Intra-tumoral bacteria in head and neck cancer: holistic integrative insight

Patterns of involvement of intra-tumoral bacteria

Most studies have shown that intra-tumoral bacteria mainly exist in tumor and immune cells in the TME and participate in immune regulation via innate components^26,27. Intracellular bacteria enter host cells by conventional modes of invasion. Intra-tumoral bacteria can directly stimulate related receptors to affect the genetic material of host cells and activate signaling pathways to affect tumor cells. Intra-tumoral bacteria can also indirectly participate in tumor-related activities and behaviors by using bacterial membrane vesicles and metabolites.

Distribution and source of intra-tumoral bacteria

With the development of sequencing technology and updated databases, increasing evidence has shown that the bacterial flora in head and neck tumors significantly differs from normal para-cancerous tissue⁴⁸. The major bacterial composition in HNSCC based on existing studies is summarized in Figure 1 and Table 1.

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

Common intra-tumoral bacteria in each major subtype of HNSCC. (A) OSCC: The common bacterial flora in OSCC is shown in the figure. P. gingivalis and F. nucleatum are the most common and most studied intra-tumoral bacteria in Porphyromonas and Fusobacterium, respectively, while Streptococcus has been observed to have reduced abundance in most studies. (B) LSCC: Fusobacterium is also the most common species in LSCC. Helicobacter pylori is a specific bacterial species found in LSCC. (C) NPC: Fusobacterium and Prevotella are common in NPC and intra-tumoral bacteria in NPC are often associated with prognosis. (D) HPSCC: The intra-tumoral bacteria in HPSCC are like other subtypes. Specifically, the Eubacterium coprostanoligenes group comprises specific bacteria that inhibit the tumor. HNSCC, head and neck squamous cell carcinoma; OSCC, oral squamous cell carcinoma; LSCC, laryngeal squamous cell carcinoma; NPC, nasopharyngeal carcinoma; HPSCC, hypopharyngeal squamous cell carcinoma.

Studies using 16S rRNA sequencing and alpha/beta analyses have shown an increased relative abundance of Fusobacterium and decreased abundance of Streptococcus and Actinomyces in tumor tissues compared to normal tissues^50,61. Streptococcus acts as an early colonizer in oral squamous cell carcinoma (OSCC), while F. nucleatum serves as a transitional bacterium between early and late colonizers and has the ability to coaggregate⁶². Moreover, Streptococcus has been shown to attenuate F. nucleatum-induced pro-inflammatory responses in oral epithelial cells⁶³. Some researchers suggest that Firmicutes and Actinobacteria are sensitive to cancer-related environments and a significant decrease in the proportion of Firmicutes and Actinobacteria, along with an increase in F. nucleatum, may indicate a pre-cancerous state⁵¹.

The origin of intra-tumoral bacteria is of great significance for research. The intra-tumoral bacterial sources found in > 10 head and neck tumors can be broadly divided into 3 categories. Some oral Streptococcus species are found in the tumors of patients with oral cancer and are derived from the patients’ oral microbiome⁶⁴. In addition, bacteria can also enter the tumor tissue through the damaged mucosal barrier or through normal adjacent tissues⁶⁵. The bacterial composition of hypopharyngeal squamous cell carcinoma (HPSCC) is dominated by Fusobacterium and Prevotella. Fusobacterium, Micromonas, and Dialister tend to be abundant in advanced T classification and TNM staging⁶⁶. Bacteria spread through the blood or lymphatic circulation in metastatic tumor tissue and migrate into the tumor. Intestinal flora, such as Klebsiella, Escherichia, and Enterococcus, have been detected in cervical lymph nodes adjacent to oral cancer tumor tissue⁶⁴.

These findings suggest the possible existence of oncogenic mechanisms associated with alterations in oral bacteria and provide new perspectives on the role of bacterial communities in head and neck tumors. Moreover, this evidence suggests that finding specific bacteria and identifying changes in the number and distribution of bacteria in tumors⁶⁷ are key links to clarify the pathogenesis of this disease.

Predominant intra-tumoral bacteria in HNSCC

Based on the available studies, the intra-tumoral bacteria associated with HNSCC have been shown to be mainly related to Fusobacterium and Streptococcus. Porphyromonas is particularly prevalent in oral-associated tumors. In addition, the role of bacteria, such as Lactobacillus and Actinomycetes, warrants further study.

Fusobacterium, which is characterized as a Gram-negative anaerobic bacteria, is frequently observed to be enriched across diverse subsets of HNSCC and these bacteria are implicated in a spectrum of oral maladies as well as anaerobic infections^68,69. The relative abundance of Fusobacterium and Streptococcus is increased in primary and metastatic tumor tissues compared to normal healthy tissues of HNSCC patients^70,71. However, other studies have reported a reduced abundance of Streptococcus in head and neck tumor samples^50,67. The inconsistent findings may potentially arise from patient-specific variables, lifestyle factors, medication usage, and variations in sample collection methods. The impact of lifestyle on microbiota abundance in HNSCC is apparent. The log abundance of Firmicutes and Peptostreptococcus is significantly decreased in smokers, while the log abundance of Fusobacterium and Actinomycetes is significantly increased^72,73. The ethanol-associated increase in the abundance of Neisseria that produce acetaldehyde and the decrease in the abundance of Lactobacillales that are involved in acetaldehyde degradation have been demonstrated in smokers and alcohol consumers⁷⁴. Oral Corynebacterium and Kingella are involved in xenobiotic degradation and are associated with various metabolic pathways, including the capacity to process several toxicants present in cigarette smoke⁷⁵. Thus, a reduced risk of HNSCC is associated with an increased abundance of Corynebacterium and Kingella species^76,77. In particular, laryngeal squamous cell carcinoma (LSCC) tends to have the strongest association with smoking and alcohol consumption, mainly because of alterations that alter the laryngeal ecologic balance and thereby affects the development of LSCC¹¹.

Impact of tobacco and alcohol on intra-tumoral bacteria

The results of tobacco and alcohol use often act synergistically with the cancer-promoting effects of intra-tumoral bacteria. The mechanisms underlying these changes may be the effects of smoking on DNA damage and repair capacity⁷⁸, as well as the effect on inflammatory responses⁷⁹. HNSCC is highly inflammatory in nature and expresses a variety of cytokines and growth factors involved in inflammation⁸⁰. Pre-treatment serum IL-6 levels predict recurrence and poor survival in HNSCC⁸¹. Smoking is strongly associated with high IL-6 levels⁸², which laterally assists intra-tumoral bacteria in promoting progression of TME inflammation⁸³. F. nucleatum inhibits ethanol metabolism by downregulating the tumor suppressor enzyme, alcohol dehydrogenase 1B (ADH1B), which eventually leads to the accumulation of ethanol and causes metabolic stress in LSCC. Local ethanol accumulation also promotes the proliferation of F. nucleatum. Such a mutual promotion pattern explains the increase of F. nucleatum in LSCC patients and the promotion of tumor development⁵⁴. In addition, several commensal bacteria metabolize ethanol to carcinogenic acetaldehyde, further exerting a role in alcohol-related carcinogenesis⁸⁴. Acetaldehyde dehydrogenase generated by Streptococcus catalyzes the formation of mutagenic levels of acetaldehyde under aerobic or microaerophilic conditions, which leads to elevated acetaldehyde concentrations and the induction of tumors by hydroxyethyl and hydroxyl radicals⁸⁵. In addition, acidogenic Streptococcus mutans synthesizes extracellular polymeric substances in the presence of sucrose to form polysaccharide reserves, thereby providing energy for tumor progression⁸⁶.

Studies have shown that P. gingivalis, together with other oral bacteria, such as Fusobacterium, affect the heterogeneity of cancer cells and change the transcriptional program, thereby affecting specific cellular behaviors^6,87,88. This evidence suggests that personalized studies should be conducted involving specific bacterial flora based on the identification of specific tumor types.

Intra-tumoral bacteria promote the occurrence and development of tumors

Intra-tumoral bacteria primarily promote cancer progression through mechanisms, such as DNA damage repair disruption, chronic inflammation, regulation of oxidative stress, and interactions with viruses. Bacterial infections induce DNA damage in host cells, increasing the susceptibility to malignant transformation. Additionally, bacterial infections activate various inflammatory signaling pathways, promoting inflammation in surrounding tissues. Disruption of reactive oxygen species (ROS) homeostasis also contributes significantly to tumor progression. Furthermore, the interplay between bacteria and viruses within the TME acts synergistically to enhance tumor initiation and progression.

Synergy with viruses

Bacterial involvement also impacts intra-tumoral viruses. A significant positive correlation was demonstrated between the abundance of oral-translocation microbes and local Epstein-Barr virus (EBV) load in NPC and an altered virus-associated microenvironment in intra-tumoral meta-transcriptome data was found⁵⁶. Earlier studies collectively showed that the F. nucleatum culture medium effectively induces EBV-associated antigens in vitro¹⁰⁵. The researchers hypothesized that the translocated oral microbiota might act synergistically with EBV in the pathogenesis of NPC, together creating favorable conditions for latent infection⁵⁶. Patients with periodontitis have a higher incidence of human papillomavirus (HPV)-positive HNSCC. We speculate that P. gingivalis and F. nucleatum in the HNSCC may synergistically interact with HPV¹⁰⁶. In addition, Streptococcus in the oral cavity contributes to the development of HPV-related HNSCC by stimulating the host inflammatory response and producing carcinogenic metabolites, such as acetaldehyde¹⁰⁷. However, these studies are still preliminary and further prospective studies with larger sample sizes are needed to confirm these findings (Figure 2).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Intra-tumoral bacteria promote tumor initiation and progression. (A) Regulation of DNA damage: ① F. nucleatum may directly cause DSB and disrupt Ku 70/80 complex in inhibiting DNA repair. ② Metabolites of F. nucleatum activate TLR4/MyD88 followed by miR-205-5p upregulation, which inhibits MMR gene expression, including MLH1, MSH2, and MSH6. ③ Intracellular bacteria may promote the formation of JUN and FOS heterodimers and the AP-1 complex for transcriptional activation of DNA damage repair genes, such as ERCC-1 and XPC. (B) Promotion of chronic inflammation: ④ Downregulation of antibacterial HBD1 coupled with IL-1β–mediated induction of oncogenic HBD2 and HBD3 facilitates bacterial persistence and contributes to tumor progression. ⑤ F. nucleatum and P. gingivalis activate the NF-κB and ERK/MYC pathways through the TLR/MyD88 axis, driving tumor progression. ⑥ Intra-tumoral bacteria induce monocyte secretion of IL-6 via metabolites and chemokines to activate IL-6/STAT3 signaling and promote effectors, such as cyclin D1 and MMP9. (C) Regulation of oxidative stress: ⑦ F. nucleatum promotes tumor growth by driving NF-κB activation, which increases ROS production, indirectly activates the AKT/mTOR pathway, and suppresses the p53 pathway. ⑧ F. nucleatum regulates oxidative stress by activating the miR-361-3p/NUDT1 axis via TLR4, relieving miR-361-3p inhibition of NUDT1 to promote autophagy and DDR. Autophagy indirectly activates DDR, while DDR alleviates DNA damage stress through autophagy, forming a feedback loop that reinforces both processes and drives tumor progression. ⑨ F. nucleatum inhibits ROS production by suppressing purine degradation and uric acid production, a key antioxidant. (D) Synergy with viruses: ⑩ Co-culture of F. nucleatum with EBV enhances EBV antigen expression. Periodontal pathogens activate and exacerbate the oncogenic potential of EBV and HPV through their metabolites, synergistically promoting malignant transformation. Streptococcus species contribute to carcinogenesis by inducing inflammation and generating carcinogens, such as acetaldehyde. AMP, adenosine monophosphate; AP-1, activator protein 1; DDR, DNA-damage response; DSB, double-strand break; EBV, Epstein–Barr virus; HBD, human beta-defense; HPV, human papillomavirus; MMP9, matrix metalloprotein 9; MMR, mismatch repair; MyD88, myeloid differentiation primary response gene 88; NUDT1, nudix hydrolase 1; ROS, reactive oxygen species; TLR, Toll-like receptor.

Intra-tumoral bacteria inhibit tumor progression

Studies suggesting a suppressive role of intra-tumoral bacteria in HNSCC are limited with intra-tumoral bacteria, often aiding in cancer progression. However, P. gingivalis has a different role, exhibiting suppressive effects in HNSCC.

Relationships between intra-tumoral bacteria and tumor metastasis

Intra-tumoral bacteria mainly initiate or promote tumor metastasis by altering mechanical stress and promoting initiation of the epithelial-mesenchymal transition (EMT) program. Intra-tumoral bacteria initiate a fluid shear stress response upon invasion of host cancer cells and this response is associated with the ability of bacterial species to promote metastasis. Cancer cells invaded by bacteria can carry the bacteria, move to distant organs, and promote cancer cell survival²⁷. The bacteria can regulate the cytoskeletal reorganization pathway, allowing cancer cells to become more resistant to mechanical stress.

In addition to physically inducing cancer cell migration, intra-tumoral bacteria also interfere with signaling pathways. F. nucleatum triggers TLR signaling to cause IL-6 production and activates STAT3, thereby promoting the production of cyclin D1 and MMP and subsequently activating EMT to promote the invasion and migration of OSCC cells⁵¹. Moreover, OMVs activate the autophagy pathway in tumor cells, promote the EMT of tumor cells, and promote the formation of lung metastasis¹¹¹. P. gingivalis increases the invasion of OSCC cells through IL-8-dependent upregulation of MMPs, enhances the expression of mesenchymal intermediate filament protein and MMP, decreases the expression of E-cadherin and β-catenin, regulates EMT, and promotes the migration of infected cells¹¹². Metastasis to cervical lymph nodes is more common in cancer cells harboring bacterial infections. Mechanistically, the compromised oral mucosa in oral cancer facilitates colonization of the oral surface by new bacterial species, which can subsequently migrate to local lymph nodes. Notably, Streptococcus oralis is the most frequently isolated bacterial species from cervical lymph nodes in these cases⁶⁴.

Interestingly, the F. nucleatum culture supernatant, which contains LPS, induces IL-8 and MMP expression, suggesting that direct bacterial contact may not be necessary for carcinogenesis¹¹³. Another study demonstrated that LPS from F. nucleatum increases miR-155-5p and miR-205-5p expression through the TLR4/MyD88 pathway, which targets ADH1B and transforming growth factor-β (TGFBR2), respectively, to promote LSCC EMP, leading to tumor development and invasion⁵⁴. S. mutans induces inflammation via IL-6, accelerates tumor progression, upregulates MMP9, promotes basement membrane degradation and invasiveness, and establishes an immune-inhibitory TME⁴⁹.

Intra-tumoral bacteria enhance anti-tumor immune responses

Intra-tumoral bacteria enhance the immune response in HNSCC primarily by recruiting immune cells through chemokines, downregulating PD-L1 expression, and boosting the anti-tumor activity of immune cells. Immunosuppressive cells and processes are simultaneously diminished or inhibited, further strengthening anti-tumor immunity.

Regulation of inflammation

Lachnoclostridium, Flammeovirga, and Luteibacter are significantly enriched in TMEs. Lachnoclostridium enhances anti-tumor immunity by promoting CD8⁺ T cell infiltration and activation, while butyric acid metabolites exert anti-inflammatory effects^115,116. Additionally, Flammeovirga and Luteibacter are positively associated with the presence of infiltrating CD8⁺ T cells and the expression of chemokines, such as CXCL9, CXCL10, and CCL5¹¹⁷. Notably, the co-existence of these three bacterial genera has been linked to poor prognosis, potentially inducing chronic inflammation. Inflammation exhibits a dual role in the context of cancer; specifically, chronic inflammation fosters tumor initiation and progression, whereas acute inflammation enhances therapeutic efficacy¹¹⁸. This duality underscores the complex and context-dependent impact of intra-tumoral bacteria on tumorigenesis and clinical outcomes.

Interestingly, F. nucleatum is more prevalent in elderly OSCC patients who abstain from alcohol consumption and is associated with reduced lymph node invasion, lower distant recurrence rates, and improved survival outcomes. F. nucleatum is negatively correlated with macrophage M2 polarization and TLR4 expression, suggesting a potential role in mitigating immunosuppressive mechanisms¹¹⁹. Similarly, F. nucleatum has been implicated in anti-tumor activity in patients with colon cancer with autoinducer-2 (AI-2), a quorum-sensing signal molecule, activating the TNFSF9/IL-1β signaling pathways in macrophages to exert immunomodulatory effects^120,121.

These findings are in contrast with the predominant evidence suggesting that intra-tumoral bacteria promotes tumor progression and is linked to a poor prognosis. This finding highlights the complex and context-dependent roles of intra-tumoral bacteria in cancer immune responses, underscoring the need for multidimensional investigations into the diverse mechanisms of action (Figure 3).

• Download figure
• Open in new tab
• Download powerpoint

Figure 3

Dual role of intra-tumoral bacteria in regulating the anti-tumor immune response. (A) Enhancement of anti-tumor immunity: ① P. gingivalis downregulates MUC1 to reduce PD-L1 and inhibit MDSC, thereby enhancing anti-tumor immunity. ② F. nucleatum has a negative correlation with M2 macrophages and a positive correlation with M1 macrophages, suggesting involvement in tumor suppression, potentially through activation of the TNFSF9/IL-1β axis. ③ Flammeovirga and Luteibacter are positively associated with chemokines, such as CXCL9, CXCL10, and CCL5, facilitating the recruitment and activation of CD8⁺ T cell. ④ Lachnoclostridium further supports anti-tumor immunity by promoting CD8⁺ T cell activity and producing the anti-inflammatory metabolite, butyrate, which exerts tumor-suppressive effects. (B) Inhibition of anti-tumor immunity: ⑤ P. gingivalis activates NOD1 in tumor cells via teichoic acid carried by OMVs, upregulating PD-L1. OMVs also stimulate monocytes to secrete IL-10 and inhibit TNF production, enhancing P. gingivalis survival and promoting immune evasion. ⑥ F. nucleatum and S. aureus utilize TLR signaling to activate ERK/MYC and NF-κB pathways, driving PD-L1 and suppressing T cell activation to facilitate tumor progression. ⑦ F. nucleatum and Selenomonas are positively correlated with FOXP3, a marker of Treg, suggesting an association with Treg. ⑧ High abundance of Capnocytophaga showed a negative correlation with Tem, further linking them to immunosuppressive effects. ⑨ Roseobacter, Streptococcus, and Clusterobacter are associated with the abundance of TN and Tcm, yet significantly reductions of TN and Tcm in tumors may impair the efficacy of these T cell subsets. ⑩ Corynebacterium, Prevotella, and members of the Peptostreptococcaceae family show positive correlations with the Th2 marker GATA3, suggesting roles as potential immunosuppressive agents in the tumor microenvironment. AI-2, autoinducer-2; CCL, C-C motif chemokine ligand; CXCL, C-X-C motif chemokine ligand; MDSC, myeloid-derived suppressor cell; MUC1, pleomorphic epithelial mucin 1; NOD1, nucleotide-binding and oligomerization domain 1; OMVs, outer membrane vesicles; PD-1, programmed death 1; PD-L1, programmed death-ligand 1; RIP2, receptor-interacting protein kinase 2; Tcm, central memory T cell; Tem, effector memory T cells; Th2, T helper 2 cell; TLR, toll-like receptor; TN, naive T cell; TNF, tumor necrosis factor; TNFSF9, tumor necrosis factor ligand superfamily member 9; TRAF1, tumor necrosis factor receptor-associated factor 1; Treg, regulatory T cell.

Intra-tumoral bacteria inhibit anti-tumor immune responses

Intra-tumoral bacteria mainly rely on activation of PD-1/PD-L1 to affect tumor immune escape. In addition, intra-tumoral bacteria promote the secretion of inflammatory factors by inhibiting relevant immune cells as well as conventional signaling pathways, thereby inducing chronic inflammation, which affects TME and inhibits anti-tumor immune responses.

Intra-tumoral bacteria and radiotherapy

A recent clinical study revealed that radiotherapy positively influences Streptococcus and Lactobacillus abundance, which provides direct evidence of oral microbiota modulation by radiotherapy¹³⁰. Oral tumors exhibit higher hypoxia levels than other head and neck tumor sites, potentially contributing to radiation resistance. Subsite-specific microbial populations are overrepresented in hypoxic tumors¹⁶.

Follow-up evaluations post-radiotherapy often reveals mucositis, xerostomia, colitis, radiation enteritis, and inflammatory bowel disease due to microbial dysbiosis. Research has increasingly focused on the interaction of radiotherapy with the intestinal and oral microbiota, including the use of probiotics to mitigate radiation reactions and alter the intestinal flora composition to enhance radiation sensitivity¹³¹. Anaerobe levels increase during radiation, suggesting the prevalence on tumor surfaces and potential involvement in radiation support. Scholars advocate the use of tailored antibacterial regimens in OSCC diagnostics to mitigate radiation mucositis deterioration caused by Gram-negative bacill¹³².

While current research primarily concentrates on the intestinal flora, it is plausible that tumor bacteria impact radiotherapy efficacy and complications via hypoxia-induced pathways affecting tumor and para-carcinoma tissue.

Intra-tumoral bacteria and chemotherapy

Research on the effect of intra-tumoral bacteria on chemotherapy is warranted in the future. F. nucleatum-induced miR-146a-5p expression via TLR2 activation inhibits downstream caspase recruitment domain family member 10 (CARD10), rendering OSCC cells resistant to cisplatin-induced chemotherapy⁵¹. Additionally, intra-tumoral bacteria may influence the cancer therapy response through direct interactions with the tumor immune microenvironment. For example, Gammaproteobacteria expressing cytidine deaminase within tumors mediate gemcitabine resistance¹⁷. These findings underscore the critical need to further elucidate the mechanisms by which intra-tumoral bacteria influence chemotherapeutic outcomes.

Intra-tumoral bacteria and immunotherapy

Intra-tumoral bacteria are widely involved in the immune response of tumors and have an impact on the survival and function of immune cells. Therefore, immunotherapies for tumors exhibit synergistic or inhibitory effects depending on the immune response effects.

P. gingivalis infection increases PD-L1 expression on the surface of DCs and significantly inhibits the cytotoxic activity of CD8⁺ T cells¹⁸. PD-L1 expression on the surface of DCs is a key factor for tumor immunotherapy. PD-L1 inhibitors achieve therapeutic effects by inhibiting PD-L1 expression on DCs, increasing the number of infiltrating T cells or increasing the activity of depleted T cells¹⁹. This finding suggests that P. gingivalis infection may lead to reduced immunotherapy efficacy.

However, intra-tumoral bacteria also have a synergistic effect on immunotherapy. In our previous work we reported that patients with OSCC had a higher abundance of Peptostreptococcus in tumors and a higher likelihood of a good prognosis. Synthetic material containing bacteria alone or in combination with a PD-1 blocker inhibited tumor growth and increased the number of CD8⁺ T cells in draining lymph nodes. The levels of Peptostreptococcus could be increased in tumors with low Peptostreptococcus content by the addition of exogenous Peptostreptococcus anaerobius, leading to an enhanced immune response and anti-tumor effect. The direct cytotoxic effect of Peptostreptococcus on tumor cells is limited and the anti-tumor effect is mainly achieved by regulating the host immune response, such as promoting maturation of DCs²⁰.

Intra-tumoral bacterial engineering

Engineering bacteria to treat cancer is an emerging therapeutic strategy. Engineered bacteria are used to localize a payload inside or outside tumor cells and release the payload through mechanisms, such as secretion, diffusion, or lysis²⁴. Based on this concept, some scholars have proposed use of the quorum sensing (QS) genetic circuits principle to enable engineered bacteria to achieve targeted drug delivery through the accumulation of the QS molecule, acyl homoserine lactone¹³³. Upon entry of engineered bacteria into the tumor, neoantigens are released into the TME through the function of antigen-presenting cells, where neoantigens recruit and activate tumor infiltrating lymphocytes (TILs) at specific sites and promote the production of pro-inflammatory cytokines and chemokines²⁴.

It is also possible to combine engineered bacteria with aggregation ultrasound to visualize the localization of bacteria within the tumor¹³⁴. The combination of engineered bacteria with magnetic guidance and interactions with 3D materials also appears to be a new therapeutic strategy¹³⁵. Moreover, engineered bacteria can act as synthetic antigens between chimeric antigen receptor T-cell and tumors to delay tumor progression¹³⁶.

Intra-tumoral bacteria BMVs

BMV engineering is an important treatment method, among which OMVs are more widely studied and applied. OMVs are more capable of triggering an inflammatory response than bacterial metabolites, such as LPS alone to stimulate the production of pro-inflammatory cytokines and chemokines by different cells in the body¹³⁷. Some studies have shown that OMVs are safe and effective delivery vectors for tumor drugs because OMVs can deliver encapsulated drugs to tumor tissues, improve drug pharmacokinetic properties, and reduce drug toxicity¹³⁸. In addition to tumor targeting, OMV-based drug delivery platforms can also delay drug release²⁵ and can be used to regulate the pharmacokinetic parameters of OMV delivery platforms.

According to the characteristics of OMVs, scholars have constructed Escherichia coli strains expressing PD-1 to produce PD-1-rich OMVs. OMV-PD-1 accumulates at tumor sites, effectively binds to PD-L1 on tumor cells, inhibits the interaction between tumor PD-L1 and PD-1 on CD8⁺ T cells, and blocks the PD-1/PD-L1 immune checkpoint axis. These findings protect the function of CD8⁺ T cells and allow CD8⁺ T cells to exert normal anti-tumor activity¹³⁹.

Intra-tumoral bacterial injection therapy

Intra-tumoral bacteria demonstrate potential for tumor ablation. For example, direct injection of lactic acid bacteria into human squamous cell carcinomas in nude mice inhibited tumor growth, although with associated risks. These findings suggest that utilizing Lactobacillus casei cell supernatants may offer a safer and effective approach for tumor treatment¹⁴⁰. Furthermore, a novel recombinant Listeria monocytogenes tumor vaccine, Lm-ActA-E7, induced E7-specific cytotoxic T lymphocytes in HPV-positive HNSCC patients, resulting in tumor regression without harming normal tissue¹⁴¹.

The expression of target genes in vitro showed that Bacillus brevis inhibits the occurrence and development of tumors by increasing IL-24 expression at the mRNA level. The experiment proved that the bacteria were only detected in the tumor tissues 7 days after injection of bacteria. The specific ability of Bifidobacterium strains to target and colonize tumor tissues in vivo was demonstrated. This new anti-cancer gene, IL-24, can be used for in vivo tumor treatment to provide a safe and acceptable clinical treatment¹⁴².

Signaling molecule inhibitor therapy

Intra-tumoral bacteria often act through a variety of signaling pathways and key cytokines, so related inhibitor studies may be potential treatment methods and strategies. IL-6 is an important therapeutic target in HNSCC. Several IL-6 receptor antagonists, such as tocilizumab and sarilumab, are currently in clinical trials. These drugs inhibit IL-6-mediated signaling by blocking IL-6 binding to its receptor. Tocilizumab has been shown to have some anti-tumor activity in an HNSCC study¹⁴³. In addition, direct targeting of IL-6 protein using neutralizing antibodies reduces IL-6 levels and slows tumor progression. Several neutralizing antibodies to IL-6 have entered clinical studies, and preliminary results have shown a promising safety profile and modest therapeutic efficacy¹⁴⁴. In addition to directly targeting IL-6 or its receptor, researchers are also exploring key molecules in the IL-6 signaling pathway, such as JAK and STAT3. JAK inhibitors, such as ruxolitinib, and STAT3 inhibitors, such as stattic, have shown modest anti-tumor efficacy in laboratory studies and early-phase clinical trials¹⁴⁵. Because IL-6 has a complex role in the TME, IL-6-targeted therapy alone may have limited efficacy. Therefore, researchers are exploring strategies to combine IL-6-targeted therapy with other therapeutic methods, such as chemotherapy and immunotherapy, to enhance the therapeutic effects¹⁴⁶.

In addition, use of inhibitors to inhibit the signaling molecules that are caused by bacteria is a strategy that cannot be ignored. P. gingivalis promotes the migration and invasion of HNSCC cells by targeting desmocollin-2 (DSC2), a small RNA released by OMVs (sRNA23392) to reduce the expression of DSC2, thereby affecting cell adhesion and invasion. Inhibition using sRNA23392 attenuates P. gingivalis-derived OMV-induced adhesion, migration, and invasion and restores DSC2 expression in HNSCC cells, further suggesting a critical role of sRNA23392 in bacteria-induced tumor progression. These findings suggest that inhibitors targeting sRNA23392 and its target (DSC2) may become a new strategy for the prevention and treatment of P. gingivalis-associated HNSCC, providing new ideas for the prevention and treatment of such cancers³⁸.

Most of the treatment strategies discussed remain at the theoretical and pre-clinical research stage. However, several scholars have already been translated into clinical applications by researchers. We identified an ongoing clinical trial combining three strategies (NCT03435952). Researchers genetically modified Clostridium novyi-NT to remove toxic substances and simultaneously injected Clostridium novyi-NT into the tumor with combined use of pembrolizumab to solve the problem of a poor chemotherapy effect due to vascular distribution in the tumor area. This finding also suggests that we should focus on multi-strategy combination therapy when considering treatment strategies.

Diagnostic role of intra-tumoral bacteria

Intra-tumoral bacteria in head and neck tumors regulate the TME through various mechanisms, which influence tumor progression. Furthermore, significant differences in the abundance of these bacteria before and after tumorigenesis suggest the potential as diagnostic markers^51,123,150.

Due to the dynamic and heterogeneous nature of predictive markers, such as PD-1, researchers are exploring bacterial populations and metabolites as potential predictive biomarkers for head and neck tumors. Bacteria produce volatile organic metabolites (VOMs) with unique biological characteristics and it has been shown that a systematic analytical model of VOMs in urine can be used as a diagnostic tool for tumors, especially HNSCC^150–152.

Changes in intra-tumoral bacterial populations can enhance the accuracy of diagnosing head and neck tumors¹⁵⁰. A study involving Fanconi anemia patients with tongue squamous cell carcinoma utilizing 16S RNA sequencing revealed distinct flora between tumor and healthy sites, with Mycobacterium salivarius predominating in tumors and potentially serving as a predictive biomarker for tumorigenesis¹⁵³. Some researchers have identified diverse oral tumor microbiota with genera, such as Lautropia, Asteroleplasma, Parvimonas, Peptostreptococcus, Pyramidobacter, Roseburia, and Propionibacterium, showing high diagnostic efficacy for OSCC metastasis⁵³. Analysis of the bacterial community data in cancer lesions and controls showed that Bacteroidetes, Proteobacteria, Firmicutes, Fusobacteria, and Actinobacteria accounted for 98.62% of all sequences¹⁵⁴. Bacteroidetes accounted for 37.6% of the sequences and could serve as an indicator of OSCC occurrence and development¹⁵⁴. In a prospective observational study by Torralba et al. the data showed a greater number of bacteria belonging to the phyla Fusobacteria, Bacteroidetes, and Firmicutes associated with tumor tissue compared to all other sample types¹⁵⁵. These data suggest that determining the number of specific bacteria within a tumor will greatly improve the diagnosis of OSCC⁵³.

It is worth noting that the use of biomarkers for diagnosing head and neck tumors faces challenges. Similarities between pathogens in these tumors and neck abscesses may cause misdiagnosis, necessitating excisional biopsies for histologic examination for confirmation¹⁵⁶.

Additionally, systemic hematoporphyrin derivative fluorescence may not distinguish between normal tissue autofluorescence and endogenous porphyrin fluorescence from bacteria within tumor tissues, potentially leading to false-positive cancer identification¹⁵⁷. In summary, while changes in bacterial abundance and metabolites hold diagnostic promise, the challenge of identifying and validating predictive biomarkers remains for future research.

Prognostic role of intra-tumoral bacteria

The distribution patterns of intra-tumoral bacterial communities allow such communities to serve as new prognostic markers due to interactions with tumors⁴⁶. Specifically, the nasopharynx tumor tissue microbiome shows a specific structure and contains a higher bacterial biomass compared with normal nasopharyngeal tissues. Patients with NPC have lower microbial diversity in tumor tissues compared to patients with chronic nasopharyngitis. The intra-tumoral bacterial burden is closely related to the prognosis of patients with NPC. A high bacterial burden is associated with worse disease-free survival and OS. In addition, an inverse correlation has been reported between bacterial biomass and T cell infiltration, suggesting that the tumor microbiome may influence the tumor immune microenvironment, which in turn affects patient outcomes^158,159. In a clinical study in which samples from > 802 NPC patients were analyzed, intra-tumoral bacterial burden was identified as a reliable prognostic tool that distinguishes the risk of malignant progression¹⁴⁹. Differences in bacterial composition were noted across tumor stages, with Fusobacterium linked to early cancer stages¹²³. High Fusobacterium abundance correlates with poor survival in HNSCC patients and its cut-off value in HNSCC specimens shows promising sensitivity and specificity in prognostic models, suggesting its potential as a meaningful microbial marker for predicting poor outcomes⁸⁹. Several studies have shown that the number of P. gingivalis bacteria within the tumor has a large impact on the prognosis of HNSCC. Higher P. gingivalis abundance is associated with increased mortality from oral cancer¹⁶⁰. If OSCC is chronically exposed to P. gingivalis, the probability of tumor migration is greatly increased (approximately 3 times that of uninfected controls)¹⁶¹.

There is also a correlation between intra-tumoral bacteria and tumor recurrence. The higher abundance of Prevotella and the lower abundance of the Eubacterium coprostanoligenes group in HPSCC are indicators of higher recurrence and metastasis rates. Specifically, the Eubacterium coprostanoligenes group from the primary tumor affected cholesterol metabolism by converting local cholesterol into fecal steroids, which could not be directly utilized by tumor cells, and further inhibited tumor recurrence and metastasis⁶⁰. Previous studies have indicated that antimicrobial therapy impacts HNSCC prognosis with increased survival observed with increased proportions of Leptotrichia, suggesting the need to avoid ciliate-targeted therapy¹⁴⁷. Radiation induces specific microbial changes in head and neck tumors, serving as biomarkers for treatment response and aiding in personalized radiation therapy planning¹³¹.

In summary, intra-tumoral bacterial load and composition differences hold promise as prognostic indicators for head and neck tumor patients, guiding therapeutic decisions based on risk levels for malignant progression.

Characteristics and diversity

The origin and distribution of bacteria within head and neck tumors are not random but rather follow a certain logic⁶. However, it is not clear if a direct relationship exists with the intra-tumoral bacterial species. It is well-known that the bacterial Gram classification is closely related to bacterial structure, but it is unknown whether the vesicles or metabolites produced by the bacterial cell wall or outer membrane structure are related to the bacterial parasitic preference for host tumor cells. At present, there is a lack of macroscopic studies at the bacterial species level in HNSCC-related fields. Moreover, there is a lack of diversity in the existing studies. OSCC and other common bacteria, such as F. nucleatum and P. gingivalis, have also been the focus of research. However, for bacterial research, the interaction between bacteria and bacteria, bacteria and tumors, and the interaction between the former two is an important cornerstone in both basic research and clinical applications.

Molecular mechanism

Given that intra-tumoral microflora can promote the occurrence and development of tumors through immunosuppression, inflammation promotion, and EMT signaling pathways but the underlying mechanism is still unclear. It is therefore necessary to elucidate the composition of the main cytokines induced by intra-tumoral microflora and the related mechanisms^6,54,162,163. F. nucleatum can change the status of the TME by promoting the secretion of pro-inflammatory factors in tumors, thereby inhibiting the proliferation of anti-tumor immune cells, promoting the immune escape of tumor cells and promoting the occurrence and development of tumors. In addition, macro-autophagy promoted by oral cancer cells is an adaptive mechanism for P. gingivalis to invade tumors and a survival mechanism to limit bacterial toxicity¹¹². As mentioned in the previous section, intra-tumoral bacteria have dual roles in tumorigenesis and tumor development and intra-tumoral bacteria do not have a single role in the tumor immune response and need to be studied from multiple perspectives. In addition, from the bacterial species perspective, F. nucleatum and P. gingivalis are both Gram-negative anaerobic bacteria, so we hypothesized that the link between HNSCC and intra-tumoral bacteria might be more focused on Gram-negative anaerobic bacteria. However, few studies have addressed the bacterial species associated with HNSCC. It is necessary to closely follow the latest progress in this field and elucidate the underlying mechanisms involved. Moreover, the existing evidence strongly suggests that intra-tumoral bacteria are closely related to PD-1/PD-L1^43,164, suggesting that researchers should not ignore the role of intra-tumoral bacteria when seeking to improve the PD-1 response rate and their role may be crucial.

Many studies on the mechanism by which bacteria affect anti-tumor immunity have made progress but the current research results are not uniform, which hinders the clinical application of bacteria-related therapeutic strategies. More attention should be given to the correlation between the specific mechanisms of various tumor-promoting and -suppressing pathways and intra-tumoral bacteria in the future.

Strategies for intra-tumoral bacterial therapy

Due to the hypoxic environment of the tumor and the metabolism of chemotherapeutic drugs, intra-tumoral bacteria can lead to drug resistance and radiation resistance in tumor cells, thus affecting the therapeutic efficacy in HNSCC patients¹⁶. Intra-tumoral bacteria influence the efficacy of radiotherapy and chemotherapy, while these treatment modalities also alter the balance of the oral microbiota in patients¹³¹. However, the mechanisms underlying how these changes impact HNSCC progression or suppression remain unclear and warrant further investigation. Immunotherapy targeting intra-tumoral bacteria will be a promising potential treatment option. According to the different flora and working mechanism, intra-tumoral bacteria have different effects on immunotherapy, which also suggests that there is still a huge space for exploration of intra-tumoral bacteria^18–20. Targeting intra-tumoral bacteria allows intra-tumoral bacteria to be engineered to deliver drugs. Therefore, imaging the location of bacteria within the tumor cannot be ignored. Some scholars have proposed that the special prodrug, NR-NO₂, can be used to detect the unique fluorescence signal effect of Gram-negative bacteria¹⁶⁵. Moreover, encapsulation of drugs in bacteria-produced OMVs appears to be more biocompatible. However, the difficulty lies in the production accuracy and efficiency, which is one of the difficulties in the translation of basic research results. Notably, intra-tumoral bacteria have tumor ablative potential. Some studies have reported that direct injection of lactic acid bacteria into human squamous cell carcinomas in nude mice retards tumor growth¹⁴⁰. However, the risk to other healthy tissues remains a significant concern. Cytokines and chemokines caused by bacteria promotes tumor progression using signal molecules inhibitors to inhibit tumor progression and treat cancer patients. This is also a part of the field of future HNSCC treatment strategy research that should not be neglected. However, whether the normal tissue related signaling pathways will be inhibited and lead to undesirable results needs to be considered.

Intra-tumoral bacteria represent an emerging and promising area of research in HNSCC. Current findings hold significant implications for future investigations and offer valuable insights into improving cancer prognosis with broad potential for clinical applications. The origin, distribution, and abundance of intra-tumoral bacteria in HNSCC patients are closely linked to tumor etiology. These bacteria actively influence tumor-associated immunity and modulate the biological behavior of tumor cells. Furthermore, intra-tumoral bacteria have been explored for potential clinical applications, including use in diagnosis, prognosis, and therapeutic strategies. The results of recent studies underscore the critical role of intra-tumoral bacteria in cancer research and highlight the expansive prospects for advancing patient care.