+1 Recommend
1 collections

      We are delighted to announce our new 2022 CiteScore (issued by Scopus) is 4.6, SNIP 0.868, ranking 22/79 in Category "Biochemistry, Genetics and Molecular Biology (miscellaneous)". Huge thanks to our authors, reviewers and editors for helping to achieve this new milestone for the journal https://www.scopus.com/sourceid/21101107165

      Interested in becoming a BIO Integration published author?

      • Platinum Open Access with no APCs.
      • Fast peer review/Fast publication online after article acceptance.

      Check out the call for papers on our website: https://bio-integration.org/call-for-papers-bio-integration-2/

      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Utilizing Bacteria-Derived Components for Cancer Immunotherapy



            Bacteria-related cancer immunotherapy, because of its mechanisms and useful applications in the induction of anti-tumor immunity, has gained substantial attention in recent decades. Bacteria can enable targeting of tumors, and specifically can colonize the core tumor area. Because they contain many pathogen-associated molecular patterns—which efficiently stimulate immune cells, even within microenvironments that suppress anti-tumor immunity—bacteria boost immunological recognition leading to the destruction of malignant cells. This Editorial highlights various bacteria with immunotherapeutic effects and their by-products used as immunotherapeutics.

            Main article text


            The complexity of bacteria, as living organisms, influences the challenges and risks associated with transforming them into anti-tumor weapons. This complexity enables scientists to fine-tune the diverse capabilities of various bacterial strains to elicit anti-tumor effects that are impossible to achieve with other medications [1]. The advantages of using bacteria for tumor targeting lies in their ability to function as vehicles transporting therapeutic agents to tumor tissue as well as to interact with the immune system ( Figure 1 ), thus resulting in recognition and elimination of malignant cells [2]. Intravenous, subcutaneous, and intra-tumoral injections are used to introduce the bacteria to the host’s body [3]. Subsequently, the bacteria spread across the noncancerous body parts of the host in addition to solid tumors [4]. The number of bacterial cells in the body that spread through the vasculature and normal tissue markedly decreases within hours or days, because of the oxygen-rich physiology of the human body and immunological elimination. The bacteria are eventually eliminated, thus preventing any possible toxic effects to the host [5]. After the bacteria arrive at a solid tumor, they move to the hypoxic necrotic core areas of tumors through various processes, including chemotaxis [6]. The hypoxic tumor microenvironment (TME), in addition to the nutrients released by expired tumor cells, facilitates the growth of anaerobic bacteria. The immune system cannot rapidly eliminate the bacteria in the tumor, owing to the immunosuppressive TME. When the bacteria multiply and arrive at the tumor cells, the immune system is stimulated through contact of many immune cells with the tumor [7]. The major categories of bacteria and their functions in cancer immunotherapy are summarized in Table 1 . Several bacterial drug-delivery methods for cancer treatment are in clinical trials, despite difficulties and restrictions regarding their production, adverse effects, stability, and mutations. Vion Pharmaceutics evaluated Streptococcus typhimurium VNP20009 in 24 patients with melanoma in phase I. However, no objective tumor shrinkage was observed, although proinflammatory cytokines were elevated [810]. Clostridium novyi-NT spores have been applied in phase I clinical trials and demonstrated good outcomes after intratumoral injection. However, the Clostridium cells cannot completely destroy the tumor cells, thereby resulting in recurrence [11]. Similarly, the safety of bacterial minicells designed to deliver paclitaxel to cancer cells has been assessed in a human phase I clinical trial on patients with advanced solid tumors. No deaths were reported during the experiment, thus indicating that the bacterial minicells are safe and have modest clinical efficacy [12]. These clinical results have demonstrated several difficulties in clinical application. However, new combinatory strategies with bacterial medication delivery are expected to improve intratumoral bacterial colonization and therapeutic output.

            Figure 1

            Illustration of the essential characteristics of bacteria and bacterial elements, including those in native and engineered bacteria. The blue box includes descriptions of each bacterial component and the interactions between the immune system and bacterial components.

            Table 1

            Major Categories of Bacteria

            Listeria Listeria initiate the death of tumor cells through immune-cell activation and stimulate CD8+ T cells to eliminate remaining tumor cells, while additionally preventing metastasis. Listeria contaminate bone-marrow-derived suppressor cells (MDSCs) at tumor sites, thus decreasing MDSCs and subsequently facilitating a shift from immunosuppressive to immunostimulatory status. Moreover, IL-12 can be obtained after the residual infected MDSCs adopt an immunostimulatory phenotype, thus enhancing T cell and NK cell activity.[1315]
            Clostridium Bacillus Clostridium activates apoptosis within tumors by facilitating the activity of tumor necrosis factor–associated apoptosis-inducing ligand (TRAIL) from polymorphonuclear neutrophils. Furthermore, the early spread of Clostridium in solid tumors facilitates granulocyte and macrophage filtration of tumor cells, thus increasing adaptive immunity and the engagement of immune cells (e.g., CD8+ T cells) at tumor sites, owing to increased chemokine secretion.[7, 1618]
            Salmonella Pathogen-associated molecular patterns such as LPS, Salmonella, and flagellin are identified by antigen-presenting cells (APCs). Flagellin facilitates the development of APCs, and upregulates pro-inflammatory cytokines (such as IL-12) and co-stimulatory molecules such as CD40, through binding and via the activation of Nod-like receptors (NLRs) and TLR5 on APCs. Because of their inflammatory activity, they further stimulate the production of interferon-gamma (IFN-γ) and the T helper type 1 (Th1) cell–mediated immune response. Macrophages and DCs secrete IL-1β and TNF-α, which are pro-inflammatory, during this process, as LPS-induced TLR4 signaling and tumor cell debris are stimulated. Moreover, the secretion of IFN-γ, after activation of NK cells via a TLR-independent pathway that includes myeloid differentiation factor 88 (MyD88) and IL-18, is facilitated by flagellin. The secreted IFN-γ decreases the frequency of CD4+ CD25+ regulatory T cells within the TME.[1924]
            Lactobacillus Lactobacillus prevents colonization by infectious agents, stimulates the immune system, and has been demonstrated to elicit direct cytotoxic outcomes in cancer cells. Some strains of Lactobacillus have demonstrated antimutagenic properties [49]. Lactobacilli modify the Th1/Th2 balance, according to several studies. Th1-type responses are beneficial in cancer immunotherapy because they activate cytotoxic T lymphocytes. Th1 cells directly kill tumor cells by secreting cytokines that trigger death receptors. Th1 development is dependent on IL-4 in the absence of IL-12 [10].[2532]

            Bacterial components as immunotherapeutics

            Bacterial outer-membrane vesicles

            Gram-negative bacteria generate nano-sized spherical vesicles called outer-membrane vesicles (OMVs). OMVs are composed primarily of cellular elements of the bacterial periplasm and the outer membrane, such as proteins, membrane lipids, peptidoglycans (PGs), lipopolysaccharide (LPS), and various virulence factors [33, 34]. OMVs contain several intracellular components, including RNA, DNA, intracellular proteins, metabolites, and ions. [3538]. The mechanisms underlying the production of OMVs remain unclear. However, three widely acknowledged hypotheses may explain how OMVs are produced. First, OMV production may be due to the accumulation of phospholipids within the external membranes of the bacteria, in addition to regulation by the VacJ/Yrb ATP-binding cassette transport system present in most Gram-negative bacteria [39]. Second, the cross-linking between the bacterial external membrane and the PG layer–lipoprotein crosslinks in the cell walls of Gram-negative bacteria—which comprise PG and the outer membrane, with covalent bonds to preserve the envelope structure—may be involved. When the PG layer decomposes, a portion of the outer layer dissociates from the PG layer and extends outside the cell, thus resulting in formation of OMVs [40]. Finally OMVs are formed because of periplasmic accumulation of misfolded proteins and abnormal envelope components, which decrease the strength of the envelope and consequently divide the PF layer and the outer-membrane layer [41].

            OMVs with many microbe-associated molecular patterns (such as LPS, PG, RNA, or DNA) facilitate interaction with host pattern-recognition receptors, thus stimulating the innate-immunity response ( Figure 2 ). Because of the abundance of natural adjuvant elements in OMVs, the administration of OMVs (packaging small interfering RNAs) obtained from a mutated E. coli strain has been found to upregulate the production of the cytokines TNF-α, IL-6, and IFN-γ, as well as the anti-tumor cytokine CXCL10, all of which promote anti-tumor immunity [42, 43]. The most important microbe-associated molecular pattern may be LPS, which comprises a core polysaccharide and lipid A, as well as O-antigen, a polysaccharide on the bacterial outer-membrane surface, and is the manifestation of bacterial cell antigens [44]. Lipid A is strongly inflammatory and regulates the immune response by prompting immune-cell production of antibodies against various antigens; it is central to the biological activity of LPS. Nonetheless, studies have shown that an excess of LPS leads to immunosuppressive reactions: blocking lipid A inhibits the activity of endotoxin, thereby decreasing immunosuppression [45, 46]. OMVs decrease the toxicity of LPS as immune adjuvants, and block lipid A function via inactivating of msbB gene; thus resulting in attenuation of immunosuppression, thus resulting in attenuation. Kim et al. have eliminated the msbB gene that encodes E. coli endotoxin, thus blocking lipid A–meditated immunosuppression [43]. Moreover, their study has indicated that use of G-bacteria inhibits tumor cell growth in a murine colon cancer model. NK cells and T cells, after stimulation by OMVs, secrete INF-γ, which inhibits tumor growth. Moreover, naturally produced OMVs have been used as carriers for drug delivery. For instance, OMVs have been loaded with immunomodulatory molecules, photosensitizers, and chemotherapy drugs, and used to transport these cargo to targeted tumor cells, thus achieving a combination of immunotherapy and phototherapy. In one study, Chen et al. have coated polymer micelles packed with drugs with DSPE-PEG-RGD-hybridized bacterial OMVs, and tested this novel nanomedicine’s efficacy in immunotherapy for cancer and the prevention of metastasis [47]. This study demonstrated that the OMV nanomedicine directly interferes with immune cells, thereby inducing cytotoxicity via activation of the inflammatory response, which in turn activates the host immune response. Moreover, OMV-coated micelles loaded with tegafur have been found to regulate chemotherapy and the immune system, thus preparing melanoma-specific cytotoxic T lymphocytes and additionally suppressing pulmonary metastasis. Thus, to augment OMV functionality and achieve better tumor suppression, two designs have been proposed. The first involves hybridization of lipid polymers (or other biological membranes) with OMVs to attain new functions or for improve efficiency. The second uses the high loading capacity of OMVs to boost the anti-tumor immune response to other treatments and the OMVs themselves by delivering therapeutic agents (such as chemotherapy drugs, immune adjuvants, or photosensitizers) to tumor sites.

            Figure 2

            An overview of bacteria-mediated immunotherapy, showing how bacteria target tumors, including how naive live bacteria trigger the immune system, how modified bacteria are used in immunotherapy, and the different bacterial components used in immunotherapy.

            Bacterial toxins

            Bacterial toxins are exceptionally toxic proteins that are generated and released by bacteria that possess specific functionality such as cell cycle arrest and apoptotic cell death etc. These toxins have been demonstrated to be a potent tool for the treatment of cancer, owing to their considerable toxicity [48]. Anti-tumor bacterial toxins are divided into two categories: those conjugated to the tumor cell antigen surface and those conjugated to ligands. Bacterial toxins that target specific antigens (which are highly expressed on the tumor surface) such as Clostridium perfringens enterotoxin, diphtheria toxin (DT), and Pseudomonas exotoxin, are used for the targeting and elimination of tumor cells [4951]. DT is used primarily for the treatment of tumors both in murine models and humans, because it has relatively few anti-tumor effects [52], possibly because of its high cytotoxicity or its simultaneous induction of anti-tumor immunity. Buzzi et al. have engineered cross-reacting non-toxic material 197 (CRM197) for the treatment of a specific group of cancer patients [53]. As a non-toxic variant of DT, CRM197 has immunological functions similar to those of DT. Like DT, CRM197 targets heparin-binding epidermal growth factor, which is commonly overexpressed in tumor cells. Moreover, subcutaneous injection of CRM197 results in inflammatory immunological reactions, thereby activating a biological anti-tumor response. On the basis of these results, the authors have suggested that TNF-α and neutrophils may be associated with the anti-tumor process. Thus, bacterial toxins not only affect tumor cells but also initiate anti-tumor immunity. Fusion proteins comprising targeting antibody fragments and bacterial toxins are also known as immunotoxins [54]. The targeting antibody fragments act on cancer cells and increase the potency of bacterial toxin fragments in eliminating targeted cells. Bacterial immunotoxins have powerful cytotoxicity through inhibition of protein translation and have been demonstrated to be highly effective in the treatment of several hematological diseases [55, 56]. In an earlier study, Ontak, a fusion protein consisting of anti-IL-2 and DT, has achieved satisfactory outcomes in the treatment of chronic lymphocytic leukemia, because chronic lymphocytic leukemia cells overexpress high-affinity IL-2 receptors [57]. For immunotoxin treatment, repeated administration of the drug is required, as in chemotherapy, to maintain the lethal concentrations necessary for optimal results. However, repeated treatment is restricted by immunogenetics, i.e., the development of anti-drug antibodies. After treatment with immunotoxins, several patients have shown a rapid immune response and the generation of anti-drug antibodies, which neutralize immunotoxins and consequently prohibit multiple administrations. To solve this problem, researchers have attempted to combine immunotoxins with chemotherapy drugs, and to modify bacterial toxins to avoid their identification by the immune system. These approaches have achieved the necessary decrease in immunogenicity. Another direct and widely accepted method is the deletion or mutation of T cell epitopes through the design of recombinant proteins to decrease immunogenicity. Mazor et al. have discovered a novel immunotoxin containing a disulfide-stabilized Fv of the anti-Tac antibody and PE38 bearing nine-point mutations in domains II and III. Furthermore, they have demonstrated that domain II is necessary for CD25-mediated cell destruction—a process distinct from CD22-mediated internalization. The recently engineered immunotoxin LMB-142 has demonstrated greater cytotoxic action in humans in vitro and a five-fold lower non-specific toxicity in murine models than LMB-2 (anti-Tac(Fv)-PE38).

            Beyond the use of immunotoxins in the treatment of T cell malignancies and other solid tumors, Tregs depleting fusion-protein toxins have great promise in cancer immunotherapy. Tregs consist of T cells, which are considered the “brakes” of the immune response mediated by effector T cells. Moreover, they have major roles in immune tolerance, the prevention of autoimmune disease, and the inhibition of anti-tumor immunity [58, 59]. Tregs, the drivers of the immunosuppressive microenvironment, through processes such as interleukin consumption and immune suppression, promote tumor growth; consequently, their use in many immunotherapies has increased [6062]. One technique for exhausting Tregs involves moving bacterial toxins to make use of their inherent cytotoxicity to directly eliminate Tregs. This method restores the normal binding of bacterial toxins to the ligands present on the receptors of Tregs. Consequently, cells rich in Treg receptors eliminate the toxins themselves. This process is beneficial, because it alleviates the TME’s immunosuppressive nature and minimizes the toxicity of bacterial toxins toward non-targeted cells. Moreover, the high expression of Foxp3 in Tregs increases CD25 expression on the surfaces of Tregs, thus leading to the formation of heterotrimeric high-affinity IL-2 receptors [63, 64]. The abundance of CD25 on Tregs results in exhaustion of IL-2 within the local microenvironment. However, a decrease in the number of cytokines results in apoptosis of activated effector T cells [65]. Thus, CD25 is an example of a targeted site. Cheung et al. have engineered a next-generation IL-2 receptor-targeted diphtheria fusion toxin with excellent anti-tumor effects in decreasing Tregs [66]. Moreover, this fusion toxin has a beneficial synergistic effect with anti-PD-1 in the treatment of melanoma. However, the clinical applications of denileukin diftitox or Ontak (fusion protein consisting of the bacterial toxin DT and anti-IL-2) are limited by a danger of vascular leakage and production issues related to aggregation and purity. One production approach has used Corynebacterium diphtheriae to directly reproduce the fully folded and biologically active s-DAB-IL-2 as a monomer within the culture medium. Moreover, the highly developed fusion protein s-DAB-IL-2(V6A) has been generated through the mutation of a single amino acid (V6A). In comparison to s-DAB-IL-2, V6A has 50 times less vascular leakage in vitro with ~3.7 times lower lethality in mice. In a murine model of melanoma, s-DAB-IL-2(V6A) monotherapy as well as combination therapy using anti-PD-1 have been found to increase inhibition of tumor cell growth The desirable therapeutic effects are associated with a decline in Tregs and the proliferation of effector T cells. Although bacterial toxins are generally considered to have high toxicity toward tumor cells, through fusion with Treg-targeted proteins, they also effectively exhaust Tregs and facilitate an anti-tumor response. More importantly, the association of bacterial toxins with immune-checkpoint blockade enables their application in cancer immunotherapy.

            Bacterial spores

            Spores are inactive forms of bacteria and are thus extremely resistant. They can live in oxygen-rich cells for extended durations without germinating. After encountering a suitable environment, such as the hypoxic/necrotic area within the tumor core, spores undergo germination and multiplication. Because no critically hypoxic microenvironments are present in normal human tissues, spores do not exhibit toxicity in human organs under physiological conditions. Many researchers have administered a Clostridium histolyticum spore suspension into tumor cells and observed effective inhibition of transplanted rat sarcomas without apparent systemic toxicity [52] In some reports, the mice died from tetanus within 48 hours after the intravascular injection of Clostridium spores in tumor-infected murine models, and this effect was not limited to intratumoral injections [67]. The healthy mice receiving the same treatment remained asymptomatic for as long as 40 days, thus establishing that spores demonstrate tumor-specific germination even after vascular administration. Clostridium novyi (C. novyi) has been widely studied because of its high sensitivity to oxygen and high mobility because of its peritrichous flagella [6]. These two factors contribute to the tumor enrichment of C. novyi even when only a small amount of spore germination occurs. The main systemic toxin (α-toxin) gene of C. novyi has been isolated and used to generate a novel attenuated C. novyi-NT, which has better application prospects because of its lower systemic toxicity [68]. Agrawal et al. have noted that systemic administration of C. novyi-NT spores in fully immune mice with tumor cells results in long-term tumor regression [7]. C. novyi-NT spores have been found to spread throughout the body after systemic injection. However, the anaerobic properties of the environment cause them to germinate in only the necrotic core of the tumor, which is relatively hypoxic. The germinated bacteria then eliminate the surrounding tumor cells through local production of lipases, proteases, and other degrading enzymes. Meanwhile, the host responds to this local infection by secreting immunostimulatory cytokines, including MIP-2, IL-6, tissue inhibitor of metalloproteinases 1 (TIMP-1), and granulocyte colony-stimulating factor (G-CSF), thus facilitating the infiltration of tumors by various immune cells. Initially, only a neutrophil response is observed, but monocytes subsequently participate. The spread of bacterial infection is inhibited by the inflammatory response, thus also providing a second layer of control beyond the initial layer supplied by the anaerobic environment. Moreover, the elimination of tumor cells is facilitated by inflammation via generation of reactive oxygen species, and proteases and other enzymes. In addition, inflammation evokes an efficient cellular immune reaction, which continues to eliminate tumor cells remaining to be destroyed by bacteria. Malignant cells have promisingly been found to be eradicated in 30% of mice with tumors. In a later study, the administration of C. novyi-NT spores into naturally occurring tumor cells in dogs was found to provoke a powerful immune response [69]. Intratumoral inoculation with C. novyi-NT spores enhances phagocytosis as well as the functions of NK-like cells. Moreover, intravenous injection of C. novyi-NT spores results in TNF-α production activated by LPS and IL-10 production triggered by LTA, and enhances NK-like-cell function. These findings have demonstrated that the administration of C. novyi-NT spores produces long-term alterations in the functions of immune cells. In a different study, Heaps et al. have injected engineered clostridial spores into the blood circulation and successfully suppressed and healed human colon carcinoma in a murine xenograft model [70]. The engineered spores germinated and became activated after reaching the hypoxic necrotic areas of the tumor core, after which they released a prodrug-converting enzyme, which converts non-toxic prodrug molecules to a potent cytotoxic forms at the tumor site, thus resulting in the death of tumor cells.


            Extensive research conducted on the interactions between tumor cells and the human immune system has indicated that immunotherapy may be one of the most promising approaches to cancer treatment. Because both bacteria and their constituents naturally stimulate the host’s immune system, they generate a strong anti-tumor immune response. Although the definitive interactions among tumors, bacteria, and the immune system remain unclear, further research will shed light on how bacteria might be adapted to regulate this interaction to achieve better outcomes. With developments in synthetic biology, many solutions are for these issues are being discovered, and this area of research is expected to be highly worthwhile. Clinicians might be able to control the engineering and release of immunotherapeutic agents by deciding on the number of gene copies, enhancing bacterial strength and metabolic rate, and adjusting the initial bacteria injection dosage. For patients with various tumors in distinct stages, a personalized immunotherapy plan could be achieved by adjusting the intensity of the therapeutic agents to achieve optimal treatment effects.


            1. , , , , , et al. Bacteria-based cancer immunotherapy. Adv Sci 2021;8(7). [PMID: 33854892 DOI: 10.1002/advs.202003572]

            2. , , , , et al. Tumour-targeting bacteria engineered to fight cancer. Nat Rev Cancer 2018;18(12):727–43. [PMID: 30405213 DOI: 10.1038/s41568-018-0070-z]

            3. . Engineering bacteria toward tumor targeting for cancer treatment: current state and perspectives. Appl Microbiol Biotechnol 2012;93(2):517–23. [PMID: 22120621 DOI: 10.1007/s00253-011-3695-3]

            4. , , , , , et al. Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium. J Infect Dis 2000;181(6):1996–2002. [PMID: 10837181 DOI: 10.1086/315497]

            5. , , , , , et al. Visualization and in situ ablation of intracellular bacterial pathogens through metabolic labeling. Angew Chem Int Ed Engl 2020;59(24):9288–92. [PMID: 31449353 DOI: 10.1002/anie.201910187]

            6. , , , , , et al. Overcoming the hypoxic barrier to radiation therapy with anaerobic bacteria. Proc Natl Acad Sci U S A 2003;100(25):15083–8. [PMID: 14657371 DOI: 10.1073/pnas.2036598100]

            7. , , , , , et al. Bacteriolytic therapy can generate a potent immune response against experimental tumors. Proc Natl Acad Sci U S A 2004;101(42):15172–7. [PMID: 15471990 DOI: 10.1073/pnas.0406242101]

            8. , , , , , et al. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 2002;20(1):142–52. [PMID: 11773163 DOI: 10.1200/JCO.2002.20.1.142]

            9. , . Continuous intravenous administration of live genetically modified Salmonella typhimurium in patients with metastatic melanoma. J Immunother 2003;26(2):179–80. [PMID: 12616110 DOI: 10.1097/00002371-200303000-00011]

            10. , . A phase I trial of genetically modified Salmonella typhimurium expressing cytosine deaminase (TAPET-CD, VNP20029) administered by intratumoral injection in combination with 5-fluorocytosine for patients with advanced or metastatic cancer. Protocol no: CL-017. Version: April 9 2001. Hum Gene Ther 2001;12(12):1594–6. [PMID: 11529249]

            11. , , , , , et al. Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses. Sci Transl Med 2014;6(249). [PMID: 25122639 DOI: 10.1126/scitranslmed.3008982]

            12. , , , , , et al. A first-time-in-human phase I clinical trial of bispecific antibody-targeted, paclitaxel-packaged bacterial minicells. PLoS One 2015;10(12). [PMID: 26659127 DOI: 10.1371/journal.pone.0144559]

            13. , , , . High efficacy of a listeria-based vaccine against metastatic breast cancer reveals a dual mode of action. Cancer Res 2009;69(14):5860–66. [PMID: 19584282 DOI: 10.1158/0008-5472.CAN-08-4855]

            14. , , , , . Potentiating antibacterial activity by predictably enhancing endogenous microbial ROS production. Nat Biotechnol 2013;31(2):160–5. [DOI: 10.1038/nbt.2458]

            15. , , , , . Myeloid-derived suppressor cells have a central role in attenuated Listeria monocytogenes-based immunotherapy against metastatic breast cancer in young and old mice. Br J Cancer 2013;108(11):2281–90. [PMID: 23640395 DOI: 10.1038/bjc.2013.206]

            16. , , , , , et al. The genome and transcriptomes of the anti-tumor agent Clostridium novyi-NT. Nat Biotechnol 2006;24(12):1573–80. [PMID: 17115055 DOI: 10.1038/nbt1256]

            17. , , , , , et al. A bacterial protein enhances the release and efficacy of liposomal cancer drugs. Science 2006;314(5803):1308–11. [PMID: 17124324 DOI: 10.1126/science.1130651]

            18. , , , , , et al. Clostridium butyricum MIYAIRI 588 shows antitumor effects by enhancing the release of TRAIL from neutrophils through MMP-8. Int J Oncol 2013;42(3):903–11. [PMID: 23354042 DOI: 10.3892/ijo.2013.1790]

            19. , , . Engineering of bacterial strains and their products for cancer therapy. Appl Microbiol Biotechnol 2013;97(12):5189–99. [PMID: 23644748 DOI: 10.1007/s00253-013-4926-6]

            20. , , , , , et al. Activation of inflammasome by attenuated Salmonella typhimurium in bacteria-mediated cancer therapy. Microbiol Immunol 2015;59(11):664–75. [PMID: 26500022 DOI: 10.1111/1348-0421.12333]

            21. , , , , , et al. Engineered Salmonella enterica serovar Typhimurium overcomes limitations of anti-bacterial immunity in bacteria-mediated tumor therapy. Oncoimmunology 2018;7(2). [PMID: 29308303 DOI: 10.1080/2162402X.2017.1382791]

            22. , , , , , et al. Therapeutic bacteria to combat cancer; current advances, challenges, and opportunities. Cancer Med 2019;8(6):3167–81. [PMID: 30950210 DOI: 10.1002/cam4.2148]

            23. , , , , , et al. Salmonella typhimurium Suppresses Tumor Growth via the Pro-Inflammatory Cytokine Interleukin-1 beta. Theranostics 2015;5(12):1328–42. [PMID: 26516371 DOI: 10.7150/thno.11432]

            24. , , , , , et al. Antitumor activity of the TLR-5 ligand flagellin in mouse models of cancer. J Immunol 2006;176(11):6624–30. [PMID: 16709820 DOI: 10.4049/jimmunol.176.11.6624]

            25. , , , , , et al. Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition. PLoS One 2018;13(2). [PMID: 29394276 DOI: 10.1371/journal.pone.0192181]

            26. , , , , , et al. Inhibition of HIV and HSV infection by vaginal lactobacilli in vitro and in vivo. Daru 2012;20:53. [PMID: 23351891 DOI: 10.1186/2008-2231-20-53]

            27. , , , , , et al. Mucosal immunity: its role in defense and allergy. Int Arch Allergy Immunol 2002;128(2):77–89. [PMID: 12065907 DOI: 10.1159/000059397]

            28. , , , , , et al. Lactobacillus acidophilus and Lactobacillus crispatus culture supernatants downregulate expression of cancer-testis genes in the MDA-MB-231 cell line. Asian Pac J Cancer Prev 2014;15(10):4255–59. [PMID: 24935380 DOI: 10.7314/apjcp.2014.15.10.4255]

            29. , , , , , et al. Normal and tumour cervical cells respond differently to vaginal lactobacilli, independent of pH and lactate. J Med Microbiol 2013;62:1065–72. [PMID: 23618799 DOI: 10.1099/jmm.0.057521-0]

            30. , , , , , et al. Differences in vaginal lactobacilli composition of Iranian healthy and bacterial vaginosis infected women: a comparative analysis of their cytotoxic effects with commercial vaginal probiotics. Iran Red Crescent Med J 2013;15(3):199–206. [PMID: 23983998 DOI: 10.5812/ircmj.3533]

            31. , , , , , et al. Antimutagenic and anticancer effects of lactic acid bacteria isolated from Tarhana through Ames test and phylogenetic analysis By 16S rDNA. Nutr Cancer 2014;66(8):1406–13. [PMID: 25330454 DOI: 10.1080/01635581.2014.956254]

            32. , . Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer Immunol Immunother 2005;54(8):721–8. [PMID: 16010587 DOI: 10.1007/s00262-004-0653-2]

            33. , . Immune modulation by bacterial outer membrane vesicles. Nat Rev Immunol 2015;15(6):375–87. [PMID: 25976515 DOI: 10.1038/nri3837]

            34. , . Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol 2015;13(10):605–19. [PMID: 26373371 DOI: 10.1038/nrmicro3525]

            35. . Outer Membrane Vesicles (OMVs) of Gram-negative Bacteria: a perspective update. Front Microbiol 2017;8. [PMID: 28649237 DOI: 10.3389/fmicb.2017.01053]

            36. , . Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol. 2010;64:163–84. [PMID: 20825345 DOI: 10.1146/annurev.micro.091208.073413]

            37. , . Biogenesis and multifaceted roles of outer membrane vesicles from Gram-negative bacteria. Microbiology-Sgm 2014;160:2109–21. [PMID: 25069453 DOI: 10.1099/mic.0.079400-0]

            38. , , , , et al. Proteomics in gram-negative bacterial outer membrane vesicles. Mass Spectrom Rev 2008;27(6):535–55. [PMID: 18421767 DOI: 10.1002/mas.20175]

            39. , , , , , et al. A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria. Nat Commun 2016;7. [DOI: 10.1038/ncomms10515]

            40. , , . NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli. Microbiologyopen 2015;4(3):375–89. [PMID: 25755088 DOI: 10.1002/mbo3.244]

            41. , , . Modulation of bacterial outer membrane vesicle production by envelope structure and content. BMC Microbiol 2014;14. [DOI: 10.1186/s12866-014-0324-1]

            42. , , , , , et al. Bioengineered bacterial outer membrane vesicles as cell-specific drug-delivery vehicles for cancer therapy. ACS Nano 2014;8(2):1525–37. [PMID: 24410085 DOI: 10.1021/nn405724x]

            43. , , , , , et al. Bacterial outer membrane vesicles suppress tumor by interferon-gamma-mediated antitumor response. Nat Commun 2017;8:626. [PMID: 28931823 DOI: 10.1038/s41467-017-00729-8]

            44. , , , , , et al. The Escherichia coli Serogroup O1 and O2 Lipopolysaccharides Are Encoded by Multiple O-antigen Gene Clusters. Front Cell Infect Microbiol 2017;7:30. [PMID: 28224115 DOI: 10.3389/fcimb.2017.00030]

            45. , , . Nanotechnology intervention of the microbiome for cancer therapy. Nat Nanotechnol 2019;14(12):1093–103. [PMID: 31802032 DOI: 10.1038/s41565-019-0589-5]

            46. , , , , , et al. Trapping of lipopolysaccharide to promote immunotherapy against colorectal cancer and attenuate liver metastasis. Adv Mater 2018;30(52). [PMID: 30387230 DOI: 10.1002/adma.201805007]

            47. , , , , , et al. Bioengineering bacterial vesicle-coated polymeric nanomedicine for enhanced cancer immunotherapy and metastasis prevention. Nano Lett 2020;20(1):11–21. [PMID: 31858807 DOI: 10.1021/acs.nanolett.9b02182]

            48. . Bacterial toxins and cancer — a case to answer? Nat Rev Microbiol 2005;3(4):343–9. [PMID: 15806096 DOI: 10.1038/nrmicro1130]

            49. , , . Targeted diphtheria toxin-based therapy: a review article. Front Microbiol 2019;10:2340. [PMID: 31681205 DOI: 10.3389/fmicb.2019.02340]

            50. , , , , , et al. Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy. BMC Cancer 2017;17:129. [PMID: 28193196 DOI: 10.1186/s12885-017-3123-x]

            51. , , , , , et al. Characterization of a re-engineered, mesothelin-targeted Pseudomonas exotoxin fusion protein for lung cancer therapy. Mol Oncol 2016;10(8):1317–29. [PMID: 27507537 DOI: 10.1016/j.molonc.2016.07.003]

            52. , . The role of bacterial toxins and spores in cancer therapy. Life Sci 2019;235:116839. [PMID: 31499068 DOI: 10.1016/j.lfs.2019.116839]

            53. , , , , , et al. CRM197 (nontoxic diphtheria toxin): effects on advanced cancer patients. Cancer Immunol Immunother 2004;53(11):1041–48. [PMID: 15168087 DOI: 10.1007/s00262-004-0546-4]

            54. , , . Immunotoxins. Cell 1986;47(5):641–8. [PMID: 3536124 DOI: 10.1016/0092-8674(86)90506-4]

            55. , , . Adverse effects of denileukin diftitox and their management in patients with cutaneous T-cell lymphoma. Clin J Oncol Nurs 2012;16(5):E164–72. [PMID: 23022942 DOI: 10.1188/12.CJON.E164-E172]

            56. , . Immunoconjugates in the management of hairy cell leukemia. Best Pract Res Clin Haematol 2015;28(4):236–45. [PMID: 26614902 DOI: 10.1016/j.beha.2015.09.003]

            57. , , , , et al. DAB(389)IL2 (ONTAK (R)) fusion protein therapy of chronic lymphocytic leukaemia. Expert Opin Biol Ther 2003;3(1):179–86. [PMID: 12718740 DOI: 10.1517/14712598.3.1.179]

            58. , , . Metabolic regulation of tregs in cancer: opportunities for immunotherapy. Trends Cancer 2017;3(8):583–92. [PMID: 28780935 DOI: 10.1016/j.trecan.2017.06.005]

            59. , , , , , et al. TCF1 and LEF1 control treg competitive survival and Tfr development to prevent autoimmune diseases. Cell Rep 2019;27(12):3629–3645. [PMID: 31216480 DOI: 10.1016/j.celrep.2019.05.061]

            60. , . Regulatory T (Treg) cells in cancer: Can Treg cells be a new therapeutic target? Cancer Sci 2019;110(7):2080–9. [PMID: 31102428 DOI: 10.1111/cas.14069]

            61. , , , , , et al. Depletion of CD4(+)CD25(+) human regulatory T cells in vivo: kinetics of Treg depletion and alterations in immune functions in vivo and in vitro. Int J Cancer 2007;120(12):2723–33. [PMID: 17315189 DOI: 10.1002/ijc.22617]

            62. , , , , , et al. Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells. J Clin Invest 2005;115(12):3623–33. [PMID: 16308572 DOI: 10.1172/JCI25947]

            63. , , , , et al. The Role of FOXP3 in Regulating Immune Responses. Int Rev Immunol 2014;33(2):110–28. [PMID: 23947341 DOI: 10.3109/08830185.2013.811657]

            64. , . Regulatory T cells in cancer immunotherapy. Cell Res 2017;27(1):109–18. [PMID: 27995907 DOI: 10.1038/cr.2016.151]

            65. , , , , , et al. Recent advances with Treg depleting fusion protein toxins for cancer immunotherapy. Immunotherapy 2019;11(13):1117–28. [PMID: 31361167 DOI: 10.2217/imt-2019-0060]

            66. , , , , , et al. Second-generation IL-2 receptor-targeted diphtheria fusion toxin exhibits antitumor activity and synergy with anti-PD-1 in melanoma. Proc Natl Acad Sci U S A 2019;116(8):3100–5. [PMID: 30718426 DOI: 10.1073/pnas.1815087116]

            67. , . Localization of the vegetative form of Clostridium tetani in mouse tumors following intravenous spore administration. Cancer Res 1955;15(7):473–8. [PMID: 13240693]

            68. , , , , , et al. Combination bacteriolytic therapy for the treatment of experimental tumors. Proc Natl Acad Sci U S A 2001;98(26):15155–60. [PMID: 11724950 DOI: 10.1073/pnas.251543698]

            69. , , , , , et al. Immune response to C. novyi-NT immunotherapy. Vet Res 2018;49. [PMID: 29690928 DOI: 10.1186/s13567-018-0531-0]

            70. , , , , , et al. Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo. Oncotarget 2014;5(7):1761–9. [PMID: 24732092 DOI: 10.18632/oncotarget.1761]

            Author and article information

            BIO Integration
            Compuscript (Ireland )
            December 2022
            04 August 2022
            : 3
            : 4
            : 180-187
            [1] 1College of Biology and the Environment, Co-Innovation Centre for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210-037, China
            [2] 2Cell Factory Research Centre, Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
            Author notes
            *Correspondence to: Long Jin, Tel: +86-25-8542-7210, Fax: +86-25-8542-7210, E-mail: isacckim@ 123456alumni.kaist.ac.kr

            aThese authors contributed equally to this work.

            Copyright © 2022 The Authors

            This is an open access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/). See https://bio-integration.org/copyright-and-permissions/

            : 27 June 2022
            : 04 July 2022
            : 14 July 2022
            Mini Review

            Medicine,Molecular medicine,Radiology & Imaging,Biotechnology,Pharmacology & Pharmaceutical medicine,Microscopy & Imaging
            Listeria ,immunotherapy, Lactobacillus , Salmonella ,Immunotherapeutics


            Comment on this article