Bacteria as Precision Tools for Cancer Therapy

ABSTRACT

The discovery at the end of the 20th century of genes that induce cell death revolutionised the biocontaintment of genetically manipulated bacteria for environmental or agricultural applications. These bacterial ‘killer’ genes were then assayed for their potential to target and control malignant cells in human cancers. The identification of the bacteriomes in different human organs and tissues, coupled with the observation that bacteria tend to accumulate near tumours, has opened new avenues for anti‐cancer strategies. This progress, along with recent insights into how cancer cells evade immune response, has prompted innovative therapeutic approaches. Tumour microenvironments are typically nutrient‐rich, characterised by low oxygen tensions and very resistant to immune responses. Two recent studies in MBT highlight the promise of using Salmonella typhimurium and Escherichia coli as vectors in novel cancer treatments. Engineered S. typhimurium strains can generate adjuvant flagellin‐antigen complexes that function as in situ vaccines, hence increasing the immunogenic responses within tumour environment. Similarly, gut E. coli can be used as vectors to targert tumour cells in colon cancer, enabling both diagnostic applications and localised treatments. Both approaches hold significant potential to improve patient survival outcomes.

The identification of the bacteriomes in different human organs and tissues, coupled with the observation that bacteria tend to accumulate near tumours, has opened new avenues for anti‐cancer strategies. This progress, along with recent insights into how cancer cells evade immune response, has prompted innovative therapeutic approaches.

The discovery of DNA as the source of genetic information in the mid‐20th century provided a significant boost to understanding DNA structure and function and opened avenues to manipulate DNA for different purposes, such as gene cloning, protein expression and constructing novel metabolic pathways. Over time, concerns about DNA manipulation raised, varying in intensity. In the 1990s, the European Commission and other funding agencies promoted developing biocontainment systems for genetically manipulated microbes. A pioneering contribution came from Soeren Molin's group in Denmark, which used genes that induce bacterial death, such as the hok/sok (host killing/save of killing) system, the Gef (gene expression fatal) protein and gene E from phage ɸX174, to create biocontainment systems. This approach led to the concept of suicide bacteria (Molin et al. 1993), where bacteria perform a specific function while alive and self‐destruct once its role is over. In collaboration with Soeren Molin, Juan L. Ramos's group in Spain envisaged a containment system for genetically modified Pseudomonas with expanded catabolic pathways. Specifically, Contreras et al. (1991) and Jensen et al. (1993) engineered Pseudomonas putida strains that survive in the presence of pollutants (i.e., methylbenzoates) and commit suicide once the substrate depletes. For agriculture applications, Ronchel and Ramos (2001) developed a dual containment system that warrants bacterial survival in plant roots but not in bulk soil.

The aforementioned killer proteins are porins, which disrupt cell membranes. This initial idea for the control of genetically manipulated microorganisms then evolved into a potential strategy for cancer treatment. Thus, Aranega's research group at the University of Granada investigated the toxicity of these prokaryotic/phage‐derived proteins in eukaryotic cells. Initially, the group examined the gef and E genes as potential killers of cancer cells. Their findings demonstrated that the expression of both proteins is also toxic for eukaryotic cells and reduces tumour cell growth rates (Prados et al. 2008; Ortiz et al. 2009). Notably, E and Gef proteins localised to the mitochondria inducing tumour cell death by activating the apoptotic pathway (Ortiz et al. 2009). Studies in colon and breast cancer cells revealed that combining these cytotoxic proteins with doxorubicin enhanced tumour cell killing (Rama et al. 2011) The E gene was also successfully targeted towards colon and lung cancer cells both in vivo and in vitro using the carcinoembryonic antigen (CEA) promoter (Rama‐Ballesteros et al. 2020). However, the practical application of these findings remained limited due to the lack of effective carriers for delivering the killer proteins to tumours in situ.

It is well known that bacteria exert immune‐stimulatory effects, and various research groups have shown their ability to colonise solid tumours by thriving in environments rich in nutrients, hypoxia and immunosuppressive conditions characteristic of tumour microenvironments (Duong et al. 2019). Bacteria administered systematically can accumulate ~10,000 times in tumours compared to other tissues. Over two decades ago, modified antibiotic‐sensitive S. typhimurium strains were designed to preferentially accumulate in tumours while gradually becoming eliminated from other organs, even without the use of antibiotics, as observed in studies involving mice and non‐human primates (Clairmont et al. 2000). However, the use of these bacteria for stimulating immunogenic responses was hindered by their inability to deliver antigens directly to the cytosol of antigen‐presenting cells, which is pivotal for generating a robust cytotoxic T‐cell response (Carleton et al. 2013).

To overcome this limitation, S. typhimurium strains capable of delivering therapeutic agents via the type 3 secretion system (T3SS), a needle‐like structure that injects effector proteins from bacteria into the cytoplasm of host cells, were used. Thus, recombinant attenuated S. typhimurium expressing heterologous protein antigens delivered via this system can induce cellular immune responses (Bai et al. 2018).

All these findings, together with recent evidence showing that most (if not all) human organs are colonised with bacteria Michán‐Doña et al. (2024), pointed towards bacteria as delivery vehicles for combating cancer, leading to the development of several bacterial therapies (Gurbatri et al. 2022; Raman et al. 2023; Redenti et al. 2024). Raman et al. (2023) reviewed the field, describing a modular ‘build‐a‐bug’ approach that focuses on five design characteristics: bacterial strain (chassis), therapeutic compound, delivery method, immune‐modulating features and genetic control circuits. Bacterial‐based therapies show promises in treating patients with untreatable tumours because they are able to (1) colonise primary and metastatic tumours, (2) deliver therapeutic molecules directly into tumours and cancer cells (3) directly kill cancer cells and (4) induce antitumor immunogenic responses (see Gurbatri et al. 2022).

In situ vaccination is a therapeutic approach aimed at exploiting tumour antigens located at the tumour site alongside immunostimulatory molecules (adjuvants) to trigger tumour‐specific adaptive immune responses (Shen et al. 2023). Antigens released from dying tumour cells are captured by activated dendritic cells and presented to T cells, which then target and destroy tumour cells. However, this process is often hindered by the tumour immunosuppressive microenvironment. Recent in situ vaccination strategies have shown promise using bacteria as natural adjuvants, functioning as cytokine factories to enhance antigen‐presenting cells within tumours. Notably, recent research used the probiotic E. coli Nissle 1917 strain as a platform for presenting tumour neoantigenic peptides, which successfully elicited immune responses in situ. Preclinical trials in mice demonstrated promising results, particularly for colorectal cancer treatment. Furthermore, the authors showed a very significant extended survival after intravenous vaccination with engineered strains in a very aggressive advanced metastasis melanoma model (Redenti et al. 2024).

Significant advances in this field have been discussed recently in Microbial Biotechnology. Firstly, Xia and Wu (2024) reported that directing bacteria to tumour is feasible and a promising approach for cancer therapy. The use of flagellated bacteria represents a novel approach to deliver antigens and to activate dendritic cells, thereby facilitating intratumoral antigen delivery and enhancing the immune activation potential of in situ vaccines. Flagellin, the primary structural protein in bacterial flagella, is an excellent adjuvant. When recognised by Toll‐like receptor 5, it enhances the phagocytic functions of dendritic cells and promotes T‐cell activation. Furthermore, bacteria capture released antigens within the tumour microenvironment, forming adjuvant–antigen complexes. This process provokes a cooperative increased immunogenic response (Figure 1). The authors also discuss the advantages and limitations of this approach, highlighting opportunities both to increase antigen capture by the bacteria and ensuring their safety to minimise potential pathogenicity.

FIGURE 1.

Formation of adjuvant flagellin‐antigen and promoting T‐cell activation against cancer cells. The figure has been prepared with images obtained from NIH Bioart (courtesy of Dr. Jose Alhama).

The importance of the gut microbiota in human health is undeniable. Related to colorectal cancer (CRC), certain bacterial species have been identified as tumorigenesis promoters, whereas others are associated with better performance of the anti‐tumour responses (Okumura et al. 2021; Minot et al. 2024). Jian et al. (2024), in their review ‘The two‐edged sword theory’ discuss the ambivalent role of E. coli in CRC screening and treatment. They highlight that the variations in abundance and subtypes of E. coli across populations could serve as a valuable biomarker.

The potential of E. coli as a genetic tool lies in its adaptability for compatible use with various technologies, including nanoparticles, imaging modalities and synthetic biology modifications. Imaging methods, such as positron emission tomography (PET) and magnetic resonance imaging (MRI), long established in cancer detection, can benefit from engineered bacteria to enhance visualisation of tumours and in situ bacterial localisation. Once inside the tumour, engineered bacteria can report on tumour presence, burden and on the local microenvironment by levering bacteria‐based sensors and circuits. Therefore, the application of E. coli and other bacteria in cancer therapy should adopt the ‘One Health’ perspective, considering its interactions with other microorganisms, the host, environmental factors and their evolutionary dinamycs (Lynch et al. 2022). In addition to image‐based detection, bacteria can produce detectable molecules in urine, blood and stool, offering additional non‐invasive diagnostic options.

Current trends suggest that combining bacteria therapies with other cellular therapies or external modalities, where interactions with these technologies are engineered in rationally designed bidirectional systems, holds significant potential for new strategies in the treatment and diagnosis of cancers.

Author Contributions

Carmen Michán: conceptualization, writing – original draft, supervision, funding acquisition, writing – final version. José Prados: writing – original draft and writing – final version. Juan‐Luis Ramos: conceptualization, writing – original draft, supervision, funding acquisition, writing – final version.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

This is a highlight.