Oncolytic bacteria: A revolutionary approach to cancer therapy

4.1 Anti-tumor immune responses (immunosuppression)

The induction of anti-tumor immune responses by oncolytic bacteria is an emerging and promising area of cancer research. Oncolytic bacteria are either modified or non-modified bacteria that selectively kill cancer cells [34,35]. These bacteria can directly kill the cancer cells and destroy them, which leads to the release of tumor-associated antigens, thus provoking a higher degree of immune response from the body. This process not only assists in the depletion of the tumor mass but also assists in the generation of long-term anti-tumor immunity by stimulating innate immunity as well as adaptive immunity. Consequently, the body’s immune system is prepared to target the remaining cancer cells to possibly reduce the chance of recurrence [36].

The process through which oncolytic bacteria stimulate anti-tumor immune responses is a multi-step process. First, the bacteria selectively adhere to the TME, which can be hypoxic and immunosuppressive, whereby conventional treatment is also ineffective. In this case, bacteria that are capable of accessing the site within tumor tissues can produce toxins or enzymes that result in the killing of tumor cells [37]. The constant secretion of tumor-associated antigens to the extracellular fluid of the tumor increases the immigration and activation of DCs. These DCs then take these antigens to internalize, process, and present them to T cells, triggering a specific adaptive immune response. This chain of events not only affects the primary tumor but also affects the progression of metastatic cancer cells [38].

According to the documented findings, current clinical and preclinical research proves the efficacy of oncolytic bacteria in the case of several types of cancers: melanoma, CRC, and glioblastoma [39,40]. In such research, investigators have revealed that oncolytic bacteria can be administered singly or together with other treatments, including immune checkpoint inhibitors, to improve the treatment outcomes. This attribute of oncolytic bacteria is to reshape the TME and recruit the immune system to the tumor site while also overcoming mechanisms that enable tumor immune evasion [40,41]. With growing research in the sector, oncolytic bacteria can effectively complement the growth of cancer immunotherapy and give fresh hope to patients with both refractory and recurrent cancers.

The key components of the immune system that have a significant impact on suppressing malignant and deformed cells associated with bacteriotherapy are macrophages, CD8+ T-lymphocytes, DCs, natural killer (NK) cells, and T-regs, which contain forkhead box P3 (FOXP3) as a biomarker. These cells have a significant impact on the propagation of cancerous cells and destruction as well as eradicating them [42].

Bacteria-induced infections (e.g., E. coli) can be employed to inhibit the growth of tumor tissues. These induced infections enhance and facilitate the differentiation of CD8+ killer T-cells, giving rise to the synthesis of IFN-γ and promoting the expression of major histocompatibility complex subtype I (MHC-I) on cancerous cells. This integrative strategy led to the targeted destruction of tumor cells, enhancing the ability of the immune system to recognize and eliminate malignant tissues (Figure 1) [43]. Tumor necrosis factor (TNF-α) also contributes to the formation of drainage in tumors. The bacterium is able to stimulate the production of tumor necrosis factor, which has been reported with S. typhimurium [44].

Figure 1

A schematic diagram illustrating the roles of innate immune response and acquired immune response during bacteria-mediated cancer therapy. Created and modified with BioRender.com.

4.2 Direct tumor cell lysis

The oncolytic bacteria use various strategies to hasten the growth of cancer cells. It has been known that direct killing of the cancer cell is one of the methods employed (Table 1) [45]. Oncolytic bacteria can directly colonize cancer-harboring tissue and they deploy their toxins that disrupt the functions of the attacked cells and enzymes that lyse the targeted cells. For instance, Clostridium novyi -NT produces alpha-toxin that has the ability to affect and break cell membranes, thereby causing cell rupture and death. Another one is based on using the S. typhimurium bacterium that synthesizes bacterial proteins, causing the apoptosis of tumor cells. These bacteria have a predilection for the tumor microenvironment, which, as we know, is characteristically hypoxic and pro-angiogenic and lacks nutrient supply for bacterial growth and function. This selective targeting reduces the probability of harming other adjacent tissues and organs while enhancing the anti-tumor outcomes [46]. Salmonella also has enviable characteristics due to its capability to propagate within the cancer cells and promote regression and death [47,48].

Bacteria can generate anti-tumor effects via the depletion of nutrients essential for the metabolism of cancer cells [49]. The tumor tissues that are deoxygenated (anoxic region) promote the aggregation of obligatory anaerobic bacteria in the region [50]. Research has demonstrated that the systemic introduction of Salmonella bacteria disseminated toward the solid tumor via the hemorrhaging area inhabited by the tumor exerted pressure and minimized the proliferation of the tumor [51]. The necrotic regions are formed due to the reduction of nutrient and oxygen supply, which leads to the breaking down of the viable blood vessels, which in turn causes the death of the tumor cells from suffocation and starvation [51].

Yam et al. (2010) demonstrated that a genetically manipulated auxotroph strain of S. typhimurium was able to selectively and effectively suppress liver metastasis in a mouse model of pancreatic cancer. To increase tumor-targeting ability and tumor-killing efficacy, the S. typhimurium strain was additionally modified by re-isolation from a tumor growing in a nude mouse. The strain obtained from this method was termed A1-R. In this study, the authors explored the efficacy of locally as well as systemically introduced A1-R on liver metastasis of pancreatic cancer. Mice who received A1-R locally via the intrasplenic route of administration or systemically via tail vein injection experienced a notable reduction in splenic and hepatic tumor burden relative to the control group. Intravenous injection of A1-R also improves survival time of the treatment group [52].

Some bacterial species, like E. coli, exhibit tropism for tumor tissue, which secretes chemical constituents such as clusterin, TGF-β2, and serglycin, while others, like facultative anaerobes (e.g., Escherichia and Salmonella), can sense the nutrient-rich environment of tumors and contribute to their accumulation in the tumor cells and exert their killing effect [53,54].

Listeria infection can directly kill tumor cells via the propagation of nicotinamide adenine dinucleotide phosphate (NADP+) oxidase and elevated levels of intracellular Ca²⁺ [55]. These mechanisms can generate cytotoxic free radical, reactive oxygen species (ROS), [56] which can promote the immunogenic death of tumor cells, then activate CD8+ T cells to clean up residual tumor cells and ultimately avert metastases. Additionally, Listeria can infect bone marrow-derived suppressor cells (MDSCs) at the tumor site, causing a significant reduction in MDSCs and, consequently, transforming the immunosuppressive microenvironment to an immunostimulatory condition, which can maximize the response of T cells and NK cells toward the cancer cells [57].

Clostridium kills the tumor cells mostly by producing some exotoxins following its colonization. For example, phospholipases and hemolysins can destroy tumor cells by attacking their membrane [58]. This organism can promote the apoptosis of tumors by activating the release of tumor necrosis factor-related apoptosis-inducing ligands from polymorphonuclear neutrophils. Furthermore, the early expansion of Clostridium in solid tumors could lead to intratumoral infiltration of macrophages and granulocytes and maximize the secretion of chemokines, which might provoke the adaptive immunity and recruit immune cells to the tumor site and facilitate the killing process [59].

According to Qi et al., P. aeruginosa 1409 triggered programmed necrosis (necroptosis) of TC-1 tumor cells through activation of TLR4-RIP3-MLKL and the HMGB1 released by the dying tumor cells. According to their report, such a mechanism reshaping the TME leads to remarkable tumor suppression in a TC-1-grafted tumor mouse model [60].

4.3 Microbial biosurfactants

Biosurfactants are chemical compounds that have both hydrophilic (polysaccharides, amino acids, peptides, and various ions) and hydrophobic (saturated or unsaturated fatty acids) properties. These compounds are known for various physiological activities, including their capability to minimize the tension exerted on the surface and interfacial between liquids [75]. Biosurfactants are mainly classified into low- and high-molecular-weight compounds. The low-molecular-weight compounds consist of fatty acids, lipopeptides, lipoproteins, phospholipids, and glycolipids, while the high-molecular-weight compounds of biosurfactants contain polymeric biosurfactants [76].

The anticancer potential of biosurfactants is attributed to their ability to hinder cancer cell growth, enhance apoptosis, trigger necrosis, and arrest the cell cycle [77,78]. These biosurfactants comprise surfactin [79], mannosylerythritol lipids (MELs) [80], sophorolipids (SLPs) [81], rhamnolipids (RLs), succinoyl trehalose lipids (STLs) [82], serrawettins [83], and monoolein [84].

Biosurfactants are chemicals that exhibit surface activity and are synthesized by various microorganisms, including bacteria, fungi, yeast, and other small algae [85]. These compounds are recognized by their ability to lower the surface and interfacial tension, rendering them useful in many sectors and processes. More recently, they have been reported to possibly act as anticancer agents because of their characteristics and action modes. In addition, biosurfactants possess several biological activities such as antimicrobial, antiviral, and anti-inflammatory properties that endow them with anticancer properties. They are able to provoke apoptosis, inhibit the spread of cancer cells, and increase the effectiveness of initial chemotherapy. Also, biosurfactants have been found to possess properties that enable them to preferentially affect cancer cells without harming normal cells, and due to this, there are fewer side effects when treating cancer. In addition, the biodegradability and low toxicity of these molecules add to the rationale for the development of such agents as therapeutics. Studies continue in an attempt to understand all aspects of their anticancer potential and the preparation of biosurfactant-based products for clinic use as the next horizon in cancer therapy [86].

Several biosurfactants produced by different bacterial strains, such as RLs, SLPs, and MELs, have shown promising therapeutic activities against cancer cells. RLs synthesized by P. aeruginosa, for instance, have a cytotoxic effect on different cancer cell lines, including breast cell lines (MCF-7, MCF-10A, and MDA-MB-231) [87], acute leukemia cells (SKW-3, BV-173), promyelocytic leukemic cells (HL-60), and murine cells. In one of the studies, RLs were found to exert a substantial effect in causing cytotoxicity to CRC cell lines, including HCT-116 and Caco-2, but the non-significant effect to normal cells, indicating that their selectivity was inclined toward the cancer cells [88]. In another study, Mishra et al. [89] investigated the properties and apoptotic effects of bacterial RLs on MDA-MB-231 breast cancer cells. Their research shed light on the fact that such RLs exert their antiproliferative action by targeting and modulating the p38 mitogen-activated protein kinase (MAPK) signaling pathway, which plays crucial roles in cell proliferation and survival.

Lipopeptide, surfactin in particular, which is a microbial metabolite of Bacillus subtilis, is a potent anticancer agent that has been found to trigger apoptosis and restrain metastasis in numerous cancer cells. Surfactin suppresses cell viability through the modulation of membrane fluidity and the induction of apoptosis through the caspase 3 and caspase 9 pathways [90]. Similarly, Zhao et al. [91] observed that lipopeptides from B. subtilis, mainly containing iturin, possess pronounced anticancer activity to chronic myelogenous leukemia cells (K562 cells) in vitro. The results revealed that these lipopeptides are capable of directly causing both apoptosis and paraptosis and, at the same time, suppressing autophagy. This multiple strategy highlights their promising role in the management of leukemia [91].

Studies revealed that a novel SLP, specifically C18:1 lactonic SLP (produced by Starmerella bombicola), showed enhanced cytotoxicity against the MDA-MB-231 breast cancer cell line. These SLPs inhibited cell migration without compromising cell viability. Furthermore, SLPs triggered the increase of intracellular ROS, which can cause stress and potentially damage cancer cells [92]. Similarly, Duarte et al. [93] showed the relevant effect of SLP against cancer cell growth in a dose-dependent manner. This was again associated with the induction of apoptosis due to a ROS and c-Jun N-terminal kinase (JNK)-mediated mechanism. This report supports that ROS is produced during the inhibition of cancer growth through SLP, producing an activation in JNK, inducing apoptosis. Table 2 provides different types of biosurfactants produced by bacterial species/strains and their anticancer effects.

4.4 Bacterial spore

Recent studies reveal that bacterial spores, especially those belonging to the Bacillus group, are beneficial to cancer treatment [105]. Bacterial spores are very crucial structural and biochemical organizations of prokaryotes that protect the agents under extremely unfavorable conditions (extreme temperatures, radiation, and desiccation). This ability to withstand unfavorable conditions, together with their capacity to stay dormant until conditions improve, makes them a favorable candidate for many uses in the medical field, especially in cancer research. Studies have demonstrated that bacterial spores can home in on cancer cells and kill them with minimal effects on the healthy cells. This selective targeting is mostly because the tumor microenvironment is mostly hypoxic and acidic, which creates a suitable environment for spore germination and subsequent growth of the agent [106].

The anticancer activity attributed to bacterial spores is based on their capacity to generate and proliferate in hypoxia and nutrient-rich tumor milieu. The spores, after entering the tumor, develop and grow and release toxic proteins and enzymes that have a direct toxic effect on cancer cells. Presumably, the appearance of these spores in the organism provokes the natural immune response in the host and improves the body’s ability to combat cancer. The spores can also produce secondary metabolisms, which possess anti-tumor properties that increase the effectiveness of this remedy [107]. In addition, these bacterial spores can be genetically manipulated and engineered to incorporate special genes, which make therapeutic proteins that can locate and be expressed within the tumor region, thus increasing the efficiency and effectiveness of anticancer therapy [108].

Research has demonstrated that the spores of B. subtilis have potential in cancer treatment. It has been reported that the spores of B. subtilis could home in on tumor cells and cause regression of tumors. The spores germinate in the hypoxic areas of the tumor, and upon contact with the tumor, the agent releases bioactive compounds, including toxins and enzymes that degrade the matrix and facilitate the invasion of cancer cells, thereby improving therapeutic benefits [109]. Similarly, C. novyi -NT is an oncolytic strain of C. novyi and is one of the most thoroughly studied strains known so far. C. novyi-NT spores selectively germinate only in the hypoxic tumor region and liberate toxins that cause the tumor cells to rupture. This bacterium has been assessed and found to have high anticancer potential in preclinical and clinical trials [110]. Listeria spores are able to infect both tumor cells and antigen-presenting cells, resulting in a direct lethal effect on tumor cells and induction of systemic immune response. This strategy has been proven to be effective in preclinical models of a variety of cancers, including pancreatic and ovarian cancers [111]. Figure 2 illustrates the application of bacterial spores as anticancer agents, highlighting their features and applications in cancer therapy. The figure underlines the opportunity to use bacterial spores in cancer therapy based on their specificity and displays the possible applications of such a strategy.

Figure 2

Schematic representation of bacterial spores and their potential as anticancer agents. Created with BioRender.com.

4.5 Bacterial toxins and metabolites

Researchers have extensively studied bacterial toxins produced by pathogenic bacteria as potential anticancer agents (Table 3). These toxins, usually proteins or peptides, can disrupt cellular processes, causing cell death. Although bacterial toxins are traditionally known for causing diseases, some have been GM to target cancer cells because of their strong biological effects. This strategy aims to use the natural ability of bacterial toxins to kill cancer cells while minimizing harm to healthy tissues. Studies in this area have led to the development of new cancer treatments that use bacterial toxins in their original form or as part of engineered structures to improve their effectiveness and specificity [112].

The role of microorganisms in combating cancer was first observed about 150 years ago following the oncolytic effect of erysipelas S. pyogenes infections observed in hospitalized cancer patients. Also, in the mid-nineteenth century, it was reported that regression of tumors was noted in patients experiencing skin infection by Streptococcal bacteria. These discoveries facilitated the development of cancer immunotherapy and the use of a vaccine for killed bacterial species, referred to as Coley’s toxins. Coley’s toxins were also found to be useful in delaying the regression of several terminal malignancies, such as myelomas, carcinomas, lymphomas, melanomas, and sarcomas. This pioneering work laid the groundwork for what became the potentially promising area associated with microbial toxin-induced cancer therapy [113].

The efficacy of bacterial toxins as anticancer agents is largely attributed to their capacity to impair crucial cellular activities in cancer cells. For example, one mechanism is the inhibition of protein translation. Diphtheria toxin (DT) produced by Corynebacterium diphtheriae inactivates elongation factor-2, thus inhibiting protein translation and leading to cell death [114]. Similarly, exotoxin A, produced by P. aeruginosa, inhibits protein translation through adenosine diphosphate-ribosylation of elongation factor-2 [115]. Other toxins, such as Clostridium perfringens enterotoxin, target a specific cell receptor and affect cytoplasmic membrane integrity, resulting in cell lysis [116]. Also, toxins can trigger apoptosis, such as anthrax toxin produced by Bacillus anthracis, by impairing intracellular signaling pathways. Collectively, these represent the multitude of mechanistic strategies bacterial toxins leverage to induce cytotoxicity in cancer cells. Given this, bacterial toxins represent an attractive modality in cancer treatment [37].

The potential benefits of using bacterial toxins as a treatment for cancer are noteworthy for many reasons. A major benefit is potency; bacterial toxins are often effective in low amounts, meaning that systemic toxicity is negligible [117]. Another benefit is that scientists could enhance the specificity of cancer cells using genetic engineering to produce fusion proteins integrating toxins with specificity, often in the form of antibodies or growth factors. This strategy would prevent unintended damage to normal tissues. For example, immunotoxins (antibodies fused with toxins) have shown promising efficacy for a range of cancers, hematological malignancies, and solid tumors in preclinical studies and could provide a pathway forward to market [118]. Additionally bacterial toxins could bypass resistance. Because bacterial toxins often engage a different cellular pathway than traditional chemotherapeutics, bacterial toxins present an opportunity for drug-resistant cancer cells [119]. In general, based on their unique mechanisms and advances in biotechnology, bacterial toxins have the ability to be both effective and versatile anticancer agents.

There are certain merits of employing bacterial toxins as anticancer agents. One of the advantages is that bacterial toxins are naturally potent biological compounds capable of inducing the death of targeted cells at low concentrations, thereby minimizing toxicity to surrounding cells. In addition, the selectivity of the toxin to the cancer cells can be further improved by GE for the development of hybrid proteins in which toxins are conjugated with targeting ligands such as antibodies or growth factors. Another advantage is the capability to counter drug resistance because bacterial toxins act through pathways that differ from those of chemotherapeutic agents [119]. In general, due to specific characteristics of bacterial toxins, as well as the rapid paradigm shift in the GE, they are promising frontiers for the emergency of effective anticancer drugs.

DT, derived from the bacterium C. diphtheriae, was chosen as the basis for the development of the first immunotoxin, denileukin diftitox (ONTAK), due to its potent cytotoxic properties and convincingly well-understood mechanisms of action. As a protein synthesis inhibitor, DT acts by ADP-ribosylating elongation factor-2, which interrupts cellular function and leads to cellular death. In denileukin diftitox, the bacterium’s toxin was also modified to contain interleukin-2 (IL-2), which targets the IL-2 receptor on the surface of IL-2-expressing cells, such as certain cancer cells. It is the strategic and functional targeting of DT that enhances the specificity of the action of DT to selectively kill cancer cells and spare normal cells. Denileukin diftitox has been especially effective in the treatment of cutaneous T-cell lymphoma with notable clinical efficacy, leading to its approval for therapeutic use [119].

C. perfringens enterotoxin (CPE) has displayed frontier anticancer properties, predominantly in cancers that developed from epithelial cells, including ovarian and CRC. CPE can target two important proteins, i.e., claudin-3 and -4 (tight junction proteins), which are commonly expressed in cancer cells. Research findings revealed that when CPE was introduced in an ovarian cancer animal model, it resulted in the binding of CPE to claudins and provoked rapid cytolysis and cell death. The binding of CPE to claudin-4 in CRC cells also demonstrates cytotoxicity by causing the immediate loss of tight junctions along with cell death. Altogether, researchers suggest that CPE can exploit the differential expression of epithelial-derived claudins in cancer cells and selectively induce cell death [120].

P. aeruginosa exotoxin A (PE) has shown very unique anticancer properties by interfering with the process of protein synthesis in cancerous cells. For instance, PE has been conjugated with other targeting ligands to synthesize PE-based immunotoxins, such as PE38 immunotoxin, an immunotoxin consisting of an antibody linked to PE38 that targets cancer cells. The SS1P immunotoxin, which consists of PE38 linked to an anti-mesothelin antibody, has shown effectiveness in treating mesothelioma and other tumors, which overexpress mesothelin in preclinical as well as clinical trials [121].

Staphylococcus aureus enterotoxin B (SEB), for instance, has also been identified to possess anticancer activity. This superantigen activates T cells and generates cytokines and enhances anti-tumor immunity. In other studies, it was found that SEB could enhance immune responses against tumor cells using murine models like melanoma and lymphoma. As potent as this toxin is in producing an immune response, it may be administered in cancer immunotherapy. Considering how powerfully this toxin induces an immune response, it could be used in cancer immunotherapy [122–125].

P. aeruginosa exoenzyme S (ExoS) is the other notable example of a multifunctional toxin, which is known for its anticancer activity, and acts by disrupting cell signaling pathways involved in cancer progression. According to documented reports, ExoS can downregulate the activity of Ras and Rho GTPases, which are significant regulators of proliferation and survival of cells, which ultimately induce apoptosis and inhibit the growth of cancer cells as observed in various models, including melanoma and lung cancer [126].

Bacillus anthracis is known to synthesize the anthrax lethal toxin (LT), and this toxin could have noteworthy anticancer properties. The LT of anthrax is composed of lethal factor (LF) and protective antigen (PA). LF works as a zinc-dependent metalloprotease that destroys the target proteins belonging to the MAPKK family by cleaving them in the cytosolic part of the host cell after its penetration inside the body. By interfering with the MAPK signaling pathways, the cancer cells lose their ability to survive, and apoptosis is triggered. The LT of anthrax has also been reported to have the ability to target the cancer cells selectively and spare the normal tissues, as observed in preclinical models [127].

Recently, scholars have identified that the secondary metabolites (lipopeptides) in B. subtilis strain Z15 (BS-Z15 SMs) exhibit an inhibitory effect on cancer cells, particularly liver cancer cells (BEL-7404). These metabolites trigger apoptosis and cause G0/G1 phase arrest, resulting in a decline in the potential of the mitochondrial membrane. The overall mechanism of action suggests that apoptosis specifically occurs through the mitochondrial pathway [128].

Niamah et al. reviewed the anticancer activity of bacteriocins derived from lactic acid bacteria. The review explores the mechanisms through which these bacteriocins operate. It also demonstrates the potential technical advancements that could maximize the efficacy of bacteriocins in the treatment of different types of cancers. In addition, the authors also displayed some of the challenges that need to be mitigated for better and effective utilization in clinical settings [129].

4.6 Bacterial biofilms

Over the years, bacterial biofilms, which cause chronic infections and are linked to medical implant-related complications, have been identified as a possible modality of cancer treatment. Biofilms are complex microbial communities formed with bacteria, and they are enclosed by a protective layer known as an extracellular matrix that hinders them from the host’s immune system or antimicrobial agents. This endurance, along with their capacity to keep on to the surface and build intricate form, has urged interest in using biofilms for targeted delivery of therapeutic agents to carcinoma cells [144].

The utility of bacterial biofilms in cancer therapy is rooted in their capacity to access and infiltrate tumors readily. Some biofilms can be engineered or naturally selected that may selectively target TME because these sites are hypoxic and acidic in nature and are common features of solid tumors [145]. This selective targeting can improve the biodistribution of therapeutic agents, including chemotherapeutic drugs or nanoparticles, to cancer cells while reducing systemic exposure, thus minimizing off-target toxicity. Additionally, encapsulation of agents within the biofilm matrix enhances their stability by protecting them from degradation by enzymes or other factors, hence enhancing the release of bioactive molecules into the TME over an extended period [145].

The development of biofilm by bacterial pathogens on cancer cells triggers metastasis disruption. Hence, bacterial biofilm demonstrates feasible applicability in the treatment of cancer [146]. It has also been realized that anticancer therapeutic agents can induce the development of biofilm during therapeutic intervention, which causes the distraction of metastasis [147]. Research findings indicated that bacterial biofilm can impact the progression and development of colon cancer via the modification of cancer metabolome to generate a regulator of cellular proliferation [146]. Besides, the bacterial macromolecules essential for the formation of biofilm, such as DNA and proteins, shield cancer cells to stop metastasis [148]. For instance, polysaccharides produced by Streptococcus agalactiae hinder the adherence of cancer cells to endothelial cells, a crucial stage in cancer metastasis [148]. In a different investigation, Kumeria et al. [149] display the prospective use of iron oxide nanowires extracted from biofilm byproducts of the bacteria Mariprofundus ferroxydans as a new multifaceted drug vehicle for the treatment of cancer and cancer hyperthermia. In alternative research, biofilm developed by Lactobacillus reuteri was utilized to generate nanoparticles (mesoporous silica) for the directed delivery of 5-FU in the colorectum, and this biofilm, when covered with zinc gallogermanate promoted CRC imaging [150].

4.7 Bacterial proteins and enzymes in prodrug therapy

Various bacterial proteins may inhibit cancer through various mechanistic avenues. One of the notable bacterial proteins that demonstrated oncolytic activity against cancer cells is Azurin. Azurin is a bacterial protein metabolically produced from P. aeruginosa. Azurin facilitates the apoptotic process in neoplastic cells through the stabilization of the tumor suppressor protein p53, which results in cell cycle arrest and subsequent cell death [151]. Mycobacterium bovis also synthesized very crucial protein with endostatin-like effects, which exert an inhibitory effect for angiogenesis and metastasis [152]. Moreover, ActA is a special protein secreted by L. monocytogenes that can elicit a more prominent immune response to elicit greater recognition and cellular attack toward neoplastic cells [153].

Bacterial enzymes play a crucial role in prodrug therapy, especially toward cancerous cells. This strategy involves using an inactive compound that is toxic only when given as a drug and accumulates at the tumor site without affecting the rest of the body. The process begins with systemic delivery of the prodrug after which bacterial enzymes are localized to the tumor site either via vectors (bacterial) or as purified enzymes. These enzymes then activate the prodrug within the TME, thus leading to the selective killing of tumor cells without affecting normal cells (Figure 3) [154,155].

Figure 3

Schematic illustration of the mechanism of bacterial enzyme prodrug cancer therapy. Created with BioRender.com.

Bacterial enzyme prodrug therapy offers several advantages. First, bacterial enzymes are very selective, so the prodrugs are activated only in the tumor region, which in turn minimizes the side effects. Second, the prodrugs are inactive and only become active when they reach the tumor sites; therefore, patients treated with prodrug-based chemotherapy suffer fewer side effects compared to those who undergo usual chemotherapy [156]. Besides, bacterial enzyme–prodrug systems can be employed in synergistic therapy with other treatments, such as immunotherapy and radiotherapy, to boost the efficiency of total treatment [157]. Nevertheless, bacterial enzyme prodrug therapy has several limitations and directions for future research. One limitation is the ability to deliver the bacterial enzymes to the tumor site and safely distribute them while avoiding harm to the body; researchers are currently exploring nanotechnology and other delivery systems to address this issue. Furthermore, the human body’s immune system may eliminate bacterial enzymes or vectors before reaching the tumor, and, therefore, strategies to alter immune response become mandatory. In addition, although there are increasing numbers of preclinical studies, more research is needed in this area to assess the safety and effectiveness of these therapies in clinical trials before such therapies can be applied in the clinical setting [158,159].

Cytosine deaminase (CD) is an enzyme that biotransforms the non-toxic prodrug 5-fluorocytosine (5-FC) into the toxic chemotherapeutic compound 5-fluorouracil (5-FU). This mechanism has proved useful in cancer treatment since the transformation of 5-FC to 5-FU is local in the case of malignant cells, resulting in the death of the cancerous cells with minimal side effects to the rest of the body. This approach has been implemented in different cancer models and has proved to have a huge potential to be used to treat cancers such as CRC as well as gliomas. Carboxypeptidase G2 (CPG2) is another enzyme that activates nitrogen mustard prodrugs by converting them into their cytotoxic forms. This enzyme–prodrug system is promising in the treatment of solid tumors, and further research focuses on increasing the targeting ability and the advantages of the enzyme and prodrug system. Research has been conducted to analyze different approaches to enhance the targeting efficacy of CPG2 so as to improve the disease benefits without effects on normal tissues [160]. Likewise, beta-glucuronidase is an enzyme that cleaves the glucuronide prodrugs to liberate active chemotherapeutic agents. This system is especially effective in tumors that express high levels of beta-glucuronidase, which includes some types of breast and colon cancers. The localized presence of the enzyme beta-glucuronidase within the TME enables the selective activation of the prodrugs, thus increasing the selectivity and decreasing the systemic toxicity of the treatment [161].

5.1 Salmonella typhimurium

Salmonella enterica serovar typhimurium (S. typhimurium) is a facultative anaerobic bacterium that is well-documented to possess potential anti-tumor properties. It shows direct cytotoxicity on tumors and is used for the selective administration of anticancer agents [29].

According to a report by Hayashi et al., a GM strain of S. typhimurium, termed A1-R, has been developed as a targeted therapy for cancer metastasis. This strain requires both arginine and leucine for growth and is capable of growing in cancer cells but not in the normal cells of the body. When injected into the lymph nodes and the bloodstream in mouse models of pancreatic cancer and fibrosarcoma, A1-R selectively localized and killed metastatic tumors over the course of 7–21 days, as visualized by real-time imaging. This approach appears quite effective in managing metastatic cancers and is completely safe without the use of chemotherapy [166].

Tan et al. investigated the anticancer activity of GM S. typhimurium against both stromal cells and cancer cells in pancreatic tumors. In this study, cytolysin A (ClyA) produced GM S. typhimurium was introduced into nude mice with either subcutaneous or orthotopic human pancreatic cancers. Following intravenous administration of the bacterium, researchers examined the markers on stromal cells, changes in the tumor surroundings, stromal cell counts, and the infiltration of immune cells. Results obtained from this experiment revealed that the GM strain selectively aggregated and grew within tumor tissues. The retarded growth in pancreatic cancer is attributed to the ClyA produced by the agent. This approach suggested the ability of the bacterium to penetrate tumor-linked stromal barriers and exhibit anticancer effects [74].

A study by Zheng and Min [167] explored the novel ability of an attenuated strain of S. typhimurium strain ΔppGpp (guanosine 5’-diphosphate-3’-diphosphate) (strains deficient in guanosine pentaphosphate and tetraphosphate) to selectively target and perpetuate within cancer-harboring tissues, resulting in a considerable reduction in the size of the tumor or being fully eliminated in the mice model. The study displays the role of ΔppGpp S. typhimurium strains as vectors for introducing therapeutic payloads selectively to tumors.

Recently, Sun et al. studied the GE oncolytic bacterium HCS1, comparing its activity to the attenuated S. typhimurium strain VNP20009. In vitro laboratory analysis showed that HCS1 had significantly lower colonization and cytotoxicity against cancer cells compared to VNP20009. The administration of HCS1 to tumor-bearing mice resulted in considerable suppression of cancer growth in a dose-dependent manner without causing adverse effects in healthy tissue, suggesting its potential in cancer treatment with an improved safety profile [168].

5.2 Listeria monocytogenes

Current and past investigations on GE L. monocytogenes demonstrate the inhibitory activity of this bacterium against various types of cancers, highlighting its potential in oncolytic therapy.

According to Vitiello et al., attenuated L. monocytogenes (Lmat-LLO) serve as a potential anticancer agent and delivery strategy for the treatment of different types of cancers. In vitro studies show that Lmat-LLO causes ROS generation and apoptotic cell death in numerous melanoma cell types, independent of stage, mutational profile, susceptibility to BRAF inhibitors, or stemness. In Braf/Pten genetically altered mice, Lmat-LLO lowers the size and volume of initial melanoma tumors while also decreasing metastatic spread [169].

Jia et al. developed a GE L. monocytogenes strain that expresses the HPV16 E7 protein. Vaccinating mice with cervical cancer intraperitoneally with the modified strain (LM4Δhly: E7) resulted in considerable tumor size reduction and even total remission of certain existing tumors. The study demonstrated that the recombinant L. monocytogenes may enter tumor tissues and promote non-specific tumor cell death, most likely via the production of ROS and raised intracellular levels of calcium ions [170].

Currently, a group of researchers indicated that L. monocytogenes mutated for oncolytic use has shown inhibitory activity toward different types of cancers. These modified bacteria are designed to selectively infect and replicate within tumor cells without affecting healthy cells. In the process of oncolytic activity caused by L. monocytogenes, the GE L. monocytogenes selectively infect tumor cells because of their capability to thrive in the hypoxic environment typical of solid tumors. Once internalized into the tumor cells, the agent induces lysis of the targeted cell through the potent toxins released for the bacterial cell [171]. Besides, GE Listeria can be organized to express tumor-linked antigens, improving the ability of the immune system to recognize and fight the cancer cells.

5.3 E. coli

Chiang and colleagues used the probiotic E. coli Nissle 1917 (EcN) strain for the treatment of CRC. In this study, the cytotoxic protein HlyE, which is regulated by the araBAD promoter (PBAD), was engineered into EcN. EcN was metabolically engineered to decouple glucose-mediated control and l-arabinose transport induction while simultaneously blocking l-arabinose catabolism in order to overcome the drawback that PBAD was sensitive to glucose and prone to all-or-no induction. EcNe, the reprogrammed strain, allowed for the effective and time-controlled expression of HlyE. At 30- to 50 μM l-arabinose concentrations, EcNes HlyE production reached its peak and was not impacted by glucose. In mice xenografted with human CRC cells, the engineered EcNe markedly decreased tumor size and specifically colonized tumors with a tumor-to-organ ratio of 106:1. This study introduced a novel approach for bacteria-mediated delivery of therapeutic proteins to tumors [172].

Apart from GE oncolytic bacterial species, naturally existing oncolytic bacteria strains have been reported for their unique capability to specifically attack and eradicate cancer cells. For instance, the two commonly occurring bacterial strains, such as C. novyi and S. pyogenes, flourish tumors cell and eliminate cancer cells without causing significant damage to normal tissues [163,173].

The most prominent benefits and successes of oncolytic therapy linked to naturally occurring oncolytic bacteria lie in their evolutionary adaptation to TMEs, where this therapeutic frontier gains an advantage over GE counterparts. The proliferation and survival of this agent within the tumor cell are partly associated with the natural selection processes, which can boost their therapeutic potential and minimize the possibility of resistance occurring in cancer cells [139].

In spite of the peculiar oncolytic characteristics of naturally occurring bacteria, these bacteria may necessitate some form of changes or improvements to enhance their therapeutic uses for clinical cases. The combination of naturally occurring oncolytic bacteria with immunotherapies or other cancer treatments is being studied to enhance their specificity, unlocking their mechanism of action and overall therapeutic outcomes [139].

5.4 Clostridium spp.

Clostridium species are known for their unique oncolytic activity as the agent targets the cancerous cells and selectively colonizes the tumor cell. This specificity is guaranteed because the hypoxic and necrotic areas, which are characteristic only for solid tumors, are suitable for the germination of Clostridium spores, thereby killing the tumor cells while having little effect on the normal cells [174]. This tumor-selective colonization not only assists in the direct destruction of tumor cells through bacterial lysis but also enables the targeted deposition of therapeutic agents synthesized by the bacteria. Such kind of approach is also very helpful in reducing the side effects often associated with conventional treatments [175].

The anticancer activity of Clostridium species and its spore, mainly C. novyi-NT, was reviewed by Staedtke et al. and Feng et al. [176,177].

5.5 Bacillus spp.

Bacillus species, such as B. subtilis and B. thuringiensis, have been recently explored for their oncolytic properties in cancer treatment [178,179]. These bacteria have inherent properties that are beneficial for use in targeted cancer therapy. Moreover, their capacity to dwell in hypoxic TMEs and their specificity to eliminate cancer cells and not harm normal cells further emphasize their therapeutic utility. Furthermore, Bacillus species can be GM to increase their ability to kill cancer cells and demonstrate a therapeutic load containing anticancer drugs or immunomodulators directly to cancer tissues. Current research remains focused on elucidating the molecular pathways through which they exert their oncolytic effect and the optimization of their use in clinical practice [90,180].

Recently, Zhao et al. assessed the anticancer potential of four bacterial strains (BY38, BY40, BY43, and BY45), which were isolated from the fecal samples of healthy persons as well as cancer patients. According to the results obtained from this study, the tested organisms significantly suppress the growth of ovarian and CRC cell lines in a dose-dependent manner, suggesting the possibility of these products being used as alternative therapeutic agents for these forms of cancer [181].

5.6 Bifidobacterium

Facultative anaerobes like Bifidobacterium are bacteria that cannot survive in an environment when the level of oxygen is high. This characteristic forces them to move toward the hypoxic areas inside the tumors where oxygen is scarce [182].

Recently, a group of researchers investigated the effects of heat-killed Bifidobacterium and Lactobacillus strains on the MKN1 human gastric carcinoma cells using animal models and in vitro assays. In this study, the MKN1 xenograft models were established by implanting BALB/c nude mice with MKN1 cells and then the mice receiving the tested bacterial strains orally as single or in combination. Western blotting and immunohistochemistry of the tumor tissues showed that the strains L. reuteri MG5346, B. bifidum MG731, and L. rhamnosus MG5200 were capable of eliciting greater apoptosis in MKN1 cells compared to other strains. Besides, these strains, especially MG731, showed a significant reduction in the growth of tumors in these animal models [183].

According to the study by Faghfoori et al., Bifidobacteria had anticancer properties on colon cancer cell lines, and the findings showed that it was able to significantly decrease the survival rate of the colon cancer cells compared to that of the control groups. The data obtained by flow cytometry and reverse transcription polymerase chain reaction proved that metabolites produced by Bifidobacteria led to apoptosis in colon cancer cells. According to the authors, the effect was attained by the suppression of anti-apoptotic genes along with the promotion of pro-apoptotic genes [184].

In a recent study published by Yoon et al., the administration of two different strains of B. breve suppressed the proliferation of tumors in mice with MC38 colon carcinoma. The results revealed that only one of these strains improved the performance of the chemotherapeutic agent used (oxaliplatin and PD-1 inhibitors). According to the immune profiling and transcriptomic analysis, this strain of B. breve increased the anticancer immune response by stimulating lymphocytes. The authors concluded that the combined effect of B. breve and the tested antibiotics could be a viable strategy to harness the potential of B. breve to enhance the effectiveness of CRC treatments [185].

Bahmani et al. isolated 17 isolates of Bifidobacterium from diverse sources and tested them against colon cancer. According to the findings of the study, Bifidobacterium was detected frequently in infant fecal samples and dairy products, with the least prevalence in local milk. In this study, both the infant feces-derived isolate (cell-free supernatant (CFS) and one from the probiotic capsule showed maximum inhibition of cancer cell growth on a colon cancer cell line. Out of all the probiotics, B. bifidum was especially effective against cancer cells and received a vast improvement in the results of gastrointestinal cancer. In conclusion, this study stated that the CFS produced by these isolates can suppress the growth of colon cancer cells; thus, it shows that probiotics have high potential as a new therapeutic strategy for colon cancer [186].

In a recent study, Fahmy and co-workers explored the modulatory impact of B. longum (BL), which was isolated from women’s breast milk samples, on selected oncomiR and tumor-suppressor miRNA, as well as on IL-1β and IL-6-targeted miRNA, employing the mice model of inflammation-induced CRC. The study observed that BL administration inhibited miR-21a and miR-155 oncomiRs that modulated IL-6 and IL-1β in both normal and CRC mice. It also upregulated tumor suppressor miRNAs, including miR-145 and miR-15a of the tumor cells. Furthermore, BL treatment downregulated the expression of miR-146a, which regulated the expression of IL-1β and IL-6. In CRC mice, the raised concentration of NF-Kb was, however, reduced by BL. Alternatively, in CRC mice, BL caused an increase of IL-1β but a decrease of IL-6. In addition, reduced levels of aberrant crypt foci in CRC mice were observed under the BL treatment, while increased necrosis and fibrosis in colon cells were also observed. The results obtained in this study suggest that BL modulation of microRNAs could provide positive therapeutic outcomes in CRC by inhibiting tumor cell proliferation, apoptosis, and related activities [187].