Probiotics lead to a new frontier in tumor fighting: tumor vaccines based on probiotics

Abstract

Cancer remains one of the leading causes of mortality, posing a significant threat to human health and consistently serving as a focal point for scientific research. In recent years, immunotherapy has garnered increasing attention as a promising approach to cancer treatment, leading to substantial improvements in patient outcomes. Among the various immunotherapeutic strategies, tumor vaccines have emerged as a particularly innovative field. Research on tumor vaccines has focused on improving their immunogenicity and safety, aiming to elicit stronger and more durable antitumor immune responses. Probiotics, as bacteria that are “foreign” to the body, can induce a variety of beneficial responses and stimulate numerous positive biological effects, holding significant promise in the field of immunotherapy. Research on probiotic-based tumor vaccines has illustrated the unique advantages of in situ vaccines, neoantigenic vaccines, and oral vaccines with specific routes of administration, each of which has its own advantages. Owing to advancements in biosynthetic technologies and the inherent plasticity of probiotics, probiotics can be used as carriers or components that offer safety benefits and a range of health-promoting effects. Moreover, accumulating evidence suggests that probiotic-based tumor vaccines may be even more effective when combined with other therapeutic modalities, such as radiation therapy.

Graphical Abstract

Background

Aberrant tissue formation in tumors results from DNA damage, which is often driven by genetic mutations and epigenetic alterations in the cellular developmental and apoptotic pathways. These changes occur in a synergistic or sequential manner, causing local tissue cells to lose normal growth regulation at the genetic level, ultimately leading to uncontrolled cell proliferation. With improvements in living standards and increased life expectancy, the likelihood of developing cancer during an individual’s lifetime has risen accordingly. Recent advancements in tumor immunotherapy have shown promising efficacy and substantial development prospects [1]. The high tumor heterogeneity, elevated mutation rates, and significant individual variability, along with the clinical translational importance of low mutation data, position tumor vaccines as one of the most promising yet challenging areas in immunotherapy [2].

The emerging frontier of therapeutic tumor vaccines is represented by in situ vaccination (ISV) and personalized neoantigen-based vaccines. A distinct advantage of in situ vaccines is their ability to utilize tumor foci as “antigen factories”, enabling tumors to autonomously release antigens and convert “cold” tumors, characterized by low immunogenicity, into “hot” tumors with high immunogenicity. This unique mechanism offers novel insights into the development of tumor vaccines and provides a new paradigm for therapeutic strategies [3–5]. Neoantigen vaccines specifically target tumor-specific antigens (TSAs) expressed exclusively on tumor cells and exhibit immunogenic properties. These vaccines require neoantigen screening to address significant interindividual heterogeneity [6].

Probiotics not only adapt to the unique environment of the gastrointestinal tract and regulate disruptions in the intestinal microbiota to prevent various diseases, including cancer but also elicit a range of anticancer immune responses. Oral vaccination induces mucosal immunity and offers enhanced safety and convenience, which further supports the widespread adoption of tumor vaccines. Consequently, probiotic-based tumor vaccines show great promise for future therapeutic strategies [7].

Probiotics play crucial physiological roles in enhancing the intestinal microecological balance and modulating the host immune system [8, 9]. As natural carriers, probiotics can also activate both innate and adaptive immune responses, functioning as active therapeutic agents. By leveraging synthetic biology techniques, such as the design of epitope arrays that regulate tumor-specific neoantigen sources or key components that modulate antitumor immunity, engineered probiotics can overcome the limitations of traditional carriers. This enables the development of more efficient and functional delivery platforms for cancer immunotherapy [3, 10].

This review focuses on the various engineered forms of probiotic-based tumor vaccines, the advantages and challenges of their foundational research and clinical translation, and the potential benefits of combining probiotic tumor vaccines with radiation therapy.

Construction of tumor vaccines based on probiotics

On the one hand, probiotics, as live microorganisms, can induce an innate immune response and elicit a series of beneficial health effects in the host without posing any potential pathogenic risk. On the other hand, probiotics act as immunomodulators, demonstrating antitumor properties, and can function as immune adjuvants in tumor vaccines [3, 7]. Some probiotics are engineered or possess inherent tumor-targeting properties, making them ideal candidates as carriers for live vaccines. Recent studies have shown that certain probiotics, such as an attenuated strain of Clostridium novyi (C. novyi-NT), can induce localized cytolysis and inflammatory responses within tumors while also possessing the metabolic capability to produce anti-inflammatory cytokines. These activities play critical roles in preventing cancer and eliminating cancer cells during the early stages of the disease [7, 11].

The safety of active vector vaccines, methods of antigen loading, and overall effectiveness of immunization are key concerns [12]. The use of probiotics as vaccine carriers improves safety and enhances immunization effectiveness to a significant extent. The rapid advancement of synthetic biology has allowed the utilization of various biotechnological approaches to further enhance and direct the antitumor immune response of selected probiotics. This can be achieved through the design of genetic engineering strategies, the optimization of specific biosynthetic pathways, and other innovative methods [13].

Probiotic-based in situ tumor vaccines

Introduction of the ISV and the antitumor mechanism

Unlike traditional tumor vaccines, in situ tumor vaccines (ISVs) utilize tumor antigens present at the tumor site itself to induce a tumor-specific adaptive immune response. This approach circumvents the labor-intensive and time-consuming process of identifying and screening for tumor-specific antigens while simultaneously triggering a more effective and comprehensive immune response [14]. In recent years, several probiotics have emerged as promising tumor-targeting drug carriers and effective adjuvants [3].

In situ vaccination (ISV) generates an immune response against tumor antigens by inducing immunogenic cell death (ICD) in tumor cells. During ICD, the tumor microenvironment (TME) reactivates a silent immune response in the immunosuppressive microenvironment. This reactivation occurs due to the release of tumor debris and the generation of tumor-associated molecular patterns, also known as danger-associated molecular patterns (DAMPs), which stimulate the immune system and restart the suppressed immune response [5]. Tumors treated with ISVs undergo a repetitive cascade cycle of “immune initiation”, “immune response”, “tumor cell death” (antigen release), “immune reactivation”, and “immune response” [15, 16].

Probiotic-based ISV construction form

The development of natural probiotics as components in ISV-based immunotherapy often involves chemical modifications, nanotechnology, genetic engineering, and other advanced techniques to optimize their efficacy.

In terms of chemical modification, probiotics can be engineered as active delivery vehicles for in situ tumor vaccination via appropriate loading methods. These methods can be broadly classified into four categories: electrostatic adsorption, covalent conjugation, antibody–antigen-specific interactions, and membrane encapsulation, along with metabolic labeling [17]. For example, photothermal reagents such as polydopamine (pDA3), lysoviruses, dopamine, and anti-PD1 antibodies (Bac Mel) can be incorporated onto the surface of probiotics to enable them to function as potent inducers of ICD in tumor cells [3, 18–20].

In nanotechnology, the primary focus is on customizing bacteria to the nanoscale, which are also called bacteria-derived nanovesicles. These nanovesicles retain pathogen-associated molecular patterns (PAMPs) while reducing pathogenicity. Bacteria-derived nanovesicles can be categorized into five groups on the basis of their origin and manufacturing process: membrane vesicles (G⁺ released by bacteria), outer membrane vesicles (OMVs) (G^- produced by bacteria), bilayer membrane vesicles (produced through chemical/physical disintegration), protoplasmic body-derived nanoparticles (derived from bacterial inner membranes), and microcells (originating from aberrant bacterial divisions) [21]. Among these, outer membrane vesicles (OMVs) are the most extensively studied, with four primary mechanisms by which they contribute to antitumor immunity: (1) OMVs can inhibit tumor progression in an IFN-γ-mediated manner; (2) OMVs can also directly bind to TLR2 on DCs to trigger their maturation; and (3) OMVs can be genetically engineered or used with other functional parts (e.g., PD-1 and CaP nanoshells) for chemical surface modification. These moieties can neutralize the tumor immunosuppressive microenvironment and work with CD8⁺ T cells generated by OMV-based in situ tumor vaccination for better therapeutic efficacy. (4) OMVs can be genetically engineered via the protein insertion and presentation system. This system allows OMVs to specifically bind to labeled neoantigens to form tumor vaccines [3, 22–25].

In the context of genetic engineering, bacteria offer customizable genetic circuits, positioning them as multifunctional platforms with significant developmental potential. Bacteria also possess metabolic pathways that can be harnessed for artificial modifications, including those that interfere with tumor metabolic processes [26].

Milestones in clinical progression

Through advanced synthetic biology techniques, bacteria can be engineered to perform a variety of antitumor functions, including the synthesis of chemical molecules in situ, secretion of stress-inducing factors, and metabolic regulation within the tumor microenvironment (TME). For the development of tumor vaccines, researchers often employ a combination of strategies to achieve multiple objectives, continuously refining their approaches to optimize tumor vaccine construction across different levels.

Liu B et al. developed an engineered, food-grade probiotic, Lactococcus lactis (FOLactis). This strain was designed to express a fusion protein comprising FMS-like tyrosine kinase 3 ligand (Flt3L) and OX40 ligand (OX40L) through synthetic biology. In various mouse models of subcutaneous and in situ tumors characterized by poor immune cell infiltration and resistance to anti-PD-1 therapy, the team reported that this Lactococcus lactis-based in situ tumor vaccine (FOLactis) led to significant tumor regression. This effect was driven primarily by an increase in the number of conventional dendritic cell type 1 (cDC1) cells and the restoration of cytotoxic T lymphocyte (CTL) responses within the TME. Furthermore, this in situ vaccination (ISV) induced an abscopal effect (AE) and synergized with anti-PD-1 antibodies [10]. Liu’s team further advanced this work by constructing a new Lactobacillus lactis (L. lactis)-based in situ tumor vaccine, OR@Lac, via chemical modifications (Fig. 1). This vaccine involves the modification of OX40 and R848 onto the Lactobacillus lactis surface. In the CT26 in situ tumor model, tumor progression was significantly reduced in the OR@Lac group, which notably prolonged the survival of the treated mice. Moreover, 40% (2/5) of the mice in this group achieved complete tumor regression (CR). In a 4T1 in situ model established in BALB/c female mice, the administration of OR@Lac more potently inhibited tumor growth than the combination of OX40 and Lactobacillus lactis alone [27]. Liu’s team is currently developing a recombinant Mycobacterium smegmatis (M. smegmatis) expressing anti-PD-L1-IL-15, using M. smegmatis as a vector.

Fig. 1.

An immunomodulator-enriched Lactococcus lactis platform for enhancing in situ tumor vaccines. Reproduced with permission [27]. Copyright 2025, Adv Healthc Mater.OX40 and R848 were modified onto the surface of Lactobacillus lactis, generating OR@Lac with reserving adjuvant properties, movement capacity, and the ability to activate DCs. Intratumoral injection of OR@Lac Lac enhances CTL tumor-killing capacity and activates mature DCs while suppressing M2-TAM polarization. This promotes efficient cross-presentation of antigens by DCs within the TME and tumor-draining lymph nodes, increases the number of mature DCs, and restores CTLs. Consequently, it induces significant tumor regression and elicits a robust systemic immune response. Notably, Ibrutinib affects the TME by inhibiting MDSCs. Thus, the synergy between OR@Lac and ibrutinib significantly enhanced the anti-tumor effect

Neoantigen vaccines based on probiotics

Introduction of the neoantigen vaccine and the antitumor mechanism

Tumor antigens are generally classified into two categories: tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). TAAs refer to a group of antigens that are expressed in both normal and tumor cells, albeit with relatively high expression levels in tumor cells. These antigens are widely used as biomarkers in clinical practice. TSAs are antigens that are not expressed in normal cells but are highly expressed in tumor cells. Compared with TAAs, TSAs exhibit greater immunogenicity and are more likely to be recognized by the host as nonself, thus eliciting a stronger, more specific immune response [28]. Among TSAs, tumor neoantigens are particularly prominent. These antigens arise from somatic mutations, are uniquely expressed in tumors and are capable of inducing tumor-specific T cells that target and kill tumor cells with high precision. Neoantigens have demonstrated substantial potential for personalized cancer vaccine development. The mechanism of probiotic-based in situ and neoantigenic vaccines is as follows (Fig. 2).

Fig. 2.

A sketch of the mechanism of probiotic-based in situ and neoantigenic vaccines.The left panel illustrates in situ vaccination (ISV), a strategy that harnesses the tumor’s own antigens to initiate an anti-tumor immune response. Reformulated probiotics attack the tumor, prompting the release of damage-associated molecular patterns (DAMPs) and tumor antigens. Simultaneously, the probiotics serve as a natural adjuvant by providing pathogen-associated molecular patterns (PAMPs). These signals work in concert to activate antigen-presenting cells (APCs), which in turn potently stimulate cytotoxic T lymphocytes (CTLs) to mount a robust anti-tumor immune response.The right panel depicts the process for developing personalized neoantigen-based vaccines. This approach begins with the identification of tumor-specific neoantigens through genomic analysis. These uniquely personal antigens are modified onto probiotics to recognize and eliminate tumor cells by initiating immune response

Probiotic-based neoantigen vaccine construction

The design and construction of an effective tumor vaccine involves five critical steps: antigen identification, antigen encapsulation, antigen delivery, antigen release, and antigen presentation to T cells [29]. Rapid and efficient screening of suitable tumor antigens is crucial in this process. Personalized neoantigen-based tumor vaccines address a major challenge in tumor immunotherapy by targeting the immunogenicity of tumor vaccines.

The delivery of neoantigens via probiotics is a promising strategy. Engineered probiotics can be used to deliver arrays of tumor-specific epitopes, enhancing both safety and immunogenicity. This approach facilitates the generation of a system capable of driving potent, antigen-specific T-cell responses that effectively control or even eradicate tumor growth [30]. The synergy between probiotics and neoantigens may lead to unexpected, enhanced therapeutic effects in tumor treatment.

Milestones in clinical progression

Chen et al. at Stanford modified Staphylococcus epidermidis (S. epidermidis) to specifically express tumor antigens and induce the production of tumor-specific T cells capable of targeting and killing tumor cells [31]. Nicholas Arpaia at Columbia University engineered a probiotic Escherichia coli strain, Nissle 1917 (EcN). The two teams worked on similar lines of research, but the modification of EcN was more refined, including optimization of the structural form of the neoantigen; removal of the cryptic plasmid and deletion of the Lon protease (LON) and outer-membrane proteases T (OmpT) to increase the accumulation of neoantigens; increased susceptibility to phagocytosis; expression of listeriolysin O (LLO), which facilitates entry of the neoantigens into the bacterial cytoplasm; presentation of the neoantigens through MHC class I molecules, which induces TH1-type immune responses; and induction of TH1-type immune responses by decreasing the time of the bacterium in the blood survival time and biofilm formation, aiming to improve the production and cytoplasmic delivery efficiency of peptide arrays containing neoantigenic peptides and enhance their susceptibility to blood clearance and phagocytosis [30]. Therapeutic efficacy was demonstrated in a colorectal cancer model (CT26), where probiotic vaccine therapy successfully achieved the design goals. Notably, the vaccine effectively targeted and eradicated distant metastases formed by colorectal cancer cells in the lungs. The therapeutic efficacy was further validated in a melanoma model (B16F10), in which similar results were observed.

The emergence of “off-the-shelf”, shared neoantigens

In addition to personalized neoantigen vaccines, targeted shared neoantigen vaccines have emerged as important developments. Shared neoantigens are immunogenic epitopes commonly present across various tumor patients. These antigens are exclusively expressed in tumor tissues, are essential for tumor growth, and function as tumor-specific antigens that trigger immune responses [32]. Shared neoantigens are particularly attractive for research because of their “off-the-shelf” availability, universal applicability, high specificity, and strong immunogenicity. Targeting shared neoantigens could provide universal therapeutic vaccines or cellular therapies applicable to a broad range of patients. A multivalent viral vector-based tumor vaccine, Nous-209, which targets shared neoantigens, has already entered clinical trials [33, 34]. As research progresses, probiotic-based vaccines targeting shared neoantigens could offer new possibilities for enhancing antitumor therapies.

Probiotic-based oral vaccines

Advantages of oral vaccines and antitumor mechanisms

Oral tumor vaccines have three main advantages. First, they can elicit a stronger immune response than other forms of vaccination can. Second, they are capable of activating mucosal immunity, leading to the production of specific antibodies, particularly immunoglobulin A (IgA). Third, oral vaccines are more convenient, safer, and demonstrate greater patient adherence when applied in clinical settings. Compared with intramuscular and subcutaneous tumor vaccines, oral vaccines tend to stimulate a greater number of lymphocytes and do not require adjuvants to increase their immunogenicity [7, 35]. Furthermore, the gut, as the largest immune organ in the body, is rich in immune cells, making oral tumor vaccines extremely promising options for antitumor immunotherapy.

The advent of probiotic-vectored oral vaccines has mitigated certain risks associated with traditional live oral vaccines, such as infection and transmission. Additionally, these probiotic-vectored vaccines have been shown to improve therapeutic efficacy by tolerating the harsh gastrointestinal environment and functioning as biological delivery systems [35]. Rapid advancements in synthetic biology have facilitated the development of various probiotic-based oral vaccine delivery vectors, where engineered probiotics or programmed bacteria are designed to recognize cancerous tissues within the intestinal tract. These bacteria can then secrete therapeutic agents locally in situ, thereby promoting tumor regression [36, 37]. Notably, oral vaccines have demonstrated significant antitumor effects in animal models, including those for nongastrointestinal tumors, and have exhibited favorable safety profiles, such as in the case of lung metastatic melanoma [35].

Probiotic-based oral tumor vaccine construction

Owing to the specific route of administration, the development of oral vaccines often requires the consideration of several critical properties, including the ability to tolerate the gastrointestinal environment, overcome the intestinal epithelial barrier, and interact effectively with the abundant immune cells in the intestinal lamina propria [38]. Achieving these properties involves a range of synthetic biology techniques, such as nanotechnology, advanced genetic engineering, and the introduction of inducible promoters. Key strategies include the customization of probiotic-derived vesicle particles to increase their interaction with the host and generate robust immune responses, genetic editing to enable targeted proliferation and drug delivery, and the use of inducers to optimize the behavioral control of the oral vaccine [35].

Milestones in clinical progression

A team led by Guangjun Nie genetically engineered Escherichia coli, one of the most prevalent commensal bacteria in the gut, to develop an oral tumor vaccine based on “bacterial robots.” This approach enables the oral administration of bacteria to achieve in situ, controlled production of tumor-antigen-carrying OMVs within the gut. Specifically, the Fc fragment of immunoglobulin G (IgG; mFc) was fused to the C-terminus of the E. coli OMV surface protein ClyA (ClyA-Ag-mFc). To prevent immune tolerance induced by prolonged antigenic exposure, researchers introduced a promoter induced by the monosaccharide arabinosean (Ara) to regulate the expression of the fusion protein. By orally administering the modified E. coli along with the Ara promoter, they successfully achieved controlled, in situ production of OMV-Ag-mFc carrying tumor antigens in the intestine. This study demonstrated that engineered E. coli (Top10 strain) could tolerate the intestinal environment with sufficient safety, control gene expression via the Ara promoter, and activate antigen-specific immune responses, thereby inducing effective immune memory [35]. Tal Danino’s team conducted an extensive series of preclinical and clinical evaluations of modified E. coli Nissle 1917 (EcN) as an oral delivery system for the treatment of colorectal cancer (CRC). These findings support the use of EcN as an oral delivery platform for the detection and treatment of CRC [39]. There have been probiotic-based cancer vaccine-related clinical trials in the past 5 years (Table 1).

Table 1.

Probiotic-based cancer vaccine-related clinical trials in the past 5 years

Vaccine types	Vaccine name	Clinical trial main ID	Vector	Experimental phase	Research time
In situ vaccination	FOLactis	NCT06512896	Engineered Lactococcus lactis	N/A	2023–2026
Neoantigen vaccines	NECVAX-NEO1	NCT06631079	Ty21a (a live-attenuated Salmonella typhi strain oral typhoid vaccine)	Phase 1/2	2024–2028
	RT201	NCT05930301	Listeria monocytogenes	N/A	2021–2024
Oral vaccine	NECVAX-NEO1	NCT06631079	Ty21a (a live-attenuated Salmonella typhi strain oral typhoid vaccine)	Phase 1/2	2024–2028
	B440	JPRN-jRCT2051220143	Recombinant Bifidobacterium longum	Phase 1	2022-
Others	EO2463	NCT04669171	OncoMimics	Phase 1/2	2021–2029
	EO2401	NCT04187404	OncoMimics	DONE	2020–2024

Probiotic tumor vaccines in combination with radiotherapy

Tumor vaccines are designed to activate the body’s immune system to target and eliminate tumor cells. However, tumor cells have evolved multiple immune escape mechanisms to counteract immune recognition and attack [10, 40]. To increase the therapeutic efficacy of tumor vaccines, combining them with other treatment modalities, such as immune checkpoint inhibitors, chimeric antigen receptor T-cell therapy (CAR-T-cell therapy), conventional radiotherapy, and chemotherapy, represents a more conventional and effective treatment strategy [41, 42].

Introduction to radiation therapy

Radiation therapy (RT) has closely followed advancements in scientific and technological developments, evolving toward precision, digitalization, and intelligence. Modern radiotherapy includes techniques such as proton therapy, image-guided radiation therapy (IGRT), stereotactic radiosurgery (SRS), stereotactic body radiotherapy (SBRT), helical tomotherapy (TOMO), and flash radiotherapy (FLASH-RT). These technological innovations enable radiotherapy to impact various stages of tumor progression. Studies have demonstrated that radiotherapy plays a crucial role in disrupting multiple steps of the cancer immunity cycle [43].

Antitumor mechanisms of radiation therapy

Ionizing radiation, a key stressor in cellular therapy, can induce immunogenic cell death (ICD) in tumor cells. This process exposes previously hidden tumor antigens to the immune system, enabling recognition and subsequent immune-mediated destruction. When tumor antigen levels are insufficient to trigger a robust T-cell response, combining tumor vaccines with radiotherapy can effectively address this limitation [44]. The release of tumor antigens through ICDs, which activate systemic immune responses, has brought attention to the abscopal effect (AE) of radiotherapy. AE is a phenomenon wherein local radiotherapy to a primary tumor leads to the shrinkage or disappearance of distant, unirradiated tumors. These findings suggest that radiotherapy triggers a systemic antitumor immune response through complex immune mechanisms. The occurrence of AE involves intricate activation of the immune system, modulation of the tumor microenvironment, and interactions between various molecular and cellular signals [45, 46].

Radiation therapy also functions as a viral mimic. Using a mechanism similar to that triggered by viral infections, radiation therapy induces interferon (IFN) production and exposes neoepitopes, which enables the host’s immune system to recognize and reject [44]. Radiation-induced cellular damage results in the release of damage-associated molecular patterns (DAMPs), including DNA fragments, in response to DNA damage. These DAMPs activate DCs, while DNA fragments, which originate from the nucleus, are translocated to the cytoplasm. In the cytoplasm, these DNA fragments activate cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), leading to a type I interferon response. The cGAS-STING pathway is a hallmark of immune defense against viral infections [44, 47].

Moreover, irradiation alters the tumor microenvironment by increasing the expression of major histocompatibility complex class I (MHC-I) antigens, death receptors, and natural killer group 2 member D ligand (NKG2DL) on cancer cells. This promotes a chemotactic milieu, facilitating T-cell homing and vascular remodeling, which alleviates tumor-induced immunosuppression [44, 48].

Progress in developing probiotic tumor vaccines combined with radiotherapy

Wu’s group conducted a study exploring the use of bacteria as tumor antigen delivery carriers in combination with local radiation therapy. The study employed positively charged nanoparticles, polyamide amine dendritic polymers, that were electrostatically adsorbed onto VNP20009, a modified bacterium. These bacteria capture tumor antigens in situ via ionic interactions. Upon radiation therapy, tumor cells undergo ICD, releasing tumor antigens and various DAMPs. The modified bacteria captured these antigens and transported them from the necrotic tumor core to the tumor margins, where a high density of normal DCs resides. This approach enhances the crosstalk between tumor antigens and peripheral DCs induced by immune response therapy (IRT), thereby increasing the body’s antitumor immune response [18] (Fig. 3).

Fig. 3.

A radiotherapy-bacterial combination therapy for antitumor immunity. Radiotherapy attacks tumor cells, inducing immunogenic cell death(ICD). This process triggers the release of tumor antigens and various damage-associated molecular patterns (DAMPs). Engineered bacteria, designedwith enhanced motility and high affinity for tumor antigens, capture these released antigens. They then migrate from the necrotic tumor core towardthe tumor periphery, which is enriched with normal dendritic cells (DCs). There, the bacteria facilitate antigen delivery to DCs, thereby activatingthe immune system and initiating a potent antitumor immune response

A novel triple in situ vaccine strategy combining local radiotherapy with intratumor injections of unmethylated CG-enriched oligodeoxynucleotide (CpG, a TLR9 agonist) and an OX40 agonist emerged. Analyses demonstrated that this triple vaccine strategy not only activates the therapeutic tumor microenvironment and upregulates multiple immune-related pathways but also enhances systemic antitumor responses, resulting in superior tumor control and survival benefits [49].

Recent studies have indicated that irradiation at multiple sites may offer greater immunological benefits, potentially even in nonablative regimens [46]. Furthermore, radiation therapy has been shown to enhance both the local and systemic effects of cancer immunotherapy (IT). Importantly, optimal sequencing of RT and IT enhances antigen-specific immune responses locally and systemically. Therefore, sequencing strategies must be optimized before initiating clinical trials [50, 51].

Conclusion

With in-depth studies of probiotics and rapid advancements in biosynthetic technologies, studies focusing on the expression and modification of probiotics themselves have demonstrated the safety and efficacy of probiotic-based antitumor immunotherapy.

Recent investigations revealed that the expression of a murine-derived antidetection-inducing IL-18 mutant protein (DR18) on the outer membrane of nonpathogenic Escherichia coli notably enhanced the immune response of natural killer (NK) cells and CD8⁺ T cells against tumors, effectively inhibiting tumor growth in various murine tumor models [52]. Novel antitumor lytic bacteria, often genetically engineered to minimize their toxicity while enhancing their antitumor capabilities (e.g., expression of cytokines), have been developed. Recent research has developed a mineralization method for growing manganese dioxide nanostructures on a bacterial surface. Mineralized bacteria exhibit potent tumor inhibitory effects and high curing rates [53].

Our understanding of tumor immunotherapy has evolved from the need for individualized antigens to the need for tailored vectors and, more recently, to the integration of combination therapies. In parallel, innovative approaches such as in situ tumor vaccines that do not require individualized antigens, “off-the-shelf” shared neoantigen vaccines, and oral vaccines where food-grade probiotic bacteria serve as the active vectors have emerged. The main research foci in the construction of probiotic-based tumor vaccines currently include in situ tumor vaccines, neoantigenic tumor vaccines, and oral vaccines, which have gained increasing attention because of their distinct immunological pathways. However, the antitumor mechanisms of these vaccines are not entirely independent, and the biosynthetic modification strategies overlap. Therefore, further exploration of shared molecular signaling pathways, along with the application of additional modification strategies, holds promise for improving the efficacy of probiotic-based tumor vaccines.

Abbreviations

ISV

In situ vaccination

TSAs

Tumor-specific antigens

C. novyi-NT

Clostridium novyi

ICD

Immunogenic cell death

TME

Tumor microenvironment

DAMPs

Danger-associated molecular patterns

OMVs

Outer membrane vesicles

FOLactis

Lactococcus lactis

Flt3L

FMS-like tyrosine kinase 3 ligand

OX40L

OX40 ligand

cDC1

Conventional dendritic cell type 1

CTL

Cytotoxic T lymphocyte

AE

Abscopal effect

L. lactis

Lactobacillus lactis

M. smegmatis

Mycobacterium smegmatis

TAAs

Tumor-associated antigens

TSAs

Tumor-specific antigens

S. epidermidis

Staphylococcus epidermidis

EcN

Escherichia coli strain, Nissle 1917

LON

Lon protease

OmpT

Outer-membrane proteases T

LLO

Listeriolysin O

IgA

Immunoglobulin A

IgG; mFc

The Fc fragment of immunoglobulin G

Ara

Monosaccharide arabinosean

CRC

Colorectal cancer

CAR-T

Chimeric Antigen Receptor T-Cell Therapy

RT

Radiation therapy

IGRT

Image-guided radiation therapy

SRS

Stereotactic radiosurgery

SBRT

Stereotactic body radiotherapy

TOMO

Helical tomotherapy

FLASH-RT

Flash radiotherapy

IFN

Interferon

cGAS

Cyclic GMP-AMP synthase

STING

Stimulator of interferon genes

MHC-I

Major histocompatibility complex class I

NKG2DL

Natural killer group 2 member D ligand

IRT

Immune response therapy

IT

Immunotherapy

DR18

Murine-derived anti-detection-inducing IL-18 mutant protein

NK

Natural killer

Author contributions

Conceptualization, JS; writing-original draft preparation, TM; writing-final manuscript, TM and JS; visualization, ZZ; supervision, LQ. All the authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by fundings for Clinical Trials from the Affiliated Drum Tower Hospital, Medical School of Nanjing University (2024-LCYJ-PY-59); Medical New Technology Development Project of Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School (XJSFZLX202321).

Data availability

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing Interests

The authors declare that they have no competing interests.