Leveraging the intratumoral microbiota to treat human cancer: are engineered exosomes an effective strategy?

Abstract

Cancer remains a leading cause of global mortality. The tumor microbiota has increasingly been recognized as a key regulator of cancer onset and progression, in addition to shaping tumor responses to immunotherapy. Microbes, including viruses, bacteria, fungi, and other eukaryotic species can impact the internal homeostasis and health of humans. Research focused on the gut microflora and the intratumoral microbiome has revolutionized the current understanding of how tumors grow, progress, and resist therapeutic interventions. Even with this research, however, there remains relatively little that is known with respect to the abundance of microbes and their effects on tumors and the tumor microenvironment. Engineered exosomes are a class of artificial extracellular nanovesicles that can actively transport small molecule drugs and nucleic acids, which have the broad prospects of tumor cell therapy. The present review offers an overview of recent progress and challenges associated with the intratumoral microbiome and engineered exosomes in the context of cancer research. These discussions are used to inform the construction of a novel framework for engineered exosome-mediated targeted drug delivery, taking advantage of intratumoral microbiota diversity as a strategic asset and thereby providing new opportunities to more effectively treat and manage cancer in the clinic.

Introduction

Cancer remains a leading cause of global mortality, with an estimated 20 million new cancer diagnoses and 10 million deaths forecast worldwide in 2024 alone according to the International Agency for Research on Cancer. Lung cancer (12.4%) has once again become more common than breast cancer (11.6%), and it accounts for the greatest fraction of cancer-related mortality (18.7%), followed by colorectal cancer (9.3%). By 2050, a 77% increase in the global cancer burden is forecast relative to 2022, with an estimated 28.4 million cases [1].In the USA alone, the American Cancer Society projects the incidence of 2 million new cancer diagnoses and 0.6 million cancer-related deaths [2]. A similarly high burden of cancer has been reported in China, with a summary of trends in the cancer burden from 2005 to 2020 based on data from 300 million individuals revealing a 21% increase in total cancer-related deaths, with lung, liver, and gastric cancers being the leading contributors to such mortality [3]. Extensive efforts have sought to prevent and treat cancer in a curative manner, but the heterogeneity and diversity of tumors at the metabolic, phenotypic, genetic, and epigenetic levels have hampered these efforts [4]. It is thus essential that accurate strategies capable of evaluating this tumor heterogeneity be devised in order to define new therapeutic strategies.

Tumors consist of a complex microenvironment that includes both tumor and non-tumor cells together with the surrounding extracellular milieu [5]. Advances in next-generation sequencing technologies have led to growing interest in tumor-associated microorganisms owing to their diversity and ability to influence tumor growth and infiltration by host immune cells. Indeed, in contrast to prior assumption that malignancies comprise a sterile environment, tumors are increasingly recognized to contain many symbiotic microbial species [6]. The host microbiota plays a central role in shaping susceptibility to tumorigenesis and consequent tumor progression. Microbes present within tumors have the potential to indirectly impact proximal and distal tumor tissues through metabolites and the stimulation of host immunity, particularly in colorectal tumors that are in close contact with the gut microbiota [7]. However, research focused on intratumoral microbes remains in its infancy, and the overall levels of microbial biomass within tumors are fairly low such that much remains unknown with respect to how these microorganisms shape the process of tumor progression.

There are many strengths and limitations to all of the physical, chemical, and biological approaches to cancer treatment that have been developed to date, with immunotherapy recently emerging as a robust option for the systemic treatment of many malignancies. Engineering-based strategies such as those that entail the use of synthesized cell-targeting exosomes are also increasingly recognized as promising therapeutic tools, as they exhibit effective spatiotemporal targeting activity [8]. Exosomes have frequently been leveraged as diagnostic and prognostic biomarkers [9], and engineered exosomes are a novel platform for drug delivery that can help improve antitumor treatment efficiency while minimizing the side effects associated with such treatment [8]. When delivered intravenously, targeted exosomes specific for αv integrin-positive breast tumors, for instance, have been demonstrated to suppress tumor growth without causing substantial adverse toxicity, making them ideal tools for clinical administration [10]. While efforts to leverage exosomes as tools for drug delivery or genetic analyses have been summarized in many recent reviews [11], the present article instead focuses on the potential utility of engineered exosomes as they pertain to the intratumoral microbiota. To that end, we provide an overview of the characteristics and distribution of microbial communities present within tumors from humans, together with a discussion of the application of engineered exosomes for drug or gene delivery in various diseases, with a focus on the potential application of this strategy to target the intratumoral microbiota by using tumor microenvironment (TME) as a “bridge” (Fig. 1), emphasizing important opportunities and challenges in this therapeutic space.

Fig. 1

Sepeculation on the relationship between intratumoral microorganisms and engineered strategy. TME: tumor microenvironment. EVs: extracellular vesicles. DC: dendritic cell. Neut: neutrophils. Mac-1: macrophages type-1. Lym: lymphocyte. PTX: paclitaxel. DOX: doxorubicin. TNF-α: tumor necrosis factor-α

Cancers and intratumoral microorganisms

Although researches have found that microorganisms are commonly present in cancer, the number of microorganisms known to directly cause carcinogenesis remains small. With in-depth research on the gut and other microbial niches, the role of distinguishing between colonized and intratumoral microbiota is crucial [12]. A clear assessment and framework construction of the microorganisms in human based on cancer biology and existing evidence is an importment task. Accordingly, we briefly elucidated the microbial composition of gastrointestinal and non-gastrointestinal cancers, and evaluated their functions and subsequent pathway regulation.

Gastrointestinal tumors

In 2006, Nilsson et al. determined that Helicobacter pylori DNA was present in the pancreas of 75% of patients with pancreatic cancer [13], and since then there have been many other studies supporting the non-sterile nature of the pancreas. Riquelme et al. found that pancreatic cancer patients who experienced long-term survival exhibited a more diverse tumor microbiota, with the abundance of Pseudoxanthomonas, Streptomyces, Saccharopolyspora, and Bacillus clausii being predictive of prolonged survival cohorts [14]. Pushalkar et al. further conducted the 16 S rRNA sequencing of 12 pancreatic ductal adenocarcinoma (PDAC) patients, ultimately detecting Proteobacteria, Bacteroidetes, and Firmicutes as the most abundant members of the intratumoral microbial community [15]. Geller et al. also determined that Gammaproteobacteria are capable of metabolizing the active ingredient of gemcitabine, 2’,2’-difluorodeoxycytidine, into the inactive 2’,2’-difluorodeoxyuridine metabolite within PDAC tumors, providing resistance to this therapeutic [16]. Alam et al. further observed that the fungal mycobiome within tumors is a key determinant of the secretion of the pro-tumorigenic Interleukin 33 (IL-33), with IL-33 gene deletion of antifungal treatment being sufficient to suppress tumor-promoting T helper 2 cell (Th2) and type 2 innate lymphoid cell (ILC2) populations, thus improving rates of pancreatic cancer survival [17]. These analyses have yielded new insight into the mechanisms that drive the progression of PDAC, emphasizing the central role of the intratumoral microbiome in therapeutic resistance and metastasis.

Relative to normal adjacent tissues, colorectal tumors contain higher levels of Fusobacterium nucleatum (F. nucleatum) enrichment [18, 19]. Intratumoral bifidobacteria are strongly associated with signet-ring cells [20]. Zhang et al. found that F. nucleatum abundance in patients with colorectal cancer was positively correlated with ALPK1 and ICAM1 expression, inducing the ALPK1-mediated activation of NF-κB signaling to drive ICAM1 upregulation. This bacteria is also capable of promoting colorectal cancer cell adhesion to the endothelium, facilitating extravasation and metastatic dissemination [21]. Conversely, F. nucleatum has also been linked to better prognostic outcomes in oral squamous cell carcinoma [22]. Better prognostic outcomes in esophageal carcinoma (ESCC) have been should to be related to higher levels of Proteobacteria, Negativicutes, and Lactobacillus abundance, while greater Clostridia and F. nucleatum levels were linked to worse outcomes [23]. High F. nucleatum levels are also associated with adverse neoadjuvant chemotherapy outcomes in patients with ESCC [24]. Wu et al. detected a positive correlation between Streptococcus enrichment and infiltration by GrzB + and CD8 + T cells within tumors, with the abundance of these bacteria serving as a valuable predictor of ESCC survival and of the efficacy of anti-PD-1 treatment [25]. Intratumoral microbes can thus play complex dual-sided roles in this oncological context, shaping tumor fate in negative or positive ways.

Chai et al. conducted the 16 S rRNA sequencing of intrahepatic cholangiocarcinoma (ICC) samples and thereby detected high levels of Burkholderiales, Pseudomonadales, Xanthomonadales, Bacillales, and Clostridiales abundance and a significant increase in Paraburkholderia fungorum (P. fungorum) levels in paracancerous tissues, with the levels of this microbe being negatively correlated with Carbohydrate antigen199 (CA199) levels. P. fungorum was further determined to regulate alanine, aspartate, and glutamate metabolism as a means of exerting antitumor effects [26]. Sun et al. performed the extensive 16 S rDNA sequencing of hepatocellular carcinoma (HCC) tissue samples and found that the phyla most common in the TME were Proteobacteria and Actinobacteria [27]. Huang et al. further determined that the phyla most closely associated with the HCC microbiota as compared to normal tissues were Patescibacteria, Proteobacteria, Bacteroidota, Firmicutes, and Actinobacteriota. Gammaproteobacteria exhibit particularly high abundance within HCC tissues [28], and have been linked to the histological severity of non-alcoholic fatty liver disease [29]. Streptococcus and Lactococcus microbes have also been found to be present at higher levels in cirrhotic HCC relative to non-cirrhotic HCC, with selective Staphylococcus and Caulobacter enrichment in HCC cases not linked to infection by Hepatitis B Virus (HBV) [28]. Gut microorganisms have been posited to influence hepatic disease through the transport of bacteria and metabolites to the liver via the entero-hepatic axis through the vascular and portal circulation, intermixing the microbiota of HCC and the normal microbiota in liver such that the HCC-specific microbiome remains poorly understood and challenging [30]. These factors can also interfere with the absorption and efficacy of many drugs, and may have implications for liver toxicity.

Non-gastrointestinal tumors

Among the non-gastronintestinal tumors, lung cancer and breast cancer (BC) have the highest incidence [1]. Unlike the gut, these tumors are in a relatively sterility environment, so that researchers are more inclined to explore the role of intratumoral microbiota in shaping TME and their effect plays in anti-tumor immunity [31]. However, there have not been any systematic studies to date focused on differentiating between the intratumoral microbiota associated with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tumors. Accordingly to reports, members of the Firmicutes phylum and the Clostridium genus have been suggested to be the predominant bacteria present in lung tumors, engaging in symbiotic relationships with normal lung tissue [32]. In lung tumors from patients who exhibit recurrence following chemotherapy, high levels of F. nucleatum can reportedly trigger the invasivity and metastasis of tumor cells through assorted mechanisms [33]. Liu et al. determined that Aspergillus sydowii is capable of promoting the progression of LUAD through the secretion of IL-1β via the β-glucan/Dectin-1/CARD9 pathway, thus triggering the expansion and activation of myeloid-derived suppressor cell (MDSC) populations in a manner that interfered with cytotoxic T cell activity and resulted in PD-1⁺ CD8⁺ T cell accumulation [34]. Jin et al. determined that pulmonary symbionts are capable of stimulating Myd88-dependent IL-1β and IL-23 production by myeloid cells, thereby inducing Vγ6Vδ1 γδ T cell activation and proliferation, leading to the release of IL-17 and a range of other effector molecules, promoting inflammatory activity and the progression of tumors [35]. Higher levels of relative Acidovorax and Comamonas abundance have also been described in patients with LUSC who were smokers and harbored mutations in TP53 [36, 37]. The MEG3 lncRNA has also been found to activate TP53, controlling the proliferation and apoptotic death of cancer cells, with the levels of this lncRNA being significantly negatively correlated with Comamonadaceae abundance such that decreased MEG3 expression is indicative of more advanced pathological staging and poorer prognostic outcomes [38]. With the widespread use of next-generation sequencing technology, genomic analysis of lung cancer microbiota has also become general. Surprisingly, the previously thought sterile lower respiratory tract actually has a highly diverse micrpbiota [39], which remains a knowledge gap with potential relationship between lung cancer progression and treatment.

By leveraging 16 S rRNA sequencing, Urbaniak et al. have determined that BC patients exhibit higher levels of DNA damage-associated bacterial abundance relative to healthy women including higher levels of relative Bacillus, Enterobacteriaceae, and Staphylococcus abundance, including increased Escherichia coli and Staphylococcus epidermidis levels [40]. Thompson et al. found that certain bacteria including Listeria and Haemophilus infleunzae are linked to BC proliferation and epithelial-mesenchymal transformation (EMT) induction [41]. Pseudomonas aeruginosa Mannose-sensitive hemagglutinin (PA-MSHA) also reportedly exhibits anti-proliferative activity in BC such that it has been explored as a tool for treating cases of HER-2 negative metastatic BC [42]. Multiomics analyses of triple-negative breast cancer (TNBC) patients have also revealed an increase in the abundance of Clostridiales and the associated metabolite trimethylamine N-oxide (TMAO) in tumors exhibiting an activated immune microenvironment, raising the possibility for treatments focused on the BC microbiota-metabolite-immunotherapy axis [43]. Ongoing research efforts are also exploring the relationship between intestinal bacteria and estrogen metabolism in the context of BC, with a particular focus on the distribution of bacteria through mesenteric lymph nodes [44]. However, many unknowns persist with respect to the composition and correlative relationships between the intestinal and intratumoral microbiota. We have provided a brief summary of the intratumoral microbiota and their activated biological effects and responsive pathways in different cancers, as shown in Table 1.

Significantly, in TME, the fluid shear stress in the systemic circulation may be unfavorable for metastasis and colonization of tumor cell clusters (CTCs), leading cell apoptosis or necrosis [45]. However, bacteria attached to these tumor cells can regulate their responses to stress, thus impacting their viability. Fu et al. utilized the MMTV-PyMT murine model of spontaneous breast cancer (BC) and determined that intratumoral microbes carried by CTCs were associated with enhanced host cell resistance to fluid shear stress in the context of tumor cell metastatic progression, influencing the actin cytoskeleton in a manner that better allows these CTCs to survival and metastasize to distant sites [46]. Intracellular bacteria are thus capable of migrating to distant organs along with host CTCs, providing an opportunity to establish new metastatic microbial communities. We speculate that compared to gut colonizing microbes, the beneficial effects of intratumoral microbes on malignant cell survival may thus be most apparent in the context of metastatic progression rather than during the growth of primary tumors, underscoring the potentiality of the intratumoral microbiota for efforts aimed at widespread metastatic tumor dissemination.

Table 1 The major effects of intratumoral microbiota in pan-cancer

However, the situation in the composition and abundance of entire microbiota in different types of cancer remain unresolved issue. On the one hand, high abundance of microorganisms does not necessarily mean the tumor-promoting pathogenic bacteria, such as Pseudoxanthomonas, Saccharopolyspora, Ptreptomyces in PDAC, which are related to a better prognosis [14]. On the other hand, certain probiotic bacteria, such as Lactobacillus, Bifidobacterium have double-sided properties. For examples, high abundance of Lactobacillus in BC and prostate cancer can stimulate the host to release proinflammatory cytokines to promote antitumor immunity, while in HCC, high abundance of Lactobacillus is related to the cirrhotic type and poor prognosis [28, 46, 64]. Bifidobacterium promotes polarization of M2-like macrophages in CRC to aid immune evasion but in melanoma, it induces NK cell–dependent tumor immunity and improves the efficacy of anti-PD-1 immunotherapy [20, 61]. F. nucleatum is considered a pathogenic bacteria that promotes progression and chemotherapy in the vast majority of cancers, but in oral cancer, it is associated with better prognosis [22]. Therefore, identifying the composition and abundance of different cancer microbiota, as well as the source of these microbiota, is very challenging.

Intratumoral microorganisms and the immune-related TME

TME is a dynamic and complex environment containing myriad immune and non-immune cell types. Using a single-cell sequencing approach, Lam et al. determined that in the absence of a microbiota the TME skews towards a phenotype dominated by pro-tumorigenic macrophages, whereas STING agonists derived from microbes (including Akkermansia muciniphilia-derived c-di AMP) are capable of inducing the production of type I interferon by monocytes within the TME, regulating cross-talk between natural killer (NK) cells and dendritic cells (DCs), and reshaping intratumoral immunity [59]. Sheng et al. determined that the prognosis of ovarian cancers exhibiting M1 macrophage infiltration is better and that such M1 infiltration was associated with the presence of five bacteria (Achromobacter deleyi, Microcella alkaliphila, Devosia sp. strain LEGU1, Ancylobacter pratisalsi, and Acinetobacter seifertii), among which Acinetobacter. seifertii was found to suppress the migration of macrophages [57]. Kras^G12D oncogene expression in PDAC can drive IL-33 upregulation and secretion in a manner dependent on intratumoral fungi, with PDAC tumors recruiting and activating Th2 and ILC2 populations to secrete protumorigenic cytokines that stimulate tumor growth and induce immune invasion such that IL-33 deletion can interfere with these processes and cause tumor regression [17]. Intratumoral fungi thus hold potential as targets for therapeutic intervention. Certain bacteria present in PDAC tissues including Saccharopolyspora, Pseudoxanthomonas, and Streptomyces may also contribute to antitumoral immunity through CD8 T cell recruitment and activation [14]. The invasion of cancer cells by bacteria can support myeloid cell recruitment, provoking JAK/STAT signaling-mediated inflammation and promoting the rejection of T cells and the growth of tumors through the secretion of particular cytokines and chemokines into the surrounding environment [7]. Members of the resident microbiota within tumors including Lachnoclostridium, Gelidibacter, Flammeovirga, and Acinetobacter exhibit positive correlations with the expression of CXCL9, CXCL10, and CCL5, and they can further impact the infiltration of tumor tissues by CD8 + T cells, thereby improving cutaneous melanoma patient survival [60]. In other studies, functional Proteus mirabilis and Rhodopseudomonas palustris have been isolated from solid tumors and found to stimulate immune cell production, particularly the production of cells that express TNF-α, thereby fostering antitumor immunity and underscoring the promise of intratumoral microorganisms in the setting of cancer treatment [61].

Certain key signaling pathways can also be activated by the microbes present within tumors, thereby modulating the immune microenvironment. Bifidobacterium can activate STING signaling within DCs, altering anti-CD47 immunotherapy responses [66]. Bacillus fragilis (B. fragilis)-derived toxins can stimulate Reactive Oxygen Species(ROS) signaling and damage the DNA of intestinal epithelial cells, inducing the upregulation of the Spermine oxidase (SMO) catabolic enzyme induced in response to inflammation and thereby supporting malignant cellular transformation [50]. Porphyromonas gingivalis is capable of activating RAS and initiating MAPK/ERK signaling activity, thereby increasing colorectal cancer cell proliferation and invasivity [52]. F. nucleatum and B. fragilis are capable of activating E-cadherin/β-catenin signaling, with the FadA virulence factor of F. nucleatum enabling it to adhere to epithelial cells and to trigger the phosphorylation of E-cadherin at the cell membrane, in turn reducing β-catenin phosphorylation and activating a range of transcription factors (including TCF and NF-κB), oncogenes (including c-Myc and cyclin D1), and chemokines (including IL-6, IL-8, and IL-18) [67, 68]. F. nucleatum is also capable of activating TLR4 (Toll-like receptors 4) and downstream NF-κB/AKT signaling, further promoting the upregulation of inflammatory mediators that include IL-1β, IL-6, IL-8, and TNF-α [69,70,71]. In addition to these signaling pathways, intratumoral microbes can activate various other pathways. Fungi such as Malassezia have been reported to be significantly enriched in PDAC, with oncogenic Kras-induced inflammatory activity resulting in fungal dysbiosis and mannose-binding lectin (MBL) activation, thus initiating the C3 complement cascade in a manner that promotes progressive disease [72]. Intratumoral bacteria can inhibit RhoA/ROCK signaling, allowing CTCs to adapt to shear stress through cytoskeletal adjustment in a manner that facilitates the distant metastasis of BC cells [46]. Certain metabolites derived from bacteria, including trimethylamine N-oxide (TMAO), are also capable of mediating PERK signaling activity and activating intracellular endoplasmic reticulum stress in cells, thereby promoting the proliferation of malignant tumor cells and associated angiogenic activity [43].

As noted above, few studies to date have probed the relationship linking the intratumoral microbiome and the TME, with the TME playing a well-established role in the proliferation of tumor cells, disease progression, and antitumor therapeutic resistance [73]. Escherichia coli was recently found to interfere with tumor gemcitabine treatment [74]. F. nucleatum can reportedly promote resistance to oxaliplain in colorectal cancer through the production of exosomes rich in miR-1246/92b-3p/27a-3p and CXCL16/RhoA/IL-8, thereby supporting normal cell infection [75]. Further advances in research focused on the microbiome present in the TME may provide new opportunities for adjuvant antitumor treatment (Fig. 2).

Fig. 2

The association between microorganisms and TME. NK: nature killer cells. Mo: monocyte. IFN-γ: interferon-γ. PERK: protein kinase RNA-like ER kinase. JAK-STAT: janus kinase-signal transducer and activator of transcription. NF-κB: nucleat factor-κ-gene binding. AKT: PI3K-Akt signal pathway, proteinkinase B. MAPK: mitogen-activated protein kinase. ERK: etracellular regulated protein kinase

Therapeutic prospects of engineered exosomes

Exosomes are extracellular nanovesicles secreted from virtually all types of cells under pathological and physiological conditions alike. These exosomes can enable the delivery of various proteins, mRNAs, microRNAs (miRNAs), lipids, and other metabolites from donor cells to their recipients [76]. Exosomes have also been suggested to support the intercellular spread of certain infectious microbes. Certain exosomes also contain microbial compounds that can activate macrophages and induce antigen presentation [77], and these vesicles can additionally influence processes including intracellular autophagy [78] and immune surveillance and evasion, impacting circulating exosomes [79]. Engineering strategies for exosomes have recently focused on the highly specific targeted delivery of drugs or gene therapies while maintaining low levels of immunogenicity and toxicity [11], providing a promising avenue for the cell-free treatment of various human diseases. These engineering strategies seek to deliver drugs or other cargoes to sites that can be difficult to access while minimizing the toxic side effects associated with their administration.

Gene delivery

The use of engineered exosomes to deliver functionalized DNA into cells through endocytic uptake has recently been explored as a cancer treatment strategy. Bai et al. devised engineered Tlyp-1-targeting exosomes to efficiently deliver siRNAs specific for the membrane receptors NRP1 and NRP2 to NSCLC cells, effectively penetrating tumor blood vessels and stromal tissues to access deep sites within these tumors [80]. Kamerkar et al. recently generated novel engineered exosomes for Antisense oligonucleotides (ASOs) delivery (exoASOs) specific for STAT6, thereby achieving robust antitumor activity in syngeneic colorectal cancer and hepatocellular carcinoma model systems [81]. Kim et al., for instance, demonstrated that exosomes derived from SKOV3 ovarian cancer cells could undergo Exosome-liposome hybrids CRISPR/Cas9 plasmid electroporation such that they were capable of effectively inhibiting PARP-1 and thereby suppressing tumor growth [82]. Kojima et al. prepared what they termed EXOsomal transfer into cells (EXOtic) devices utilizing an archaea-derived L7Ae peptide capable of binding to the C/D_box RNA structure and fusing this with CD63, which is an exosomal marker protein, thereby allowing for the recruitment of functional C/D_box-containing mRNAs into budding exosomes such that they could be delivery to the cytosol of target cells [83]. Wang et al. utilized exosomes for the delivery of HChrR6-encoding mRNAs to HER2 + ve BC cells, largely arresting BC cell growth [84]. Engineered exosomes therefore hold promise for the delivery of DNA and other nucleic acids as an avenue for cancer treatment.

These optimistic research findings have led us to made reasonable conjecture about the prospects of gene delivery in the microbial community, like packaging overexpressed or low-expressed functional genes in the form of exsomes, which are delivered to the destination by tool cells. By changing the imbalance of the microbiota, inhibiting the growth of drug-resistant microbiota and other disadvantaged community, combining traditional gene delivery modes to enhance the anti-tumor effect.

Drug delivery and vaccination

The technology of delivering small molecule drugs or compounds in extracellular form to target areas has also emerged in recent years. Imatinib (a selective inhibitor of BCR-ABL) and BCR-ABL siRNA-loaded IL-3 exosomes have been utilized for the effective killing of chronic myeloid leukemia cells, prolonging the survival of mice bearing these tumor xenografts [85]. Separately, HER2-binding affibody zHER exosomes were reported to bind with high levels of affinity to colon cancer cells expressing HER2, thereby allowing for the delivery of 5-Fluorouracil and anti-miRNA-21 [86]. Macrophage-derived exosomes loaded with paclitaxel (PTX) also reportedly exhibit robust antitumor activity in a murine model of pulmonary metastasis [87]. Mesenchymal stromal cells (MSCs) have also been suggested to serve as a platform for PTX delivery through the use of targeted microvesicles [88]. The lysosome-associated LAMP-2B membrane protein exhibits high levels of expression on exosomes derived from DCs, and it supports the specific and efficient delivery of doxorubicin to BC tissues, thus suppressing the growth of these tumors [10]. Ma et al. also found that by using tumor cell-derived microparticles loaded with antitumor drugs, they were able to enhance drug utilization rates while overcoming therapeutic resistance in tumor-repopulating cell populations [89].

Tumor cells are also capable of releasing exosomes that can disrupt immune functionality, and these tumor-derived exosomes have potential value as customized vaccine tools for cancer immunotherapeutic treatment. In addition to fostering the antitumor activity of CD8 + T cells, tumor-derived exosomes have been found to suppress the growth of other tumors exhibiting the expression of matching rejection antigens [90]. DC-targeting vaccines have been explored as tools for the immunotherapeutic treatment of cancer in certain clinical trials (NCT00683670; NCT00769704) [91, 92]. Huang et al. utilized α-lactalbumin (α-LA)-engineered BC-derived exosomes loaded with the TLR3 agonist Hiltonol and human neutrophil elastase (ELANE) as inducers of immunogenic cell death, thus producing an in situ DC vaccine designated [93]. Exosomes derived from pancreatic cancer can stimulate NK cell migration and lysis of tumor cells exhibiting surface positivity for HSP70 [94]. These immunotherapeutic vaccines, however, are fairly weak and are currently the subject of early-stage clinical trial efforts focused on the further modification of exosomes with the goal of bolstering their immunogenicity.

Table 2 Engineered exosomes for targeted gene and drug delivery

Table 2 has summarized some existing studies on gene or drug delivery for targeted therapy. However, the engineering exosome strategies still have significant limitations. Except for some preclinical studies that utilize tool cells to reach the target area and exert antitumor effects, oral colon-targeted nanomedicine delivery system is considered as a promising approach to cancer therapy [99, 100]. For examples, Li et al. have established, for the first time a nanocarrier containing hollow MnO2 loaded with the chemotherapeutic drug Oxa (Oxa@HMI), which can promote chemotherapy efficiency and activate antitumor immunity by intervening in the intratumoral microbial environment [99]. Furthermore, Lang et al. have shown that the nanoparticles (NPs) using the prebiotic xylan-stearic acid conjugate (SCXN) loaded with capecitabine can improve the effects of chemotherapy in CRC [100]. Additionally, magnetic natural lipid NPs have been used to improve antitumor immunity and regulate microbiota metabolites in CRC [101]. Although the above techonologies are against the background of the microbiota for treatmeny of intestinal tumors, there are still very few intratumoral microbiota engineering strategies targeting other cancers. Moreover, a series of physical, chemical, and microbial barriers in the intestine will hinder the delivery and absorption of nanomedicine so that oral colon-targeted drug system still faces significant challenges and development opportunities.

Connections and perspectives

In the hypoxic TME, tumor-derived exosomes can support immune evasion while also enhancing the survival, growth, angiogenic activity, autophagy, EMT, therapeutic resistance, and metastasis of tumor cells [102]. PD-1/PD-L1 are key immune checkpoint molecules that influence the expression of transcription factors including HIF, PTEN, CDK5, p53, BRD4, and STAT, modulating signaling through the PI3K/Akt/mTOR and MAPK pathways, among others [103]. PD-1/PD-L1 expression is observed on many subsets of immune cells including MDSCs and MDSC-induced PD-1⁻PD-L1⁺B-cell subsets [104]. Exosomal PD-L1 has been advanced as one mechanism through which tumors can evade or resist immune-mediated elimination, repressing the function of effector T cells while triggering the suppression of antitumor functionality throughout the TME [105]. As noted above, A. sydowii within tumors is capable of driving LUAD progression through MDSC activation and PD-1 + CD8 + T cell accumulation, thereby suppressing T cell activity [33]. Hypoxia and HIF-1 also play a role in the activation of the tumor cell surface recognition protein CD47, with PD-1 blockade driving CD47/SIRPα pathway induction and inhibiting the growth of tumors through the inhibition of immunosuppressive mediators including MDSCs, tumor-associated macrophages, DCs, and effector T cells [106]. Bifidobacterium can reportedly activate DCs and alter anti-CD47 immunotherapy responses [66]. Engineering strategies for the administration of anti-PD-1/PD-L1 may thus provide dual efficacy as a means of targeting host cells and the microbiota.

Nejman et al. determined that tumor-associated bacteria are largely found in the cytosol of tumor and immune cells [6]. Microbes found within cells are capable of disrupting signaling and the exchange of substances amongst host cells. For example, Listeria spp. can infect antigen-presenting cell populations including monocytes/macrophages, DCs, and MDSCs, supporting selective bacterial delivery to tumors wherein they can be shielded from immune-mediated elimination [107]. The precise effects that the intratumoral microbiome has on antitumor immune activity are dependent on the composition of this microbial community, interactions between these microbes and the cancer, as well as the characteristics of the tumor in question. The microbes present within tumors can provoke enhanced antitumor immune responses by activating T and NK cells, producing microbe-derived antigens, in addition to activating the ROS, β-catenin, TLR, ERK, NF-κB, and STING signaling pathways [31, 108]. A variety of approaches has been leveraged with the goal of improving engineered exosomal targeting. Phospho-activatable silencing exosomes, for instance, have been designed by loading siPAK4 and photo-activatable ROS-sensitive polymer nanocomplexes into M1 macrophage-derived extracellular vesicles, thereby facilitating PAK4 silencing and TME modulation [109]. Pan et al. prepared Exo-PMA/Fe-HSA@DOX nanocomplexes carrying tumor cell membrane-associated antigens including E-cadherin and CD47, targeting the EGFR/AKT/NF-kB/IKB pathway to achieve improved prostate cancer targeting [110]. F. nucleatum is found in many tumor tissues [18, 22, 24, 32], wherein its virulence factor FadA can modulate E-cadherin and β-catenin phosphorylation, activate NF-κB/AKT signaling, induce inflammatory activity, and drive the production of metabolites [67, 69]. In some recent studies, researchers have found that encapsulate antibiotic silver-tinidazole complex in liposomes can effectively eliminated F. nucleatum in primary CRC and liver metastases [111]. Additionally, in the mouse model of CRC, phage-guided NPs can target the delivery of irinotecan to tumor cells and eliminate intratumoral F. nucleatum to enhance the chemotherapeutic efficacy [112]. There is also an new F. nucleatum-mimetic nanomedicine by fusing F. nucleatum cytoplasmic membrane (FM) with Colistin-loaded liposomes, which selectively kills colonizing F. nucleatum without affecting other gut microbes [113]. In view of these, F. nucleatum may thus hold value as a microbial target for the development of engineered exosomes, especially in gastrointestinal diseases. Table 3 shows the crosstalk between intratumoral microorganisms and potential pathways in TME, as well as the existing engineering strategies for these related signaling pathways. In conclusion, complex and intricate interconnected relationships thus exist between exosomes or NPs and the microbial communities present in hosts (Fig. 3). We have reason to believe that microorganisms play a crucial role in the evolution of tumors, activation of related pathways, and alterations of immune TME. Conversely, host nutrition and immune TME may reshape microbial function, metabolism, and composition during their colonization. As a communication deveice between living cells, microbiota, and the outside world, exosomes can stably release and assimilate durgs, therapeutic microRNAs and proteins, what hold tremendous promise in targeted microbial therapy.

Table 3 The crosstalk between pathways, intratumoral microbiota, engineering exosomes or nanoparticles strategies

Fig. 3

Engineered strategies may affect cancer cells and microbiota. Exo-PD-L1: exosome-programmed cell death-ligand 1. Exo-ASO: exosome-antisense oligonucleotides. Exo-GELNs: exosome-Ginger like nanoparticles. PASEV: photoactivatable silencing extracellular vesicle. APCs: antigen presenting cells. EGFR: epidermal growth factor receptor. mTOR: mammalian target of rapamycin signaling pathway. CDKs: cyclin-dependent kinases. IKB: I-kappa-B-alpha. HIF-1: hypoxia inducible factor-1. SIRPα: signal regulatory protein α. PTEN: phosphatase and tension homology deleted on chromosome ten. B.fragilis: bacteroides fragilis. F. nucleantum: fusobacterium nucleatum

Future challenges

While these past studies highlight certain tantalizing opportunities, there remain several roadblocks to the clinical application of these findings. For one, the sensitivity of intratumoral bacteria to antibiotics and other drugs generally differs from that of gut microbes such that they may be able to resist gentamicin or ampicillin [31], which have certain difficulties for selection of specific antibiotics. Although most of the current research focuses on oral colon-targeted delivery system, the ability of the gut microbiome to regulate cancer immune responses and drug absorption can also complicate cancer treatment efforts. Moreover, some intratumoral microbiota may colonize in the tumor through decreased intestinal permeability or blood [127], so tools for ultimately distinguishing between intestinal and intratumoral microbes are lacking, as are any approaches to conclusively differentiating between in situ versus contaminating microbes. The necessary technologies to synthesize or extract exosomes are also limited, and preloaded exosome engineering strategies are not suitable for all cargoes as exosomes are roughly 30–200 nm in diameter and exhibit a composition similar to that of the cell membrane [128]. As a result, they are subject to volume limitations and are not compatible with some NPs or nanomedicines that can cause membrane damage. Although animal model work in this therapeutic space has yielded some promising findings [85, 87], a large amount of work focused on cargo selection, preloading strategies, and dose selection will be vital to broadly employ these engineered tumor treatments. Additionally, it is noteworthiness that the distribution of microorganisms within the tumors and other normal tissues is flexible and dynamic. It is curious whether there is the engineering strategy for bidirectional delivery that can change the microbial distribution and enhance the effectiveness of chemotherapy or targeted drugs in terms. However, concerns regarding the safety of this interventional strategy will also need to be addressed, and additional studies will be necessary to clarify whether intratumoral microbes and exosomes are causes or effects of oncogenesis, and what mechanisms underlie these relationships. It is clear that engineered exosomes and the microbiome are likely to have a profound bearing on the future treatment of human cancer.

Conclusion

In summary, this review offers a high-level overview of the current status of the intratumoral microbiome and engineered exosomes in cancer-relates research, with a further discussion of the therapeutic potential for their interconnection. Despite the challenges that persist in this setting, there remain many important avenues for future research, and the ability of the microbiome and exosomes to reshape the TME as a means of enhancing the immunotherapeutic sensitivity of tumors is a key area for future research. We believe that this review will provide an evidence-based foundation to fuel further research efforts aimed at improving innovation in the cancer treatment field, thereby aiding the eradication of malignant cells without harming normal healthy cells.