaccine, and tumor growth in B16F10 mice was subsequently inhibited by significant T cell activation (116). In a similar experiment, the Salmonella Typhi vaccine strain CVD915 was given orally to breast cancer mice to assess its effectiveness against tumors and ability to prevent liver metastasis (115). Such heartening preclinical outcomes supported the creation of the first oral cancer vaccine, VXM01, whose antitumor immune reaction was evaluated and authorized in patients with prostate cancer (115). More well-established preclinical studies explored oral doses suitable for humans. For example, the recombinant Bifidobacterium longum oral vaccine is precisely quantified for the treatment of castration-resistant prostate cancer (CRPC) (118). The rich treatment mode has broadened the range of applications for bacterial vaccines in the challenging clinical tumor treatment environment.

Intravenous bacterial vaccine
Due to their massive dosages, quick medication effects, and minimal toxicity and side effects, intravenous vaccinations have been widely used for the treatment of cancer. For example, cancer vaccines based on bacterial-plant hybrid vesicles (BPNs) were injected intravenously into CT26 and Luc-4T1 tumor models to study the immunotherapy effect (122). This intravenous vaccine still has some limitations in terms of clinical use, such as rapid clearance and increased bacterial toxicity due to systemic administration. However, there is mounting proof from considerable preclinical studies that intravenous bacterial vaccines have an antitumor effect. For example, based on genomic detection of bone tumors after intravenous red blood cell membranes encapsulated Lmo (Lmo@ RBC), researchers found that the therapeutic agent activated cell pyroptosis to reverse the immunosuppressive TEM and enhance systemic antitumor immunity (123). The discovery of emerging mechanisms also has lain a theoretical foundation for the upcoming creation of multifaceted bacterial vaccines. Preclinical studies using transplanted tumor models in mice and clinical trials using human patients produce different results. A genetically modified S.typhimurium can be intravenously colonized into mice and humans with breast cancer to the same extent to overcome this significant limiting factor in clinical translation (124). Besides, preclinical experimental research is now being conducted on numerous intravenous bacterial agents. The main innovative parts are immunotherapy systems based on engineered bacteria and OMVs, such as spatiotemporal control of engineered bacteria and self-Blockade of PD-L1 with bacteria-derived OMVs (126, 158). Their excellent immune antitumor effects have been demonstrated in a variety of tumor models, such as melanoma, 4T1 tumor, and colon tumor models. The blind zone in the development of antitumor bacterial vaccines is supplemented and optimized from all directions.

Intratumoral injection of bacterial vaccine
Cancer immunotherapy can be realized by directly injecting bacterial therapeutic agents into the tumor site, which has the advantage of lower tolerability and higher safety over intravenous injection. Preclinical studies with significant antitumor effects are now available for reference. For example, engineered E. coli SYNB1891 was colonized in situ into the four tumors, including melanoma, EL4 lymphoma, A20 lymphoma, and 4T1 breast tumor. It was revealed in detail that this intratumoral therapeutic agent can activate complementary innate immune pathways by stimulating the STING channel (134). This kind of work aims to achieve antitumor immunity while building biocontainment capabilities to meet producibility and regulatory standards (134). Besides, glycoproteins can be recognized by the immune system to stimulate innate and humoral immunity (159). Novel vaccines using polysaccharide-encapsulated bacteria can exert a key inhibitory effect on malignant tumors (159). However, several clinical trials have fallen short due to related immune tolerance. More advanced, E. coli NISSLE1917 is programmed to regulate the dynamic expression of surface capsular polysaccharides, resulting in high immune tolerance in situ colon and breast tumor encapsulation (135). The novel finding sheds new light on the tolerable dose of novel antitumor bacterial vaccines (136). Overall, it paves the way for the development of a multimodal, versatile bacterial vaccine.

Intraperitoneal injection of bacterial vaccine
Because of the large surface area of the peritoneum, intraperitoneal injection absorbs quickly, is easy to use, and offers  a wide range of uses. The use of this colonization model for novel antigens is expected to further overcome the limitations of tumor immunosuppressive microenvironment. For example, a flagellinadjuvanted HPV E7 vaccine was engineered to express the long peptide E7-LP35 which shows excellent immunogenicity (140). It was injected intraperitoneally into a mouse TC-1 tumor model to enhance the effective immune response (140). Besides, it is possible to isolate immunological components from bacteria to obtain effective vaccination antigens. For example, recombinant Nap protein (HP-Nap) derived from Helicobacter pylori was loaded into synthetic chitosan to form nanoparticle complexes, which were injected intraperitoneally into 4T1 mice to regulate cytokine production and activate the immune system to kill tumor cells (141). For the study focused on breast cancer metastasis,a vaccine  (154) developed based on Listeria expressing the tumor-associated antigen Mage-b, which effectively inhibited tumor metastasis but left serious hepatotoxicity (142). The newly designed galactosyl ceramide complex of Listeria almost abolished cancer cell metastasis without concomitant toxicity (142). In general, recombinant antigens with different modifications can be colonized intraperitoneally to achieve cancer targeting with bacterial vaccines.

Subcutaneous injection of bacterial vaccine
The therapeutic effect can be prolonged by using subcutaneous injection absorption, which is slower than intravascular absorption but faster and more precise than oral and enema methods. Numerous studies have found that using less risky subcutaneous injections rather than intravascular ones, tailored bacterial nanovaccines can reduce the intrinsic immunogenicity of bacteria. For example, a biomimetic vaccine based on a demi-bacteria (DB) from Bacillus achieves synergistic induction of cellular and humoral immune responses in a subcutaneous injection mode (144). Besides, OMVs are considered suitable vaccine candidates with their unique immunity (77,147). Recently some powerful OMV-based vaccines have been subcutaneously colonized into different cancer models. For example, the OMV-LL-mRNA vaccine can protect mice by inducing a long-lasting adaptive immune response while also quickly inhibiting the growth and metastasis of colon and melanin tumors (148). More advanced, a self-assembled protein nano vaccine (BMC-OVA) has been formed by fusion in hydrogelcoated engineered E. coli (145). After the engineered bacteria microcapsule was subcutaneously colonized into the mouse breast tumor model, the active release of BMC-OVA triggered specific immunity to lyse tumor cells B16-OVA (145). These are promising strategies to produce clinic subcutaneous long-acting immune vaccines.

Intradermal injection of bacterial vaccine
Bacteria have been extensively used in the creation of active immune vaccines for the treatment of cancer. The long-established antitumor vaccine Bacille Calmette-Guerin (BCG) is usually administered intradermally to prevent and treat noninvasive bladder cancer (160,161), and is known for high efficiency in small doses. Other newly developed recombinant BCG (rBCG) has been subcutaneously colonized in mouse models. They have reduced toxicity and enhanced the expression of tumor-associated antigens (TAAs) (162)(163)(164). Additionally, several bacterial cancer vaccines are presently undergoing clinical development (165). For example, clinical trials of recombinant CyaA vaccines for intradermal injection cervical cancer treatment (166). CyaA is a vital virulence protein of Bordetella pertussis that suppresses the adaptive immune response by regulating dendritic cells. Certain recombinant CyaA toxins have been used as vaccination carriers to spread the HPV virus and cause Th1-polarized CD8 (+) cytotoxic T-lymphocyte responses (154). The effectiveness of the pertussis vaccine in mice can also be increased by co-injecting CyaA with pertussis bacillus anti-donation. Further expected that the number of powerful bacterial vaccines could be expanded in the future.

Development of bacteria-based combination treatment
The inability to deeply eradicate tumors and adverse side effects are caused by the toxicity of traditional cancer treatment to normal cells and the tolerance of tumor cells (167). Some of the drawbacks of conventional therapy have been effectively addressed by the creation of novel bacterial antitumor immunotherapies by the development of novel bacterial antitumor immunotherapies. Even more intriguing is the preclinical study on various combination immunotherapies for cancer ( Figure 3 and Table 2). Bacterial immunotherapy and other single antitumor therapies make up for each other's deficiencies (22,207), such as chemotherapy, photodynamic therapy, photothermal therapy, immune checkpoint inhibitor therapy, and oncolytic virus therapy. The bioavailability of various combination therapies is enhanced by the distinctive compatibility of engineered bacteria (5,208,209). The tumor-targeting and immune response properties of bacteria are also significantly improved by combined therapeutic techniques. By exploring the innovative and optimal capabilities of these versatile tumor immunotherapy systems, researchers are attempting to surmount the numerous challenges of clinical translation.

Modulated with photodynamic therapy
The therapeutic effect of bacterial cancer therapy and photodynamic cancer therapy is still limited. Through a clever fusion of these two methods, more innovative cancer immunotherapy is actively sought. Looking back at numerous relevant preclinical studies, the most basic combination is the direct co-delivery of engineered bacteria and photosensitizers to the tumor site. For example, E. coli was designed to express the fluorogenic-activated protein (FAP), and the photosensitizer PS was colonized together in colon cancer models (168). On the one hand, intratumoral bacteria invade the vascular system to form a thrombus, which darkens the tumor site to facilitate NIR absorption. On the other hand, the activation of PS by a specific laser promotes the production of ROS, thereby lysing and releasing antigens to activate immune cells (168). In addition to boosting the immune response triggered by the bacteria against the tumor, the synergistic effect also lessens the harmful side effects of bacteria. Besides, photosensitizers can be designed to load bacteria into biological combinatorial therapeutics, like TPApy was loaded on Clostridium butyrate (169). This wrapped combination prevents the photosensitizer from being destroyed to a certain extent and keeps the photodynamic effect stable.
A better overall antitumor immune effect was demonstrated by PD-L1 antibody-modified attenuated Salmonella vesicles-loaded self-assembled nanocomplexes (CAT-Ce6), which can alleviate tumor hypoxia, promote the aggregation of free Ce6 to enhance the photodynamic killing effect, as well as improve the exposure of tumor-associated antigens to elicit an immune response, eventually, prolong the synergistic effect by prolonged release (170). Another triple combination therapy is that the laser simulates engineered bacteria to produce INF-g, which directly inhibits tumor growth as a third-step synergistic treatment platform (171). However, using this new class of processualized combination therapies, the efficacy of immunotherapy has only been confirmed in a few tumor models. In one study, an attenuated strain of S. typhimurium was transformed into bioluminescent bacteria by the addition of a plasmid expressing firefly luciferase as an internal light source (17). In melanoma and rabbit tumor models, Luc-S. T (DeltappGpp) together with Dluciferin localization on tumors by hydrogel and continuous luminescence, activating Ce6 to exert significant antitumor immune (17). In comparison to external lasers, bio-spontaneous PDT is a highly efficient and adaptable treatment with no penetration restrictions. Overall, rapid immune response and rapid progression are generally advantages of bacteria-based photodynamic immunotherapy, but the translation to a clinical immunotherapy combo platform remains a challenge.

Strengthen with photothermal therapy
Photothermal therapy (PTT) is critical for the elimination of advanced malignant tumors. The fundamental process involves increasing NIR absorption near the tumor, which raises warmth and causes tumor ablation (178). Existing antitumor studies have found that the near-infrared chemotaxis of natural bacteria is highly desirable. For example, natural active photosynthetic bacteria (PSB) gather in hypoxic tumor areas with hypoxia targeting, and then strongly absorb NIR to generate heat to kill cancer cells (173). Besides, PSB enhances the immune response of T lymphocytes, thereby realizing synergistic effects of PTT antitumor therapy and bacterial immunity. Its dual efficacy is a key reference idea for biointelligent cancer treatment. For example, E. coli was engineered as a heat-inducible bacterium (TIB) expressing PD1, which can both exert photothermal effects and specifically recognize PD-L1 on the surface of tumor cells, thus increasing the role of alleviating the tumor immunosuppressive microenvironment (174). Similarly, E. coli-based tellurium nanorods rely on photothermal characteristics and reprogrammed macrophages to generate the same bacterial immune-PTT synergy (175). Bacteria-based combination antitumor immunotherapy. Bacteria immunotherapy in combination with other therapies promotes antitumor treatment, generally including photodynamic therapy, photothermal therapy, radiation therapy, chemotherapy, immune checkpoint inhibitors, and oncolytic viruses. The illustration is drawn on the BioRender website. Due to the non-pathogenic nature of EcN and the nonproliferative character of TeNRs, relatively high dosages of Te@ EcN can be swiftly metabolized and eliminated in normal tissues (175). These complementary approaches have clinical conversion value and collectively enhance the treatment of individually mediated tumors. Moreover, exogenous photothermal agents are designed to be loaded on bacteria and then co-colonized into the tumor. In an in situ glioblastoma mouse model, rojan bacteria equipped with photosensitive ICG silicon nanoparticles were verified to have the synergistic effect of photothermal tumor destruction and immunological antitumor, which was reflected in the prolongation of survival of GBM mice (176). The cleverly designed activated immune response leads to the production of NO, eventually leading to the extension of vasodilation-promoting  (206) therapeutics to metastatic triple-negative breast tumors (177). A wide range of prospects for antitumor immunotherapy has been made possible by this self-driving synergistic treatment approach based on bacterial characteristics.

Improved with radiation therapy
Since the TEM is in a state of immunosuppression, it is difficult for single radiotherapy to release sufficient antigens to activate the antitumor immune response (183,210). Bacteria could provide distinct benefits as natural carriers for tumor-targeted administration and immune activation (184). Engineering changes enable bacteria to deliver specific tumor antigens that can be employed to boost immune activation during radiation therapy. For example, attenuated Salmonella VNP20009 coated with antigen-adsorbing cationic polymer nanoparticles combined with irradiation, greatly increased the activation of DCs and systemic antitumor effect (185). Moreover, therapeutic radiation can be given endogenously to the tumor site by bacteria, for example, by inactivating microbes with radioactive 125I/131I labeling (184). This ingenious combination allows for a robust and long-lasting internal radioimmune response against malignancies while maintaining a high biosafety profile. The clinical value of bacteria-based immunotherapy with radiation has been further confirmed in anal cancer patients. An engineered live attenuated Listeria monocytogenes (Lm) bacterium(ADXS11-0011 Lm-LLO), is safe in combination with standardized chemoradiotherapy for anal cancer (183). However, the specific therapeutic impact needs to be validated further. Overall, bacteria-based radioimmunotherapy is a promising and transformable combination antitumor strategy (211).

Combined with chemotherapy drugs
Chemotherapy can cause negative physiological reactions in cancer patients and only partially suppress tumor growth (38). Cancer immunotherapy mediated by bacteria in combination with chemotherapy has the potential to overcome typical antitumor obstacles. Preclinical data have shown that specific lactic acid bacteria mixtures with adjuvant 5-fluorouracil chemotherapy inhibit 4T1 breast cancer tumor growth and associated side effects (186). More profoundly, interferon IFN-g has been shown to promote attenuated Salmonella to kill mouse colon tumor cells by interrupting tumor recruitment concentration of granulocytes (187). Besides, IFNg stimulated M1-like macrophages and CD4(+) and CD8(+) T cells to infiltrate tumors to weaken bacterial immunogenicity (187). It has been discovered that the combination of chemotherapy and bacterial therapy is a promising antitumor immunotherapy.
Synergistic treatments for cancer have evolved thanks to bioengineering technology. For example, genetically modified bacterial protoplast-derived nanovesicles (PDNV) were used to load doxorubicin/idarubicin and deliver them to tumor tissue, precisely targeting chemotherapy for tumors (75). Another engineered Salmonella-driven drug delivery system allows natural ursolic acid UA to attract bacterial accumulation in CT26 colon tumors and accelerates tumor lysis (188). The natural antitumor immune activity of the bacteria was unaffected by these engineering techniques, which opens a new type of chemical-bacterial immunosynergistic therapy. This kind of preclinical combination therapy research fills the shortcomings of monotherapy and the safety has been fully confirmed, which is evolving into a clinical treatment for cancer patients.

Promoted with immune checkpoint inhibitors
The current obvious defect of immune checkpoint inhibitors (ICIs) is that they bind to other immune cells expressing PD-L1, resulting in inefficient blockade of tumors and adverse effects. The shortcomings of the therapy could be reduced by the creation of particular engineered bacterial agents (208). For example, the selfblocking of PD-L1 in tumor cells was accomplished by a LyP1 polypeptide-modified outer membrane vesicle (LOMV) loaded with a PD-1 plasmid (158). Considerable preclinical research results to improve the treatment of bladder cancer are accumulating. For example, to promote dual immunity and the elimination of tumors, some researchers combined the use of attenuated mycobacterium MTBVAC with an anti-progressive cell death ligand (anti-PD-L1) (192). Furthermore, in therapeutically relevant cancer models, the inhibitory effects of targeted ultrasound-controlled bacteriotherapy in situ tumors have been established (126). What's more attractive is that ultrasound-controlled E. coli can be combined and controlled the release of ICIs to optimize A20 tumor immunotherapy (201). The spatiotemporal controllability of ICIs in 4T1 tumors can also be improved by altering E. coli, which expresses photothermal melanin, and ICIs enhance melanin-induced immune response (200). But this combined technique may only be a useful tool in the treatment of solid malignancies. Furthermore, EcN was comodified by ICIs, tumor-specific antigens, and polymeric dopamine to create agents for multimodal colon carcinoma immunotherapy. Then cytotoxic T lymphocytes were recognized to be activated by ICIs bound to bacterial surfaces (85). Dendritic cell maturation was directly induced by connected antigens, which in turn stimulate tumor-specific immune responses (85). Polydopamine exerted photothermal effects to stimulate the polarization of tumorassociated macrophages into pro-inflammatory phenotypic (85), hence the formation of a bacteria-based triple cancer immunotherapy platform. In summary, ICIs has demonstrated their value in synergistic therapy, mainly in the form of optimized and innovative bacterial antitumor immunotherapy.

Combined with the oncolytic virus
Oncolytic viruses use the characteristics of unlimited reproduction of cancer cells to replicate offspring, thereby specifically targeting and destroying cancer cells. Oncolytic virotherapy and bacterial therapy are clinically translational components of novel cancer therapies (206), which both exert durable antitumor immune responses without little off-target toxicity (212). A new development in clinical treatment may result from the fusion of the two therapies. Traces back to the first preclinical example of using complementary combinations of microorganisms to improve tumor treatment outcomes, researchers used non-pathogenic E. coli expressing B18R to overcome innate immunity and deplete bioactive antiviral cytokines in the TEM, which greatly enhances the ability of subsequent intravenous hydro stomatitis virus (VSV) to infect and destroy tumors (203). In Murine Lewis Lung Carcinoma and HT29 adenocarcinoma model, evidence of effective treatment is a decrease in tumors and increased mouse survival. Besides, due to E. coil BL21's ability to bind to OA through lipids, oncolytic adenovirus (OA) enrichment in non-small cell lung tumors is 170 times higher than that of intravenous naked OA (204). The ingenious combination of bacteria and viruses significantly enhances antitumor immunity. Similarly, the assembly of magnetostrophic bacteria and herpes virus (HSV1716) guided the virus to avoid the harm of neutralizing antibodies, promoting not only active viral targeting to tumors but also the enrichment of activated immune cells in tumors enabling dual immunological destruction of tumors (205). Overall, the unique role of microbiomes in cancer immunotherapy has the potential to optimize clinical roles.

Safety and transformation evaluation for clinical applications Engineering sources and trends
The engineered bacterial model used for tumor immunotherapy is mainly derived from E. coli and Salmonella (213). These bacteria strains can tend to hypoxic TEM of their facultative anaerobic properties. When bacteria are enriched in the tumor region, attenuated treatment is usually used to reduce toxicity to the body (214), such as various attenuated S.typhimurium mutants (e.g., DppGpp, VNP20009, A1-R, SL7207, FAP-encoding S. typhimurium) formed by genetic engineering (168, 215-217). Based on the robust compatibility of the bacteria, they can then be modified to optimize tumor-specific localization (117), as an illustration, a metabolically engineered attenuated Salmonella treatment system was developed, which using the aggregationinduced emission (AIE) photosensitizer MA to localize Salmonella in tumor tissue (172). In addition, numerous natural immune adjuvants or immunotherapeutic agents have been identified for the tumor-suppressing immune environment, many of which are derived from the release of E. coli and Salmonella (195). For example, EcML, a mixture of 4'-monophosphoryl lipid A (MPLA) produced by engineered E. coli, activates DCs to initiate an antitumor adaptive immune response (196). The more striking optimization procedure is that of bacteria intended to be transformed into exogenous antitumor drug delivery vehicles (196) (Figure 4A). Bacterial immunotherapy is now more feasible in combination with other cancer treatments thanks to the engineered approach (191), for example, the addition of Salmonella immunotherapy has led to a safer prognosis for lymphoma patients treated with CHOP (190). Overall, S. typhimurium and E. coli dominate the antitumor development trend with unique advantages, including high tumor targeting specificity, deep tissue penetration, host immune system induction, and strong antitumor activity (132) ( Figure 4B). Many bioengineering technologies further amplify the immunostimulation of bacteria to tumors, superimpose new antitumor immunity on bacteria, and may enhance the biosafety of bacterial immunotherapy agents during the clinical development process (218). The systems of engineered bacterial immunotherapy for cancer entering clinical trials are constantly being updated and are anticipated to produce effective results to optimize clinical treatment.

Evaluation of colonization treatment modes
What cannot be ignored in clinical transformation is the effective persistence and toxicity control of therapeutic agents (219). In preclinical research, the modes of colonization of bacterial therapeutic agents are systemic colonization and local colonization. Systemic colonization, which includes oral and intravenous injections, enters systemic tissues and organs via gastrointestinal absorption or intravenous tracts (127, 220). Local colonization adopts intratumoral injection, subcutaneous injection, and other methods (143, 149,197). Many bacterial agents, such as the exotoxin Pseudomonas rCCK8PE38 and the E. coli enterotoxin STa, are known to stimulate antitumor immune responses by releasing or expressing proteins (221), which have proven potential for cancer immunotherapy (152, 222). The layers of modification or coating result in an increase in the particle size of a bacterial therapeutic agent, for example, Mycobacterium tuberculosis loaded on Zn-, and Mg-containing tricalcium phosphates (TCPs) form potential immune-enhancing adjuvants with particle sizes up to 700 nm (138). Therefore, when systemic colonization occurs, it may exacerbate difficulties such as slowing the rate of tumor targeting and weakening the efficacy of killing them.
It is equally critical to concentrate on biosafety assessment. Rather than local colonization, systemic colonization permits more bacterial treatments to accumulate in healthy tissues. For example, the lactococcal vaccine (FOLactis) peaked in the core of colon tumors after 3 hours of orthotopic colonization (139) ( Figure 5A). But 3 hours after intravenous injection, engineered Salmonella vesicles-coated polymer nanodrugs accumulate mainly in tumors, livers, and kidneys (128) ( Figure 5B). As a result, undesirable side effects such as an immunogenic attack, rejection, allergy, and toxicity are more prevalent. More consideration is the removal of the body after the therapeutic agent kills the tumor. Since local colonization is aimed at the tumor site, high-concentration doses are prone to produce local drug retention and thus evolve into toxic effects. Besides, it may be more susceptible to tolerability due to the rapid achievement of saturation therapeutic effects. To make the therapeutic agent uniformly and durably distributed in the tumor, a double-modified bacterium was designed to simultaneously control the localization of photothermal melanin and ICIs, to achieve a spatiotemporal and controllable dual immune antitumor effect (200). The minimization of adverse occurrences provides hints for solutions to practical issues in clinical translation.

Applicability of tumor models
Spontaneous tumor models do not form quickly or generate huge amounts of tumor material in a short period (150, 223). Besides, there are always discrepancies between artificial tumor models and actual malignancies, making extrapolating outcomes from animal clinical trials to people challenging. The use of orthotopic murine models humanized for CD40 and Fc-g receptors demonstrated a promising new immune mechanism for the treatment of NMIBC, which has drawn significant attention to the humanization of preclinical animal models (164). To optimize the value of clinical translation, uniform model evaluation criteria should be established when using humanized and animal-derived tumor models to explore potential bacterial therapeutics (178) (Figure 6). In addition, multimodal animal models should be established to comprehensively evaluate tumor metastasis and recurrence, for example, while examining the curative effect in high metastatic orthotopic 4T1 tumor model and postsurgical recurrence model (224).
Whether in preclinical trials or clinical trials, cancers designed with novel bacterial therapeutics always favor solid tumors that are readily available and easy to achieve therapeutic effects (225). For example, mouse tumor models of 4T1, CT26, B16F10 (151), and gastrointestinal cancer are frequently used in preclinical studies (226), which have the advantages of relatively high induction rate, high degree of artificial control, and obvious efficacy. Recent ongoing clinical trials have selected patients with bladder cancer, prostate cancer, liver cancer, etc. (178,(227)(228)(229). Non-solid tumors are mainly hematopoietic system tumors that exist in the blood circulation, which are difficult to detect, grow quickly, and are ambiguously staged, so chemotherapy is currently the main treatment. This significantly restricts the application of common bacterial immunotherapy-based combination therapies for the treatment of hematological tumor models. Because of the extensive and complex immune mechanisms involved in various hematologic tumors such as leukemia and various lymphomas, the progression of a bacterial immune platform for nonsolid tumors remains critical.

Production feasibility
One of the main challenges in achieving clinical production is to suppress the high immunogenicity of bacteria in humans. When combating tumors, it must be ensured that normal tissues, organs, and so forth are not affected. The Neiser-derived OMV of meningitidis is now regarded as safe and has the potential to be developed as the ideal antitumor vaccine vector (230). Besides, the absence of standardization of manufacturing settings and preparation processes makes bacteria prone to heterogeneity, limiting clinical application (231). The size, component, and content of the same batch of bacteria and derivatives varied greatly, for example, the component of OMV derived from Helicobacter pylori varied with the growth stage (232). Innovative quality control measures, such as better criteria for characterization parameters, are necessary for future bacteria-based medical products (231)(232)(233). Another significant issue is the control of antigen loading in bacterial vaccines. It is challenging to quantify the expression of endogenous antigens and the degree of modification of exogenous antigens (147). Achieving the optimal amount of antigen delivery is also challenging due to differences in vaccine colonization techniques and patient body composition (148). Therefore, the quantity of bacterial vaccine antigens should be accurately measured in preclinical evaluation, for example, the optimized auxotrophic Salmonella vector strain SF200 as a quantity of 5*106 injected into colorectal tumor models (136). Furthermore, promoting bacterial medical products needs to ensure economic viability. Poor extraction yield, a complicated manufacturing process, and the requirement for cutting-edge disinfection equipment are among the constraints restricting large-scale and inexpensive production of bacterial therapeutics (234). Fortunately, researchers have developed improvements such as detergent-free processes and continuous production methods (235-237). As the development of engineered bacterial therapeutics becomes more novel, it is essential to provide controllable stability and yield at the same time (238).

Conclusion
Taken together, bacteria play an essential role in the tumor environment with their distinct intrinsic immune characteristics. They are crucial in triggering the antitumor response of nonspecific cells and specific immune cells. Conventional natural bacterial immunotherapy has been improved by combining bioengineering techniques and multidisciplinary novel inventions. A series of preclinical antitumor bacterial vaccines and novel combination cancer therapies have shown versatile therapeutic effects (71). Several hurdles remain in the direct implementation of bacterial-based immunotherapies in clinical practice. To expedite the clinical translation of novel bacteria-based immunotherapy, we evaluated the engineering sources and trends of bacterial therapeutics, colonization treatment modes, suitable animal tumor models, as well as the feasibility of production. We expect that this review provides a reference for the optimization of bacterial-mediated tumor immunotherapy to enhance its value in clinical application.

Author contributions
MZ, DX, and JW defined the focus of the review. MZ and JW summarized papers. MZ drafted the manuscript. All authors contributed to the article and approved the submitted version.