Bacteria-based immunosuppressive tumor microenvironment reprogramming: a promising dawn in cancer therapy

Abstract

Traditional chemotherapy, a prevalent cancer treatment modality, is associated with significant side effects and often leads to treatment failure. Non-specific drug distribution and chemoresistance are the main factors contributing to this failure. Certain distinctive characteristics of the tumor microenvironment (TME), including hypoxia, acidic pH, and increased interstitial fluid pressure, render cancer cells resistant to conventional treatments. Multiple approaches have been devised to enhance the treatment efficiency of neoplasms and overcome chemoresistance. Nowadays, bacteria-based cancer therapy has garnered significant interest in both preclinical and clinical research, owing to its distinctive mechanism and various applications in eliciting host antitumor immunity. Due to their inherent tumor tropism, elevated motility, and capacity for quick colonization in the conducive TME, bacteria are increasingly being considered for targeted tumor treatment. Bacteria, rich in pathogen-associated molecular patterns (PAMPs), can efficiently stimulate immune cells even inside the immunosuppressive TME, boosting the particular immune detection and eradication of tumor cells. Furthermore, outer membrane vesicles (OMVs), cytoplasmic membrane vesicles (CMVs), and their derived physiological components exhibit analogous functionalities to their parental cells. This review article is representative of the latest innovations in bacteria-based immunosuppressive TME reprogramming. Additionally, the article discusses future directions in this research area, drawing on current advances.

Graphical abstract

Highlights

• Salmonella enhances the activity of CTLs and NK cells while reducing Tregs populations within the TME, thereby promoting antitumor immune responses.

• Listeria infects suppressive myeloid cells, boosting IL-12 and antitumor immunity.

• Engineered bacteria selectively colonize tumors and deliver immunostimulatory agents, reprogramming the TME to enhance antitumor immunity.

• Engineered OMVs act as targeted nanocarriers, accumulating in tumors and activating immune responses through PAMP delivery.

• Bacteria-based therapy exploits tumor hypoxia, overcoming chemo- and radio resistance.

Introduction

The aberrant growth and proliferation of cells are the result of the accumulation of genomic and epigenomic changes in cancer, a heterogeneous and multifactorial illness [1]. Recent integrative analyses have revealed that genetic and epigenetic regulatory mechanisms, particularly those mediated by microRNAs, play pivotal roles in driving tumor heterogeneity and facilitating immune evasion across diverse cancer types [2]. According to demographic-based estimates, the incidence of cancer is expected to reach 35 million by 2050. Therefore, allocating resources towards prevention, early diagnosis, and using innovative cancer therapies has the potential to save millions of additional fatalities [3]. Surgery, chemotherapy, and radiotherapy, as traditional cancer therapies, present patients with both physical and psychological difficulties [4–6]. As most radiotherapy and chemotherapy specifically target fast-proliferating cells, regardless of their malignancy, they have significant adverse consequences. It is imperative to develop strategies that specifically target tumor sites without causing harm to healthy tissues [7–9].

Considering this, the use of microorganisms for cancer treatment is a distinctive therapeutic alternative worth exploring [10–12]. The origins of bacterial-based cancer treatment can be traced back to the early 19th century, when Vautier documented his discovery of tumor regression in patients co-infected with gas gangrene caused by Clostridium perfringens [13]. In the mid-19th century, Busch observed a reduction in the tumor size of a cancer patient after inoculation of erysipelas [14]. Twenty years later, a physician called Fehleisen determined that Streptococcus pyogenes is the etiological agent of erysipelas [15]. In the late 19th century, William Coley, a surgeon from the United States, reported the therapeutic intervention of insemination Streptococcus erysipelatous to a patient suffering from sarcoma [16]. By injecting lytic substances derived from bacteria into the tumor tissues, this physician successfully inhibited tumor proliferation and halted the progression of the illness [17]. Pearl noted in the first half of the 20th century that cancer survivors had a higher incidence of active or healed tuberculosis compared to cancer patients, indicating that it inhibits the development of cancer [18]. The anticancer properties of Bacillus Calmette-Guérin (BCG), a live attenuated vaccine strain of Mycobacterium bovis, were thoroughly investigated throughout the 20th century. This investigation unveiled the principles and components of bacterial-based immunotherapy [19, 20]. Researchers have conducted extensive studies on the use of BCG vaccination in cancer treatment. The FDA has officially authorized the widespread use of intravesical BCG to treat non-invasive bladder malignancies [21, 22]. The BCG vaccination specifically aims to combat non-invasive bladder cancers by potentially stimulating both innate and adaptive immunity. Immune cell pattern recognition receptors (PRRs) can be activated by this vaccination as pathogen-associated molecular patterns (PAMPs), leading to the production of cytokines and activation of the immune system versus malignant cells [23, 24]. BCG exerts its ability to eliminate cancer cells by targeting phagocytic cells, including natural killer (NK) cells and neutrophils [24]. Follow-up studies have shown that BCG induces tumor cell death by activating the caspase-8 signaling pathway [25]. The 210-year history of bacteria-based cancer therapy discloses the immense capacity of these microorganisms to combat a wide range of cancer types (Fig. 1).

Fig. 1.

The timeline details the progression of bacterial and OMV applications in cancer therapy, emphasizing significant advancements from initial attenuated bacterial treatments to modern engineered OMV systems

This review article investigates the direct interactions of bacteria, outer membrane vesicles (OMVs), and cytoplasmic membrane vesicles (CMVs) with cancer cells, as well as their impacts on host immune reactions against cancer cells. We subsequently illustrate the significant role of engineered bacteria and engineered OMVs in reprogramming the immunosuppressive tumor microenvironment (TME), which affects the growth and migration of tumors. Recent advancements in understanding and technology suggest that engineered bacteria and engineered OMVs can serve as achievable therapeutic options for cancer treatment.

Mechanisms of bacteria-based cancer therapy

Targeting tumor cells by bacteria

Microorganisms that are facultative anaerobic, including Salmonella and Listeria, as well as obligate anaerobes, such as Clostridium and Bifidobacterium, can infiltrate and accumulate inside tumor tissue (Fig. 2). These bacteria become trapped within the tumor’s chaotic vascular system, causing inflammation, and passively infiltrate the tumor tissue [26]. Findings indicate that tumor necrosis factor alpha (TNF-α) triggers vascular disruption in the early stages of tumor colonization by Salmonella typhimurium, enabling bacteria to enter the tumor through the bloodstream. Studies have shown that TNF-α levels rose considerably with intratumoral colonization of bacteria and peaked 120 min post-injection. This increase leads to more vessel disruption, providing an optimal condition for bacteria to germinate [27].

Fig. 2.

A schematic representation of bacterial-mediated tumor targeting. Gram-negative and Gram-positive bacteria, along with their derivatives such as OMVs and CMVs, are used in modern cancer therapy due to their ability to selectively target hypoxic tumor regions. These bacteria colonize tumors by crossing inflamed, permeable vessels and responding to tumor-derived chemical cues. Their components, including LPS and vesicles, initiate immune activation. Anaerobic bacteria induce direct cytotoxicity via ROS generation and nutrition deprivation while concurrently using chemotaxis and vascular disruption via TNF-α/LPS-dependent mechanisms to target hypoxic areas inside tumors. OMVs of Gram-negative bacteria activate DCs via TLR4/NF-κB signaling, thereby augmenting antigen presentation and the recruitment of CTLs. The CMVs of Gram-positive bacteria modulate immunological responses and induce apoptosis by inhibiting the PDK1/AKT/Bcl-2 pathway. Notably, Gram-negative bacteria and their OMVs activate the TLR4/NF-κB pathway, leading to dendritic cell maturation and enhanced antigen presentation. This, in turn, promotes the recruitment and activation of CTLs. Activated CTLs and NK cells secrete IFN-γ, perforin, and granzyme B, which induce the destruction of tumor cells. On the other hand, bacterial derivatives like CMVs can induce apoptosis in tumor cells by inhibiting the PDK1/AKT/Bcl-2 signaling pathway, thereby reducing tumor cell survival. Moreover, by suppressing Tregs, the immunosuppressive pressure of the tumor microenvironment is diminished, effectively reprogramming it in favor of robust antitumor immune responses

Lipopolysaccharides (LPS), among bacterial components, stimulate a significant migration of innate immune cells to colonized tumors. Upon inflammasome activation, these cells generate interleukin-1 beta (IL-1β) via two different processes. Salmonella LPS and toll-like receptor 4 (TLR4) are directly activated, whereas injured tumor cells with Salmonella are indirectly activated [27–29]. LPS leads to increased secretion of TNF-α through interaction with TLR4 [30, 31]. Furthermore, Salmonella typhimurium flagellin enhances the cytotoxicity of CD8⁺ cytotoxic T lymphocytes (CTLs) and NK cells against tumors while reducing the frequency of CD4⁺ CD25⁺ regulatory T cells (Tregs) [32, 33]. NLRC4 inflammasomes regulate the secretion of IL-1β and IL-18, cytokines that stimulate CTLs and NK cells to produce interferon-gamma (IFN-γ). The involvement of Salmonella flagellin in this mechanism is evident [34]. The presence of flagella allows these bacteria to efficiently gain access to the tumor center by overcoming high interstitial fluid pressure. Hence, motility is an essential attribute of bacteria [26, 35]. Crucially, both obligatory and facultative anaerobic bacteria can utilize both active and passive tactics to preferentially target malignant cells [36]. Moreover, Salmonella species stimulates the generation of the protein known as connexin 43 in cancer cells. This protein increases the transmission and cross-presentation of processed tumor antigenic peptides across cancer cells and dendritic cells (DCs), while simultaneously diminishing the production of indoleamine 2,3-dioxygenase (IDO) [37, 38]. Salmonella species exert an additional negative effect on cancer cell growth by consuming nutrients and absorbing energy inside the TME. In other words, cancer cells undergo a condition of extreme nutritional deprivation [39]. Other direct interactions between bacteria and tumor cells occur when bacteria come into contact with substances generated by tumors. Studies have conclusively shown that Salmonella typhimurium responds to metabolites generated by tumor cells and hence triggers apoptosis [40]. A study was performed using a microfluidic device to investigate the role of signaling molecules in the bacterial chemotaxis mechanism for tumor targeting. The findings were unexpected due to the secretion of substances such as transforming growth factor-beta 2 (TGF-β2), clusterin, and serglycin in the tumor tissue, which is the preferred region for bacterial colonization [41].

Interestingly, the selective targeting of malignancies by Listeria species is achieved through the intracellular infection of tumor-infiltrating myeloid-derived suppressor cells (MDSCs). Listeria not only infiltrates the TME but also escapes the host’s immune system by infecting MDSCs [42, 43]. In contrast, the TME significantly suppresses the immune response, hindering the eradication of microorganisms [44, 45]. Through this approach, they are subsequently transferred from MDSCs to cancer cells via a distinct cell-cell dissemination process. Additionally, Listeria-infected MDSCs form an immune-stimulating phenotype that generates the cytokine IL-12, which boosts T and NK cell responses [42, 43]. Listeria species degrade tumors by directly eliminating cancer cells and stimulating CTL reactions to Listeria antigens. Its mechanism of action involves activating nicotinamide adenine dinucleotide phosphate (NADP+) oxidase and elevating intracellular calcium levels by generating reactive oxygen species (ROS), thereby directly eradicating cancer cells [46].

Other research has identified exotoxins from Clostridium species that harm cancer cell membranes, impair key biological activities, and destroy tumors [47–49]. Clostridium-infected tumors induce the migration of CTLs, granulocytes, and macrophages to the infection site, thereby enhancing cancer regression [50]. Neutrophils release TNF-related apoptosis-inducing ligand (TRAIL) due to immune cell migration, representing a unique mechanism of anticancer activity [51].

Based on the aforementioned characteristics, by selectively targeting the TME, microorganisms have the capacity to hinder the development of malignancies. By leveraging their anaerobic survival trait, these bacteria are capable of specifically targeting hypoxic solid tumors. These bacteria have the ability to selectively colonize and proliferate, thereby eradicating the hypoxic regions [52]. One of the primary causes of chemotherapy and radiotherapy failure is often attributed to the presence of hypoxic regions inside the tumor. Hypoxic cells, which resist ionizing radiation more than normal cells, demonstrate that radiotherapy’s efficacy relies on tissue oxygen partial pressure [53, 54]. Thus, hypoxic tumors reduce the effectiveness of radiotherapy. In contrast, bacterial-based immunotherapy functions as a radio enhancer by not altering the inherent susceptibility of tumor cells to radiation [55]. The unique properties of bacteria can be used as a synergistic therapy in combination with conventional methods such as chemotherapy and radiation to further improve the effectiveness of treatment for malignant tumors.

Targeting tumor cells by outer membrane vesicles (OMVs)

Gram-negative bacteria generate nanoscale spherical vesicles known as OMVs [56]. These vesicles are primarily composed of cellular components derived from the bacterial periplasm and outer membrane, such as membrane lipids, nucleic acids, proteins, LPS, peptidoglycans, and virulence factors [57, 58]. Furthermore, they can encapsulate specific intracellular components like DNA, RNA, proteins, ions, metabolites, signaling molecules, and enzymes [59].

Research indicates that OMVs, similar to their parental cells, enter the bloodstream following administration, infiltrate tumor vasculature, and persist at the tumor site (Fig. 2). This suggests the potential of drug-loaded OMVs as a nanoplatform for targeted tumor therapy [60]. Consequently, OMVs possess all the behavioral traits of their progenitors in targeting tumors and have a notable capacity to actively localize tumor tissue in hypoxic conditions [61]. The enhanced permeability and retention (EPR) effect is the phenomenon that causes the accumulation of OMVs in the TME [58]. Numerous studies have demonstrated that OMVs are recognized by innate immune cells through the activation of surface TLRs, leading to the secretion of pro-inflammatory cytokines and chemokines [62–64]. Research has revealed that systemically delivered bacterial OMVs precisely target and aggregate in tumor tissue, ultimately promoting the production of the anticancer cytokines C-X-C motif chemokine ligand 10 (CXCL10) and IFN-γ [65]. PAMPs serve as crucial elements of immunogenicity, delivered by OMVs and recognized by PRRs, such as TLRs on immune cells, which subsequently initiate cytokine production, inflammation, and programmed cell death (apoptosis) [66]. Furthermore, OMVs can facilitate PRR-independent host immunity to enhance tumor therapy [67]. Neutrophils efficiently identified and internalized the PAMPs on Escherichia coli OMVs during revascularization, as demonstrated by Min Li and colleagues. Neutrophils subsequently passed through the blood vessels and directed OMVs toward inflammatory tumors [68]. The immaturity of DCs under the specific conditions of the TME prevents them from effectively recognizing the inflammatory environment, leading to immune inactivation. The administration of OMVs facilitates the interaction between PAMPs on their surface and TLR4 on immature DCs, promoting their maturation. The maturation of DCs by OMVs enables the metabolization of antigens and the presentation of antigenic epitopes on their surface, employing the major histocompatibility complex (MHC) for this function. The T cell antigen receptor, located on the surface of T cells, detects the transformed antigen as a result of this modification. This activation of T cells initiates an immune response that involves both helper T cells and CTLs [69]. DCs play a crucial role in coordinating the immune response between the innate and adaptive immune systems [70, 71]. Studies have shown that OMVs can infiltrate DCs, resulting in the up-regulation of CD86 and MHC-II molecules on their surface. This internalization induces the production of cytokines, including TNF-α and IL-12, which are involved in immune response activation or the regulation of tumor immune contexture [72, 73].

Targeting tumor cells by cytoplasmic membrane vesicles (CMVs)

The CMVs of Gram-positive bacteria measure between 10 and 500 nm in diameter and originate straight from the cytoplasmic membrane [74, 75]. Historically, the presence of a dense peptidoglycan cell wall was believed to prevent Gram-positive bacteria from releasing extracellular vesicles, which explains their recent identification [74]. Alongside peptidoglycan, the vesicle membrane contains lipoteichoic acid fragments, which are essential components of the Gram-positive cell wall [76]. Similar to OMVs, Gram-positive CMVs facilitate the transport of various cargo molecules, including nucleic acids, viruses, lipids, enzymes, proteins, and toxins [77]. The CMVs deliver several cargo chemicals that interact with bacteria, host cells, and the environment. These interactions affect bacterial host-pathogen interaction, survival, invasion, immunomodulation, and infection [75]. CMVs have presented novel opportunities in a variety of medical applications [78]. In recent years, they have attracted substantial attention due to their potential to treat a diverse range of diseases, including cancer and systemic disorders [76] (Fig. 2).

In Gram-positive bacteria, evidence primarily derives from studies on probiotic strains within the Lactobacillus genus. These studies illustrate approaches to regulate human gut microbial ecology, focusing on alternative methods to mitigate damage and enhance the efficacy of cancer therapy [79]. It is important to note that Lactobacillus rhamnosus GG, a widely used probiotic supplement, produces CMVs with lethal effects on hepatic cancer cells. This is accomplished, in part, by the downregulation of the Bcl-2 and Bax genes in neoplastic cells [80]. Additionally, CMVs from Lacticaseibacillus paracasei PC-H1 have the ability to inhibit the proliferation of colorectal cancer cells in vivo and in vitro, thereby inducing apoptosis through the PDK1/AKT/Bcl-2 signaling pathway [81]. Kim and colleagues reported that CMVs from Lactobacillus acidophilus and Staphylococcus aureus show anti-tumor properties. The absence of tumor growth expansion in mice administered these CMVs can be associated with the stimulation of IFN-γ production by cytoplasmic membrane vesicle surface proteins [65].

Therefore, the emergence of Gram-positive CMVs, predominantly consisting of probiotics, represents a promising approach in anticancer therapy. Probiotics facilitate the restoration of healthy microbiota, mitigating complications from radiation and chemotherapy, and improving the efficacy of cancer treatment [82].

Reprogramming the immunosuppressive TME

The TME consists of diverse tumor stromal cells, such as fibroblasts, endothelial cells, and immune cells, along with non-cellular elements of the extracellular matrix (ECM) [83]. Tumor cells, as the command center of the TME, regulate the functions of both cellular and non-cellular constituents via intricate signaling networks, manipulating non-malignant cells to their advantage [83]. A complex network of cytokines, chemokines, growth factors, inflammatory mediators, and matrix remodeling enzymes makes this communication between cells possible [84]. The consequences of this interaction are apparent in tumor formation and maintenance, as well as insufficient treatment response. The non-malignant cells inside the TME are recognized for facilitating carcinogenesis across all stages of cancer progression and metastasis [83, 85]. Non-malignant cells exhibit genomic stability; however, their transcriptomes and phenotypes are influenced by interactions with cancer cells and other cells within the TME [86]. Bilateral interactions between tumor cells and the ECM components, including collagen, fibronectin, hyaluronan, and laminin, together with cellular constituents of the TME, result in the degradation of tissue integrity and cancer progression [87]. Recent developments in tumor-targeting and microenvironment-responsive nanoplatforms offer innovative strategies for precise modulation of this complex and immunosuppressive TME, aiming to enhance therapeutic outcomes [88, 89].

Tumors are recognized for their capacity to evade the immune system and establish an environment that facilitates their survival and expansion [90]. Research points out that the TME predominantly consists of M2-like tumor-associated macrophages (TAMs), which support tumor growth, angiogenesis, metastasis, and immune evasion [66]. They prevent T cell functions by expressing immune checkpoint ligands such as programmed death-ligand 1 (PD-L1), secreting inhibitory cytokines like IL-10 and TGF-β, and recruiting immunosuppressive cells, including Tregs [91–93]. On the other hand, M1-like TAMs exhibit anti-cancer properties by releasing nitric oxide (NO) and stimulating naive T cells to produce a type 1 helper T cell/cytotoxic response [92]. In cancer research, reprogramming M2-like TAMs to the M1-like phenotype is regarded as a promising strategy for targeting cancer cells [94–96]. In the next section, we discuss the extraordinary potential of engineered bacteria to reprogram the immunosuppressive TME (Table 1).

Table 1.

Strategies of engineered bacteria and engineered OMVs in reprogramming the immunosuppressive TME

Types of strategies for reprogramming the immunosuppressive TME	Bacteria type	Constitution	Samples studied	Cancer type	Function	References
Engineered bacteria	Salmonella typhimurium A1	Leu− and Arg−	Nude mice and PC-3 cell line	Prostate cancer	Strain A1 specifically colonized the PC-3 tumor and inhibited its proliferation. - The tumor was entirely eradicated 15–26 days after the initiation of therapy. - Twelve hours after infection, PC-3 showed the cytopathic effects of strain A1. - Elevated levels of IL-1β and TNF-α. - Inhibition of tumor proliferation.	[133]
	Salmonella typhimurium ΔppGpp	relA− and spoT−	BALB/c mice and CT26 cell line	Colon adenocarcinoma	Elevated levels of IL-1β and TNF-α. - Inhibition of tumor growth.	[134]
	Salmonella typhimurium	Containing human gene for IL-2	Clinical trials (Phase I)	Metastatic gastrointestinal cancer	There was a statistically substantial elevation in circulating NK and NK-T cells.	[135]
	Listeria monocytogenes	actA− and inlB−	BALB/c mice and CT26 cell line	Lung metastases tumor model by using the colon tumor-derived CT26 cell line	Stimulate robust innate and adaptive immunity. - Long-term survival.	[136]
	Listeria monocytogenes	prfA−	Clinical trials (Phase II)	Cervical cancer	Modify the TME. - Facilitate T-cell infiltration. - Reduce immune suppression mediated by regulatory T cells and MDSCs. - Safety and long-term survival.	[137]
	Escherichia coli (DH5-alpha)	Combined with chemotherapy (oxaliplatin)	MC-38 cell line	Colon adenocarcinoma	Macrophages were repolarized to the M1 phenotype, and APCs were recruited. - Cytotoxic T cell recruitment and tumor suppression.	[138]
Engineered OMVs	Escherichia coli Nissle	ECHy− and combination with lapatinib (tyrosine kinase inhibitor)	Mice	Breast cancer and colon cancer	Reduction in tumor growth rate compared to lapatinib monotherapy. - Immune cell infiltration. - Modulating the TME to overcome chemoresistance.	[139]
	Escherichia coli Nissle 1917	Combination with perhexiline	CT26 cell line	Colon adenocarcinoma	Macrophage repolarization (M2 to M1). - Promoting apoptosis. - Inhibiting invasion and migration.	[141]

Engineering bacteria for reprogramming the immunosuppressive TME

The engineering of pathogenic bacteria is essential for enhancing the efficacy of immunotherapy in the TME while simultaneously decreasing their pathogenicity [97] (Fig. 3). Some bacterial virulence factors may contribute to their intrinsic antitumor activity. Consequently, reducing pathogenicity should be achieved without compromising their antitumor efficacy. The deletion of key virulence genes in human pathogenic microorganisms has transformed fatally toxic strains into predominantly safe strains [39]. Research indicates that an engineered attenuated strain of Salmonella typhimurium can effectively inhibit tumor progression and migration in animal models of colon and melanoma cancer by secreting Vibrio vulnificus flagellin B (FlaB). TLR4 signaling plays a crucial role in inhibiting tumor progression through the activation and infiltration of immune cells facilitated by FlaB-secreting bacteria. FlaB generated by engineered bacteria effectively elicited robust anti-tumor immunity in the TME and extended the survival of the animal models [98].

Fig. 3.

Engineered bacteria modulate the immunosuppressive TME via six synergistic mechanisms: Induction of apoptosis via TRAIL/checkpoint inhibitors; Inhibition of angiogenesis through HSulf-1/Tumstatin; Activation of cytotoxic agents under hypoxic conditions; Tumor-targeted delivery of immunomodulators employing sialic acid recognition; Metabolic modulation by converting ammonia to L-arginine; and Augmentation of chemotherapy efficacy. Furthermore, OMVs enhance efficacy by promoting macrophage repolarization from M2 to M1, delivering IFN-γ, and inhibiting PD-1/PD-L1 interactions; hence, they surmount treatment resistance

Researchers employed an engineered non-pathogenic Escherichia coli strain, designated SYNB1891, for cancer treatment in another study. The stimulator of interferon genes (STING) agonists were delivered to intratumoral antigen-presenting cells (APCs) using SYNB1891. The SYNB1891 therapy led to the formation of immunological memory and an effective type I IFN-mediated anti-tumor immune response in B16.F10 melanoma tumor-bearing mice [99]. Chowdhury et al. effectively engineered a non-pathogenic Escherichia coli strain to selectively release a CD47 nanoantagonist inside the TME. In a lymphoma murine model, this intervention led to expedited tumor shrinkage, increased activation of tumor-infiltrating T cells, inhibition of metastasis, and extended survival [100].

Engineered bacteria to induce apoptosis

Several researchers have engineered bacteria to induce apoptosis in the TME [101, 102]. For instance, Zhang and colleagues revealed that intratumoral and intravenous injection of soluble TRAIL-expressing Escherichia coli DH5α resulted in targeted release in the TME, triggering biological molecules that significantly reduced tumor proliferation and extended the survival of tumor-bearing mice [103]. Additionally, engineered bacteria could be designed to specifically release checkpoint-blocking nanobodies, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and PD-L1, in the TME to enhance antitumor effectiveness [104].

Engineered bacteria to inhibit angiogenesis

Enhanced cancer treatment may benefit from techniques that uniformly distribute antitumor agents throughout a tumor and inhibit angiogenesis. To improve cancer therapy, researchers have investigated bacteria-mediated synergistic therapies for increased specificity, temporal and spatial control, and modulation of the immune microenvironment [105]. In this context, Yang and colleagues created an engineered Escherichia coli delivery system (GDOX@HSEc) using synthetic biology and surface chemical techniques. The engineered bacteria were altered to express heparin sulfatase 1 (HSulf-1) in HSEc for attaching doxorubicin-loaded glycogen nanoparticles (GDOX NPs) to their surface. The findings indicated that HSEc could effectively target and colonize tumor locations, leading to the upregulation of HSulf-1, which in turn inhibited angiogenesis and metastasis in a melanoma mouse model. Concurrently, GDOX nanoparticles penetrated tumor cells, causing intracellular DNA damage [105]. Wei et al. developed a delivery system for Tumstatin, an antiangiogenic protein, using Bifidobacterium longum as a vector. The Tumstatin delivery method markedly decreased the microvessel density of the tumor, triggered death in vascular endothelial cells, and suppressed tumor development in CT26 tumor-bearing mice [106]. Recent reports indicate that bacteria inhabiting tumors provoke local inflammation and lead to tumor thrombosis [107, 108]. A therapeutic platform, AIB@ClyA, was developed using engineered Escherichia coli K-12 strains in research. Upon administration of the engineered strains to animals with CT26 tumors, they initially aggregated and multiplied inside the tumors, resulting in intratumoral thrombosis and nutritional restriction. The expression of the ClyA protein induced membrane perforation and subsequently intensified tumor thrombosis [109].

Engineered bacterial promoters

Engineering the activity of inducible bacterial promoters under defined circumstances is an alternative strategy that could improve specificity in targeting the TME [110]. To enhance the selective survival of Salmonella in the hypoxic TME and minimize toxicity to normal tissues, a strain known as Salmonella YB1 was engineered by combining a critical gene under the control of a hypoxia-responsive promoter [111]. In addition, Salmonella YB1 has shown the ability to prevent liver tumor proliferation and lung metastasis [111, 112]. Researchers engineered a Salmonella typhimurium promoter that effectively expresses cytolysin A (ClyA). An L-arabinose promoter controls the bacterial ClyA gene, enabling activation only in the presence of L-arabinose. The bacteria induce the expression of ClyA, which is essential for tumor cell destruction, by activating the promoter via L-arabinose injection upon reaching the tumor site. Engineering the promoter reduces the cytotoxic effects of ClyA on healthy tissue cells [113].

Genetically engineered bacteria for targeted delivery system

It is feasible to use the engineering capabilities of bacteria in therapeutic delivery systems to selectively release therapeutic cargo into the TME [100]. Chen et al. illustrated the attachment of sialidases to the surface of genetically engineered bacteria for the recognition and targeting of tumor sialoglycans. Indeed, they engineered Escherichia coli (MG1655) to express the cytolysin of hemolysin E (HlyE) through a regulatory gene circuit sensitive to sialic acid. The expression of HlyE not only lysed tumor cells but also reversed immunosuppression within the tumor’s immune microenvironment, enhancing the efficiency of solid tumor treatment [114]. Consequently, genetically engineered bacteria may serve as a multifaceted platform for transporting cargo to tumor cells using genetic engineering methods [115].

Metabolic modulation by engineered bacteria

Engineered bacteria have also achieved metabolic modulation of the TME to enhance the effectiveness of immunotherapy [116, 117]. The intratumoral injection of an engineered Escherichia coli Nissle 1917 strain in mouse MC38 altered metabolic modulation by converting ammonia to L-arginine. The elevation of L-arginine levels in the TME facilitated the infiltration of CD4 + T cells and CD8 + T cells, producing synergistic antitumor effects in combination with anti-PD-L1 treatment [118]. Recent advancements have demonstrated that these bacteria can be engineered to modulate critical molecular regulators implicated in metabolic adaptations and immune evasion, including transcription factors such as SOX13, which is associated with resistance to ferroptosis [119]. These approaches hold significant potential to circumvent intrinsic tumor resistance mechanisms and thereby facilitate the development of more efficacious combinatorial cancer therapies.

Engineered bacteria-mediated tumor chemotherapy

Given the significant inhibitory potential of engineered bacteria against diverse cancer cell types, oncologists proposed investigating the effects of traditional chemotherapy in combination with these bacteria in cancer patients [120]. The systemic administration of the majority of chemotherapy compounds is likely to diminish treatment efficacy due to non-specific drug distribution, significant toxicity, insufficient tumor permeability, molecular instability, poor aqueous solubility, chemoresistance, and prolonged side effects [121, 122]. Numerous studies have so far addressed bacteria-mediated tumor chemo-immunotherapy [123]. Clinical trials have revealed that engineered Listeria monocytogenes (CRS-207) combined with chemotherapy (pemetrexed and cisplatin) produced substantial alterations in the TME. Importantly, 89% of patients with malignant pleural mesothelioma clearly showed a decrease in tumor size [124]. Furthermore, the intratumoral injection of SYNB1891, an engineered strain of Escherichia coli designed to activate STING within the TME, in conjunction with the anti-PD-L1 antibody atezolizumab, demonstrated both local and systemic safety in patients with metastatic cancers [125].

Engineering OMVs for reprogramming the immunosuppressive TME

OMVs have the remarkable ability to modulate the immunosuppressive TME by repolarizing M2-like TAMs into M1-like phenotypes [67] (Table 1; Fig. 3). A recently published study engineered a bacterial mimetic vesicle displaying surface-exposed IFN-γ, which effectively targeted TAMs in the TME and reprogrammed them from M2 to M1 phenotype, thereby markedly suppressing tumor progression and invasion [126]. In immune-competent mice, intravenous administration of Akkermansia muciniphila-derived OMVs diminished the tumor load of prostate cancer without impacting normal cells. The increase in the ratio of Granzyme B (GZMB+) and IFN-γ + lymphocytes within CD8 + T cells led to the recruitment of tumoricidal M1 macrophages, which inhibited the growth and migration of prostate cancer cells [127]. In this context, Guo and colleagues developed sequentially activated OMVs encapsulating paclitaxel and DNA damage response-1 (Redd-1)-siRNA to regulate macrophage metabolism and inhibit tumor spread [128]. Liu and colleagues constructed an innovative platform using OMV-like multifunctional synthetic bacterial vesicles (SBVs) that induce cell death inside the TME. This engineered OMV facilitates the encapsulation of bacterial intracellular components, including catalase, to alleviate tumor hypoxia and activate the host cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS)/STING signaling pathway. Beyond that, the engineered OMVs included the photosensitizer indocyanine green to trigger apoptosis and enhance the effectiveness of immunotherapy. As a result, the OMVs-based therapeutic platform can effectively induce targeted antitumor responses via the combination of immunotherapy and phototherapy [129]. Qing and colleagues engineered OMVs as potent immunostimulants to reprogram the immunosuppressive TME. In an effort to neutralize the acidic pH of the TME, they functionalized the surface of OMVs with calcium phosphate. Following pH neutralization, the TME was reprogrammed via the polarization of M2 to M1 macrophages [130]. Researchers have found that cancer cells with high levels of the CD47 “don’t eat me” marker protect themselves from host immune cell responses, especially macrophage-mediated phagocytosis. Feng et al. developed a programmable two-way adapter based on OMVs, where a CD47 nanobody (CD47nb) is conjugated to the OMV surface (OMV-CD47nb), and the exterior is covered with a polyethylene glycol (PEG) layer embedded with diselenide bonds (PEG/Se). Engineered OMVs primarily activate TAMs through two mechanisms: promoting M1 polarization and inhibiting the “don’t eat me” signal. The activation of TAMs enhances T cell-mediated antitumor responses via efficient antigen presentation [131]. Recently, researchers introduced genetically engineered OMVs with the ectodomain of PD1 incorporated into their surface. This engineered OMV-PD1 can bind to PD-L1 on tumor cells, leading to its internalization and reduction. This protects T cells from the PD-1/PD-L1 immune suppressive pathway. The mechanism of action involves immune system activation and checkpoint inhibition, resulting in the accumulation of effector T cells within the tumor, thereby further disrupting tumor growth [132]. This highlights the potential of engineered OMVs in reprogramming the immunosuppressive TME.

Conclusion

Engineered bacteria and engineered OMVs present significant potential as innovative strategies for cancer therapy, providing distinct benefits in targeting the TME and improving immune responses. Bacterial-based therapies offer a distinct advantage over conventional treatments by precisely modulating immune responses and effectively addressing the challenges associated with hypoxic tumor regions, which often lead to relapse and systemic toxicity. Research indicates that the application of engineered bacteria and engineered OMVs in conjunction with radiation therapies can substantially enhance treatment outcomes, including the attainment of complete remission in preclinical models. These bacteria improve treatment efficacy by utilizing the specific conditions present in tumors, such as hypoxia, which are frequently overlooked by conventional therapies. This facilitates improved recruitment and function of immune cells, thus aiding in tumor destruction. The combination of bacterial components with immune checkpoint inhibitors offers a promising strategy for improving the immune system’s capacity to combat cancer, highlighting the potential of bacterial therapies to enhance current treatment modalities. Despite this advancement, challenges persist that must be addressed prior to the widespread implementation of these therapies in clinical practice. The long-term safety of engineered bacteria, reduction of off-target effects, and optimization of delivery methods are critical areas necessitating further research. The necessity for thorough preclinical and clinical testing is paramount, as it is crucial for establishing the efficacy and safety of these innovative therapies. In conclusion, engineered bacterial therapeutics signify a significant breakthrough in cancer treatment, potentially addressing the shortcomings of traditional therapies. Continued research and advancements in bacterial engineering and combinatorial strategies are essential for achieving their full potential. Ongoing innovation in bacterial-based therapies has the potential to enhance patient outcomes and provide new prospects in cancer treatment.

Author contributions

M.A: Conceptualization, writing-original draft, data curation, writing-review & editing; P.K.Z: Writing-original draft, data curation, writing-review & editing; A.A: Writing-original draft, data curation; M.M.M: Reviewing and editing; A.A.I.F: Project administration, supervision, writing, reviewing, and final editing. All authors contributed to the manuscript and approved the submitted version.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Competing interests

The authors declare no competing interests.