Colorectal cancer (CRC) is a common malignant digestive tract tumor. Currently, CRC has the world’s third highest new cancer incidence rates, and is one of the major life-threatening cancers (Siegel et al., 2020; Miller et al., 2022). In recent years, overall CRC incidence rates have increased in individuals under 50, which is a worrying trend (Spaander et al., 2023). At present, surgery, radiotherapy, and chemotherapy are the main CRC treatment methods. Additionally, targeted therapies and immunotherapies have generated some therapeutic effects in metastatic CRC (Kuipers et al., 2015). However, these new treatment options have had limited impact on CRC cure rates and long-term survival.

The human gut is a complex ecosystem where trillions of microorganisms (bacteria, fungi, and viruses) live. These microflora exert a significant impact on human health and disease (Fan and Pedersen, 2021). Considerable evidence now suggests that the gut microbiota is strongly associated with CRC development and prognosis outcomes (Meng et al., 2018; Wong and Yu, 2019; Song et al., 2020; Liu et al., 2022c). Microbiota dysbiosis modulates host metabolic and immune pathways, disrupting intestinal homeostasis, and promoting cancer development and progression (Weiss and Hennet, 2017; Gopalakrishnan et al., 2018). Also, modulating the gut microbiota and reversing established microbial dysbiosis may help prevent and treat CRC (Fong et al., 2020).

In the last decade, research interest in bacterial extracellular vesicles (BEVs) has increased exponentially, with BEV-based cancer therapies attracting major attention in the biomedical field (Chronopoulos and Kalluri, 2020; Sartorio et al., 2021). Of these, outer membrane vesicles (OMVs) are spherical, bilayered nanostructures released by Gram-negative bacteria, and are natural bacterial component carriers. These vesicles regulate intercellular communications by transferring lipopolysaccharide (LPS), lipids, proteins, nucleic acids, and toxins (Nagakubo et al., 2019; Toyofuku et al., 2019, 2023). Gut microbiota-derived OMVs, as key mediators of bacteria-host interactions, have reportedly multiple functions, including but not limited to, horizontal gene transfer (Domingues and Nielsen, 2017), immune response modulation (Kaparakis-Liaskos and Ferrero, 2015), regulating intestinal epithelial barrier integrity, and gut homeostasis (Díaz-Garrido et al., 2021). These properties provide specific and unique advantage in cancer diagnostics, vaccine development, targeted drug delivery, and other clinical tumor treatments (Chronopoulos and Kalluri, 2020; Sartorio et al., 2021).

In recent years, the pathological impact of BEVs on gastrointestinal cancers has been partially revealed. BEVs and contents have been promoted as biomarkers for early gastrointestinal cancer diagnosis (Barteneva et al., 2017). However, specific mechanisms and potential effects underlying interactions between gut microbiota OMVs and CRC remain unclear. Therefore, we reviewed research progress in OMV composition and function, and summarize the gut dysbiosis in CRC. Then, from a microbiota-host communications perspective, we focused on potential roles and related mechanisms of gut microbiota OMVs in regulating CRC progression. Finally, OMV prospects and limitations for CRC diagnosis and treatment were discussed.

Gut microbiota refers to about 40 trillion microbes living in the human intestinal tract. Normal gut microbiota have vital roles in gut homeostasis, host nutrient metabolism, immune regulation, and pathogen defenses. In contrast, gut microbiota dysbiosis exerts adverse effects on human health, resulting in many chronic diseases (Jandhyala et al., 2015; Chen et al., 2021; Liu et al., 2022c).

A growing body of evidence suggests a link between dysbiosis of the human gut microbiota and colorectal cancer. The gut microbiota of CRC patients showed Intestinal dysbiosis with lower abundance of potentially protective taxa and increased abundance of procarcinogenic taxa compared to healthy individuals (Gagnière et al., 2016; Yu et al., 2017; Wong and Yu, 2019). High-throughput microbiome sequencing has indicated consistent correlations between CRC and several specific bacteria, such as Fusobacterium nucleatum, Escherichia coli, Bacteroides fragilis, and Enterococcus faecalis were increased in CRC patients, whereas Bifidobacterium, Lactobacillus, and Faecalibacterium prausnitzii were absent (Zou et al., 2018; Table 1). For example, Liu et al. used 16S ribosomal RNA (rRNA) gene amplicon sequencing to analyze the composition of the gut microbiota in healthy controls, polyp patients, and colorectal cancer patients. They found that Bacteroidetes, Firmicutes, and Proteobacteria were enriched in CRC patients compared to healthy individuals, and many other researchers have come to the same conclusion (Liu et al., 2020). This study not only confirmed the significant differences in diversity and composition between the microbiota of CRC patients and healthy controls, but also found that the proportion of microorganism composition varies at different stages of CRC development. In addition, metagenomic studies in humans have identified novel associations between CRC and other bacteria. By performing metagenome-wide association studies on fecal samples, Yu et al. not only confirmed the known association of Fusobacterium nucleatum and Peptostreptococcus anaerobius with CRC, but also identified other members of specific microorganisms (such as Parvimonas micra and Solobacterium moorei) that are significantly associated with CRC (Yu et al., 2017).

In the imbalance of intestinal flora, pathogenic bacteria replace harmless commensal bacteria, causing inflammation in the host, promoting cell proliferation, and inducing carcinogenic signals and metabolites, thus contributing to the onset and progression of CRC. This process may occur through different mechanisms, including bacterial-derived genotoxins, microbial-derived metabolism, regulation of host defenses and inflammatory pathways, oxidative stress induction, and antioxidant defense regulation (Gagnière et al., 2016; Wang and Li, 2022). For example, Fusobacterium is one of the most common bacterial species in CRC tissues and metastases with CRC cells. Fusobacterium nucleatum has been shown to modulate several immune responses against colorectal cancer progression, induce the production of inflammatory mediators, and affect the tumor microenvironment (Castellarin et al., 2012). By using the Apc (Min/+) mouse model treated with human isolates of F. nucleatum, Kostic et al. (2013) found that F. nucleatum can generate a pro-inflammatory microenvironment by recruiting tumor-infiltrating myeloid cells, which is conducive for carcinogenesis. They also found that F. nucleatum can facilitate CRC progression by down-regulating anti-tumor T cell-mediated adaptive immunity (Kostic et al., 2013). In addition, related studies have found that colorectal carcinogenesis caused by F. nucleatum involves a variety of other mechanisms, including virulence factors, metabolism products, and miRNAs, and so on (Castellarin et al., 2012; Zhang et al., 2018).

Table 1 summarizes the changes in gut bacterial abundance and possible carcinogenic mechanisms in CRC. These studies have broadened our understanding of the role of gut microbiota in CRC pathogenesis.

Under the background that the role of gut microbial communities in CRC occurrence and development has been widely studied, microbe-derived OMVs have emerged as novel players in the biomedical field. They were first isolated from Gram-negative bacteria (Escherichia coli) in the 1960s (Knox et al., 1967). Subsequently, they were observed in different Gram-negative bacteria, including Vibrio cholerae, Salmonella typhimurium, and Pseudomonas aeruginosa (Chatterjee and Das, 1967; Rothfield and Pearlman-Kothencz, 1969; Kadurugamuwa and Beveridge, 1995). Previous studies have reported that two main vesicle formation routes exist in Gram-negative bacteria: (1) OMV generation via outer membrane blebbing and (2) explosive cell lysis, where outer-inner membrane vesicles (OIMVs) and explosive outer-membrane vesicles (EOMVs) are formed (Toyofuku et al., 2019; Tulkens et al., 2020a).

In this review, we mainly focus on OMVs, which are spherical membrane particles measuring 20–300 nm in diameter. They are produced via outer membrane blebbing of gram-negative bacteria and are rich in outer membrane proteins and lipids, which act as multifunctional secretion and transport mechanisms for bacteria (Guerrero-Mandujano et al., 2017; Sartorio et al., 2021). Additionally, OMVs carry many cargo molecules, including membrane-bound and periplasmic proteins, LPS, nucleic acids (DNA and RNA), and toxins (Toyofuku et al., 2019; Chronopoulos and Kalluri, 2020). Both cargo quantity and content are affected by environmental and genetic factors, and vary among strains (Orench-Rivera and Kuehn, 2016; Volgers et al., 2018; Godlewska et al., 2019). The general OMV structure and composition is shown (Figure 1).

Nowadays, several mechanisms of OMV biogenesis have been proposed, including a loss of cross-linking between outer membrane (OM) and peptidoglycan (PG) layers, accumulated PG fragments or misfolded proteins in the periplasmic space, LPS remodeling, and bilayer couple modeling (Sartorio et al., 2021).

Specific OMV cargo molecules and biogenic pathways of different bacterial OMVs generate different biological functions. OMVs participate in intracellular and intercellular communications and mediate many physiological processes, including quorum sensing, gene transfer, toxin delivery, stress responses, microbiota homeostasis, pathogenesis, and immune regulation (Díaz-Garrido et al., 2021; Sartorio et al., 2021; Toyofuku et al., 2023; Figure 2).

OMVs are closely related to pathogenesis because they carry adhesins, toxins, and immunomodulatory compounds (Koeppen et al., 2016; Tian et al., 2023). OMVs directly deliver multiple virulence factors to the host cytoplasm and protect their contents from host lyase and antibodies, thus ensuring toxin activity (Macion et al., 2021). For example, the enterotoxigenic Escherichia coli (ETEC) secretes heat-labile enterotoxin via OMVs, resulting in strain pathogenicity (Horstman and Kuehn, 2000; Kesty et al., 2004). Similarly, uropathogenic Escherichia coli (UPEC) OMVs deliver cytotoxic necrotizing factor type 1 (CNF1), thereby attenuating neutrophil antimicrobial activity and chemotaxis (Davis et al., 2006). S. typhimurium also uses OMVs to transport virulence factors. Unlike other bacteria, they produce OMVs during intracellular life and escape the infected host cell. Then, bystander cells internalize OMVs containing cytolethal expansion toxin via dynamin-dependent endocytosis (Guidi et al., 2013).

In addition to toxin transfer, OMVs serve as microbial defense mechanisms contributing to immune evasion by bacteria. OMVs provide protection for parent bacteria against harmful substances, including antibiotics, phages, and reactive oxygen species (Díaz-Garrido et al., 2021). Horizontal gene transfer through OMVs can facilitate antimicrobial resistance in bacteria (Domingues and Nielsen, 2017). In 2011, OMVs were first identified as antimicrobial resistance gene carriers, and by extension resistance mechanisms, by Rumbo et al. The authors demonstrated a horizontal carbapenem resistance gene transfer mechanism via OMV release in Acinetobacter baumannii (Rumbo et al., 2011). Also, as OMVs have similar surface structures to the bacteria they originate from, they can neutralize phage and prevent direct interactions with bacterial cells (Azam and Tanji, 2019). For example, V. cholerae OMVs acted as natural decoys against three unique virulent phages (ICP1, ICP2, and ICP3) and formed defenses protecting bacteria from phage predation (Reyes-Robles et al., 2018).

OMVs also facilitate bacterial nutrient acquisition which may serve as a mechanism for competition with other bacteria. Biofilm formation protects diverse bacterial communities and facilitates nutrient acquisition and survival (Flemming et al., 2016), with evidence now suggesting that OMVs are essential in biofilm formation and stabilization (Wang et al., 2015; Brameyer et al., 2018; Begić and Josić, 2020). For example, Seike et al. (2020) observed that OMVs released by Aeromonas strains dose-dependently promoted biofilm formation. Some OMVs are rich in hydrolytic enzymes, such as Bacteroidetes OMVs, which break down complex polysaccharides in the intestinal lumen into simple carbohydrates, thus facilitating nutrient acquisition for microbial communities (Elhenawy et al., 2014; Liang et al., 2022). Other studies have shown that OMVs from different species contain iron acquisition proteins and bacterial cell surface receptors that recognize heme groups, suggesting pivotal roles for OMVs in iron acquisition in different bacteria (Schwechheimer and Kuehn, 2015). In a recent study, Dhurve et al. (2022) found the presence of TonB-dependent transporters (TonRs) capable of transporting various nutrients in the outer membrane vesicles (OMVs) of Acinetobacter baumannii DS002. They capture and transport ferric iron complexed with enterobactin into A. baumannii DS002 cells. In addition to the TonRs, A. baumannii OMVs also carry proteins with pathogenesis, immune evasion, and biofilm formation (Dhurve et al., 2022).

In terms of microbial competition, some OMVs carry degradative enzymes (lipases and endopeptidases) which kill other bacteria. Evidence exists that some OMVs in the Myxococcus xanthus contain antimicrobial factors and hydrolases, which lyse target bacterial cells and enhance predation activity (Marshall and Whitworth, 2019).

Since microbial access to the intestinal epithelium is limited by the inner mucin layer, host communications mainly depend on microbiota-secreted factors (metabolites, proteins, and vesicles) which cross mucin layers and reach intestinal mucosa surfaces (Díaz-Garrido et al., 2021). Currently, growing evidence suggests that OMVs from gut microbiota origins are effective communication vehicles between the gut microbiota and hosts. OMVs produced by gut bacteria are detected in human body fluids and fecal samples and may critically influence host pathophysiology by regulating the gut microenvironment (Villard et al., 2021).

The main mechanisms whereby gut bacterial OMVs interact with human host cells include, OMV internalization by host cells, host immune response modulation, and the maintenance of gut environmental homeostasis (Kaparakis-Liaskos and Ferrero, 2015; Chronopoulos and Kalluri, 2020; Díaz-Garrido et al., 2021; Sartorio et al., 2021).

In addition, many studies support the beneficial effects of probiotics in CRC prevention and treatment (Ryu et al., 2022; Saeed et al., 2022; Huang et al., 2023). For CRC, the most common probiotics are Lactobacillus and Bifidobacterium, including other genera such as Enterococcus, Streptococcus and Leuconostoc (Zou et al., 2018; Wong and Yu, 2019). Like OMVs, probiotics can also regulate the gut microbiota through immunomodulation, inhibition of pathogenic bacteria colonization and enhancement of intestinal barrier function, thus improving host health (Liu et al., 2016). Importantly, some of these probiotics, such as Escherichia coli Nissle 1917 (ECN) and the intestinal commensal strain ECOR63, are able to protect the physical and biological barriers of the gut through the production of OMVs in addition to the secreted factors (the secreted protein TcpC) (Alvarez et al., 2016, 2019). Taking cues from these studies, probiotics may also be developed to modulate host intestinal function, providing new insights into the combined application of probiotics and OMVs for CRC management.

We examine the above perspectives and fouse on OMV interactions between the gut microbiota and the host.

Although gut microbiota roles in CRC occurrence and progression have been widely studied, few studies have focused on microbial-derived OMV effects on CRC. For example, it has been shown that Bacteroides fragilis and its secreted toxin are associated with CRC carcinogenesis (Chung et al., 2018; Zamani et al., 2019), however, for B. fragilis OMVs, it is unclear if they affect CRC occurrence and progression. Thus, researchers are expected to pay more attention to the relationship between OMVs of intestinal bacterial origin and gastrointestinal cancer. Recently, Park et al. (2021) performed a metagenomic OMV analysis in stool samples from CRC patients and controls using 16S ribosomal DNA gene amplicon sequencing. The authors identified significant differences in microbial composition, homogeneity, and diversity in gut microbiota-derived OMVs from CRC patients when compared with controls. Additionally, OMV composition and diversity in stool from late-diagnosed CRC subjects were significantly changed when compared with early-diagnosed CRC subjects (Park et al., 2021). Therefore, gut bacterial origin OMVs may have essential regulatory roles in CRC development and progression, similar to gut bacteria. Although there are no comprehensive studies demonstrating that OMVs directly affect CRC development and progression, some studies on the interaction of OMVs with CRC cells or TME could provide hints for their putative role in CRC (Table 2).

In recent years, OMVs have received considerable attention as emerging cancer treatment strategies (Chronopoulos and Kalluri, 2020; Li et al., 2020a). Due to their many properties, in particular high levels of immunogenic components, they have tremendous advantages as antitumor immunotherapies and drug-targeted therapies (Xie et al., 2022; Won et al., 2023). Several studies have examined bacterial OMVs for CRC treatment. However, like many new therapeutic approaches, more prospective studies are required in this field. In this section, we discuss the possibility of gut microbiota OMVs in CRC treatments from several perspectives (Figure 4).

As described, gut microbiota OMVs are important mediators of microbial-host communications and regulate host immune responses, gut homeostasis, and metabolism. They have great potential in biomedical applications, such as tumor vaccines, adjuvants, and drug carriers.

While considerable progress has been made in understanding the impact of OMVs on tumors, further studies are required on the role of gut microbiota OMVs in CRC occurrence and development. Currently, most studies on OMVs are at the initial stage of using cell lines without the use of animal models, and the conclusions of these studies may be less convincing or misleading. Therefore, in future research, more clinical trials must be conducted to better elucidate interactions between OMVs and CRC.

Additionally, limitations exist when translating OMVs from the laboratory to the clinic: (1) OMVs purification and isolation techniques are time-consuming and complex laboratory procedures. (2) Some OMVs have toxic effects on hosts as they carry cytotoxic components (e.g., LPS). (3) OMVs have low levels of protective antigen expression. (4) OMVs may carry uncontrollable components which interfere with and suppress host immune responses. To address these issues, we need to further refine the techniques isolating and detecting complex components from OMVs and incorporate the latest technologies (metagenomics, proteomics, metabolomics, and bioinformatics) to guide engineered OMV development and applications toward CRC therapy.

In conclusion, with relevant technology advances and some key challenges addressed, further research will help us understand the complex relationships between OMVs and the host. Undoubtedly, gut microbiota OMVs can become powerful novel cancer treatment tools, and provide new pathways establishing effective CRC prevention, diagnosis, and treatment strategies.

RM: Writing – original draft, Writing – review & editing. MZ: Writing – review & editing. YJ: Writing – review & editing. XH: Writing – review & editing. MX: Writing – review & editing.