
==== Front
Chin Med J (Engl)
Chin Med J (Engl)
CM9
Chinese Medical Journal
0366-6999
2542-5641
Lippincott Williams & Wilkins Hagerstown, MD

39118214
CMJ-2023-1139
10.1097/CM9.0000000000003234
00004
3
Review Article
Bacterial outer membrane vesicles in the fight against cancer
Meng Yiming 1
Kong Cuicui 1
Ma Yushu 1
Sun Jing 2
Zhang Guirong 1
Pan Xiangxiang
1 Department of Central Laboratory, Cancer Hospital of Dalian University of Technology, Liaoning Cancer Hospital & Institute, Shenyang, Liaoning 110042, China
2 Department of Biobank, Cancer Hospital of Dalian University of Technology, Liaoning Cancer Hospital & Institute, Shenyang, Liaoning 110042, China
Correspondence to: Dr. Yiming Meng, Department of Central Laboratory, Cancer Hospital of Dalian University of Technology, Liaoning Cancer Hospital & Institute, No. 44, Xiaoheyan Road, Dadong District, Shenyang, Liaoning 110042, China E-Mail: cacamym@163.com
09 8 2024
20 9 2024
137 18 21692181
08 12 2023
Copyright © 2024 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0

Abstract

Bacterial outer membrane vesicles (OMVs) are diminutive vesicles naturally released by Gram-negative bacteria. These vesicles possess distinctive characteristics that attract attention for their potential use in drug administration and immunotherapy in cancer treatment. Therapeutic medicines may be delivered via OMVs directly to the tumor sites, thereby minimizing exposure to healthy cells and lowering the risk of systemic toxicity. Furthermore, the activation of the immune system by OMVs has been demonstrated to facilitate the recognition and elimination of cancer cells, which makes them a desirable tool for immunotherapy. They can also be genetically modified to carry specific antigens, immunomodulatory compounds, and small interfering RNAs, enhancing the immune response to cancerous cells and silencing genes associated with disease progression. Combining OMVs with other cancer treatments like chemotherapy and radiation has shown promising synergistic effects. This review highlights the crucial role of bacterial OMVs in cancer, emphasizing their potential as vectors for novel cancer targeted therapies. As researchers delve deeper into the complexities of these vesicles and their interactions with tumors, there is a growing sense of optimism that this avenue of study will bring positive outcomes and renewed hope to cancer patients in the foreseeable future.

Keywords:

Bacterial outer membrane vesicles
Tumor microenvironment
Immunotherapy
Phototherapy
Nanoparticle
OPEN-ACCESSTRUE
==== Body
pmcIntroduction

The tumor microenvironment (TME) encompasses an intricate assemblage of cells and extracellular matrix constituents, exerting a pivotal influence on cancer biology. This entity includes malignant cells and a diverse array of non-malignant cells, such as immune cells, fibroblasts, bacteria, and endothelial cells.[1] Interactions occurring inside the TME are characterized by their intricate and diverse nature, as many cell types play distinct roles in facilitating tumor development and progression. Recognizing the importance of the TME in cancer research, there is a growing emphasis on developing approaches to target this intricate system. Recent studies have highlighted the role of the microbiome, a collection of microorganisms inhabiting the human body, in cancer development and progression.[2] Bacterial organisms have been found to promote tumor growth and metastasis, but they also hold potential for therapeutic applications, such as the utilization of bacterial outer membrane vesicles (OMVs).[3]

OMVs are fragments of the bacterial membrane released by Gram-negative bacteria spontaneously during their development. Typically ranging in size from 20 to 200 nanometers, the small size of OMVs allows them to swiftly distribute throughout the intercellular gap and even enter into the cell’s interior.[4] OMVs possess distinct biological functions while maintaining the primary components of the original bacteria, including lipids, proteins, and pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharides (LPS), lipoproteins, peptidoglycans, DNA, RNA, and more.[5] The lipid bilayer constitutes the OMVs, with phospholipids and LPS accounting for the majority of OMVs’ lipid composition [Figure 1]. The structure of OMVs confers stability and facilitates drug delivery. Simultaneously, it also offers the potential for cell interaction, enabling OMVs to effectively perform various functions in tumor treatment such as directly killing tumor cells, regulating the tumor microenvironment, and activating the immune system.[6]

Figure 1 Gram-negative bacterial outer membrane vesicle structure and content.

Nonetheless, new evidence suggests that OMVs may promote tumor growth and spread by facilitating the processes of inflammation and angiogenesis.[7] The study conducted by Lamprinaki et al[8] showed that strains of Fusobacterium nucleatum and their OMVs could promote the growth of colorectal cancer (CRC) by inducing the development of a pro-inflammatory milieu. An inflammatory milieu can facilitate tumor formation and their metastasis to distant locations. Moreover, it has been discovered that OMVs could suppress the functioning of immune cells, possibly enabling tumor cells to evade immune monitoring and propagate.[9] Thus, researchers are endeavoring to investigate and resolve thought-provoking concerns and speculations surrounding the connection between OMVs and the TME.[10] As scientific research progresses and our understanding of these vesicles deepens, we are edging closer to a future where cancer treatment becomes more efficient and targeted. This review aims to illuminate this promising frontier in cancer research, emphasizing the potential benefits that bacterial OMVs could offer in improving the well-being of cancer patients.

Technology for OMVs Isolation, Identification, and Characterization

The International Society for Extracellular Vesicles (ISEV) has issued guidelines for the separation and characterization of OMVs.[11] Differential ultracentrifugation is widely regarded as the predominant technique for extracellular vesicle (EV) separation, as stated by ISEV. Various methods, including density gradient, filtration, and immunoisolation, are often used to separate OMVs. Recent studies have shown that relying only on ultracentrifugation may not provide the purified OMVs. Therefore, the ISEV suggests that employing multiple separation techniques is more advantageous than relying solely on a single approach for producing OMV with low contaminant.[12]

Comprehensive analysis of the structures and content of OMVs is critical for utilizing OMVs. Multiple complementary strategies are often employed for identifing OMVs.[13] These methods involve the use of nanoparticle-tracking analysis (NTA), which enables to determine the size distribution and concentration of vesicles, and transmission electron microscopy that offers incredibly detailed, high-resolution images of the vesicles. And, biochemical assays, which can determine the presence of specific proteins or lipids that constitute the OMVs, can also be used to identify OMVs.[14] Besides, sophisticated techniques such as proteomics and lipidomics, can also identify the different proteins and lipids present in the vesicles.[7] In addition, advanced techniques like next-generation sequencing were used to analyze the genetic material inside the vesicles.[15] These methodologies enable us to obtain insight into OMVs, which makes available for using OMVs in medical and biotechnological areas, such as drug delivery systems (DDS) and vaccines. In addition, studying OMVs will greatly promote our understanding of bacterial physiology and the intricate relationship between bacteria and their environment.[16]

OMVs are known for their ability to transport a wide range of substances. Molecules can be delivered directly to specific target cells by encapsulating into OMVs. OMVs can enter a cell through various pathways, including clathrin-mediated endocytosis, lipid-raft-mediated uptake, caveolin-mediated endocytosis, macropinocytosis, and phagocytosis [Figure 2].[17] These mechanisms enable OMVs to evade the immune systemand protect encapsulated compounds from enzymatic degradation. One instance involves the use of OMVs to transport bacterial toxins or other virulence factors to host cells that play a role in the progression of bacterial infections. However, OMVs can also activate the immune system and trigger the production of antibodies targeting bacterial antigens, offering a valuable defense mechanism against pathogenic microorganisms.[6] In summary, the investigation of bacterial OMVs is promising in several disciplines.

Figure 2 Mechanisms of entry of OMV into host cells. OMV: Outer membrane vesicle.

OMVs in Tumor Immunotherapy

In recent years, with the emergence of immunotherapy in the field of tumor treatment, there has been increasing attention on the synergistic effect of bacterial OMVs and immunotherapy. As a natural nano-carrier, OMVs possess unique biological activity and immunomodulatory function, which can be integrated into immunotherapy strategies to achieve more significant anti-tumor effects. This is expected to provide safer and more effective treatment options for cancer patients.[6]

Immunomodulatory functions of OMVs in cancer treatment

OMVs can express various PAMPs allowing them to exhibit unique immunomodulatory functions in tumor treatment.[18] The PAMP component of OMVs interacts with host pattern recognition receptors (PRRs) and triggers proinflammatory signaling pathways that result in the synthesis of cytokines, chemokines, and antimicrobial peptides. These molecules have been extensively studied for their capacity to enhance the immune response against cancer.[19] OMVs induce robust anti-tumor effects in mice by activating interferon (IFN)-γ and T cell-mediated mechanisms.[20] The observed immunogenicity has sparked considerable interest in exploring the potential use of OMVs as immunization agents. A study has shown the strong anti-tumor effects of mass-produced Escherichia coli OMVs, making them a promising choice for immunotherapeutic agents.[21] These OMVs could activate cancer antigen-specific stem-like cluster of differentiation 8 positive (CD8+) T cells, facilitating a synergistic combination of immunotherapy with anti-programmed cell death protein 1 (PD-1) treatment. In a clinical investigation, peripheral blood mononuclear cells (PBMCs) derived from healthy individuals were subjected to co-cultivation with E. coli OMVs, and then co-cultured OMV-activated γδ T cells and several cancer cell lines to evaluate their cytotoxic effects. The results found the stimulatory effects of the OMV challenge on the proliferation of Vγ9Vδ2 T cells, ultimately leading to anti-tumor properties.[22]

To mitigate toxicities and side effects like cytokine storms and antibody-specific clearance caused by simple direct intravenous injection of OMVs, some research groups have employed biomimetic mineralization to coat bare OMVs.[23] A calcium phosphate (CaP) “stealth shell” is grown to shield the danger signals and antibody recognition sites of OMVs, enhancing the system’s safety and prolonging its circulation time in the body. When the mineralized particles reach the tumor with the help of nanoparticle (NP) size, the slightly acidic environment at the tumor site dissolves the CaP shell. Then, the exposed OMVs improve the immunosuppressive microenvironment by promoting the infiltration of cytotoxic T cells. Furthermore, the CaP shell dissolves and neutralizes H+ in the microenvironment, thereby improving the polarization of M2-type macrophages to M1-type.[24] This synergistic effect disintegrates the tumor immunosuppressive microenvironment, enhances the killing function of immune cells, and effectively inhibits tumor growth. The incorporation of functional components like folic acid or photosensitizer agents into the outer shells allows for combination treatments, boosting therapeutic outcomes synergistically. To this end, numerous studies have proposed using OMVs as a tumor immunotherapy platform to regulate the tumor microenvironment safely and efficiently without the need for additional immune-stimulating drugs. Table 1 lists the recent research on OMVs as an immunostimulatory agent for modifing TME.[2122242526272829]

Table 1 The TME may be modified by OMVs as an immunostimulatory agent.

OMV sources	Constitution	Combination	Model	Disease	Results	Ref.	
Escherichia coli	OMVs	Anti-PD-1 antibody immunotherapy	Mice	Bladder cancer, breast cancer, CRC, and melanoma	OMV treatment increases infiltration and activation of CD8+ T cells, demonstrating synergistic antitumor activity when combined with anti-PD-1 antibody immunotherapy.	[21]	
Escherichia coli	OMVs	–	Human	Breast cancer and leukemia	The OMV challenge induces the proliferation of Vγ9Vδ2 T cells, which exhibit anti-tumor properties.	[22]	
Escherichia coli	OMVs	–	Cell lines and mice	Neuroblastoma	The suppression of malignancies by OMVs may be attributed to their direct impact on reducing cell stemness, DNA damage, apoptosis, and cell cycle arrest.	[25]	
Escherichia coli	OMVs	–	Mice	Colon cancer	OMVs have been shown to have robust anti-tumor effects upon intratumoral administration in three distinct animal models.	[27]	
Salmonella typhimurium	OMVs	–	Cell lines and mice	CRC, breast cancer, and hepatocellular carcinoma	OMVs exhibites a notable cytotoxic activity.	[26]	
Vibrio cholera, Shigella flexneri, and Escherichia coli	CaP shells cover the surface of OMVs	–	Mice	Colon cancer	OMVs have been shown to possess significant immunostimulatory properties, making them effective agents for reprogramming the TME.	[24]	
Salmonella typhimurium	OMVs	PTT	Mice	Colon cancer	OMVs have emerged as a potential novel therapeutic modality for cancer treatment, exhibiting diverse functionalities.	[29]	
Escherichia coli	AuNPs and OMVs	Radiotherapy	Mice	Glioblastoma	The combination of AuNPs and OMVs, in conjunction with radiation, yields notable radiosensitizing and immunomodulatory effects, effectively inhibiting the proliferation of tumors.	[28]	
AuNPs: Gold nanoparticles; CaP: Calcium phosphate; CD8+T cells: Cluster of differentiation 8 positive T cells; CRC: Colorectal cancer; Ref.: Reference; OMVs: Outer membrane vesicles; PD-1: Programmed death protein-1; PTT: Photothermal therapy; TME: Tumor microenvironment; –: Not available.

OMVs: a promising vaccine platform on the rise

OMVs have the ability to be recognized and taken up efficiently by dendritic cells (DCs). They also contain a high number of PAMPs, which can effectively activate various Toll-like receptor (TLR) signaling pathways. OMVs stimulation helps with antigen presentation and T cell activation, thereby charactering OMVs as excellent nanocarriers for vaccines.[30] Researchers have extensively utilized genetic engineering and molecular glue technology to modify OMVs and create an OMV-based nanocarrier platform. This platform is particularly well-suited for the rapid development of a personalized cancer vaccine.[31] In a recent study, researchers utilized native OMVs to develop functionalized OMVs capable of disrupting programmed death ligand-1 (PD-L1)-mediated immunosuppression.[32] By combining the coding region for the mouse programmed PD-1 ectodomain with that of cytolysin A (ClyA), a surface protein of OMV, researchers engineered OMVs that displayed the ectodomain of PD1 on their surface (OMV-PD1). This modification did not compromise the immunostimulatory properties of the OMVs. The surface-modified OMVs not only bound to PD-L1 on tumor cell membranes but also were internalized by the cells, effectively blocking the inhibition of T cell proliferation by tumor cells. This strategy was tested in two solid tumor models, where the functionalized OMVs demonstrated superior anti-tumor efficacy compared to native OMVs or commonly used PD-L1 antibodies, leading to increased levels of effector T cells in the tumor tissue.

Scientists have successfully armed OMVs with RNA-binding protein technology to make the “Plug-and-Display” strategy work for mRNA antigens in the OMV-based platform.[33] Putting an archaeal RNA-binding protein (L7Ae) on the C-terminal of the surface protein ClyA on the OMVs made it possible for the two proteins to join together. Additionally, the 3′-untranslated region (UTR) of the in vitro transcribed mRNA was modified by adding the matched binding sequence, box C/D. The L7Ae protein specifically acknowledges the box C/D sequence’s typical k-turn conformation, enhancing the stem-loop’s stability and forming a standard L7Ae-k-turn complex. Due to the powerful and precise interaction between L7Ae and the box C/D sequence, the box C/D-labeled mRNA (box C/D-mRNA) quickly attached to the surface of the OMVs. To improve endosomal escape and mRNA translation, the C-terminal of OMV ClyA was fused with listeriolysin O (LLO). Using an engineered OMV-based vector, the mRNA antigens were effectively displayed and delivered to DCs. This led to efficient translation, followed by antigen processing and presentation. Furthermore, certain molecules in the OMVs stimulated the body’s natural defense mechanisms, resulting in stronger activation of T cells that specifically target antigens. This powerful immune response effectively suppressed the growth of tumors in both cases of metastatic melanoma and colon cancer.

Scientists have recently shown, however, that short peptides may be concentrated in OMVs even without an anchoring sequence, which is very unexpected.[34] The peptides were biosynthesized in bacteria and then released extracellularly using the natural secretion system of the bacteria, allowing for stimultaneous biosynthesis and delivery of the peptide. By leveraging the innate properties of bacterial OMVs to enhance antigen uptake and DCs maturation, the researchers were able to induce human papillomavirus (HPV) tumor-specific CD4+ Th1 and CD8+ cytotoxic T-lymphocyte (CTL) responses in tumor-bearing mice, effectively inhibiting the growth of HPV tumors. The technique described in the study only requires one strain of bacteria to produce an HPV tumor vaccine that automatically encapsulates the antigen within bacterial OMVs. This approach eliminates the need to artificially synthesize peptide antigens and streamlines the complex processing of attaching antigens to OMVs. Overall, this research fosters innovation and expands the potential applications of peptide-based vaccine biosynthesis technology. Importantly, the study introduces a novel method for personalized cancer immunotherapy by utilizing screened peptides as antigens for future applications. Consequently, bacterial OMVs are highly effective therapeutic vaccine delivery systems with the potential to enhance cancer therapy.

OMVs that carry a large amount of PAMPs also have the ability to promote trained immunity.[35] However, the OMV-based vaccination, which aims to regulate the activity of tumor-associated macrophages (TAMs), faces numerous challenges such as the development of LPS resistance, the requirement for systemic injection, the requirement for long-term training of both the central and peripheral systems, and the potential interference from macrophage-mediated phagocytosis.[36] To tackle these problems, it is necessary to include extra vaccine elements to enhance the effectiveness of OMV-based trained immunity-related vaccines (TIrV). Some researchers genetically modified OMVs to incorporate the fusion protein SIRP-Fc, then coated them with CaP to create this vaccine.[37] Granulocyte-macrophage colony-stimulating factor (GM-CSF) was added during this procedure to complete the OMV nanohybrids, which are OMV-SIRPα@CaP/GM-CSF. In the end, the researchers discovered that these nanohybrids could make the immune system work better against tumors. This shows that OMVs could be used to help make broad-spectrum vaccines and next-generation tumor vaccines.

In order to initiate macrophage phagocytosis in the TME via numerous routes, an alternate research team built a programmable, two-way adapter based on modified OMVs.[38] The researchers first combined the neutralizing CD47 nanobody (CD47nb) with the surface protein, ClyA, on OMVs (OMV-CD47nb). Then, they covered the OMV-CD47nb with a layer of polyethylene glycol (PEG) that had diselenide linkages added to it (PEG/Se; PEG/Se@OMV-CD47nb). Following intravenous administration, the alteration of the PEG/Se layer effectively prevented the immunogenicity of OMV-CD47nb, resulting in a significant increase in the safe intravenous dosage. Additionally, when radiation therapy was applied directly to the tumor site, the diselenide links were broken. This let go of the PEG layer and allowed OMV-CD47nb to enter the TME. The use of radiation as a trigger for regulated release provided safe and effective transport of OMV-CD47nb throughout the body, leading to efficient accumulation in tumors. OMV-CD47nb, functioning as an immunostimulant, altered the TME by promoting the differentiation of M1-polarized TAMs and attracting a diverse range of immune cells, including B cells, natural killer (NK) cells, DCs, macrophages, neutrophils, and CTLs. Furthermore, OMV-CD47nb enabled TAMs to engulf tumor cells, resulting in the release of tumor antigens. These antigens were then processed and transmitted by antigen-presenting cells (APCs) to the nearby lymph nodes, therefore triggering a T-cell-mediated immune response against the tumor.

Enhancement of antitumor immunity by immunostimulatory microrobots

Microrobotic platforms utilize several actuation techniques to provide active delivery, thereby enhancing the speed and precision with which a payload may be localized to a specified target. Furthermore, these systems can be engineered using diverse biocompatible materials, such as magnesium or zinc, to exploit the propulsive properties of the body’s inherent fluids.[39] To advance the area of microrobotics and broaden its therapeutic applications, researchers have developed an approach that utilizes microrobots to simultaneously destroy tissue and stimulate biological processes at tumor sites to improve cancer immunotherapy.[40] More specifically, they used an Mg-based micromotor system that reacts with aqueous solutions to cause disruption of the surrounding tumor tissue in solid tumors. Micromotors are equipped with OMVs to improve their immune recruitment capacity significantly. These data demonstrated that motor-OMV successfully contributed to the recruitment of immune cells to the tumor site, enabling the processing of tumor antigens derived from deceased cancer cells. This results in developing a strong systemic anticancer immune response, which is crucial for achieving tumor regression. This study presents a novel use of microrobots in cancer immunotherapy and proposes a compelling approach to enhance the effectiveness of cancer treatment when used in conjunction with traditional medicines.

Table 2 lists recent studies on antitumor immunotherapies based on OMVs.[273233343738404142434445464748495051] The findings from these investigations have laid the foundation for innovative strategies in utilizing OMVs for tumor immunotherapy, creating a range of promising opportunities in cancer treatment. These breakthrough represent a significant advancement in our understanding of how these small vesicles can be leveraged for the benefit of patients globally. With further research and development, OMVs have the potential to revolutionize the field of cancer immunotherapy.

Table 2 Antitumor immunotherapies based on OMVs.

OMV sources	Constitution	Model	Disease	Results	Ref.	
Escherichia coli	HPV16 E7 peptide and OMVs	Mice	Cervical cancer	The technique uses OMVs’ inherent qualities to increase antigen absorption and inhibit tumor development.	[34]	
Escherichia coli	M03, M20, M26, M27, M68, and OMVs	Mice	Colon cancer	Incorporating five CT26 neo-epitopes into OMVs demonstrates a synergistic effect with the adjuvanticity of the vesicles, leading to a robust anti-tumor response.	[27]	
Escherichia coli	Cell membrane originated from the tumor (mT) and OMVs (mTOMV)	Mice	Colon cancer	The activation of the adaptive immune response by the mTOMV can potentially augment personalized immunotherapy in the bilateral tumor model.	[41]	
Escherichia coli	Tumor antigen and OMVs	Mice	Colon cancer, pancreatic cancer, and melanoma	The bioengineered platform that uses OMVs makes it possible to present tumor antigens and get the immune system to respond specifically to tumors.	[42]	
Escherichia coli	PD1 and OMVs	Mice	Melanoma	OMVs as carriers for PD-1 in cancer immunotherapy could enhance immune activation and checkpoint inhibition.	[32]	
Escherichia coli	EGFRvIII and OMVs	Mice	Melanoma	In immunocompetent mice, tumor antigens may be incorporated into OMVs, leading to the induction of antigen-specific and protective antitumor responses.	[44]	
Escherichia coli	PD-1 and LyP1 polypeptide-modified OMVs (LOMV)	Mice	Breast cancer and melanoma	LOMVs can recruit cytotoxic lymphocytes and natural killer cells to tumor tissues, stimulating their secretion of IFN-γ and improving the antitumor activity of PD-1/PD-L1 self-blocking therapy.	[45]	
Escherichia coli	Tumor antigen and OMVs (NanoVac)	Mice	Melanoma, pancreatic adenocarcinoma, and CRC	Combining primary tumor excision with NanoVac has potential as a holistic therapy strategy for severely metastatic cancers.	[46]	
Escherichia coli	mD8-FAT1 and OMVs (mD8-FAT1 OMVs)	Mice	CRC and melanoma	The mice immunized with mD8-FAT1 OMVs developed anti-mD8-FAT1 antibodies, providing some protection against challenges from CT26 and EGFRvIII-B16F10 cell lines.	[47]	
Escherichia coli	ClyA-Hy and OMVs	Mice	Breast cancer, pancreatic cancer, and colon cancer	The designed bacterial OMVs have unique qualities that make them a promising choice for peptide delivery.	[51]	
Escherichia coli	EGFRvIII B cell epitope or five tumor-specific CD4+ T cells neoepitopes and mD8-FAT1 OMVs	Mice	CRC and melanoma	mD8-FAT1 OMVs conferred robust protection against tumor challenge in mice when combined with OMVs decorated with the EGFRvIII B cell epitope or with OMVs bearing five tumor-specific CD4+ T cell neoepitopes.	[47]	
Escherichia coli	Whole BFGF molecule and OMVs	Mice	Melanoma	The BFGF-OMVs were shown to elicit the development of anti-BFGF-autoantibodies in mice, subsequently inhibiting tumor cell proliferation.	[43]	
Escherichia coli	Polybia–MPI fusion peptide into OMVs	Mice	Bladder cancer	The bioengineered OMVs exhibited robust inhibition of bladder cancer and demonstrated excellent biocompatibility.	[49]	
Escherichia coli	MerTK inhibitor UNC2025 loaded into OMVs, and then modified with maleimide (mU@OMVs)	Mice	Breast cancer	The in situ vaccine that was acquired demonstrates effective transport to lymph nodes due to the inherent characteristics of OMVs. Subsequently, it elicits robust immune responses that successfully inhibit mouse cancer development, metastasis, and recurrence.	[50]	
Escherichia coli	PEG/Se@OMV-CD47nb	Mice	Colon cancer	The activation of macrophage phagocytosis via numerous mechanisms led to the modification of TME and the fostering of immunity mediated by T cells.	[51]	
Escherichia coli	L7Ae, boxC/D sequence-labeled mRNA antigens and OMVs	Mice	Colon cancer and melanoma	This approach is well-suited for expeditiously developing mRNA vaccines targeting diverse and intricate tumor antigens.	[48, 33]	
Escherichia coli	OMV-SIRPα@CaP/GM-CSF	Mice	Colon cancer and melanoma	The trained immunity and activated T cell response are the main drivers of the TIrV-mediated anticancer mechanism in the colon cancer model (TAM-hot and T cell-cold). In contrast, trained immunity is the primary driver in the melanoma model (T cell-hot and TAM-cold).	[37]	
Escherichia coli	Motor-OMV	Mice	Colon cancer	This study presents a novel use of motor-OMVs in cancer immunotherapy and proposes a compelling approach to enhance the effectiveness of cancer treatment.	[40]	
ClyA: Cytolysin A; CRC: Colorectal cancer; BFGF: Basic fibroblast growth factor; EGFRvIII: Epidermal growth factor receptor variant III; Hy: Hyaluronidase; MPI: Mastoparan I; OMVs: Outer membrane vesicles; Ref.: Reference; TAM: Tumor-associated macrophage; TME: Tumor microenvironment.

OMVs for Targeted Drug Delivery in Tumor Therapy

Nanoparticle DDS have gained significant attention in recent years due to their potential to improve drug stability, enhance bioavailability, and enable targeted delivery. Nevertheless, challenges such as potential toxicity and production hinder its widespread utilization.[9] OMVs are natural nanoscale vectors used in DDS and have notable capabilities for effectively transporting drugs to targeted cells while evading immune system recognition and enzymatic degradation. They can also be designed to release their cargo in a controlled manner, which can help sustain their effects and improve their efficacy.[52] Consequently, numerous studies have been conducted using OMVs to deliver siRNA, micro RNA (miRNA), and other drugs in tumor therapy [Figure 3].

Figure 3 Targeted delivery of therapeutic medications involves using OMVs. One approach involves engineering OMVs to encapsulate precise therapeutic compounds, enabling targeted delivery to the desired location. This strategy has the potential to enhance the effectiveness of these medicines while minimizing the likelihood of adverse reactions. OMVs may be engineered to include a mix of immunomodulatory molecules and tumor antigens, eliciting a synergistic effect that augments the immune response and facilitates the destruction of tumor cells. This approach can revolutionize cancer therapy by providing a more efficient and targeted DDS that can potentially overcome the limitations of tumor immunosuppressive microenvironments and drug resistance. AuNPs: Gold nanoparticles; mRNA: Messenger RNA; pre-miRNA: Precursor-micro RNA; siRNA: Small interfering RNA; DDS: Drug delivery systems; OMVs: Outer membrane vesicles.

Novel approach for precise miRNA delivery to cancer cells

Pre-miRNAs are precursors of mature miRNAs, tiny RNA molecules that can bind to and silence the translation of target messenger RNAs (mRNAs).[53] In recent years, notable research has emphasized developing therapeutic interventions that specifically target pre-miRNAs to impede the proliferation of cancer cells. It has been revealed that prokaryotic cells may produce recombinant human pre-miRNAs using a “tRNA scaffold”. This method allows miRNA to be efficiently biosynthesized in bacterial cells. However, the transfer of pre-miRNAs from their source cells to the target tissues via OMVs remains an area requiring further investigation to determine their potential utility in cancer therapy. Enlightened by these methodologies and complexities, several researchers have used OMVs that bear varying tRNALys-pre-miRNA molecules.[54] These vesicles were introduced into four distinct cancer cell lines to substantiate the inhibitory impact on the target genes and assess their universal applicability. The findings indicate that OMVs can be released and inherit the overexpression of tRNALys-pre-miRNA from the mother, E. coli, which may be directly used for tumor treatment. These results suggested that OMV may be an efficient and universal carrier to instantly block the target genes for tumor treatment by transporting the appropriate pre-miRNA.

These benefits have inspired scholars working on breast cancer treatment and produced OMV-coated ZIF-8 biomimetic NPs containing miRNA.[50] The pre-miR-34a is transcribed in vitro and incorporated into ZIF-8 to create ZIF-8@miR-34 NP, which is then coated with OMVs to form OMV@ZIF-8@miR-34 NPs. The deliberate modification of the OMV membrane to express PD1 enables effective tumor targeting of miRNA delivery vectors. A study involving four groups (ZIF-8, ZIF-8@miR-34a, OMV-PD1, and OMV-PD1@ZIF-8@miR-34a) showed that intravenously administered OMV-coated ZIF-8 nanocarriers efficiently deliver miRNA with precise targeting. Additionally, these nanocarriers exhibited the potential to boost the effectiveness of cancer therapy via a synergistic interaction with the immunostimulatory properties of OMVs.

Co-delivery platform for chemical pharmaceuticals and genetic medicines

Metabolic interactions inside the TME often induce the reprogramming of malignant cell metabolism, leading to a distinct departure from typical physiological processes. This metabolic adaptation is crucial for accelerating tumor development. It has been proposed that intratumoral metabolic communication pathways function symbiotically to facilitate tumor maintenance and development, while competitively suppressing antitumor immunity.[55] Thus, blocking glycolysis in tumor cells is a viable strategy for suppressing tumor growth. However, novel research has indicated that tumor cells may promote tumor metastasis in a hypoxic environment by blocking macrophage glycolysis, which lessens the competition for glucose between macrophage and neovascularized epithelial cells.[56] As a result, there is a need for a DDS that is sensitive to the TME and specifically engineered to target tumor cells and macrophages differently.

By leveraging the drug-loading characteristics of OMVs, a system (siRNA@M-/PTX-CA-OMVs) has been designed to modulate the tumor metabolic microenvironment and inhibit tumor growth compared with the other groups (siRNA@OMVs, siRNA@M-OMVs, siRNA@M-/PTX-OMVs, and siRNA@PTX-CA-OMVs group).[57] By incorporating the pH-sensitive linker cis-aconitic anhydride (CA) between paclitaxel (PTX) and 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly (ethylene glycol) (DSPE- PEG), the system achieved the sequential pH-triggered release of chemotherapeutic drugs. Upon reaching the tumor site, the acidic pH triggered the release of PTX, followed by the recruitment of M2 macrophages to enhance their glycolytic activity. The siRNA@M-/PTX-CA-OMVs demonstrated promise in repolarizing TAMs, suppressing tumor growth, stimulating immune responses against tumors, and altering TME in a triple-negative breast cancer (TNBC) model.

The current state of scientific research is actively exploring the use of OMVs as a co-delivery system [Table 3].[33374854575859606162] Despite the promising potential, the research is still in its early stages and faces challenges like optimizing loading and release of therapeutic agents, as well as targeting specific cells or tissues. With ongoing research and technological advancements, the application of OMVs as a co-delivery system could greatly advance drug delivery and therapeutics, leading to the development of innovative treatment strategies.

Table 3 OMVs served as co-delivery systems.

OMV sources	Constitution	Model	Disease	Results	Ref.	
Escherichia coli	pre-miRNA and OMVs	Mice	Breast cancer and liver cancer	The findings indicate that OMVs can be released and inherit the overexpression of tRNALys-pre-miRNA from the mother, Escherichia coli, which may be directly used for tumor treatment.	[54]	
Escherichia coli	L7Ae, boxC/D sequence-labeled mRNA antigens, and OMVs	Mice	Colon cancer and melanoma	OMVs are used as an mRNA delivery platform.	[48, 33]	
Escherichia coli	OMV-SIRPα@CaP/GM-CSF	Mice	Colon cancer and melanoma	The trained immunity and activated T cell response are the main drivers of the TIrV-mediated anticancer mechanism in the colon cancer model (TAM-hot and T cell-cold). In contrast, trained immunity is the primary driver in the melanoma model (T cell-hot and TAM-cold).	[37]	
Escherichia coli	Perhexiline and OMVs (OMV@Perhx)	Cell lines	Colon cancer	The internalization of OMV@Perhx by macrophages effectively modified the phenotype of TAMs from an M2 to an M1, enhancing the effect of tumor suppression.	[58]	
Salmonella	DOX and OMVs	Mice	Glioma	When administered to glioma patients, OMVs/DOX were detected specifically by neutrophils, resulting in tumor accumulation 18 times greater than with conventional passive targeting.	[59]	
Escherichia coli	siRNA@M-/PTX-CA-OMVs	Mice	Breast cancer	The OMV has yielded valuable knowledge in developing a co-delivery platform for chemical pharmaceuticals and genetic therapies.	[57]	
Klebsiella pneumoniae	DOX and OMVs	Mice	Non-small-cell lung cancer	OMVs are promising for tumor chemoimmunotherapy since they can act as biological nanocarriers for chemotherapeutic drugs and trigger appropriate immune responses.	[60]	
Escherichia coli	5-FU, MSN, and OMVs (OMVs-MSN-5-FU)	Mice	Colon cancer	The research presented a potentially effective nanotechnology-based platform for the targeted treatment of colon cancer.	[61]	
Salmonella	PEG, tumor-targeting ligand Arg-Gly-Asp (RGD) peptide, 5-FU, and OMVs	Mice	Melanoma	The administration of bioengineered polymeric nanomedicines covered with OMVs has significant potential for enhancing the effectiveness of cancer immunotherapy.	[62]	
5-FU: 5-fluorouracil; DOX: Doxorubicin; MSN: Mesoporous silica; OMVs: Outer membrane vesicles; PEG: Polyethylene glycol; Ref.: Reference; TAMs: Tumor-associated macrophages; TIrV: Trained immunity-related vaccines.

Multifunctional OMV nanoplatform in photothermal therapy (PTT) is a promising local therapeutic modality for tumors, wherein the tumor tissue is efficiently ablated by the conversion of near-infrared light (NIR) into heat using photothermal transduction agents (PTAs).[63] As tumor cells are destroyed by heat, they generate tumor antigens, which can activate tumor-specific CTLs that are essential for eliminating lingering tumor cells and distant lesions. Nevertheless, the immune-mediated elimination of tumors resulting from local PTT is consistently constrained. Several studies have highlighted the potential use of NPs to capture tumor antigens generated during radiation, microwave ablation, or PTT. These NPs can then effectively transfer the captured antigens to APCs, stimulating a robust immune response against the tumor. To improve immune-mediated tumor clearance after PTT, several researchers have reported OMV-based multifunctional in situ vaccinations with antigen capture and immune modulation characteristics.[64] An in situ multifunctional vaccine (1-MT@OMV-Mal) based on bacterial OMVs was developed by the surface conjugation of maleimide groups (Mal) and interior loading with 1-methyl-tryptophan (1-MT), an inhibitor of indoleamine 2,3-dioxygenase (IDO). After PTT, 1-MT@OMV-Mal could bind to the released tumor antigens and be efficiently taken up by DCs. Additionally, the direct administration of 1-MT@OMV-Mal directly at the tumor site effectively counteracted the immune suppression caused by IDO on the effector T cells that infiltrated the tumor. This significantly inhibits the primary tumor and any cancers that metastasize to distant sites.

In addition to the beneficial effects of photodynamic treatment, the potential of OMVs as a platform for optoacoustic applications, which could improve contrast enhancement and therapeutic applications, is worth considering. Gujrati et al[65] used a bacterial strain expressing a tyrosinase transgene to create OMVs encapsulating biopolymer-melanin (OMVMel) to study bioengineered OMVs as contrast agent in photoacoustic imaging. Their findings demonstrate that the OMVMel produces robust optoacoustic signals suitable for imaging when exposed to near-infrared light. They further explained that OMVMel was responsible for mediating photothermal effects in vitro and in vivo, indicating that it generated significant heat from the absorbed laser energy. Multispectral optoacoustic tomography can non-invasively track the temporal and spatial distribution of tumor-associated OMVMelin vivo. This study illustrates the utilization of bioengineered vesicles as substitutes for synthetic particles often used in optoacoustic imaging. These vesicles can boost the imaging quality and facilitate photothermal applications.

Current research is focusing on determining the best design and formulation for OMV-based phototherapeutics. Scientists are investigating the optimal loading of photosensitizers into OMVs and the most efficient ways to target these nanoparticles to specific tissues or cells [Table 4].[2329646566676869707172]. In summary, the use of OMVs phototherapy shows great promise for creating new and effective treatment approaches. With ongoing research and advancements, OMV-based phototherapeutics could emerge as a valuable tool in modern medicine.

Table 4 The therapeutic application of OMVs in the field of phototherapy.

OMV sources	Constitution	Phototherapy	Combination	Model	Disease	Results	Ref.	
Escherichia coli	Maleimide groups (Mal), 1-MT, IDO, and OMVs (1-MT@OMV-Mal)	PTT	–	Mice	Colon cancer	A potentially effective in situ vaccination using OMVs has shown promise in enhancing the immune-mediated elimination of tumors after PTT by coordinating antigen collection and immune regulation.	[64]	
Escherichia coli	Copper sulfide and OMVs	PTT	–	Mice	Breast cancer	CuS-OMVs exhibit significant effectiveness in inhibiting tumor growth, both at the initial tumor site and in untreated tumors located at a distance, when exposed to NIR-II light.	[66]	
Escherichia coli	Coating OMVs on NPs	PTT	–	Mice	Breast cancer and colon cancer	The rapid release of NPNs from neutrophils in response to inflammatory stimuli is followed by their uptake by tumor cells, where they exert anticancer effects.	[67]	
Escherichia coli	DOX, Ce6, and OMVs	PDT	–	Mice	Breast cancer	OMVs-based technique with bioengineering has the potential to function as a robust therapeutic platform for a synergistic approach to anticancer treatment.	[68]	
Salmonella	Ce6, catalase, PD-L1, and OMVs	PDT	–	Mice	Breast cancer	The CAT-Ce6@OMV-aPDL1 compound has remarkable effectiveness in PDT and immunotherapy, leading to significant anticancer effects.	[69]	
Salmonella	PTSLs, siRNA, and OMVs (siRNA@PLOV)	PTT	Anti-PD-1 therapy	Mice	Colon cancer, hepatocellular carcinoma, breast cancer, brain astroblastoma, and non-small cell lung cancer	The results of this study show that administering anti-PD-1 on the fifth day after siRNA@PLOV therapy was more effective at getting rid of tumors than other experimental groups.	[23]	
Salmonella typhimurium	OMVs	PTT	Photoacoustic imaging	Mice	Colon cancer	OMVs have emerged as a potential novel therapeutic modality for cancer treatment, exhibiting diverse functionalities.	[29]	
Escherichia coli	αvβ3 integrin peptide, indocyanine green, and OMVs (I-P-OMVs)	PTPT	Thermographic imaging	Mice	Melanoma	I-P-OMVs are the first nanoplatforms with improved antitumor efficacy and good safety to generate transdermal photo-TRAIL-programmed treatment in melanoma.	[70]	
Escherichia coli	B16-F10 CC membrane, OMVs, and HPDA NPs (HPDA@[OMV-CC] NP)	PTT	Photothermal imaging	Mice	Melanoma	The antitumor immune response and PTT mutually enhance therapeutic efficacy and effectively eliminate melanoma without significant side effects.	[71]	
Escherichia coli	The thylakoid nanovesicles (NTs) of spinach and OMVs (BPNs)	PDT	–	Mice	Colon cancer	The BPNs have a synergistic impact, resulting in powerful therapeutic outcomes that eliminate primary solid tumors and hinder the spread of cancer to other locations.	[72]	
Escherichia coli	Biopolymer-melanin and OMVs (OMVMel)	PTT	Optoacoustic imaging	Mice	Breast cancer	These vesicles can boost imaging quality and facilitate photothermal applications.	[65]	
1-MT: 1-methyl-tryptophan; CC: Cancer cell; Ce6: Chlorin e6; DOX: Doxorubicin; HPDA: Hollow polydopamine; IDO: Indoleamine 2,3-dioxygenase; NIR: Near-infrared light; NPs: Nanoparticles; OMVs: Outer membrane vesicles; PD-1: Programmed death protein-1; PD-L1: Programmed death ligand-1; PDT: Photodynamic therapy; PTPT: Photo-TRAIL-programmed treatment; PTSLs: Photothermal sensitive liposomes; PTT: Photothermal therapy; Ref.: Reference; –: Not available.

Prospect of Using OMVs

Researchers have recently begun utilizing biotechnology to manipulate OMVs and customize them to fulfill specific requirements.[6] Targeted drug delivery can be achieved by genetically modifying the surface of vesicles to exhibit particular ligands. The capacity to specifically target the cells of interest enhances the effectiveness of drugs and reduces the risk of harm to healthy cells. Engineered OMVs may also enhance treatment efficacy and minimize adverse effects on the whole system.[73]

OMVs in cancer virotherapy

Oncolytic adenovirus (Ad) infection induces intracellular autophagy inside tumors, eliminating cancer cells and enhancing anticancer immunity mediated by Ads.[74] The clinical results of possible Ads have failed to meet expectations for several reasons. One of the most important challenges is the efficient viral replication and dissemination inside neoplastic cells. Another significant challenge for resistance to Ads-based immunotherapy is the presence of an immunosuppressive TME.[75] Hence, it is evident that the early and accurate induction of heightened autophagy at tumor locations employing Ads-based treatment is essential. To allow autophagy-cascade-augmented antitumor immunotherapy, some researchers have suggested a microbial nanocomposite concept that encapsulates oncolytic viruses (OVs) inside pyranose oxidase (P2O)-engineered and biomineral CaP-camouflaged OMVs.[76] The findings of this study indicate a significant enhancement in the replication efficiency of Ads in cancer cells infected with OVs, with an increase in the number of autophagy-induced autophagosomes. OMVs are potent immunostimulants that reshape the immunosuppressive TME and promote anticancer immune responses.

OMVs in multimodal cancer therapy

There has been significant interest within the biomedical community in microrobots that can convert various forms of energy, such as chemical, magnetic, acoustic, and light, into mechanical motion. Microrobots have garnered attention owing to their potential applications in many fields, including biosensing, imaging, detoxification, and drug delivery.[77] However, endogenous microrobots with inherent targeting functionality and enhanced therapeutic pathways will provide unparalleled advantages in illness management. Researchers presented a biogenic macrophage-based microrobot laden with NPs and bioengineered bacterial OMVs that, upon magnetic manipulation, possesses multimodal targeted cancer therapy.[78] Magnetic-induced aggregation, in conjunction with the tumor tropism shown by macrophages, significantly augments the in vivo anticancer efficacy of cell robots that contain a repertoire of therapeutic modalities. These modalities include macrophage-mediated tumor suppression, immunotherapy, and the administration of antitumor peptides through OMVs. This design has significant promise for a dynamic medical vehicle that can effectively fulfill the demanding criteria associated with clinical trials.

The unique features of OMVs have positioned them as potential therapeutic agents for cancer treatment. Nevertheless, it is essential to acknowledge the drawbacks associated with OMV use. The primary drawback is their limited cargo capacity. OMVs are restricted in their ability to transport therapeutic substances owing to their small size. This can limit their use in clinical settings where high doses of therapeutic agents are needed.[79] The risk of immunogenicity is yet another drawback to OMVs.[80] OMVs are obtained from bacteria and can elicit an immunological response in humans. This phenomenon may have unfavorable consequences and limit its effectiveness as a therapeutic intervention. Additionally, it is crucial to consider the difficulties associated with OMV production and isolation. The successful execution of this procedure necessitates using sophisticated methodologies to guarantee the integrity and uniformity of OMV samples.[52] It is essential to acknowledge that the intrinsic variability of OMVs may increase the complexity and uncertainty of their application. OMVs exhibit variations in size, composition, and functional characteristics, even when originating from the same bacterial strain.[81] The wide range of variations among these entities poses a significant challenge to developing a universally accepted and defined methodology for medical and biotechnological contexts. The existence of various OMVs, which may produce different results, further complicates the characterization of vesicles.

Despite these limitations, OMVs have a significant therapeutic potential for cancer treatment. Researchers continue to investigate strategies to overcome these limitations and enhance the efficacy of OMVs in cancer therapy. One potential approach to mitigate the challenge of limited cargo capacity is the integration of OMVs with other drug delivery methods, such as NPs, to generate hybrid systems capable of accommodating a greater quantity of therapeutic agents. In addition, it can be selectively modified to target specific types of cancer, enhancing their effectiveness and mitigating their potential for negative consequences.

Although some drawbacks are associated with using OMVs in cancer therapy, their distinctive characteristics render them up-and-coming candidates for cancer treatment and medication administration. Researchers continue to investigate strategies to overcome the limitations associated with OMVs and enhance their efficacy in cancer therapy. OMVs hold the potential to revolutionize cancer treatment by providing a more efficient and targeted DDS capable of overcoming challenges posed by tumor immunosuppressive microenvironments and drug resistance.

Acknowledgements

We sincerely thank the article’s anonymous reviewers, editors, and contributors for their time and thoughtful comments.

Funding

This research work was supported by the Fundamental Research Funds for the Central Universities, Natural Science Foundation (Nos. 2022-YGJC-86 and 2020-ZLLH-38 to Yiming Meng) of Liaoning Province, and Excellent Talent Fund of Liaoning Province Cancer Hospital of Yiming Meng.

Conflicts of interest

None.

How to cite this article: Meng YM, Kong CC, Ma YS, Sun J, Zhang GR. Bacterial outer membrane vesicles in the fight against cancer. Chin Med J 2024;137:2169–2181. doi: 10.1097/CM9.0000000000003234
==== Refs
References

1. Xiao Y Yu D . Tumor microenvironment as a therapeutic target in cancer. Pharmacol Ther 2021;221 :107753. doi: 10.1016/j.pharmthera.2020.107753.33259885
2. Sepich-Poore GD Zitvogel L Straussman R Hasty J Wargo JA Knight R . The microbiome and human cancer. Science 2021;371 :eabc4552. doi: 10.1126/science.abc4552.33766858
3. Ding Y Wang L Li H Miao F Zhang Z Hu C , . Application of lipid nanovesicle drug delivery system in cancer immunotherapy. J Nanobiotechnology 2022;20 :214. doi: 10.1186/s12951-022-01429-2.35524277
4. Dhital S Deo P Stuart I Naderer T . Bacterial outer membrane vesicles and host cell death signaling. Trends Microbiol 2021;29 :1106–1116. doi: 10.1016/j.tim.2021.04.003.34001418
5. Deo P Chow SH Han ML Speir M Huang C Schittenhelm RB , . Mitochondrial dysfunction caused by outer membrane vesicles from Gram-negative bacteria activates intrinsic apoptosis and inflammation. Nat Microbiol 2020;5 :1418–1427. doi: 10.1038/s41564-020-0773-2.32807891
6. Long Q Zheng P Zheng X Li W Hua L Yang Z , . Engineered bacterial membrane vesicles are promising carriers for vaccine design and tumor immunotherapy. Adv Drug Deliv Rev 2022;186 :114321. doi: 10.1016/j.addr.2022.114321.35533789
7. Rueter C Bielaszewska M . Secretion and delivery of intestinal pathogenic Escherichia coli virulence factors via outer membrane vesicles. Front Cell Infect Microbiol 2020;10 :91. doi: 10.3389/fcimb.2020.00091.32211344
8. Lamprinaki D Garcia-Vello P Marchetti R Hellmich C McCord KA Bowles KM , . Siglec-7 mediates immunomodulation by colorectal cancer-associated Fusobacterium nucleatum ssp. animalis. Front Immunol 2021;12 :744184. doi: 10.3389/fimmu.2021.744184.34659241
9. Jahromi LP Fuhrmann G . Bacterial extracellular vesicles: Understanding biology promotes applications as nanopharmaceuticals. Adv Drug Deliv Rev 2021;173 :125–140. doi: 10.1016/j.addr.2021.03.012.33774113
10. Aytar Çelik P Derkuş B Erdoğan K Barut D Blaise Manga E Yıldırım Y , . Bacterial membrane vesicle functions, laboratory methods, and applications. Biotechnol Adv 2022;54 :107869. doi: 10.1016/j.biotechadv.2021.107869.34793882
11. Théry C Witwer KW Aikawa E Alcaraz MJ Anderson JD Andriantsitohaina R , . Minimal information for studies of extracellular vesicles 2018 (MISEV2018): A position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 2018;7 :1535750. doi: 10.1080/20013078.2018.1535750.30637094
12. Wang S Song A Xie J Wang YY Wang WD Zhang MJ , . Fn-OMV potentiates ZBP1-mediated PANoptosis triggered by oncolytic HSV-1 to fuel antitumor immunity. Nat Commun 2024;15 :3669. doi: 10.1038/s41467-024-48032-7.38693119
13. Mosby CA Perez Devia N Jones MK . Comparison of methods for quantifying extracellular vesicles of Gram-negative bacteria. Int J Mol Sci 2023;24 :15096. doi: 10.3390/ijms242015096.37894776
14. Lopez C Delmonti J Bonomo RA Vila AJ . Deciphering the evolution of metallo-beta-lactamases: A journey from the test tube to the bacterial periplasm. J Biol Chem 2022;298 :101665. doi: 10.1016/j.jbc.2022.101665.35120928
15. Diallo I Provost P . RNA-sequencing analyses of small bacterial RNAs and their emergence as virulence factors in host-pathogen interactions. Int J Mol Sci 2020;21 :1627. doi: 10.3390/ijms21051627.32120885
16. Toyofuku M Schild S Kaparakis-Liaskos M Eberl L . Composition and functions of bacterial membrane vesicles. Nat Rev Microbiol 2023;21 :415–430. doi: 10.1038/s41579-023-00875-5.36932221
17. Wang H Tao Z Zhao X Wang G Peng Y Chen Y , . Enrichment and delivery of target proteins into the cell cytosol via outer membrane vesicles. ACS Appl Mater Interfaces 2023;15 :29743–29751. doi: 10.1021/acsami.3c00427.37336754
18. Giordano NP Cian MB Dalebroux ZD . Outer membrane lipid secretion and the innate immune response to Gram-negative bacteria. Infect Immun 2020;88 :e920–e919. doi: 10.1128/IAI.00920-19.
19. Micoli F Alfini R Di Benedetto R Necchi F Schiavo F Mancini F , . Generalized Modules for Membrane Antigens as Carrier for Polysaccharides: Impact of Sugar Length, Density, and Attachment Site on the Immune Response Elicited in Animal Models. Front Immunol 2021;12 :719315. doi: 10.3389/fimmu.2021.719315.34594333
20. Kim OY Hong BS Park KS Yoon YJ Choi SJ Lee WH , . Immunization with Escherichia coli outer membrane vesicles protects bacteria-induced lethality via Th1 and Th17 cell responses. J Immunol 2013;190 :4092–4102. doi: 10.4049/jimmunol.1200742.23514742
21. Won S Lee C Bae S Lee J Choi D Kim MG , . Mass-produced Gram-negative bacterial outer membrane vesicles activate cancer antigen-specific stem-like CD8(+) T cells which enables an effective combination immunotherapy with anti-PD-1. J Extracell Vesicles 2023;12 :e12357. doi: 10.1002/jev2.12357.37563797
22. Firth J Sun J George V Huang JD Bajaj-Elliott M Gustafsson K . Bacterial outer-membrane vesicles promote Vgamma9Vdelta2 T cell oncolytic activity. Front Immunol 2023;14 :1198996. doi: 10.3389/fimmu.2023.1198996.37529036
23. Zhai Y Ma Y Pang B Zhang J Li Y Rui Y , . A cascade targeting strategy based on modified bacterial vesicles for enhancing cancer immunotherapy. J Nanobiotechnology 2021;19 :434. doi: 10.1186/s12951-021-01193-9.34930285
24. Qing S Lyu C Zhu L Pan C Wang S Li F , . Biomineralized bacterial outer membrane vesicles potentiate safe and efficient tumor microenvironment reprogramming for anticancer therapy. Adv Mater 2020;32 :e2002085. doi: 10.1002/adma.202002085.33015871
25. Jin L Zhang Z Tan X Wang Z Tang B Wang Z , . Antitumor effect of Escherichia coli-derived outer membrane vesicles on neuroblastoma in vitro and in vivo. Acta Biochim Biophys Sin (Shanghai) 2022;54 :1301–1313. doi: 10.3724/abbs.2022127.36148954
26. Aly RG El-Enbaawy MI Abd El-Rahman SS Ata NS . Antineoplastic activity of Salmonella typhimurium outer membrane nanovesicles. Exp Cell Res 2021;399 :112423. doi: 10.1016/j.yexcr.2020.112423.33338480
27. Caproni E Corbellari R Tomasi M Isaac SJ Tamburini S Zanella I , . Anti-tumor efficacy of in situ vaccination using bacterial outer membrane vesicles. Cancers (Basel) 2023;15 :3328. doi: 10.3390/cancers15133328.37444437
28. Chen MH Liu TY Chen YC Chen MH . Combining augmented radiotherapy and immunotherapy through a nano-gold and bacterial outer-membrane vesicle complex for the treatment of glioblastoma. Nanomaterials (Basel) 2021;11 :1661. doi: 10.3390/nano11071661.34202555
29. Zhuang Q Xu J Deng D Chao T Li J Zhang R , . Bacteria-derived membrane vesicles to advance targeted photothermal tumor ablation. Biomaterials 2021;268 :120550. doi: 10.1016/j.biomaterials.2020.120550.33278684
30. Kashyap D Panda M Baral B Varshney N R S Bhandari V Parmar HS , . Outer Membrane Vesicles: An Emerging Vaccine Platform. Vaccines (Basel) 2022;10 :1578. doi: 10.3390/vaccines10101578.36298443
31. Wang S Guo J Bai Y Sun C Wu Y Liu Z , . Bacterial outer membrane vesicles as a candidate tumor vaccine platform. Front Immunol 2022;13 :987419. doi: 10.3389/fimmu.2022.987419.36159867
32. Li Y Zhao R Cheng K Zhang K Wang Y Zhang Y , . Bacterial outer membrane vesicles presenting programmed death 1 for improved cancer immunotherapy via immune activation and checkpoint inhibition. ACS Nano 2020;14 :16698–16711. doi: 10.1021/acsnano.0c03776.33232124
33. Li Y Ma X Yue Y Zhang K Cheng K Feng Q , . Rapid surface display of mRNA antigens by bacteria-derived outer membrane vesicles for a personalized tumor vaccine. Adv Mater 2022;34 :e2109984. doi: 10.1002/adma.202109984.35315546
34. Chen H Zheng X Li L Huang L Huang W Ma Y . Peptide-based therapeutic HPV cancer vaccine synthesized via bacterial outer membrane vesicles. Int J Nanomedicine 2023;18 :4541–4554. doi: 10.2147/IJN.S416706.37576463
35. Ochando J Mulder WJM Madsen JC Netea MG Duivenvoorden R . Trained immunity – Basic concepts and contributions to immunopathology. Nat Rev Nephrol 2023;19 :23–37. doi: 10.1038/s41581-022-00633-5.36253509
36. van der Ley PA Zariri A van Riet E Oosterhoff D Kruiswijk CP . An Intranasal OMV-Based Vaccine Induces High Mucosal and Systemic Protecting Immunity Against a SARS-CoV-2 Infection. Front Immunol 2021;12 :781280. doi: 10.3389/fimmu.2021.781280.34987509
37. Liang J Zhu F Cheng K Ma N Ma X Feng Q , . Outer membrane vesicle-based nanohybrids target tumor-associated macrophages to enhance trained immunity-related vaccine-generated antitumor activity. Adv Mater 2023;35 :e2306158. doi: 10.1002/adma.202306158.37643537
38. Feng Q Ma X Cheng K Liu G Li Y Yue Y , . Engineered bacterial outer membrane vesicles as controllable two-way adaptors to activate macrophage phagocytosis for improved tumor immunotherapy. Adv Mater 2022;34 :e2206200. doi: 10.1002/adma.202206200.35985666
39. Gong D Celi N Zhang D Cai J . Magnetic biohybrid microrobot multimers based on chlorella cells for enhanced targeted drug delivery. ACS Appl Mater Interfaces 2022;14 :6320–6330. doi: 10.1021/acsami.1c16859.35020358
40. Zhou J Karshalev E Mundaca-Uribe R Esteban-Fernández de Ávila B Krishnan N Xiao C , . Physical disruption of solid tumors by immunostimulatory microrobots enhances antitumor immunity. Adv Mater 2021;33 :e2103505. doi: 10.1002/adma.202103505.34599770
41. Zou MZ Li ZH Bai XF Liu CJ Zhang XZ . Hybrid vesicles based on autologous tumor cell membrane and bacterial outer membrane to enhance innate immune response and personalized tumor immunotherapy. Nano Lett 2021;21 :8609–8618. doi: 10.1021/acs.nanolett.1c02482.34661419
42. Cheng K Zhao R Li Y Qi Y Wang Y Zhang Y , . Bioengineered bacteria-derived outer membrane vesicles as a versatile antigen display platform for tumor vaccination via plug-and-display technology. Nat Commun 2021;12 :2041. doi: 10.1038/s41467-021-22308-8.33824314
43. Huang W Shu C Hua L Zhao Y Xie H Qi J , . Modified bacterial outer membrane vesicles induce autoantibodies for tumor therapy. Acta Biomater 2020;108 :300–312. doi: 10.1016/j.actbio.2020.03.030.32251780
44. Grandi A Tomasi M Zanella I Ganfini L Caproni E Fantappiè L , . Synergistic protective activity of tumor-specific epitopes engineered in bacterial outer membrane vesicles. Front Oncol 2017;7 :253. doi: 10.3389/fonc.2017.00253.29164053
45. Pan J Li X Shao B Xu F Huang X Guo X , . Self-blockade of PD-L1 with bacteria-derived outer-membrane vesicle for enhanced cancer immunotherapy. Adv Mater 2022;34 :e2106307. doi: 10.1002/adma.202106307.34859919
46. Ma N Chen Z Liu G Yue Y Li Y Cheng K , . Normalizing the immune macroenvironment via debulking surgery to strengthen tumor nanovaccine efficacy and eliminate metastasis. ACS Nano 2023;17 :437–452. doi: 10.1021/acsnano.2c08880.36534945
47. Grandi A Fantappiè L Irene C Valensin S Tomasi M Stupia S , . Vaccination with a FAT1-derived b cell epitope combined with tumor-specific B and T cell epitopes elicits additive protection in cancer mouse models. Front Oncol 2018;8 :481. doi: 10.3389/fonc.2018.00481.30416985
48. Gao X Li Y Nie G Zhao X . mRNA delivery platform based on bacterial outer membrane vesicles for tumor vaccine. Bio Protoc 2023;13 :e4774. doi: 10.21769/BioProtoc.4774.
49. Ren C Li Y Cong Z Li Z Xie L Wu S . Bioengineered bacterial outer membrane vesicles encapsulated Polybia-mastoparan I fusion peptide as a promising nanoplatform for bladder cancer immune-modulatory chemotherapy. Front Immunol 2023;14 :1129771. doi: 10.3389/fimmu.2023.1129771.36999028
50. Cui C He Q Wang J Kang J Ma W Nian Y , . Targeted miR-34a delivery with PD1 displayed bacterial outer membrane vesicles-coated zeolitic imidazolate framework nanoparticles for enhanced tumor therapy. Int J Biol Macromol 2023;247 :125692. doi: 10.1016/j.ijbiomac.2023.125692.37414322
51. Thomas SC Madaan T Kamble NS Siddiqui NA Pauletti GM Kotagiri N . Engineered bacteria enhance immunotherapy and targeted therapy through stromal remodeling of tumors. Adv Healthc Mater 2022;11 :e2101487. doi: 10.1002/adhm.202101487.34738725
52. Zhu Z Antenucci F Villumsen KR Bojesen AM . Bacterial outer membrane vesicles as a versatile tool in vaccine research and the fight against antimicrobial resistance. mBio 2021;12 :e0170721. doi: 10.1128/mBio.01707-21.34372691
53. Lee YY Kim H Kim VN . Sequence determinant of small RNA production by DICER. Nature 2023;615 :323–330. doi: 10.1038/s41586-023-05722-4.36813957
54. Cui C Guo T Zhang S Yang M Cheng J Wang J , . Bacteria-derived outer membrane vesicles engineered with over-expressed pre-miRNA as delivery nanocarriers for cancer therapy. Nanomedicine 2022;45 :102585. doi: 10.1016/j.nano.2022.102585.35901958
55. Xia L Oyang L Lin J Tan S Han Y Wu N , . The cancer metabolic reprogramming and immune response. Mol Cancer 2021;20 :28. doi: 10.1186/s12943-021-01316-8.33546704
56. Li M Yang Y Xiong L Jiang P Wang J Li C . Metabolism, metabolites, and macrophages in cancer. J Hematol Oncol 2023;16 :80. doi: 10.1186/s13045-023-01478-6.37491279
57. Guo Q Li X Zhou W Chu Y Chen Q Zhang Y , . Sequentially triggered bacterial outer membrane vesicles for macrophage metabolism modulation and tumor metastasis suppression. ACS Nano 2021;15 :13826–13838. doi: 10.1021/acsnano.1c05613.34382768
58. Jiang S Fu W Wang S Zhu G Wang J Ma Y . Bacterial outer membrane vesicles loaded with perhexiline suppress tumor development by regulating tumor-associated macrophages repolarization in a synergistic way. Int J Mol Sci 2023;24 :11222. doi: 10.3390/ijms241311222.37446401
59. Mi Z Yao Q Qi Y Zheng J Liu J Liu Z , . Salmonella-mediated blood–brain barrier penetration, tumor homing and tumor microenvironment regulation for enhanced chemo/bacterial glioma therapy. Acta Pharm Sin B 2023;13 :819–833. doi: 10.1016/j.apsb.2022.09.016.36873179
60. Kuerban K Gao X Zhang H Liu J Dong M Wu L , . Doxorubicin-loaded bacterial outer-membrane vesicles exert enhanced anti-tumor efficacy in non-small-cell lung cancer. Acta Pharm Sin B 2020;10 :1534–1548. doi: 10.1016/j.apsb.2020.02.002.32963948
61. Shi J Ma Z Pan H Liu Y Chu Y Wang J , . Biofilm-encapsulated nano drug delivery system for the treatment of colon cancer. J Microencapsul 2020;37 :481–491. doi: 10.1080/02652048.2020.1797914.32700606
62. Chen Q Bai H Wu W Huang G Li Y Wu M , . Bioengineering bacterial vesicle-coated polymeric nanomedicine for enhanced cancer immunotherapy and metastasis prevention. Nano Lett 2020;20 :11–21. doi: 10.1021/acs.nanolett.9b02182.31858807
63. Gupta N Malviya R . Understanding and advancement in gold nanoparticle targeted photothermal therapy of cancer. Biochim Biophys Acta Rev Cancer 2021;1875 :188532. doi: 10.1016/j.bbcan.2021.188532.33667572
64. Li Y Zhang K Wu Y Yue Y Cheng K Feng Q , . Antigen capture and immune modulation by bacterial outer membrane vesicles as in situ vaccine for cancer immunotherapy post-photothermal therapy. Small 2022;18 :e2107461. doi: 10.1002/smll.202107461.35152555
65. Gujrati V Prakash J Malekzadeh-Najafabadi J Stiel A Klemm U Mettenleiter G , . Bioengineered bacterial vesicles as biological nano-heaters for optoacoustic imaging. Nat Commun 2019;10 :1114. doi: 10.1038/s41467-019-09034-y.30846699
66. Qin J Yang T Li J Zhan G Li X Wei Z , . Bacterial outer membrane vesicle-templated biomimetic nanoparticles for synergistic photothermo-immunotherapy. Nano Today 2022:46. doi: 10.1016/j.nantod.2022.101591.
67. Li M Li S Zhou H Tang X Wu Y Jiang W , . Chemotaxis-driven delivery of nano-pathogenoids for complete eradication of tumors post-phototherapy. Nat Commun 2020;11 :1126. doi: 10.1038/s41467-020-14963-0.32111847
68. Li Y Wu J Qiu X Dong S He J Liu J , . Bacterial outer membrane vesicles-based therapeutic platform eradicates triple-negative breast tumor by combinational photodynamic/chemo-/immunotherapy. Bioact Mater 2023;20 :548–560. doi: 10.1016/j.bioactmat.2022.05.037.35846843
69. Zhang J Li Z Liu L Li L Zhang L Wang Y , . Self-assembly catalase nanocomplex conveyed by bacterial vesicles for oxygenated photodynamic therapy and tumor immunotherapy. Int J Nanomedicine 2022;17 :1971–1985. doi: 10.2147/IJN.S353330.35530972
70. Peng LH Wang MZ Chu Y Zhang L Niu J Shao HT , . Engineering bacterial outer membrane vesicles as transdermal nanoplatforms for photo-TRAIL-programmed therapy against melanoma. Sci Adv 2020;6 :eaba2735. doi: 10.1126/sciadv.aba2735.32923586
71. Wang D Liu C You S Zhang K Li M Cao Y , . Bacterial vesicle-cancer cell hybrid membrane-coated nanoparticles for tumor specific immune activation and photothermal therapy. ACS Appl Mater Interfaces 2020;12 :41138–41147. doi: 10.1021/acsami.0c13169.32830477
72. Zhuang WR Wang Y Lei Y Zuo L Jiang A Wu G , . Phytochemical engineered bacterial outer membrane vesicles for photodynamic effects promoted immunotherapy. Nano Lett 2022;22 :4491–4500. doi: 10.1021/acs.nanolett.2c01280.35605283
73. Gao J Su Y Wang Z . Engineering bacterial membrane nanovesicles for improved therapies in infectious diseases and cancer. Adv Drug Deliv Rev 2022;186 :114340. doi: 10.1016/j.addr.2022.114340.35569561
74. Kaverina NV Kadagidze ZG Borovjagin AV Karseladze AI Kim CK Lesniak MS , . Tamoxifen overrides autophagy inhibition in Beclin-1-deficient glioma cells and their resistance to adenovirus-mediated oncolysis via upregulation of PUMA and BAX. Oncogene 2018;37 :6069–6082. doi: 10.1038/s41388-018-0395-9.29991800
75. Hemminki O Dos Santos JM Hemminki A . Oncolytic viruses for cancer immunotherapy. J Hematol Oncol 2020;13 :84. doi: 10.1186/s13045-020-00922-1.32600470
76. Ban W Sun M Huang H Huang W Pan S Liu P , . Engineered bacterial outer membrane vesicles encapsulating oncolytic adenoviruses enhance the efficacy of cancer virotherapy by augmenting tumor cell autophagy. Nat Commun 2023;14 :2933. doi: 10.1038/s41467-023-38679-z.37217527
77. Soto F Karshalev E Zhang F Esteban Fernandez de Avila B Nourhani A Wang J . Smart materials for microrobots. Chem Rev 2022;122 :5365–5403. doi: 10.1021/acs.chemrev.0c00999.33522238
78. Li Y Cong Z Xie L Tang S Ren C Peng X , . Magnetically powered immunogenic macrophage microrobots for targeted multimodal cancer therapy. Small 2023;19 :e2301489. doi: 10.1002/smll.202301489.37300342
79. Richter M Vader P Fuhrmann G . Approaches to surface engineering of extracellular vesicles. Adv Drug Deliv Rev 2021;173 :416–426. doi: 10.1016/j.addr.2021.03.020.33831479
80. Avila-Calderón ED Ruiz-Palma MDS Aguilera-Arreola MG Velázquez-Guadarrama N Ruiz EA Gomez-Lunar Z , . Outer membrane vesicles of Gram-negative bacteria: An outlook on biogenesis. Front Microbiol 2021;12 :557902. doi: 10.3389/fmicb.2021.557902.33746909
81. Assoni L Girardello R Converso TR Darrieux M . Current stage in the development of Klebsiella pneumoniae vaccines. Infect Dis Ther 2021;10 :2157–2175. doi: 10.1007/s40121-021-00533-4.34476772
