Intratumoral Microbiome: Impact on Cancer Progression and Cellular Immunotherapy

1. Introduction

Immunotherapy represents a rapidly advancing field that has demonstrated significant progress in recent years with regard to the treatment of oncological diseases. The findings of research conducted to date indicate that this therapeutic method is effective in the suppression of tumor growth and the improvement of clinical outcomes. The treatment approach has been demonstrated to stimulate the host’s immune system, thereby restoring immunological functions and eliminating tumor cells over an extended period of time [1]. The employment of immunotherapy can be considered as a standalone treatment strategy or in combination with traditional methods, thereby demonstrating the potential for a synergistic effect [2]. Cellular immunotherapy can be defined as the administration of living immune cells to patients for therapeutic purposes. It may be active, as in dendritic cell vaccines that stimulate an antitumor immune response, or passive, as in adoptive cell transfer (ACT), where autologous or allogeneic lymphocytes with intrinsic antitumor activity are administered, either in their natural form or after modification [3]. Non-specific OCI encompasses a variety of cell types, including dendritic cells (DCs), natural killer cells (NKCs), cytokine-induced killer cells (CIKs), tumor-infiltrating lymphocytes (TILs), lymphocyte-activated killer cells (LAKs), and killer-induced macrophages (MAKs), among others. In specific OCI, immune cell activation is mediated by tumor antigens and specific stimulating factors. These methods include TIL-based therapy, T-cell receptor-transfer (TCR-T) technologies, and chimeric antigen receptor-positive T-cell (CAR-T) immunotherapy [4,5,6,7,8,9,10]. The U.S. Food and Drug Administration (FDA) has granted regulatory approval to several cell-based therapies. In the domain of immune cell therapies, a number of CAR-T products have received approval for the treatment of hematologic malignancies, including certain forms of leukemia, lymphoma, and multiple myeloma. Examples include tisagenlecleucel (Kymriah), axicabtagene ciloleucel (Yescarta), and idecabtagene vicleucel (Abecma) [11]. Furthermore, the TIL drug lifileucel (Amtagvi) has been approved for the treatment of adult patients with unresectable or metastatic melanoma [12]. However, immunotherapy still faces many challenges. These include relatively low efficacy in solid tumors, in part due to heterogeneity and the difficulty of targeting specific antigens. Other challenges include tumor microenvironment immunosuppression and complications in cellular drug delivery [13]. Furthermore, there are also safety concerns [14,15]. Immunotherapy has been shown to be associated with a risk of adverse effects, including unfavorable outcomes [16,17]. Nevertheless, the development of next-generation drugs and additional approaches aimed at increasing the effectiveness and safety of cell therapy is ongoing [18].

The human microbiome both positively and negatively contributes to carcinogenesis by influencing signaling pathways involved in inflammation, DNA repair, and stability [19]. Moreover, the gut microbiome is widely recognized as a factor that significantly influences the regulation of local and systemic immune responses in mouse models and human studies [20]. It has been demonstrated that the composition of the microbiota, particularly the levels of abundance of individual bacterial taxa, exerts a substantial influence on the efficacy of the host’s immune response [21]. The relationship between the microbiome and immune cells used in immunotherapy is a relatively new and understudied area of research [22]. A substantial body of research has evidenced the correlation between specific taxa of microbiota and the manifestation of a response to immunotherapy. However, the comparability of these findings is constrained by the paucity of shared species among studies. Clinical data underscores the deleterious effect of antibiotics administered prior to treatment outcomes and survival. Broad-spectrum antibiotics reduce microbial diversity, disrupt the balance between beneficial and unfavorable taxa, promote the growth of immunosuppressive fungi, and alter the intestinal mucosa, which disrupts host-microbiome interactions and modulate the immune response [23].

The long-standing hypothesis that microorganisms may inhabit tumor tissues of organs outside the gastrointestinal tract has only recently gained support through advanced technological methodologies, revealing that organs and tissues traditionally considered sterile can in fact harbor diverse microbial communities [24]. A range of molecular biology techniques are employed in the study of the intratumoral microbiome, including next-generation sequencing (NGS) methods such as the sequencing of the 16S rRNA gene and whole-genome shotgun sequencing (WGS). To validate NGS results and confirm the presence, localization, and metabolic activity of bacteria directly within the tumor, methods such as fluorescence in situ hybridization (FISH) and fluorescently labeled D-alanine incorporation technologies are used [25]. The handling of tumor tissues presents significant challenges due to the low concentration of bacterial DNA, rendering samples highly susceptible to contamination from reagents, the laboratory environment, and adjacent tissues [26]. Moreover, the analysis of bioinformatics data presents a considerable challenge, given the possibility of false-positive results in the identification of bacterial reads [27]. A substantial body of evidence suggests a close association between a number of malignant neoplasms and infection by microorganisms, including bacteria, viruses, and fungi. Intratumoral microbiota is regarded as an important element in the development of cancer, exhibiting both stimulatory and inhibitory effects on tumor progression [28,29].

The aim of this review is to summarize data on the role of the intratumoral microbiome in the development and progression of cancer, as well as the interaction of microorganisms in tumor tissue with immune cells, especially in the context of cellular immunotherapy.

2. Origin of the Intratumoral Microbiota and Relationships with Other Human Microbiomes

The human body contains a multitude of ecological niches that are populated by microorganisms. Furthermore, these distinct microbiomes have the capacity to influence each other [30]. For instance, the oral cavity is in direct connection with the gut, and members of the oral microbiome can enter the gut via the enteral route [31]. While the oral-intestinal barrier is generally considered to be effective in preventing the translocation of most microorganisms, there are instances in which these first lines of defense are compromised, allowing for oral-intestinal transfer. Low gastric acidity, particularly due to the use of proton pump inhibitors, has been demonstrated to shift the composition of the gut microbiota toward oral profiles [32]. A recent study showed that F. nucleatum resists gastric acid due to the presence of erucic acid in the cell membrane [33]. In addition, intestinal bacteria themselves may be a potential barrier to the growth of oral microorganisms in the intestine, providing colonization resistance [34]. However, some studies have not found significant colonization of the gut microbiome by oral bacteria in either healthy individuals or patients taking long-term antibiotics [35,36]. The hematogenous route of bacterial transmission from the oral cavity to the intestine involves oral bacteria, particularly pathobionts entering the bloodstream during transient bacteremia induced by daily activities such as toothbrushing or chewing, as well as by dental procedures that disrupt periodontal barriers [37]. These microbes survive in circulation by resisting immune clearance and interacting with endothelial cells, then translocate to intestinal vascular beds, where inflammation enhances adhesion and mucosal seeding, contributing to diseases like colorectal cancer and inflammatory bowel disease [38]. Other microbiomes, such as the skin or genital microbiome, can interact with the gut and oral microbiomes primarily through various metabolites [39,40]. Gut-derived metabolites, such as short-chain fatty acids (SCFAs), secondary bile acids, and tryptophan catabolites, reach the skin via the circulatory system. There, they modulate cutaneous barrier function, local immune responses, and the composition and activity of resident microbial communities. This shapes susceptibility to inflammatory dermatoses and ageing-related dysbiosis [41]. A meta-analysis found that, compared with low fiber intake, high fiber intake was consistently associated with an improved therapeutic response to cancer immunotherapy (pooled odds ratio: 5.79). In experimental models, restricting methionine and cysteine, as well as reducing leucine and glutamine intake, slowed tumour progression. Meanwhile, a combination of checkpoint inhibitors with intermittent fasting or a fasting-mimicking diet significantly reduced tumour volume in mice bearing melanoma. In patients, higher concentrations of short-chain fatty acids and lactic acid-producing bacteria (Faecalibacterium prausnitzii and Akkermansia muciniphila) correlated with increased objective response rates [42]. In an in vivo study, it has been demonstrated that skin damage can result in alterations to the intestinal microbiome, particularly through the increased expression of Reg3 and Muc2 in intestinal epithelial cells [43]. Through the gut–lung axis, the same classes of metabolites influence pulmonary immunity (for example by altering alveolar macrophage and regulatory T-cell function), which in turn modifies the ecological niche in the respiratory tract and can drive shifts in lung microbial composition and infection susceptibility [44].

The origins of intratumoral microbiota remain a subject of investigation. However, it has been demonstrated that microorganisms capable of penetrating the intestinal mucosal barrier and entering the bloodstream can disseminate to other organs within the gastrointestinal tract, thereby contributing to tumor development [45]. Therefore, bacteria that have migrated from the intestine to the liver have been demonstrated to play a role in the development of hepatocellular carcinoma [46]. Furthermore, the presence of intestinal bacteria has been shown to promote the dissemination of colorectal cancer metastases to the liver by enhancing the formation of a premetastatic niche and attracting metastatic cells [47]. The study showed that Fusobacterium is preferentially associated with cancer cells in metastases, and treatment with metronidazole reduces bacterial load, cell proliferation and tumor growth [48]. Another study demonstrated that in patients with colon cancer, compared with the control groups and patients with adenomatous polyps, there was an enrichment of oral biofilm bacteria (Fusobacterium, Gemella, Parvimonas, Granulicatella, Leptotrichia, Peptostreptococcus, Campylobacter, Selenomonas, Porphyromonas, Prevotella) and a number of intestinal taxa (Phascolarctobacterium, Bacteroides, Tyzzerella, Desulfovibrio, Eubacterium, Lachnospiraceae), with the species F. nucleatum dominating [49]. It was shown that alpha diversity in tumor samples was lower than in healthy tissue, in addition to a decrease in the representation of Bacteroides, Lachnospiraceae, Clostridiales and Clostridium and an increase in Enterococcus, Streptococcus in tumors [50]. The Bacilli class showed a bidirectional positive association with malignant melanoma. The Betaproteobacteria and Gammaproteobacteria classes demonstrated a causal relationship with an increased risk of developing malignant melanoma and basal cell carcinoma, respectively. In a reverse Mendelian analysis, malignant melanoma was associated with reduced abundance of members of the Bacteroidetes (Bacteroidota) phylum [51]. Veillonella parvula was identified as the most common taxon driving this association. In the lung cancer model, V. parvula-induced lower respiratory tract dysbiosis resulted in decreased survival, increased tumor burden, and the development of an inflammatory phenotype mediated by the cytokine IL-17 [52]. Experimental data have shown that intestinal bacteria, including Enterococcus faecalis and Escherichia coli, are able to migrate to the pancreas, forming an intrapancreatic microbiome primarily represented by the phyla Proteobacteria (Pseudomonadota), Bacteroidetes (Bacteroidota), and Firmicutes (Bacillota). Mice with oncogenic pancreatic mutations showed a progressive enrichment of Bifidobacterium pseudolongum and Actinobacteria, indicating the involvement of intestinal dysbiosis in the development and progression of pancreatic adenocarcinoma [53].

3. The Role of the Intratumoral Microbiome in Cancer Progression

The intratumoral microbiome is constituted by a consortium of microorganisms, comprising bacteria, fungi, and viruses, that inhabit tumor tissue [54]. The potential mechanisms of microbes’ entry into the tumor include damaged mucosal membranes, direct migration from adjacent normal tissue, and hematogenous dissemination [55]. It is noteworthy that bacteria are predominantly localized inside cells [56]. The accumulation of microorganisms in tumor tissue is multifactorial. Immune evasion by cancer cells creates an immunosuppressive environment within the tumor, thereby allowing the tumor to evade immune clearance [57]. The process of hematogenous transport is facilitated by increased vascularization and vascular permeability of tumor tissue. Tumor tissue is characterized by a high nutrient content and the presence of specific metabolites (e.g., ribose, aspartic acid, etc.), which attract bacteria [58].

The tumor microbiome demonstrates low biomass. However, this parameter is influenced by various factors, including cancer type, microenvironmental conditions, and the implementation of antibiotic treatment [59,60]. The bacterial composition of tumors in various organs has been demonstrated to have distinct characteristics. The study revealed that the predominant taxa at the phylum level in esophageal carcinoma were Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria, and Actinobacteria. A substantial increase in the abundance of Firmicutes was observed, accompanied by a concurrent decrease in Proteobacteria [61]. The composition of the taxa Alistipes, Blautia, Pasteurellales, and Porphyromonas was correlated with the clinical characteristics of patients with gastrointestinal cancer, particularly colorectal cancer [62]. Studies have demonstrated that the composition of the lung microbiota undergoes significant alterations in lung cancer. An increased abundance of Thermus has been observed in patients with advanced-stage tumors, whereas Legionella is more prevalent in those with metastatic disease [63]. Moreover, metabolic pathways linked to smoking are enriched in the lung microbiota of cancer patients, with specific taxa such as Acidovorax showing notable associations with these changes [64]. Increased abundance of intratumoral Neisseriaceae has been demonstrated to promote accelerated lymph node metastasis in squamous cell carcinoma of the oropharyngeal tonsils [65]. Furthermore, the presence of certain tumor microorganisms, such as Brevundimonas and Staphylococcus, has been identified as a contributing factor to the development of distant metastases in breast cancer cases [66]. F. nucleatum, a bacterium detected in both primary tumors and metastatic lesions across various cancer types, is of particular interest, as it substantiates its potential role in the maintenance and spread of tumorigenesis [60].

The intratumoral microbiome exerts multifaceted effects on the processes of carcinogenesis. Extensive research has explored the mechanisms through which microbial communities contribute to tumor initiation and progression. To date, four principal mechanisms have been identified: induction of DNA damage with a consequent increase in mutation rates, modulation of tumor-associated signaling pathways, alteration of host immune responses, and the initiation or amplification of inflammatory processes [67]. The process of DNA mutation induction serves as a key mechanism in the context of microbial carcinogenicity. Some bacteria have been shown to cause DNA damage and mutations. E. coli that expresses the genotoxin colibactin (PKS locus) has been shown to induce double-strand DNA breaks and promote the development of colorectal cancer. It has been demonstrated that other pathogenic E. coli strains are capable of injecting the genotoxin UshA via the type III secretion system, thereby inducing DNA damage in intestinal epithelial cells [68]. Bacteroides fragilis-produced toxin (Bft) has been reported to increase levels of reactive oxygen species (ROS) in intestinal epithelial cells, leading to oxidation and DNA damage to host tissues and, consequently, malignant transformation of cells [69]. The signaling pathways that facilitate cell movement, growth, survival, and metabolism are critical to the fundamental functioning of normal and tumor tissues. The intratumoral microbiome may play a role in tumor-associated signaling cascades. The modulation of various signaling pathways by microbes is a critical factor in the development and progression of cancer [70]. Representatives of the intratumoral microbiome have been shown to modulate cascades of signaling pathways such as WNT/β-catenin, NF-κB, Toll-like receptors (TLR), ERK, PI3K, and RhoAROCK in tumor cells, thereby influencing carcinogenesis [67].

The Wnt/β-catenin signaling pathway is a conserved signaling pathway that regulates embryonic development and tissue homeostasis. β-catenin has been observed to bind to the TCF transcription complex and translocate to the nucleus, where it has been shown to stimulate transcription of downstream target genes such as c-Myc, cyclin D1, MMP, or survivin. This process contributes to the development of various types of cancer. [71]. It has been demonstrated that several intratumoral microorganisms possess the capacity to activate the Wnt/β-catenin signaling pathway. Annexin A1, a regulatory protein that has been recently implicated in this pathway, has been shown to enhance the expression of FadA, a secreted protein produced by F. nucleatum in colorectal tumours, via E-cadherin. [72]. The study demonstrated that bacteria belonging to the genus Veillonella upregulate the ERK and PI3K signaling pathways in lung cancer. Activation of the PI3K pathway promotes cancer cell proliferation and is considered an early event in lung tumorigenesis [73]. Moreover, stimulation of Toll-like receptor 4 (TLR4) by bacterial lipopolysaccharides induces the expression of proinflammatory genes, contributing to the progression of tumor-promoting inflammation across various cancer types [74]. The NF-κB signaling pathway plays a pivotal role in the development of chronic microbial-induced inflammation. It has been demonstrated that microorganisms can trigger the NF-κB pathway by activating the β-catenin and TLR signaling cascades. This activation results in the release of proinflammatory mediators and the development of a persistent inflammatory state. This process is bidirectional: immune cells recruited to the site of inflammation release cytokines and chemokines, which in turn increase NF-κB activation, creating a positive feedback loop that promotes tumor development and progression [75]. A recent study demonstrated that Faecalibacterium prausnitzii and the intestinal metabolite tyrosol exert antitumor effects by inhibiting the activation of the HIF-1α/NF-κB signaling pathway, resulting in reduced levels of reactive oxygen species and inflammatory factors [76]. The presence of bacteria within tumor cells has been shown to suppress the RhoA/ROCK signaling pathway, thereby facilitating the adaptation of circulating tumor cells to fluid shear stress through cytoskeletal remodeling and enhancing their potential for distant colonization [77]. Elevated concentrations of Staphylococcus, Lactobacillus, and Streptococcus have been detected within breast cancer cells, where these bacteria inhibit the RhoA/ROCK pathway, a key regulator of cytoskeletal organization. This inhibition enables tumor cells to withstand mechanical stress encountered in the circulatory system, reducing cellular damage and promoting metastatic dissemination [78].

However, it has been demonstrated that certain microorganisms can exert a beneficial influence by impeding the progression of cancer. Bacterial peptides are presented on tumor and antigen-presenting cell HLA molecules, creating neo-like targets that can be recognized by T cells and potentially broaden antitumor repertoires. Intracellular tumor bacteria and their microbe-associated molecular patterns can activate pattern-recognition pathways, interfacing with innate sensing hubs that shape T cell priming in the tumor microenvironment [79]. Microbial-derived metabolites (e.g. short-chain fatty acids (SCFAs) can reprogram local immunity and myeloid states; colorectal models show that leveraging microbiota metabolism can increase intratumoral SCFAs and augment chemotherapy and immune activation [80]. Therefore, Lactobacillus casei and Lactobacillus reuteri have been documented to impede the proliferation and migration of pancreatic cancer cells by attenuating TLR4 signaling. Furthermore, these bacterial species counteract the induction of the M2 macrophage phenotype by pancreatic cancer cells and promote the differentiation toward the pro-inflammatory M1 macrophage subtype [81]. Besides, a study employing a mouse glioma model demonstrated that a mixture of four Bifidobacterium species impeded tumor growth by suppressing the MEK/ERK signaling pathway [82].

Figure 1. The key mechanisms and signaling pathways that explain the influence of the intratumoral microbiome on cancer development.

Figure 1. The key mechanisms and signaling pathways that explain the influence of the intratumoral microbiome on cancer development.

5. Current limitation and Future Direction

Despite mounting evidence that the intratumoral microbiome plays a pivotal role in cancer progression and therapeutic response, several critical limitations impede the translation of these findings into clinical practice. A significant deficit in the extant literature pertains to the paucity of research addressing the influence of immune-cell therapies, including adoptive T-cell transfer, CAR-T or TIL therapies, on outcomes across diverse cancer types within the context of intratumoral microbial communities. To date, the majority of studies have been of an observational nature, having been limited to particular tumor types or immune checkpoint inhibitors, as opposed to cell therapies. For example, while the intratumoral microbiome has been implicated in shaping responses to immunotherapy, data specific to immune cell therapy remains scarce [108].

Mechanistically, the pathways by which intratumoral microbes modulate immune responses are only beginning to be elucidated. The complexity of microbial-immune-tumor cross-talk remains under-characterized, particularly in the setting of treated tumors, immune cell infusion, or following therapy-induced modifications of the tumor microenvironment. Although studies report involvement of pathways such as STING, TLR/NF-κB, β-catenin, and ROS signaling in microbe-driven immune modulation [109]. Furthermore, the relationship between extratumoral microbiomes (gut, oral cavity, and respiratory tract) and the intratumoral microbiome remains to be elucidated. The sources of the tumor-resident microbiome—whether via translocation, circulation, lymphatic spread, or local colonization—vary across tumor types and clinical contexts. In addition, the impact of these sources on intratumoral immune modulation is remains unclear [80].

These limitations indicate several key avenues for future research. In order to elucidate the manner in which specific microbial taxa or their metabolites modulate the behavior of transplanted immune cells, including their persistence, exhaustion, phenotype, trafficking, and interactions with the host immune microenvironment, mechanistic studies employing advanced experimental systems are required. Examples of such systems include tumour organoids, humanized mouse models, spatial transcriptomics, and multi-omics approaches. [110]. Prospective clinical trials incorporating longitudinal microbiome sampling (tumor-intrinsic, gut, and oral) in patients receiving immune cell therapy are essential to delineate associations between microbial community states and therapeutic outcomes (response, persistence, and toxicity) and to evaluate the efficacy of microbiome-modulating interventions [111]. Comprehensive mapping of the interrelationships among systemic microbiomes (gut, oral, and skin), the intratumoral microbiome, and the immune landscape is necessary to identify upstream microbial drivers and causal pathways underlying immune modulation. The validation of microbial signatures as biomarkers or companion diagnostics for immune-based therapies, including immune cell therapies, should be prioritized, with an emphasis on assessing their additive predictive value beyond existing genomic, immunological and clinical parameters [112]. Therapeutic innovation should focus on the rational development of microbiome-targeted adjuncts to immune cell therapy, such as engineered bacterial strains, bacteriophage-based strategies, or microbial metabolite modulators, while ensuring rigorous evaluation of their safety, off-target effects, and regulatory compliance. Translational progress in this field will depend on the standardization of microbiome sampling and sequencing protocols, stringent contamination control, and the establishment of open data-sharing frameworks to enhance reproducibility and accelerate biomarker discovery [113].

6. Conclusions

The intratumoral microbiome is a critical and functionally active component of the tumor microenvironment that significantly impacts cancer development, progression, and response to therapy. Microorganisms can either promote tumorigenesis through mechanisms like inducing genomic instability and fostering an immunosuppressive state or inhibit it by enhancing anti-tumor immunity. Future research should focus on mechanistic studies using multi-omics approaches, well-designed clinical trials to validate microbial biomarkers, and the rational development of microbiome-targeted interventions. Harnessing the therapeutic potential of the intratumoral microbiome could lead to innovative strategies to overcome treatment resistance and improve patient outcomes.