Microbial medicines: Unlocking the therapeutic potential of the microbiome in cancer treatment

Abstract

Microorganisms have had an established relationship with the maintenance of human health, and recent advancements in genetic engineering and synthetic biology have allowed the development of engineered microbes as targeted disease treatment. This review evaluates the rising use of microorganisms as therapeutic agents, particularly in the treatment of cancer. These engineered microbes are able to detect disease markers and precisely deliver therapeutic payloads to affected sites. These bacterial-based therapeutics show remarkable promise against cancers like pancreatic, breast, lung, and colorectal cancers by targeting tumor microenvironments and enhancing antitumor immunity. While obstacles still remain, such as interindividual variability, safety concerns, and regulatory barriers, the medicinal potential of microbial therapeutics is promising. Future research should focus on furthering the understanding of microbe-host interaction mechanisms and refining bacterial engineering techniques to continue to develop more precise and effective therapies. Ultimately, harnessing the power of microorganisms has the potential to revolutionize the treatment of complex diseases, such as cancer.

1. Introduction

The rapid advances in synthetic biology are driving the development of microorganisms as therapeutic agents [1,2]. These microbes are often thought of as something to avoid; however, emerging research suggests that they could help us fight a variety of enigmatic diseases, including cancer [3]. The mutualistic relationship between the microbiota and the human host has long been known, and as the understanding of the interaction and mechanisms behind the relationship is further understood, the therapeutic potential further reveals itself [4]. The fact that the microbiome has implications in a wide variety of diseases, ranging from cancer to cardiovascular disease to dermatological diseases, is generally accepted, yet native microbe-based therapies, aside from some forms of fecal microbial transplants (FMT), have not been widely created [4]. Currently, the manipulation of native microbial species used in healthcare is limited by the inconsistent data coming from its research. Several engineered live bacterial therapeutics (LBTs) are currently entering early or mid-stage clinical development and may provide the proof of concept needed to determine the future of this new class of therapeutic agents [5,6]. Results of clinical trials will determine the practical application of microbial therapy in the clinics [7]. Upon reaching these milestones, developers of engineered bacterial therapeutics using synthetic biology will be able to establish optimal safety, administration, and manufacturing processes, as well as determine how these agents can be effectively combined with existing therapies [3,5,6,8]. Optimizing the application of this new category of drugs will address the unmet needs of patients. While preclinical and early clinical results have been encouraging, regulatory hurdles remain that must be addressed to be able to apply these findings into clinical practice.

This review aims to provide a comprehensive overview of the emerging paradigm of microorganisms as therapeutic agents, with a focus on their application in addressing cancer. Most reviews focus on native microbiome–cancer associations or on engineered bacteria separately. Our review uniquely integrates both, and further emphasizes synthetic biology payload-delivery strategies, which are rarely synthesized in a single article. We provide cancer-type-specific engineering approaches, comparative discussion, and mechanistic context that existing reviews lack. Thus, although a growing body of research has explored use of microbes to treat diverse conditions, this review will focus specifically on the utilization of microbes to combat cancer. Furthermore, experimental and analytical techniques spanning genomics, transcriptomics, proteomics, metabolomics, and advanced bioinformatics that have propelled this field forward are examined. As omics technologies become increasingly accessible and affordable, and as the mechanistic understanding of microbial biology deepens, it is becoming evident that microorganisms represent an untapped reservoir of therapeutic potential. A comprehensive and interdisciplinary approach will be essential to fully harness the capacity of microbial therapies to address some of the most challenging diseases facing modern medicine.

1.1. Microbiome foundations: Biology, host interactions, and relevance to cancer

There are millions of microbes from thousands of different species that live in and have evolved with the gut, in both humans and animals, that participate in shaping the host’s health. These microbes are not limited to bacteria, but also viruses, archaea, protozoa, and fungi [4]. The healthy gut microbiota is primarily made up of two phyla, Firmicutes and Bacteroidetes, but may also have the phyla Actinobacteria and Verrucomicrobia [4]. The composition of the microbiome varies along the gastrointestinal (GI) tract. In the esophagus and stomach, there are about 100 bacteria per gram of content, and it increases along the tract up to 10¹² bacteria per gram of content in the colon [9]. The most abundant genus of bacteria also differs along the GI tract; Streptococcus is dominant in the esophagus, duodenum, and jejunum, and Helicobacter is dominant in the stomach [4,10,11]. It is generally accepted that the symbiotic relationship between the gut microbiota and the host individual contributes to the overall health of the human [4]. In healthy humans, the normal gut microbiota has beneficial functions in metabolism, immune response, and barrier protection [4]. The metabolic benefits from the gut microbiota include digestion of nutrients that are not normally able to be digested by human cells, Vitamin K and Vitamin B synthesis, and drug metabolism [4]. The immunological benefits include the synthesis of antimicrobial proteins, mucus production, and immunomodulation [4,12]. The normal gut microbiota also contributes to maintaining the gut barrier and structure by promoting intestinal epithelial growth and contributing to normal intestinal surface area, villi size, and peristalsis. Impairment to the healthy human gut microbiota has been implicated with a wide variety of diseases like irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), obesity, diabetes, allergies, neurodevelopmental diseases, and cancer [4,13–15]. In fact, it was found that individuals with a more diverse gut microbiome respond best to bacterial-based cancer therapy and immunotherapy [16]. These conditions in which bacterial therapy would be more likely to work are called microbiome signatures, and their ability to predict the effectiveness of treatment options could help with the patient stratification process [17]. For this to be put into clinical practice, however, companion diagnostics tests evaluating individuals’ characteristics such as their tumor microenvironment must be implemented. The implication of the disturbances to the gut microbiota in these diseases indicates that healthy gut microbiota is necessary for human health, thus emphasizing the importance of research into its mechanisms of interaction with the host. Over the years, several analytical tools and models have been developed to further advance this field of biology (Fig. 1).

Fig. 1.

Outline of the scientific progress made from the discovery of the gut microbiome to its current clinical applications [18–21].

Before examining how microbes are engineered for cancer therapy, it is essential to understand the analytical tools that have enabled this field to advance. The properties of the mutualistic microbes that inhabit the human gut microbiome have largely been identified due to the advancement in sequencing techniques for microorganisms [22–24]. There are three overarching types of analyses that the sequencing techniques for single-celled organisms aim for: single-celled genomic sequencing, single-cell transcriptomic sequencing, and single-cell proteomic sequencing [22,25]. In single-cell genomic sequencing, genetic heterogeneity can be analyzed [22,25]. This is done by amplifying the entire genome of the microbe [22]. Various methods can be performed for this, including multiple displacement amplification, multiple annealing and looping-based amplification cycles, and degenerate oligonucleotide-primed PCR, each method having advantages and disadvantages [22]. This allows for the detection of mutations in microbes or differences in genomes between microbes [22]. In single-cell transcriptomic sequencing, the levels of RNA that are transcribed from microbes are analyzed [22,26,27]. In these techniques, RNA is extracted from the microbes and converted to cDNA using reverse transcriptase [22]. From here, relative quantities of microbial RNA can be determined by reverse transcriptase – quantitative polymerase chain reaction (RT-qPCR) [22]. Specific primers can be made to selectively bind to the target cDNA, thus relative gene expression in the microbe can be accurately analyzed [22]. In single-cell proteomic sequencing, the translation of microbial proteins is analyzed [22]. Imaging techniques such as PET, CT, and MRI are often used to accurately access the bacterial cell population in vivo [3,5]. In addition to the Western blot, mass spectrometry and flow cytometry are two commonly used procedures for analyzing protein expression [22,28]. The relative abundance of specific target proteins can be determined, thus providing insight into the expression of microbial genes [22]. In addition, multiple sequencing techniques can be integrated to get a more in-depth analysis of the microbial expression pathways for genes of interest [22].

Interestingly, artificial intelligence (AI) and machine learning have been increasingly helpful in the discovery, analysis, and optimization of bacterial designs [29]. New algorithms have been developed that aid in bacterial research in two ways: predicting complex interactions and enhancing bioengineering and synthesis [29]. These AI models have been used to accurately predict relationships and interactions between pathogens and hosts, pathogens and therapeutic bacteria, and therapeutic bacteria and hosts [29]. By using simulations of these interactions, the development of new technologies, therapeutic bacterial strains, and payload delivery mechanisms is much more efficient [29]. In terms of the synthesis of these bacteria, AI has been useful in predicting metabolic reactions and developing cell factories that allow for sustained manufacturing and storage of these bacteria [30]. For example, Srivastava et al. created an AI or machine-based learning tool to accurately predict the inhibitory activity of certain small molecules against biofilm production by bacteria [31]. In another study, Gupta et al. developed an machine-based learning tool that can categorize bacterial proteins and assess their pathogenicity, which can be helpful in determining whether certain bacteria strains or drugs would have pathogenic adverse effects [32]. These examples reflect the growing use of AI in the field of biotechnology, and they highlight how these models can be useful in the development of bacterial therapeutics. Understanding these fundamental microbiome-host interactions and analytical tools and models provides the foundation for engineering therapeutic bacteria to combat complex diseases such as cancer, as discussed in the following sections.

2. Role of microbiome in cancer treatment

The use of microbes in treating cancer is a growing field of study, and the results are promising [1]. Historically, there’s precedent for the concept dating back to the 1800’s when researchers observed tumor regression in patients infected with certain types of bacteria [6]. Since the 1970’s, a weakened bacterial strain, Bacillus Calmette-Guerin, has been effectively used to treat bladder cancer [6,33]. Currently, scientists and researchers are exploring new approaches of engineering bacteria to stimulate antitumor immunity or to deliver cancer-killing drugs [3,6]. Even with modern breakthroughs, cancer continues to pose a global health challenge, necessitating the development of innovative and effective treatment approaches [34]. Conventional cancer therapies, such as chemotherapy and radiation, have limitations in terms of efficacy, systemic toxicity, and the emergence of drug resistance [35]. Therefore, there is a critical need for novel therapeutic strategies that can overcome these challenges and improve patient outcomes [36]. In recent studies, genetically reprogrammed bacteria were able to boost antitumor immunity or enhance the effect of other cancer therapies which suggest that these organisms could be allies in the war against cancer [5]– [7,37]. There are several properties of microorganisms that make them suitable agents for cancer treatment. For example, bacteria have a unique form of motility which allows them to easily penetrate tumors [38,39], The utilization of engineered microbes for cancer treatment has emerged as a promising approach in the field of cancer therapeutics [5]. The bacteria can be delivered precisely to the tumors by either direct intratumoral injection or manipulation of self-guided movements based on gradients (oxygen, pH, chemotaxis, temperature) [5,40]. This strategy involves the delivery of genetically modified bacteria directly into the tumor microenvironment, where they can exert their anticancer effects [6]. They thrive in the hypoxic and immune-deficient environment of the tumors [5,6,41]. They can deplete the tumor microenvironment (TME) of nutrients required for cancer cell survival [5]. These features, along with an easily modifiable genome and the power to induce an immune response, have led to the exploration of bacterial potential for cancer treatment [6,7]. Engineered microbes can be designed to selectively target tumor cells, induce direct cytotoxic effects, modulate the immune response, and enhance the efficacy of existing therapies [5,6]. For example, researchers engineered a non-pathogenic E. coli strain (MG1655) to express cytolysin A (ClyA) under acidic (pH-5) tumor microenvironment conditions [42]. This engineered bacterium, AIB@ClyA, specifically targets tumors, induces tumor-localized thrombosis by damaging vasculature, and expresses ClyA to enhance cytotoxicity via membrane perforation [42]. In mouse models, this approach significantly inhibited tumor growth (79% reduction), prevented metastasis, and demonstrated minimal systemic toxicity, offering a promising and precise anti-cancer treatment [42].

The appeal of this research is due to the unique advantages that bacterial vectors offer compared to existing treatment methods [1]. There are four main advantages [1]. First, the genome of the microbes used in the treatment can be manipulated to decrease their pathogenicity, thus limiting the amount and severity of adverse side effects [1]. Second, microbes already have the capacity to proliferate in human tissue, especially if the microbes used are modified strains that are typically found in humans [1]. Third, the population size of the microbes used for the treatment can be controlled via the use of antibiotics [1]. Finally, the genome of the microbes can be easily manipulated to introduce, remove, or change the expressed virulence factors, which widens the range of cancers they can treat [1]. Also, microbial therapy has shown potential in cancer treatment due to its oncolytic and immunomodulatory characteristics [43]. However, there are some limitations to this approach [1]. Some of these limitations include potential cytotoxicity (depending on the strain of bacteria used), possibility of mutation in the bacterial genome, heterogeneity and complexity of cargo, and inability to completely lyse tumor cells [1,44]. A 2014 study tested the use of microbes in treating tumors by injecting Clostridium novyi into tumors in dogs [45]. Overall, there were good responses to the treatment, and the most common side effects were those consistent with a bacterial infection [45]. The results of this study prompted the researchers to inject Clostridium novyi into the tumor of a human patient with advanced stage leiomyosarcoma [45]. Following the intratumoral injection, the size of the tumor within and around the bone was reduced [45]. In another interesting study, Clostridium tetani was injected into healthy tissue in unafflicted mice and into tumors of mice with cancer [46]. It was found that only in the mice in which the Clostridium tetani was injected into the tumor died of tetanus, indicating that Clostridium tetani only germinates in cancerous tissue [46]. This finding offers potential for future research into manipulating the genome of Clostridium tetani to release antitumor factors in the presence of cancerous tissue rather than tetanus toxin. Because the Clostridium tetani would only germinate in tumors, it would reduce the risk of affecting healthy human tissue. More recently, researchers have been focusing on the mechanisms by which engineering the microbiome could be used to combat cancer.

3. Mechanisms of gut microbiome-cancer interaction

The mechanisms by which the gut microbiota modulates tumor physiology are complex [47,48]. Evidence has shown that the microbiota plays a role in tumor physiology via three mechanisms: tumori-genesis, tumor cell metabolism, and tumor angiogenesis [47,49]. The gut microbiota exerts its influence on tumorigenesis in many ways [47]. First, microbial dysbiosis in the intestines can lead to an increased abundance of pathogenic E. coli and C. jejuni, which have been found to produce and release the carcinogens colibactin and cytolethal distending toxin (CDT), respectively [47]. Both of these compounds can damage DNA, which could lead to the development of cancer [47]. Enterococcus faecalis and B. fragilis overproduce reactive oxygen species (ROS) causing oxidative DNA damage and genomic instability in gastrointestinal cells [50]. Typically, this is prevented by either DNA repair mechanisms or the cyclic GMP-AMP synthase (cGAS)-Stimulator of Interferon Genes (cGAS-STING) pathway. However, these defense mechanisms are not always effective [51]. The cGAS-STING pathway occurs when DNA is released to the cytoplasm as a result of DNA damage [51]. The cytoplasmic DNA is detected by cGAS which activates the STING protein, leading to the production of Type I interferons that suppress tumor development [51]. Similarly, dysbiosis in the gut microbiota has been associated with a depletion of MSH2 and MLH1, which are proteins involved in mismatch repair (MMR) – a pathway that corrects DNA repair [47]. Without the proper regulation of DNA repair, cancer-causing mutations are much more likely to occur and persist [47]. The gut microbiota have been implicated in tumor cell metabolism via glycolysis and lipid metabolism [47]. Toll-like receptor (TLR) signaling between the gut microbiota and immune system causes an upregulation of glycolysis and downregulation mitochondrial activity in macrophages, which then leads to an increase in proinflammatory cytokine release that can target tumors [47]. Bile acids, which are modified by the gut microbiota, have also been shown to have a significant role in modulating tumor advancement [47]. Finally, a healthy gut microbiota has been correlated with balanced levels of pro- and antiangiogenic compounds [47]. Studies have shown that microbial dysbiosis is linked with increased levels of IL-6, IL- 1β, and VEGF-A, which all have the potential to amplify the already upregulated angiogenesis in growing tumors [47] (Fig. 2).

Fig. 2.

Mechanisms by which disturbances in the gut microbiota can lead to the development and progression of tumors.

Understanding how the native gut microbiota influences tumorigenesis, tumor metabolism, and angiogenesis has laid the foundation for a new frontier in cancer therapy-engineering microbes as precision tools to counteract these same tumor-promoting mechanisms. With advances in synthetic biology and microbial engineering, researchers are now reprogramming beneficial bacteria to sense, target, and treat specific types of cancer [47]. These engineered microbes can be designed to selectively colonize tumor microenvironment (TME), deliver immuno-therapeutic payloads, disrupt cancer-associated metabolic pathways, or modulate immune responses in a controlled manner [47]. To better understand the potential in using microorganisms to combat cancer, it is essential to understand how microorganisms, whether native or engineered, can influence human health in general.

4. Engineered microorganisms for disease detection and treatment: Hijacking microbes for human healthcare

Microbes have long been used for human healthcare, and the advent of genetic engineering increases their potential in therapeutics [52,53]. This potential is seen in the diagnosis, treatment, and management of diseases as well as promoting good health and preventing disease [52,54]. One such example of the potential of genetically engineering microbes is the engineered probiotic [52]. An engineered probiotic, termed a “smart probiotic”, is a native microbe that has been hijacked to sense the levels of biomarkers of a given disease [52]. In response to an elevated level of disease biomarkers, the smart probiotic can determine whether the individual is affected (diagnosis) and can release anti-disease therapeutics (which can be altered depending on the disease being targeted) [52]. This has the combined effect of treating a disease that is already present and preventing disease by recognizing and halting its spread early on [52]. There are many advantages to smart probiotics. First, the delivery of probiotics is made to be highly selective both in their location and in their response [52,55]. Second, the expense and time needed for the production and purification of traditional therapeutics will not be needed as the microbe-produced drugs are made in situ [52]. Finally, the design and implementation of the smart probiotic can be easily altered as the understanding of the disease advances, or the disease evolves [52]. Research has already started to highlight the potential benefit of genetically engineering lactic acid bacteria (LAB) such as Lactococcus lactis [54]. L. lactis has been modified to be used as a vector for delivering DNA vaccines, and the advancements in the CRISPR-Cas system provides even more promise for the therapeutic use of engineered microbes [54,56]. An example of this is a strain of Aci-netobacter baylyi that has been engineered to have resistance to a drug only when in the presence of DNA with a specific oncogenic mutation, effectively serving as a diagnostic tool for specific types of cancer [57]. Probiotic-guided chimera antigen receptor (CAR) T cells have even been developed to effectively direct CAR-T cells to tumors [58]. In this model, EcN engineered to express specific antigens colonize the tumor, and CAR-T cells modified to recognize these antigens are then administered and infiltrate that tumor, where they effectively kill the cancerous cells [58]. Various other phages have also been engineered to overcome multidrug-resistant bacteria (MDR) that may result from post-surgical or other infections [59,60]. These advancements are also making it possible for the development of vaccines for specific complex diseases, such as cancer [61] (Fig. 3)

Fig. 3.

The direct (left) and indirect (right) mechanisms by which engineered probiotics can protect against diseases.

5. Bioengineering strategies for payload delivery

Another way that microorganisms can be manipulated is through their mechanism of releasing anti-cancer products, also known as their payload delivery mechanism [62]. There are several payload delivery mechanisms currently used, one of them being exclusive OR (XOR) gate amplifying gene switches. In this mechanism, a DNA sequence that halts transcription (a transcription terminator) is surrounded by two recombination sites, each one activated by a different signal of the engineer’s choice [63]. When one of the two signals is present, the transcription terminator is inverted, preventing its effects and allowing gene expression [63]. If neither or both signals are present, the transcription terminator will be in its original direction, and gene expression will be inhibited [63]. Essentially, an XOR gate amplifying gene switch causes bacteria to deliver its payload only when one of two signals are present [63]. Zhou et al. integrated an XOR gate amplifying gene switch into a strain of EcN to target colorectal cancer. In this model, there was approximately a twofold increase in signal output, and there was a 30-50% reduction in cell viability of the colorectal cancer cells in vitro. Additionally, there was a 50% inhibition in tumor growth when tested on mouse models [64]. XOR gate amplifying gene switches are just one of many mechanisms that have been implemented in anti-cancer bacterial therapeutics.

Quorum sensing is a technique that can be used to ensure a large enough bacterial population before payload delivery begins [65]. In this mechanism, each bacteria produce and release signaling molecules known as autoinducers [65]. These autoinducers activate a signal transduction pathway typically involving phosphorylation cascades that end in the payload being produced, but bacteria only respond to autoinducers when autoinducer levels reach a certain threshold [65]. This leads to coordination between cells, allowing a group of bacteria to act as a unit, and deliver their payload only when they reach a threshold population density [65]. Swofford et al., developed a strain of Salmonella that selectively delivers its therapeutic agent via quorum sensing to reduce unintended side effects in healthy tissues. It was found that in mouse models, this Salmonella initiates drug expression in cancerous tissue where inducer molecules reach a specific threshold, but does not produce any drug in the liver [66].

Building off quorum sensing, bioengineers have designed synchronized lysis circuits as a way for therapeutic bacteria to deliver their payload [38]. In this pathway, quorum sensing is used to ensure a sufficient population density in the engineered bacteria, but rather than the autoinducers leading to payload release, the autoinducers activate cell lysis pathways [38]. This is advantageous for two reasons. First, it eliminates the possibility of errors or challenges resulting from the bacteria releasing the payload. Once the population density reaches the threshold, the cells simply lyse, and any products produced by the bacteria are released to the tumor microenvironment [38]. Second, a few of the bacteria will survive, allowing them to repopulate, and the cycle repeats again [38]. This allows for a cyclic payload delivery mechanism in which the therapeutic product is released repeatedly [38]. A variation of this synchronized circuit is a bacterial consortium. Bacterial consortiums are a gene circuit in which two separate strains of bacteria are mixed into a single population, and each bacteria produces an inhibitor of the other bacteria’s signaling molecule [67]. If one strain is engineered to proliferate in cancerous tissue and the other in healthy tissue, a system can be created in which a payload is only released inside a tumor, thus minimizing adverse side effects [67]. Din et al. engineered a strain of bacteria that uses this synchronized lysis circuit to effectively follow a pulsatile release cycle of anti-cancer drugs. With this strain, there was both a reduction in tumor activity and an increase in survival rate in mice with liver cancer.

Finally, researchers have developed mechanisms for bacteria to deliver their payload in response to changes in their environment, such as through temperature-dependent and pH dependent pathways or in response to light. Bioengineers can clone environment-dependent promoters into bacteria that respond to changes the external environment by changing gene expression [68,69]. In the case of temperature dependence, researchers at Washington University have developed a promoter that activates a cell lysis pathway when there is a decrease in temperature [70]. This model can be manipulated to prevent bacteria from delivering its payload to healthy tissue or to create a system in which temperature is intentionally changed only in the target region to induce localized payload delivery [70]. For pH-responsive promoters, these can be manipulated to prevent the release of the payload unless there is a sufficient drop in pH, which is a characteristic of the tumor microenvironment [71]. Recent studies have found that biofilms, or a matrix of attached bacteria, can be responsive to light [72,73]. This mechanism, termed optogenetic control, can be harnessed to deliver anti-tumor drugs directly to tumor sites via biofilms in response to changes in light [72]. Each of these mechanisms allow for a localized delivery of the payload directly to a tumor and a lower risk of affecting healthy tissue (Fig. 4).

Fig. 4.

Schematics illustrating engineering logic of bacterial payload delivery systems.

6. Combination therapies and synergistic approaches

The use of tumor-targeting bacteria in combination with other cancer therapies has been demonstrated to improve clinical outcomes by addressing the limitations standard cancer treatments cannot overcome, such as immune suppression, drug resistance, and poor tumor penetration [34]. One application of using tumor-targeting bacteria is in conjunction with nanoparticles to carry cancer treatments to tumor sites and target the immune system as a whole [74–76]. Nanoparticles are becoming popular in use for cancer treatment because they can be used for drug delivery, gene therapy, and imaging [76,77]. However, for nanoparticles to be effective, they need to go through biological barriers, such as the endothelial, cellular, skin, and mucosal barriers, which prevent nanoparticles from getting accumulated in the tumor site [78]. Tumor-targeting bacteria, such as Salmonella and Clostridium, are attracted to oxygen-deficient conditions and necrotic regions on the tumor that do not have proper vascularization, which makes it harder for drugs to reach the tumor [79]. Tumor-targeting bacteria are loaded with nanoparticles that have chemotherapeutics and can behave as vehicles to migrate and penetrate deeper into the tumor to release the therapeutics, resulting in higher drug concentration and reduced systemic toxicity [77]. Oncologists must go through a decision tree to decide which therapy or combination of therapies would be best for their patient, and bacterial-based therapeutics show the potential to become a viable treatment option in the future, whether it be on its own or in conjunction with another therapy.

Another application of tumor-targeting bacteria in conjunction with standard therapies is their use alongside immune checkpoint inhibitors. This approach can improve the effectiveness of immune checkpoint therapy by overcoming obstacles such as poor drug delivery and limited immune activation [79,80]. Tumor-targeting bacteria have been shown to enhance immune checkpoint blockade (ICB) therapy [79–81]. Immune checkpoint inhibitors (ICIs), such as anti-PD-1 and anti-PD-L1 antibodies, are used to block proteins that inhibit T cells and keep the immune system active to recognize and attack cancer cells [79]. However, their effectiveness is limited in “cold” tumors, which are tumors that lack sufficient immune infiltration and therefore do not produce an effective immune response [82]. Some bacteria can change the tumor microenvironment by activating the innate immune system, producing cytokines, and drawing immune cells into the tumor [83]. The inflammation induced by the bacteria can turn cold tumors into “hot” tumors, which have active immune infiltration and are responsive to ICBs [82]. One example is the production of inosine by the probiotic Bifidobacterium pseudolongum [84]. It was found that the administration of B. pseudolongum in conjunction with immune checkpoint inhibitors simultaneously increases the effectiveness of the immunotherapy while reducing side effects, and while the exact underlying mechanism is unclear, it is likely due to the production and release of inosine [84].

Third, tumor-targeting bacteria can be paired with photothermal therapy (PTT), which uses heat generated from light-absorbing materials to destroy cancer cells [85]. In this approach, bacteria are genetically engineered to make gold nanoparticles, which are minuscule particles of gold that can absorb and scatter light, making them a useful tool for cancer therapies and imaging [85]. The gold nanoparticles can absorb near-infrared (NIR) light. Bacteria carry gold nanoparticles to the tumor site, and once the NIR light causes nanoparticles to create localized heat, they kill the cancer cells without damaging nearby tissue [85]. PTT used alongside tumor-targeting bacteria can cause precise and effective tumor destruction while also initiating anti-tumor immune responses via the release of tumor antigens after killing the tumor with heat [86]. Bacterial vectors for PTT can be used to ensure that heat is generated exactly where it is needed, even in deep areas of tumors [86].

Furthermore, tumor-targeting bacteria have been proven to be beneficial when used alongside chemotherapy [34]. Standard chemotherapy struggles to fully kill tumors due to their hypoxic and necrotic regions that are not accessible by blood vessels, meaning chemotherapeutic drugs cannot reach the entirety of the tumor [34]. Tumor-targeting bacteria, however, can reach the harsh, oxygen-deprived regions in a tumor, making them a good candidate for localized drug delivery [34]. One study showed the use of bacteria to deliver doxorubicin, a commonly used chemotherapeutic drug, directly to the tumor, achieving tumor reduction and reduced side effects [87]. The application of tumor-targeting bacteria alongside chemotherapeutics improves drug distribution within tumors and effectively minimizes systemic toxicity [87].

Finally, bacteria have also been shown to work effectively in conjunction with radiotherapy [88]. Radiotherapy functions by damaging the DNA of cancerous cells, causing cell death and increased visibility of tumor cells to the immune system via the release of tumor-associated antigens [88]. Radiotherapy, however, is not of much benefit in certain regions of the tumor due to the oxygen dependence of the radiation-induced DNA damage [89]. Tumor-targeting bacteria, especially anaerobic strains, can integrate themselves into these oxygen-deprived regions and stimulate the release of cytokines to activate more immune cells [90]. The bacterial colonization of regions of the tumor inaccessible by radiation compensates for the lack of efficiency of radiotherapy in hypoxic areas [89,90]. The combination of radiotherapy and tumor-targeting bacteria can lead to the direct killing of tumor cells, radiation-induced tumor cell DNA damage, and immune activation [91].

7. Efforts in engineering microbiome to fight specific types of cancer

Understanding the diverse mechanisms by which the native gut microbiota influences tumor biology - ranging from DNA damage to metabolic reprogramming and angiogenic modulation - has catalyzed a paradigm shift in therapeutic strategies. This has led to the emergence of engineered microbial therapeutics capable of precisely targeting tumors, remodeling the TME, and enhancing anti-cancer immunity. Leveraging advances in synthetic biology, researchers have designed bacterial platforms to deliver cytotoxic agents, immunomodulatory proteins, and even radiosensitizers directly to tumors. The following examples demonstrate how engineered microbes are being tailored to combat specific cancer types through novel mechanisms (Table 1).

Table 1:

Table summarizing recent efforts in engineering microbes and their applications in cancer therapy.

Study	Bacterial Platform	Target Cancer Type	Therapeutic Mechanism	Delivery Strategy	Outcome
Chull An et al., Molecules and Cells, 2019	P. pentosaceus	DLD-1 and HT-29 Colorectal Cancer	Secretion of Lactic acid bacteria protein p8 to downregulate expression of Cyclin B1 and Cdk1 in tumor cells	Intraperitoneal (IP) injection and oral administration	IP injection showed 59% reduction in tumor mass, oral administration showed 64% reduction in tumor mass, overall anti-proliferative effect IP injection showed 59% reduction in tumor mass, oral administration showed 64% reduction in tumor mass, overall anti-proliferative effect IP injection showed 59% reduction in tumor mass, oral administration showed 64% reduction in tumor mass, overall anti-proliferative effect [92]
Thomas et al. Adv. Healthcare Mater. 2021	Hypervesiculating E. coli Nissle 1917	4T1 Breast Orthotopic	CHy OMV release to degrade tumor hyaluronic acid (HA), enhancing drug and immune cell penetration	Intratumoral or systemic (with TKIs)	Enhanced response to lapatinib, ECM remodeling, improved T cell infiltration [5]
Li et al., ACS Synth. Biol. 2022	E. coli Nissle 1917	4T1 Breast	TNF-α release in response to sufficient temperature increase	Intravenous injection	4T1 tumor growth effectively inhibited 4T1 tumor growth effectively inhibited 4T1 tumor growth effectively inhibited [93]
Deb et al., Sci. Rep. 2022	S. typhimurium	H1819 Lung, H460 Lung	Theta-toxin release causing pore formation in cancer cells	Intratumoral injection	Significant increase in cell death in vitro, significant reduction in tumor growth in vivo Significant increase in cell death in vitro, significant reduction in tumor growth in vivo Significant increase in cell death in vitro, significant reduction in tumor growth in vivo [94]
Xiao et al., J Nanobiotechnol. 2022	B. infantis	4T1 Breast	Release of DOX nanoparticles in response to tumor microenvironment	Intravenous injection	Increased DOX concentration in tumor, prolonged survival of mice with tumor Increased DOX concentration in tumor, prolonged survival of mice with tumor Increased DOX concentration in tumor, prolonged survival of mice with tumor [87]
Qingqing et al., Adv Mater. 2022	E. coli BL21	MC38-OVA Colon	Radiation-triggered release of CD47 nanobodies fused to outer membrane vesicles	Intratumoral and intravenous injection	Macrophage stimulation by CD47 inactivation Macrophage stimulation by CD47 inactivation Macrophage stimulation by CD47 inactivation [95]
Li et al., Chem Eng J. 2022	B. infantis	A549 Lung Carcinoma	Release of DOX nanoparticles in response to reductive stress of tumor microenvironment	Intravenous injection	Increased tumor cell apoptosis, inhibited tumor growth, reduced systemic side effects of DOX Increased tumor cell apoptosis, inhibited tumor growth, reduced systemic side effects of DOX Increased tumor cell apoptosis, inhibited tumor growth, reduced systemic side effects of DOX [96]
Savage et al., Sci. Advances, 2023	E. coli Nissle 1917	A20 B Cell Lymphoma	Solute carrier-mediated release of human cytokines CXCL16 and CCL20	Intratumoral injection	Effective recruitment of both adaptive (CD8+ T cells) and innate (dendritic cells) immune systems Effective recruitment of both adaptive (CD8+ T cells) and innate (dendritic cells) immune systems Effective recruitment of both adaptive (CD8+ T cells) and innate (dendritic cells) immune systems [97]
Mi et al., Acta Pharmaceutica Sinica B, 2023	S. typhimurium	Glioma	Expression and excretion of flagellin B (FlaB) to activate M1 macrophages and recruit neutrophils to the tumor in combination with DOX delivery	Intravenous injection	Nearly complete eradication of glioma tumors with minimal risk of bacterial infection Nearly complete eradication of glioma tumors with minimal risk of bacterial infection Nearly complete eradication of glioma tumors with minimal risk of bacterial infection [98]
Chen et al., Cancer Immunology, 2023	S. epidermidis	B16-F10 Melanoma	Expression of tumor-associated antigens used to induce anti-tumor response in CD4+ and CD8+ T cells	Subcutaneous injection	Effective T cell activation and decreased tumor progression Effective T cell activation and decreased tumor progression Effective T cell activation and decreased tumor progression [99]
Siddiqui et al., Adv	E. coli Nissle 1917	MC38 Colon, 4T1	Copper radionuclide uptake via FyuA	Intratumoral +67Cu-	Enhanced CD8+:Treg ratio, prolonged
Healthcare Mater. 2023	(EcN)	Breast	receptor; in situ radionuclide delivery	YbT injection	survival, tumor-specific radionuclide accumulation [6]
Shahid et al., ACS Synth. Biol. 2023	E. coli BL21	JIMT1 Breast	HlyE release causing pore formation in cancer cells	In vitro	Effective cytotoxicity against JIMT1 cells Effective cytotoxicity against JIMT1 cells Effective cytotoxicity against JIMT1 cells [100]
Study	Bacterial Platform	Target Cancer Type	Therapeutic Mechanism	Delivery Strategy	Outcome
Shi et al., Int J Nanomedicine. 2023	B. infantis	A549 Lung Carcinoma	Paclitaxel-loaded nanoparticles released in response to reductive stress of tumor microenviro-nment	Intravenous injection	Inhibition of tumor growth, prolonged survival of mice with tumor Inhibition of tumor growth, prolonged survival of mice with tumor Inhibition of tumor growth, prolonged survival of mice with tumor [101]
Li et al., Adv Funct Mater. 2023	B. infantis	4T1 Breast	Tumor microenvironment disintegrates PDA layer of B. infantis releasing CaO2 nanoparticles and doxorubicin	Intravenous injection	Chemotherapeutic effects, enhanced apoptosis due to increased Ca2+ and •OH production Chemothera-peutic effects, enhanced apoptosis due to increased Ca2+ and •OH production Chemotherapeutic effects, enhanced apoptosis due to increased Ca2+ and •OH production [102]
Vaziri et al., Adv. Science, 2024	Bacillus Calmette- Guéerin	Hepatocellular Carcinoma (HCC)	Promotion of immune cell infiltration into tumors (CD4+ and CD8+ T cells, M1 macrophages) and induction of IFN-γ signaling	Subcutaneous injection	Tumor cell apoptosis, prevention of fibrosis, increased metabolism, and overall anti-HCC effects Tumor cell apoptosis, prevention of fibrosis, increased metabolism, and overall anti-HCC effects Tumor cell apoptosis, prevention of fibrosis, increased metabolism, and overall anti-HCC effects [103]
Gurbatri et al., Nat. Commun. 2024	E. coli Nissle 1917	Orthotopic Colorectal Cancer	Local release of GM-CSF, PD-L1, and CTLA-4	Oral administration	Significant reduction in burden from CRC, T cell infiltration into tumor core Significant reduction in burden from CRC, T cell infiltration into tumor core Significant reduction in burden from CRC, T cell infiltration into tumor core [104]

Building on the mechanistic understanding and engineering strategies described above, researchers have begun developing bacteria specifically tailored to combat individual cancer types [105]. The heterogeneity of cancer from tumor microenvironment characteristics to immune landscapes necessitates cancer-specific approaches. The following sections examine engineered microbial therapeutics for four prominent cancer types: pancreatic, breast, lung, and colorectal cancers [94,100,104,106].

7.1.1. Engineered microbiome in pancreatic cancer

Pancreatic cancer, with its high mortality and morbidity rates, has one of the worst prognoses of any type of cancer [106]. Recently, the notion that both healthy and cancerous pancreatic tissue are sterile sites has been challenged [106]. This microbiome, especially in the tumors, has been shown to be implicated in all aspects of pancreatic cancer, including diagnosis, progression, treatment, and treatment resistance [106]. In pancreatic ductal adenocarcinoma (PDAC) tumors, the bacterial phyla Proteobacteria, Bacteroidetes and Firmicutes and the fungal phyla Ascomycota and Basidiomycota are the most prevalent, which could potentially serve as biomarkers for the screening or diagnosis of PDAC [106]. By modulating the immune system, both innate and adaptive, the pancreatic microbiome in PDAC tumors contributes to the progression of PDAC [106]. Activation of the lectin pathway of the complement system, release of neutrophil chemokines, stimulation of tumor-associated macrophage 2, and reduction of CD8+ T cells by the PDAC microbiome create an immunosuppressed tumor microenvironment that allows the progression of PDAC [106]. The role of the PDAC microbiome in cancer treatment is demonstrated with both antibiotics and probiotics [106]. When antibiotics were administered to mice with PDAC, tumor progression was inhibited [106]. Also, antibiotic treatment in PDAC patients resulted in an improved prognosis [106]. Probiotic supplementation, Lactobacillus in particular, has had the effect of reducing pancreatic precancerous lesions and slowing the proliferation of PDAC cancerous cells [106]. It has also been shown that bacteria in the PDAC microbiome can confer resistance to chemotherapy in PDAC [106]. In another example, engineered Salmonella typhimurium expressing cytolysin A was administered via intravenous injection, leading to the release of cytolysin A within the tumor microenvironment in nude mice bearing subcutaneous or orthotopic human pancreatic tumors [107]. Salmonella typhimurium vector expressing collagenase Streptomyces omiyaensis Trypsin (SOT) was developed to target collagen-rich extracellular matrix in PDAC [107]. In aggressive PDAC models, this therapy degraded intratumoral collagen, reduced tumor cell proliferation and immunosuppressive cells, and reshaped the tumor microenvironment to support potential immunotherapy [108]. Tumor-targeting Salmonella typhimurium expressing bacterial hyaluronidase (bHs-ST) was developed to degrade hyaluronic acid (HA) in pancreatic tumors. bHs-ST effectively colonized PDAC tumors, depleted tumor HA, and enhanced bacterial diffusion within the dense tumor matrix [109]. This strategy shows promise for improving drug delivery while reducing off-tumor toxicity [109].

7.1.2. Engineered microbiome in breast cancer

Breast cancer, one of the most common forms of cancer, has two problems with its current treatment: lack of specificity and inefficient targeting [100]. A 2024 study aimed to overcome these obstacles by designing and engineering E. coli BL21 to express a nanobody that selectively binds to HER2+ receptors in breast cancer cells. In this study, the surface-expressed nanobody was used to help facilitate the binding of the E. coli to the breast cancer cell, and following attachment, the E. coli released HlyE, which is a pore-forming toxin that kills breast cancer cells [100]. The results showed that their engineered E. coli displayed effective and efficient treatment of cancer cells [100]. However, these experiments were only performed in vitro [100]. Another study, published in 2022, tested an engineered E. coli Nissle 1917 (EcN) that was sensitive to heat [93]. In this design, the EcN would release TNF-α in response to a specific temperature in vivo [93]. Temperature regulation was chosen as it has many advantages compared to other current regulation methods (chemicals or light) such as being more controllable and being better able to diffuse into deep tissues [93]. The results showed that there was tumor suppression upon heat-stimulation of the engineered EcN [93]. In addition to in vitro and rodent testing, there is currently a phase I trial in which genetically engineered measles virus expressing Helicobacter pylori neutrophil-activating protein (NAP) is administered to metastatic breast cancer patients to determine shedding and immune response rate [110]. Another strategy utilized hyper-vesiculating EcN (ΔECHy) to deliver ClyA-Hyaluronidase (CHy) fusion protein via outer membrane vesicles (OMVs) [5,111]. This design targeted and degraded the tumor stroma, particularly hyaluronic acid (HA), which is a major barrier to immune and drug penetration [5]. Delivery of CHy remodeled the dense extracellular matrix (EcM), enhancing tumor accessibility to checkpoint inhibitors and tyrosine kinase inhibitors (TKIs) [5]. When used in combination with lapatinib in 4T1 tumor-bearing mice, ΔECHy significantly improved therapeutic response and prolonged survival, thus emphasizing the role of bacteria-mediated stromal modulation [5].

7.1.3. Engineered microbiome in lung cancer

Lung cancer, which accounts for the most cancer-related deaths, is also beginning to be studied as a target of engineered microbes [94]. A 2022 study researched the use of modified S. typhimurium to help treat non-small cell lung cancer (NSCLC), which is the most common subtype of lung cancer [94]. The researchers designed a bacterium that releases theta-toxin, which is a pore-forming toxin, to kill NSCLC cells [94]. They found that the S. typhimurium expressing the theta-toxin caused more cell death [94]. Also, the researchers found that NSCLC spheroids treated with the modified S. typhimurium had significantly reduced cell viability [94]. These promising results, while in vitro, warranted exploration of these bacteria in live mouse models [94]. Mice were given an NSCLC tumor, and the experimental group was then treated with the engineered S. typhimurium [94]. The experimental group not only showed a drastic reduction in tumor growth, but the mice’s peripheral organs were unaffected by the treatment [94]. The peripheral organs did not have any detectable S. typhimurium, did not show signs of apoptosis, and the mice did not show signs of morbidity during treatment [94]. This means the engineered S. typhimurium is very effective at treating NSCLC while not showing any signs of systemic toxicity. The researchers also point out that the efficacy of the treatment with S. typhimurium could potentially be improved if used in combination with other standard anti-cancer therapies [94]. A phase I study was performed on patients having a wide variety of cancer types, one of which tested was NSCLC [112]. CRS-207, a mesothelin-releasing strain of Listeria monocytogenes that has been extensively studied, was given to patients with NSCLC, and it was found that these patients had an induced innate and adaptive T cell response [112]. This success in trials on human cancer patients is a significant advancement in bacterial cancer therapy, and once again highlights the immense potential these therapies could have in the future of cancer treatment.

7.1.4. Engineered microbiome in colorectal cancer

Colorectal cancer is another type of cancer that is being studied as a target for bacterial therapy [104,113,114]. One study in particular tested engineered EcN on colorectal adenomas in both mice and human groups [104]. This study found many promising results. First, the oral administration of EcN manipulated to release cell cycle inhibitors and cytokine GM-CSF showed a reduction in the burden from microsatellite stable (MSS) colorectal cancer in mouse models [104]. This is especially interesting because this subtype of colorectal cancer in humans is normally unresponsive to systemic administration of the same inhibitors [104]. Also, while the underlying mechanism is unclear, the administered EcN appeared to remodel the tumor microenvironment [104]. T cells usually cannot thrive in the tumor microenvironment due to its low-oxygen conditions, leading to T cell exclusion, characterized by the presence of T cells only at the periphery of the tumor [104]. However, the treatment group showed an increase in the infiltration of T cells into the core of the tumor, which is evidence that the EcN contributes to a more hospitable microenvironment for T cells to thrive in [104]. These researchers also found that this EcN can be used to aid the diagnosis of colorectal cancer [104]. They used the ability of the EcN to colonize the colorectal tumors to show the possibility of diagnosing colorectal adenomas via non-invasive stool and urine assays [104]. In addition to cancer treatments, microbial-based cancer vaccines are under development as well. In this study, Vaaben et al. engineered the EcN to produce indole-3-acetic acid (IAA), an aryl hydrocarbon receptor (AhR) agonist, and evaluated its therapeutic potential in colorectal cancer models [2]. The engineered strain (EcNIAA) was intratumorally injected into mice bearing CT26 and MC38 CRC tumors, leading to significant reductions in tumor growth, improved survival, and increased infiltration of CD4+ and CD8+ T cells [2]. IAA production activated AhR within the tumor microenvironment, promoting a pro-inflammatory immune response marked by elevated IFN-γ and CXCL9 and reduced IL-17A, without offtarget effects or systemic toxicity [2]. Notably, EcNIAA-treated mice also demonstrated long-term tumor immunity upon rechallenge, highlighting the potential of this microbiome-based therapeutic to enhance anti-tumor immunity in CRC [2]. In another approach, EcN was engineered to express the FyuA-receptor, allowing selective uptake of copper-based radionuclides (⁶⁷Cu-YbT) [6]. These bacteria colonize tumors and serve as in situ bioreactors, enabling a pre-targeted theragnostic platform for solid tumors [6]. This system bypasses the need for tumor-specific antigens, offering a powerful strategy for treating epitope-independent cancer, including MC38 colon tumors [6]. Tumor-bearing mice treated with engineered EcN and ⁶⁷Cu-YbT showed enhanced survival and favorable CD8⁺ T cell: Treg ratios, underscoring the immunomodulatory benefit of the platform [6] (Fig. 5).

Fig. 5.

Two innovative approaches to using EcN for anti-cancer therapeutics. A) EcN was engineered to express FyuA, allowing the uptake of radionuclides. B) EcN was engineered to hypervesiculate, delivering anti-cancer drugs via OMVs.

7.1.5. Comparative evaluation of engineered microbial strategies across cancer types

Engineered microbial therapeutics exhibit diverse mechanisms of action across pancreatic, breast, lung, and colorectal cancers, reflecting the profound biological differences in tumor microenvironments, stromal structure, immune infiltration, and resident microbiota [6,47]. While each cancer type presents promising examples of engineered microbial interventions, the suitability and efficacy of bacterial strategies depend heavily on tumor-specific constraints. Below, we synthesize key engineering approaches, highlight their shared principles, and critically evaluate their advantages and limitations across cancer contexts.

Pancreatic ductal adenocarcinoma (PDAC) presents one of the most challenging environments for microbial therapies due to its extensive fibrotic stroma, dense extracellular matrix, extreme hypoxia, and immunosuppressed niche [6,108,109]. Engineering efforts in PDAC have therefore emphasized ECM-degrading enzymes (e.g., collagenase, hyaluronidase) to improve bacterial penetration and drug diffusion, alongside intratumoral colonization by Salmonella to deliver cytotoxic proteins [109]. These strategies effectively remodel stroma and counteract immune exclusion but require careful control to avoid off-tumor matrix degradation and inflammatory toxicity [108].

In contrast, breast tumors, particularly HER2-positive subtypes, benefit from surface-displayed nanobodies and targeted receptor-binding mechanisms that enable high-precision bacterial localization [100,115]. Thermal- or stimulus-responsive gene circuits have shown promise in regulating cytokine release (e.g., TNF-α) within breast tumors [93]. However, breast cancers possess less consistent hypoxic gradients than PDAC, making hypoxia-dependent strategies less reliable [116]. Here, OMV-based delivery systems and receptor-targeted approaches offer superior specificity, though their in vivo persistence and biodistribution remain challenges [5].

Engineered microbes used in lung cancer, especially NSCLC, often prioritize controlled cytotoxic protein delivery (e.g., theta-toxin) and systemic safety, given the high risk of pulmonary inflammation [89]. S. typhimurium strains have demonstrated strong tumor selectivity and safety in rodent models without peripheral organ colonization [98]. However, lung tumors feature highly variable oxygenation, irregular immune infiltration patterns, and complex architectural barriers, necessitating tightly regulated gene circuits and fail-safe mechanisms to mitigate risks of systemic toxicity [96].

Colorectal cancer (CRC) differs markedly from the other cancer types because the gut lumen naturally harbors rich bacterial populations. This makes CRC uniquely amenable to oral delivery of engineered probiotics such as E. coli Nissle 1917 (EcN) [104,111]. Strategies such as secretion of GM-CSF, delivery of cell-cycle inhibitors, induction of T-cell infiltration, in situ tumor diagnosis, and aryl hydrocarbon receptor agonist production leverage the native colonization capacity of EcN [2,7]. CRC also benefits from innovative theranostic platforms (e.g., radionuclide-loaded bacteria), which are feasible due to the tumor’s accessibility and bacterial permissiveness [6,117]. However, the gut microbiome’s complexity may reduce colonization consistency, and immune interactions remain variable across patients.

Across all cancer types, several shared themes emerge. Tumors with dense stroma and hypoxia (PDAC) require penetration-enhancing and metabolism-adapted bacteria [108]. Tumors with defined cell-surface targets (breast cancer) favor receptor-specific binding and OMV delivery systems [5,116]. Tumors with high risk of systemic inflammation (lung cancer) demand tightly regulated circuits to minimize off-target toxicity [94,118]. Tumors with native bacterial permissiveness (CRC) support oral bacterial administration and complex therapeutic payloads [98]. Ultimately, no single strategy is universally optimal; instead, the most effective therapeutic designs integrate multiple modalities such as ECM degradation with OMV-based delivery or metabolic auxotrophy with logic-gated payload release to account for the distinct ecological and immunological landscapes of each tumor type [5] (Table 2).

Table 2:

Comparative strengths and limitations of major engineering approaches.

Cancer Type	Key Tumor Microenvironment Features	Most Suitable Engineered Microbial Strategies	Advantages	Limitations / Challenges
Pancreatic Cancer (PDAC)	Dense ECM, fibrosis, extreme hypoxia, immune exclusion	ECM-degrading enzymes (collagenase, hyaluronidase), hypoxia-targeted Salmonella, intratumoral protein delivery	Enhances drug penetration; overcomes stromal barriers; strong colonization under hypoxia	Risk of off-tumor ECM degradation; severe immunosuppression; difficult systemic delivery
Breast Cancer (HER2+, TNBC)	Variable hypoxia, moderate immune infiltration, accessible surface markers	Nanobody-mediated targeting, temperature-controlled circuits, OMV-mediated toxin/enzymes	High specificity for receptor-positive tumors; externally controllable circuits; good for localized delivery	Limited penetration depth; heterogeneity in receptor expression; in vitro-to-in vivo translation gaps
Lung Cancer (NSCLC)	Irregular oxygenation, fragile immune balance, high risk of inflammation	Toxin-secreting Salmonella, tightly regulated logic circuits, systemic but controlled delivery	Strong tumor targeting with minimal systemic toxicity; effective in orthotopic models	Risk of pulmonary inflammation; variable tumor architecture; safety constraints in clinical use
Colorectal Cancer (CRC)	Gut-accessible, bacterial-permissive, immune-excluded tumor core	Oral EcN-based delivery, immune-modulating circuits, radionuclide-loaded bacteria, in situ diagnostics	Non-invasive oral delivery; excellent colonization; theranostic applications; strong T-cell infiltration	Microbiome competition may inhibit colonization; variability across patients; mucosal immunity complexity

7.2. Novel cancer vaccine developments using microorganisms and their products

Microorganisms and microbial products have long been fundamental to the development and use of vaccines [119]. Most current vaccines are immunogens derived from either killed or attenuated microorganisms [119]. Microbial products, such as toxins and antigens, have also been used in vaccines [120]. One example of this is the Diphtheria-tetanus toxoids-pertussis vaccine [120]. The use of inactivated diphtheria and pertussis toxins in the vaccine has been shown to be effective in the induced production of antitoxin [119]. An interesting breakthrough in microbial-based vaccines is the development of vaccines for cancer. The body’s natural attack on cancer is based on the ability of antigen-presenting cells to receive signals from antigens on cancerous cells then activate other various parts of the immune system such as cytotoxic and helper T cells [61]. However, the hypoxic environment of tumors makes it difficult for this crucial first step to occur [61]. By using engineered bacteria, which thrive in oxygen-poor environments such as the tumor microenvironment, as delivery vehicles, anti-tumor vaccines can be delivered far more effectively and efficiently [61]. The antitumor drugs are typically produced inside the engineered bacteria, though, so three types of delivery systems were developed to overcome this barrier [61]. First, engineered bacteria have been made to express the drug internally, then release it via lysis in response to a sufficient population density [61]. Second, the engineered bacteria can be made to present the drug on its surface to elicit a more robust humoral and cell-mediated immunity [61]. Third, the engineered bacteria can be made to simply secrete the drug upon production [61]. As of now, development of successful microbial-based cancer vaccines is a matter of honing the understanding of the exact mechanisms by which these microbes can be used to target cancer, which will be discussed in the following sections. Although these studies are promising, there is still much more research to be done.

8. Clinical pipeline and regulatory landscape

In addition to the in vitro and in vivo studies discussed previously, several notable clinical trials have begun to take place. In a Phase I trial, an engineered strain of EcN (SYNB1891) was made to produce cyclic dinucleotides that activate the STING pathway when in hypoxic conditions, inducing an immune response in tumors [121]. This study found that in 24 patients, repeated administration of SYNB1891, both on its own and in conjunction with atezolizumab, was considered safe and tolerated by the participants [121]. Also, the upregulation of genes regulated by interferon (IFN), cytokines, and T cells indicates that the STING pathway was activated as intended [121]. This study, however, is no longer in development. In another clinical trial, this one in Phase II, Salmonella was engineered to release human interleukin-2 (IL-2) locally to tumors to induce Natural Killer cells and T cells in patients with pancreatic cancer [122]. This strain of Salmonella, Salmonella-IL2 or Saltikva, was administered in combination with FOLFIRINOX, which is a standard chemotherapy regimen [122]. This trial was successful, showing a 73% partial response rate (31% in controls) and a 24-month median survival time (11.1-13.1 months in controls), meaning a combination of Saltikva and FOLFIRINOX resulted in an approximately doubled median survival [122].

While successes have been found in clinical trials using bacteria as cancer therapies, it is important to discuss failed trials as well, including why they failed and what there is to learn from them. An example of this was the Phase II trial on CRS-207, a modified strain of Listeria monocytogenes, which produced mesothelin in tumors to activate an immune response [123]. The results of Phase I trials with this strain were promising with a 7.5- and 14.7-month progression-free survival and overall survival, respectively, and evidence of immune cell infiltration into tumors [123]. However, in the Phase II trial, while it was shown to be safe, no clinical activity was observed in combination with pembrolizumab, an immunotherapeutic drug, and further evaluation resulted in the discontinuation of CRS-207 development [123]. Similarly, a Phase I trial investigated the effects of VNP20009, a modified strain of Salmonella typhimurium shown to inhibit tumor growth in mice, was tested on patients with metastatic cancer [124]. In clinical trials, VNP20009 was shown to be safe and well-tolerated, but it did not show any antitumor effects [124]. In each of these trials, it is important to note that the bacterial therapy did not cause severe adverse reactions, and they were even considered to be tolerated well by the patients. The downfalls of these studies were in the limited effect on the overall cancer treatment, suggesting that researchers should experiment more with understanding and refining the bacterial anti-tumor mechanisms and bacteria-host interactions.

The regulation of the development and testing of these bacterial-based therapeutics is another unique hurdle for bioengineers. Regulatory bodies such as the U.S. Food and Drug Administration (FDA), and specifically its Center for Biologics Evaluation and Research (CBER) department oversee these studies [125]. Some considerations that researchers must take into account while developing new bacterial treatments include the safety and containment, manufacturing and standardization, environmental risk, and clinical trial designs of the bacteria [125]. Additionally, bacteria-based therapeutics can fall into one of two categories depending on their mechanism of action: live biotherapeutic products (LBPs) or gene therapy products [126]. LBPs are live microorganisms excluding vaccines, viruses, and gene-transfer vectors, whereas gene therapy products are designed to simply introduce new genetic material into the cells of patients [126]. Each of these classifications have their own regulatory processes, the biggest difference being stricter guidelines for gene therapy products due to their complexity and long-term safety concerns [127]. There are documents that outline these regulations and guidelines for research on bacterial-based therapeutics such as the Pharmaceutical Microbiology Manual (PMM) and the Investigational New Drug (IND) application [128,129]. However, pathways such as the Regenerative Medicine Advanced Therapy (RMAT) designation by the FDA allows for particularly innovative medicines and therapies to have an accelerated timeline for testing and approval [130] (Fig. 6).

Fig. 6.

Timeline illustrating notable clinical trials of bacterial cancer therapies.

9. Manufacturing and scale-up challenges

While the recent developments in microbial-based cancer therapies hold promise, it should also be noted that the translation of their therapeutic potential into viable clinical products comes with several manufacturing and scale-up challenges, which can be divided into three groups: upstream processes, downstream processes, and quality control [131–133].

The two major scale-up challenges in the upstream process, which involves cultivating and producing the bacteria, are media optimization and process control [132]. Advancing from small-scale laboratories mass production of these bacterial therapeutics will require substantially more materials. Because of this, it is important to develop highly effective and cost-efficient media that support optimal activity for the bacteria while also following safety guidelines, which is a significant challenge for researchers [132]. Additionally, having a substantially larger number of bacteria to be grown, and a larger volume of media, makes it more challenging to maintain optimal environmental conditions for the bacteria, including pH, temperature, and nutrient availability [132].

The challenges associated with downstream processing, which involves isolating the bacteria, include the purification, stability, and environmental safety of the bacteria [131]. Effectively removing impurities from growing conditions and host cells without affecting the viability of live therapeutics, especially when producing large amounts of bacteria, is another significant challenge posed by scaling up [131]. Another obstacle to be overcome is developing safe and environmentally friendly ways to store these bacteria [131]. When mass producing bacteria, it is important to create storage formulations that ensure a long shelf-life, maintain their stability, and have a limited effect on their safety and viability [131]. Environmental considerations are also a concern in the production of live therapeutics, as there need to be safety measures to ensure their proper containment [134]. Currently, several strategies are employed; specialized air handling systems with HEPA filters and controlled access points are used in these facilities, aseptic sealed packaging is used to transport live bacteria, and proper waste disposal techniques are put in place [134].

Finally, quality control challenges mostly come in the form of maintaining batch consistency [133]. It is crucial that each batch of bacteria produced are consistent with each other, both to maintain their clinical effects and to ensure safe use in patients, but it is often difficult to ensure consistency in bacteria due to small mutations and genetic variation [133]. Some strategies used to maintain batch consistency include standardizing environmental conditions for the growing bacteria and constantly monitoring and adjusting bacteria colonies, which allows for the quick detection and response to colonies that may be growing irregularly [135]. Also, practicing and abiding by good manufacturing process (GMP) guidelines helps to maintain consistency [136]. GMP is a framework that developers and producers of live therapeutics follow to ensure the proper quality and safety of their products, and it includes guidelines on facility designs, product characterization and validation, and quality management [136]. One of the biggest challenges for adhering to GMP guidelines with LBTs is the complexity of bacterial systems, which can be overcome by creating and adapting more robust biological assays to test these bacteria [136].

Economic considerations should also be taken into account when discussing the manufacturing of bacterial cancer therapeutics as they become more clinically relevant. As LBT production scales up, operation costs will also increase [123]. This comes from a need for the construction and maintenance of manufacturing facilities, specialized equipment, increased raw materials such as media and nutrients, and adequately trained personnel [123]. However, the demand for LBTs are expected to increase, especially as they are further understood and as they continue to show clinical success [137–140]. Various analytics predict that the compound annual growth rate (CAGR) of the LBT market will be between seven and fifteen percent for the coming years [137–140].

10. Discussion

The relationship between humans and microorganisms has evolved from one of fear and avoidance to sophisticated appreciation of their therapeutic potential. As demonstrated across diverse disease models from cancer to cardiovascular diseases to metabolic, respiratory, and neurological disorders - microbial therapeutics represent a transformative shift in modern medicine. The clinical landscape and incorporation of bacterial-based cancer therapy into medical practice seems to be advancing rapidly with several clinical trials underway. The successes of these trials show the promise held by therapeutic bacteria while the unsuccessful trials help to refine and further understand the mechanisms by which they work.

Recent technological breakthroughs have provided further opportunities for the development of anti-cancer microbes. The advancements in AI and the development of algorithms to simulate biological pathways and interactions will be incredibly helpful in predicting and creating new payload delivery mechanisms. Also, the progression of gene editing systems such as CRISPR-Cas will make engineering bacteria more efficient and accurate. Finally, the refinement of standard omics techniques and sequencing analyses will aid in the production and testing of safe live bacterial therapeutics in both small-scale laboratories and largescale manufacturing facilities.

Despite these advances, several challenges persist. First, the complexity and interindividual variability of the human microbiome complicate efforts to standardize microbial therapeutics. Differences in microbiome composition based on geography, diet, genetics, and prior medical history can affect therapeutic outcomes, leading to in-consistencies across preclinical and clinical studies. Furthermore, much remains unknown about the mechanistic underpinnings of microbe-host interactions, including which specific microbial strains and metabolites drive health benefits or disease progression [47,141]. Without this mechanistic clarity, therapeutic applications risk being nonspecific or unpredictable. In terms of production and manufacturing, scaling up and maintaining consistency between batches of bacteria while adhering to strict safety guidelines proves to be another obstacle. Finally, preventing spontaneous mutations and evolution of bacteria will be a challenge that requires constant monitoring.

To fully realize the potential of microbial therapeutics, two major directions must be prioritized in future research. First, deeper mechanistic studies are essential to unravel the molecular interactions between microbes and host tissues. Leveraging multi-omics technologies - including single-cell genomics, metabolomics, and spatial transcriptomics - will be a key to deciphering how microbial metabolites, extracellular vesicles, and surface molecules modulate host pathways [22]. Identifying reliable microbial biomarkers predictive of therapeutic efficacy will allow for more personalized microbiome-based interventions. Second, innovations in synthetic biology might continue to refine microbial engineering platforms. Developing programmable probiotics capable of sensing, responding, and adapting to dynamic disease microenvironments while minimizing unintended systemic effects will be critical. Integration of microbial therapies with traditional treatments (e.g., immunotherapy, radiation, chemotherapy) may offer synergistic benefits, particularly in complex diseases like cancer. The application of microbial therapeutics in preventive healthcare is another exciting frontier, and taking these directions could fundamentally shift healthcare paradigms.

11. Conclusion

The rapid evolution of microbial science and synthetic biology has unveiled microorganisms as powerful allies in the fight against a broad spectrum of diseases. The advancements in bioengineering techniques, the creation of various payload delivery techniques, and the success of bacterial therapeutics in conjunction with other therapies and on their own in both laboratories and clinical trials provide the foundation that enables clinical translation of microbial medicine in the future. Although current studies are performed in prominent cancer types, some understudied subtypes such as leiomyosarcoma, neuroendocrine cancers, or thyroid cancer, to name a few, could be benefited by the progression of bacterial therapy research in the future. The next steps in the field might further unravel the mechanisms by which microorganisms interact with tumor and host physiology and to continue to refine synthetic biology platforms utilized in the creation and production of novel therapeutic bacteria. Although significant challenges remain, particularly regarding mechanistic understanding, safety, and regulatory approval, the early successes in both preclinical and clinical studies are compelling. As research advances, microbial therapeutics are poised to redefine the landscape of modern medicine, offering novel solutions for some of the most complex and enigmatic diseases facing humanity.

Data availability

No data was used for the research described in the article.