Dumont-Leblond et al. (15) profiled the lung microbiota of twenty-nine patients with non-small cell lung cancer (NSCLC) using a 16S rRNA sequencing approach. Diaphorobacter, Micrococcus, Paracoccus, Phascolarctobacterium and Ralstonia were overabundant in both healthy and cancerous tissues in NSCLC patients. Enteric, inflammatory or pathogenic bacteria, including Alloprevotella, Brevundimonas, Escherichia-Shigella, Faecalibacterium, Pseudomonas and unclassified Enterobacteriaceae, were only detected in cancerous tissues. Another study also revealed that pathogenic microbes, such as Acinetobacter Jungii, Enterococcus, Haemophilus haemolyticus, Haemophilus parainfluenzae and Streptococcus pneumoniae, were prevalent in lung tumor tissues (16). The common pathogenic microbes may cause lung infections due to the compromised immune system. It is intriguing whether these bacteria lead to pulmonary carcinogenesis or whether their enrichment is a result from the alteration of the pulmonary environment in cancer patients. Intratumoral pathogens could cause direct damage or induce chronic inflammation, contributing to lung cancer pathogenesis. Future work should concentrate on unraveling the involvement of intratumoral microbiota in lung carcinogenesis. The effect of NSCLC microbiota on clinical outcomes necessitates thorough exploration. Pathogenic microbes may also shape the tumor immune microenvironment. The crosstalk between intratumoral microbiota and host immune system is worthy of further research. In addition, there was an association between intratumoral microbiota and the microbial component in the bronchoalveolar lavage fluid (BALF) (16). Actinomyces Neesii, Haemophilus, H. haemolyticus, Haemophilus influenzae, human herpes virus type 7 (HHV-7), Neisseria lactose, Prevotella II, Streptococcus constellatus, Streptococcus crista, Streptococcus gordonii and S. pneumoniae were commonly present in the tumor tissue and BALF of NSCLC patients. It is reasonable to infer that exploration of microbial diversity and abundance in the BALF could provide hints regarding the NSCLC tumor condition.

The bacterial phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria were abundant in tumor tissues of patients with primary liver cancer (PLC) (17). The relative abundance of Agrobacterium and Rhizobiaceae was dramatically increased in tumor tissues, while that of Pseudomonas was significantly reduced. Different histopathological subtypes (combined hepatocellular-cholangiocarcinoma (cHCC-CCA), intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC)) of PLC possessed their respective intratumoral microbial signatures. The HCC group exhibited special bacterial biomarkers (e.g., Enterobacteriaceae). The microbial biomarkers may provide a potential tool for the subtype stratification of PLC. Nevertheless, more clinical studies are warranted to track intratumoral microbiota dynamics in PLC patients. Upper and lower gastrointestinal tumors had different microbiota signatures (18). The five phyla Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria and Proteobacteria dominated the microbial composition in upper gastrointestinal tumors including esophageal carcinoma (ESCA) and stomach adenocarcinoma (STAD). The level of Bacteroidetes was higher while that of Firmicutes was lower in lower gastrointestinal tumors including colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) than upper gastrointestinal tumors. The Bacteroidetes/Firmicutes ratio was higher in lower gastrointestinal tumors than that in upper gastrointestinal tumors. The genera Capnocytophaga and Helicobacter were more abundant in upper gastrointestinal tumors than lower gastrointestinal tumors. Faecalibacterium and Porphyromonas were enriched in READ. The high levels of intratumoral Alistipes and Blautia correlated with better survival probability in COAD patients. The high abundances of Pasteurellales and Porphyromonas were associated with the tumor stage in COAD and READ, respectively. Profiling tumor-resident microbiota can be helpful in discovering prospective microbial biomarkers for cancer progression and prognosis. A great deal of further work needs to be done to elucidate the contribution of dysregulated microbiota in the pathogenesis of gastrointestinal cancer. Members of the genus Bifidobacterium were detected in CRC tissues (19). The amount of intratumoral Bifidobacteria DNA was associated with the extent of signet ring cells, alluding to a possible role of Bifidobacteria in regulating tumor microenvironment (TME) and tumor differentiation during CRC progression. Bifidobacteria, a natural proportion of the intestinal flora, are producers of lactic acid and acetate (30). The impairment of intestinal barrier function might lead to the entry of Bifidobacteria into CRC tissues (31). In the gut, Bifidobacteria prevented colorectal carcinogenesis (32). Nevertheless, Bifidobacteria seemed to behave oppositely in the TME. It was likely that Bifidobacteria-produced lactic acid and acetate served as an energy source for CRC cells, allowing for cancer growth and immune escape. The divergent roles of Bifidobacteria during CRC development remain to be explored in the future.

Intratumor pancreatic microbiome was characterized through large-scale sequencing data from The Cancer Genome Atlas (TCGA) (20). Members of the phylum Proteobacteria represented the dominant bacteria in pancreatic adenocarcinoma (PAAD) and were associated with tumor metastasis. The abundance of Citrobacter freundii, uncultured Pseudomonadales bacterium HF0500_12O04, Acidovorax ebreus TPSY, Shigella sonnei Ss046, the primary endosymbiont of Sitophilus zeamais, Toxypothrix sp. PCC 7601, Mycoplasma hyopneumoniae and uncultured bacterium HF0500_10F10 exhibited an association with the upregulation of oncogenic pathways, the downregulation of tumor-suppressive pathways, and immunosuppression. The high abundance of A. ebreus was linked with decreased levels of M2 macrophages, memory T cells and CD8⁺ T cells. Altogether, these pancreatic microbes might be involved in PAAD occurrence and development by reprograming the TME. It is still elusive how microbes colonize the pancreas. Permeability of intestinal barrier may be a cause of bacterial dissemination into the pancreatic duct, which requires further corroboration. Intratumoral microbes could affect tumor susceptibility to chemotherapy and patient outcome. Gammaproteobacteria acted to induce gemcitabine resistance via the bacterial enzyme cytidine deaminase (33). Oppositely, Bifidobacterium potentiated the efficacy of immune checkpoint inhibitor (ICI) (34). It is plausible to propose that antibiotic/probiotic administration in a tumor type-specific manner could sensitize cancer cells to anticancer treatments. More extensive studies will be required to evaluate the therapeutic benefits of microbial manipulation combined with conventional treatments in cancer patients.

The compositions of bacterial communities differed between renal cell carcinoma (RCC) tissues and adjacent normal tissues (21). The species diversity was reduced in RCC tissues compared to normal tissues. The phyla Proteobacteria (64.97%) and Firmicutes (14.09%) dominated the microbiota composition of RCC tissues. The levels of Burkholderiales and Comamonadaceae were lower in RCC tissues than those in normal tissues. The relative abundance of Actinomyces, Amycolatopsis, Brevundimonas, Deinococcus, Gordonia, Microlunatus, Nitriliruptor, Phyllobacterium, Pseudoclavibacter and Weissella was increased in RCC tissues compared with normal tissues. Deinococcus was considered as a factor contributing to GC development (35). The role of Deinococcus in RCC is worthy of more detailed investigation. Moreover, reduced levels of Chloroplast, Klebsiella and Streptophyta could differentiate RCC tissues from normal tissues with high sensitivity and specificity. The functional analysis of RCC-associated microbiota showed that pathways responsible for cell growth and death, membrane transport and transcription were enriched in RCC tissues. Dysregulated microbes might promote RCC development by regulating these intracellular pathways, which needs to be further deciphered. Substantial research efforts should be direct toward illustrating the genuine association between intratumor microbiota and RCC pathology.

The microbes belonging to the families Bacillaceae, Halobacteriaceae and Prevotellaceae were abundant in cervical cancer tissues from 121 patients (22). Cervical cancer tissues had increased levels of Fusobacterium, Peptoniphilus and Prevotella and decreased levels of Lactobacillus relative to healthy uterine cervix and vagina. Co-incubation experiments showed that Prevotella bivia increased the expression of three immunoregulatory proteins lysosomal associated membrane protein 3 (LAMP3), signal transducer and activator of transcription 1 (STAT1) and antigen peptide transporter 1 (TAP1) in cervical cancer cells under hypoxic conditions. LAMP3 acted as a tumor promoter contributing to cervical cancer metastasis (36). It is assumed that P. bivia could infiltrate cervical cancer tissues and propel cervical carcinogenesis through regulation of immunological pathways. This study suggested an indirect communication between cervical microbiota and cancer cells. It is intriguing whether intratumoral microbiota can affect the functionality of infiltrating immune cells. However, additional studies are necessary to decipher the host-microbiota interaction in cervical cancer.

Intratumoral microbes have been identified as key regulators of cancer progression ( Table 2 ). Liu et al. (45) characterized the microbial communities within CRC or precancerous adenoma. The levels of CRC-associated genera Bacteroides, Fusobacterium, Parvimonas and Prevotella were markedly different between CRC and adenoma tissues. The abundance of these genera varied across different sites of a single neoplasia, indicating the heterogeneity of microbial communities within a single CRC or nonmalignant adenoma. The amount of intra-neoplasia microbes with high variation such as Proteobacteria diminished along the adenoma-carcinoma sequence, suggesting the progressive alteration in abundance and diversity of microbes in the course of colorectal carcinogenesis. Firmicutes species (e.g., Clostridium, Parvimonas and Peptostreptococcus) were the most abundant members that displayed strong association with Kirsten rat sarcoma viral oncogene homologue (KRAS) mutation in CRC tumors and adenomas. Genera belonging to Proteobacteria (e.g., Dechloromonas and Gallionella) were the most abundant microbes that positively correlated with microsatellite instability (MSI) in CRC patients. The majority of intratumoral microbes that were related to CRC-associated genetic markers involving KRAS mutation and MSI showed high variation in abundance. The interplay between gut microbiota and host genetic mutation could accelerate cancer pathogenesis (68) ( Figure 2 ). Host genetic factors in turn alter the microbial composition (69). More attention should be paid to the complex interrelation between gut microbiota and host genetic factors. Intratumoral microbiota heterogeneity may affect the metabolism of precancerous or cancer cells. Additional studies are suggested to elucidate the capricious compositional and functional profiles of intra-neoplasia microbiota as well as its contribution to colorectal carcinogenesis.

The liver is physiologically connected to the gut via the hepatic portal vein and is considered as the predominant organ site of primary CRC metastasis. However, gut vascular barrier (GVB) forms a critical barrier restricting intestinal bacteria dissemination. The high level of plasmalemma vesicle-associated protein-1 (PV-1) in primary tumors, a marker of disrupted GVB, was found to correlate with distant liver metastasis and poor prognosis in CRC patients (40). Importantly, CRC patients with high PV-1 expression in primary tumors possessed an increased amount of bacteria in metastatic liver lesions compared with paired healthy hepatic parenchyma, suggesting potential microbiota translocation through impaired GVB. Mechanistic investigation indicated that CRC-resident bacteria E. coli C17 directly broke down the GVB and caused PV-1 upregulation through a type III secretion system (TTSS) virulence factor (Virf)-dependent mechanism. Once GVB dysfunction, intestinal bacteria could disseminate to the liver where they fostered the recruitment of immune cells including macrophages, neutrophils and inflammatory monocytes. These events resulted in the creation of a pre-metastatic microenvironment that drove cancer metastasis to the liver. These findings revealed that intestinal bacteria targeted vascular gatekeepers to migrate into the secondary organ, contributing to the establishment of a favorable immune niche that supports the growth of disseminated cancer cells. PV-1 might act as a prognostic biomarker of gut vascular impairment and CRC distant recurrence, leading to liver metastasis formation. The clinical relevance of E. coli C17 for CRC progression should be further addressed. Furthermore, some beneficial bacteria (e.g., Lactobacillus paracasei) had the ability to restore the GVB. Therefore, there is a necessity of further research to dissect the functional link between intestinal bacteria and the metastatic process in CRC.

Spontaneous murine breast tumor displayed an expansion of commensal microorganisms that included Enterococcus, Lactobacillus, Staphylococcus and Streptococcus (46). The intratumoral microbes mainly colonized in tumor cell cytoplasm. Intracellular bacteria (e.g., Staphylococcus xylosus) together with host tumor cells, could travel through the circulation system and reside in distal organs. The invasion of various bacteria (e.g., Lactobacillus animalis, Streptococcus cuniculi and S. xylosus) promoted lung metastasis without affecting primary tumor growth. The intracellular bacteria reorganized actin cytoskeleton in circulating tumor cells and promoted host tumor cell survival by enhancing resistance to mechanical stress in the circulation system during metastatic colonization. Depletion of tumor-resident microbes suppressed the metastasis of breast tumor cells in vivo. Aerobic bacteria were increased in the lung metastasis, while the facultative anaerobes were decreased, implying a dynamic oxygen microenvironment in the tumor. The human and murine breast tumors possessed a similar microbial community profile, as characterized by high amounts of Enterococcus and Streptococcus. It was plausible to infer that intratumoral microbiota played an important role in the pathogenesis of human breast cancer. Intratumoral microbes may be an integral constituent of the tumor and act as pivotal drivers of tumor progression. Intratumoral microbiota could be a prospective target for better breast cancer management. It remains ambiguous how the bacteria enter tumor cells. The role of intratumor bacteria in tumor cell intravasation, extravasation and dissemination needs to be adequately delineated. Whether intratumoral microbiota, gut microbiota and the immune system cooperatively act to regulate cancer progression is an important question that emerges.

Cancer metabolism is key and intimately linked to cancer progression. In recent years, the role of intratumoral microbiota in reprogramming of cancer metabolism has been increasingly recognized ( Table 2 ). Bacillales, Burkholderiales, Clostridiales, Pseudomonadales, Sphingomonadales and Xanthomonadales were abundant in ICC tissues (58). Klebsiella pneumoniae, Paraburkholderia fungorum, Pseudomonas azotoformans and Staphylococcus capitis were verified to be present in ICC tissues. Intratumoral bacteria mainly resided in malignant cells of cancerous tissues, while they also localized to T cells, hinting that intratumor microbiota might be involved in tumor immunity. The amount of P. fungorum was noticeably higher in paracancerous tissues and had an inverse association with the carbohydrate antigen 199 (CA199) level, alluding to the potential antitumor potency of this bacterium. As expected, P. fungorum could repress the growth of human cholangiocarcinoma cells in vivo by regulating the metabolism of alanine, aspartate and glutamate ( Figure 4 ). The hepatic-intestinal circulation may contribute to the crosstalk between intestinal bacteria and intratumoral microbes. The impact of intratumoral microbes on cancer metabolism should be an important subject for future investigation. Akkermansia muciniphila exhibited an inhibitory activity against lung cancer growth in vivo (59). This intestinal commensal bacterium could translocate to lung cancer tissues via systemic circulation. A. muciniphila altered the intratumoral metabolic pathways including amino acid metabolism, glycolysis/gluconeogenesis and fatty acid biosynthesis. Tumor-associated A. muciniphila also modified the composition of intratumor microbiota by increasing the abundance of Acidobacteriae, Akkermansia, Bacteroides, Bifidobacterium, Gammaproteobacteria, Lactobacillus, Sphingomonadales and Staphylococcus. A. muciniphila-influenced symbiotic microecology showed an association with the metabolic network in tumor tissues. Continual efforts should be made to clarify intratumoral microbe-mediated complicated cancer metabolic reprogramming. The specific intracellular microorganisms that are involved in the antitumor action of A. muciniphila should be identified.

Bacterial communities were present in HCC cells and immune cells (78). Microbial diversity and richness were dramatically increased in HCC tissues compared with paracancerous tissues. The levels of commensal Enterobacteriaceae, Fusobacterium and Neisseria were higher in HCC tissues than in paracancerous tissues, whereas Agathobacter, Chryseobacterium, Dietzia, Faecalibacterium, Hydrogenophaga, Megamonas and Pseudomonas exhibited the opposite trend. It was reported that HCC-enriched Enterobacteriaceae utilized inflammatory byproducts within the microniches as energy sources, which eventually led to inflammation (79, 80). Likewise, the proinflammatory property of Fusobacterium was also documented (81). Since inflammation has been acknowledged as a risk factor for carcinogenesis, these bacteria may play a role in HCC formation (82). The high infiltration of pro-tumorigenic bacteria (e.g., Neisseria) and the reduced abundance of the tumor-suppressive bacteria (e.g., Pseudomonas) potentially propel HCC development (83, 84). Importantly, intratumoral microbes could alter cancer metabolic pathways. Fatty acid and lipopolysaccharide biosynthesis, and bacteria and disease pathways were remarkably enriched in HCC tissues. The resultant products of microbial metabolic activities may be a key source of fatty acids and lipids for cancer cells, thereby facilitating HCC cell proliferation and invasion. Intestinal permeability may be an explanation for bacterial translocation into HCC, calling for further verification. The mechanisms of action of intratumoral microbes in HCC carcinogenesis should be further explored.

Tumor microbiota communities were markedly different between PDAC patients with short-term survival (STS) and those with LTS (53). The STS tumors showed a predominance of Bacteroidea and Clostridia, while PDAC LTS cases were dominated by Alphaproteobacteria, Flavobacteria and Sphingobacteria. Actinobacteria (Saccharopolyspora and Streptomyces) and Proteobacteria (Pseudoxanthomonas) were more abundant in LTS patients versus STS patients. The heterogeneous tumor microbiota signatures contributed to different enrichment of metabolic functional pathways between STS and LTS patients. For instance, the STS cases were enriched in the pathways related to nucleotide metabolism, replication and repair, protein synthesis and processing, while the LTS cases demonstrated enrichment in the pathways associated with metabolism of amino acid, lipids and polyketides. The divergence in metabolic pathways may be an explanation for different clinical outcomes in PDAC patients, which necessitates additional research.

Intratumoral microbiota functions as key players in cancer-associated signaling cascades. The pathogens Acinetobacter, Actinomycetales, Ochrobacterium, Pseudomonas and Streptococcus were enriched in epithelial ovarian cancer (EOC) tissues (60). Intratumoral inoculation of Propionibacterium acnes propelled tumor growth and shortened the survival rate of EOC-bearing mice. The P. acnes strain could enhanced the expression of TNF-α and IL-1β, suggesting the proinflammatory potency of this bacterium. These two proinflammatory cytokines that were excreted by necrotic tumor cells could enhance Hedgehog (Hh) transcription (85). As expected, the Hh signaling pathway was abnormally motivated in P. acnes-treated mice, as evidenced by increased expression of glioma-associated oncogene 1 (Gli1), Gli2, patched homolog 1 (PTCH1), sonic hedgehog homolog (Shh) and smoothened homolog (SMO). Conversely, blockade of the Hh signaling prevented EOC progression caused by P. acnes. The proinflammatory P. acnes facilitated EOC pathogenesis by activating the Hh signaling cascade ( Figure 5 ). Complementary in vitro and in vivo studies are warranted to delve into how intratumoral microbes interact with intracellular signaling pathways, providing important clues for improving cancer management.

The fungal species including Metarhizium acridum CQMa 102, Phaffia rhodozyma and S. cerevisiae YJM1338 were enriched in tumor tissues of patients with papillary thyroid carcinoma (PTC) (61). Three PTC subtypes, Classical (CPTC), Follicular Variant (FVPTC) and Tall Cell (TCPTC), showed a similar microbial composition and richness. For instance, Botrytis cinerea, Pichia cephalocereana and Trematosphaeria pertusa were overabundant in these subtypes. Rhizopus arrhizus and uncultured Uromyces were underabundant in both CPTC and TCPTC. The level of Chaetomium globosum CBS 148.51 was related to increasing pathologic stage in PTC patients. C. Albicans, Eremascus albus and Thanatephorus cucumeris were positively associated with a higher pathologic M stage, while Spiromyces aspiralis, uncultured Cryptomycota and Wickerhamiella pararugosa had a linkage with a higher pathological N stage. Further studies are required to characterize the influence of these fungi on PTC tumor metastasis. Importantly, the relative abundance of fungal microbes was related to PTC-specific oncogenic pathways. For instance, Metschnikowia santaceciliae, Pacynthium nigrum, Spriromyces aspiralis and T. cucumeris were associated with the downregulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in CPTC. Brevicellicium exile, E. albus and Zoophthora occidentalis correlated with the upregulation of the p53 signaling in TCPTC. Uncultured Glomus was linked with increased activity of BRAF kinase and the activation of the mitogen-activated protein kinase (MAPK) signaling in FVPTC. Additional investigation is needed to identify intracellular signaling pathways that mediate the tumor-promoting effects of intratumoral fungal species. The intricate signaling network underlying PTC development merits further study.

The momentous role of intratumoral microbiota in cancer development and tumor immunity highlights its potential clinical implications in oncology. The diagnostic values of intratumoral microbiota have been explored. Wang et al. (88) revealed that head and neck squamous cell carcinoma (HNSCC)-depleted Actinobacteria/Actinomycetales/Actinomyces, Firmicutes, Lactobacillales, Rothia, Streptococcus and Veillonellales had solid predictive ability in differentiating tumor tissues from normal tissues. This intratumoral microbiota signature was associated with the clinicopathological characteristics such as age, gender, tumor stage and neoplasm histological grade. A microbial signature consisting of HCC-depleted Acidobacteriae and Parcubacteria as well as HCC-enriched Bacilli, Gammaproteobacteria and Saccharimonadia exhibited great discriminative accuracy and performance in HCC prediction (44). HCC-enriched Actinobacteriota, Firmicutes, Gammaproteobacteria, Proteobacteria and Saccharimonadia correlated with clinicopathological characteristics of HCC patients including tumor volume, cirrhosis grading (inflammation activity) and histological severity. These microbes might contribute to the onset and development of HCC. Intratumoral microbial signatures may be a valuable resource for unearthing prospective biomarkers for cancer diagnosis ( Figure 7 ). The potential utility of specific intratumoral microbes in clinical settings should be illuminated in the future. Further study on the association between specific microbes and clinicopathological features is required to support the aforementioned results.

A microbial signature comprising Actinobacteria, Bacilli, Bacillales, Epsilonproteobacteria, Fusobacteria, Fusobacteriaceae, Lactobacillaceae, Negativicutes, Pasteurellales and Streptococcaceae could be a potential biomarker for subdividing different ESCA subtypes (ESCC and esophageal adenocarcinoma (EAD)) (89). ESCA-enriched Fusobacteriales positively correlated, while the microbial abundance of Lactobacillales inversely correlated with the tumor stage. Fusobacteria, present in multiple cancer types, promoted the establishment of a proinflammatory niche that supported cancer development (90, 91). Therapeutic manipulation of Fusobacteria could be a promising treatment option in cancer patients. High levels of Lactobacillus, Negativicutes and Proteobacteria reflected better prognosis, while the enrichment of Clostridiales and Fusobacteriales was associated with worse prognosis. Clostridia, Clostridiales, Fusobacteriia and Fusobacteriales might be independent prognostic biomarkers for ESCA. Orally derived Fusobacterium species including Fusobacterium animalis, F. nucleatum, Fusobacterium polymorphum and Fusobacterium vincentii were prevalent in tumor tissues of CRC patients (92). The Fusobacterium species were enriched in right-sided, microsatellite instability-high (MSI-H) and BRAF-mutant tumors. Fusobacterium might have prominent specificity for the inflamed CRC subtype. F. animalis was likely to correlate with disease progression and reduced survival in consensus molecular subtype 4 (CMS4) CRC patients, highlighting that intratumoral microbes might represent promising biomarkers for CRC subtype stratification and prognosis. GC tissues had a lower diversity of microbiota than adjacent nonmalignant tissues (93). MSI-H subtype might have a tendency toward an increased microbial diversity relative to other subtypes (diffuse and intestinal GC). The diversity of intratumoral microbiota may be a potential biomarker for the classification of GC subtypes. Further investigation of intratumoral microbial signatures will help to better understand key pathological processes and biological mechanisms during cancer pathogenesis, as well as facilitate the identification of new microbial biomarkers for cancer.

The high levels of Fusobacterium and Haemophilus correlated with the T3/T4 stage and lymphatic metastasis in patients with oral cancer (94). Bacillales and Gemella were more abundant in T1/T2 stage patients and the non-lymphatic metastasis cases. These results demonstrated that intratumoral microbiota could be connected with oral cancer progression and metastasis. Future work should concentrate on delineating the characteristics of the microbiota in metastatic lymph nodes and the mechanisms through which intratumoral microbes affect lymphatic metastasis. Acinetobacter, Pseudomonas, Ralstonia, Rhodococcus and Sphingomonas were the dominant bacteria in PTC tissues (95). PTC patients with advanced lesions had a higher microbiota diversity than patients with mild lesions. The abundance of Pseudomonas, Rhodococcus and Sphingomonas was high in mild lesion cases. Granulicatella and Streptococcus were more abundant in patients with advanced lesions than those with mild lesions. The eight-genera microbiota signature that was composed of g_norank_F_norank_o_Coriobacteriales, g_unclassified_o_Rhizobiales, Granulicatella, Haemophilus, Pseudomonas, Rhodococcus, Sphingomonas and Streptococcus had the potential to discriminate PTC invasion status. Intratumoral microbes potentially interacted with thyroid hormones and autoimmune antibodies, which remodeled the TME, contributing to PTC invasion and progression. The mechanism by which tumor-resident microbiota affects PTC development and outcome necessitates substantial attention. The increased abundance of Blumeria graminis f. sp. Hordei was strikingly associated with early cancer pathological stages in HNSCC, while the decreased number of Aspergillus flavus, Coccidioides immitis RS and Gaeumannomyces tritici R3-111a-1 correlated with the absence of perineural invasion (96). The high level of Podospora anserina S mat⁺ and S. cerevisiae EC1118 was linked with tumor neoplasm presence and advanced pathological N-stage. These altered microbes might give rise to cancer metastasis and progression. Moreover, Inosperma fulvum, Phlyctochytrium arcticum and uncultured Cryptomycota had a relationship with clinical characteristics of patients with HPV⁺ HNSCC. Punctularia strigosozonata HHB-11173 SS5 was linked to the upregulation of oncogenic pathways and poorer patient prognosis. These fungal microbes might be clinically relevant to patient outcome, but their genuine roles in HNSCC development needs further study.

Pathogenic oral microbes, including Firmicutes, Fusobacteriia, Fusobacteriales, Fusobacteriaceae and Fusobacterium, exhibited great predictive efficacy in OSCC diagnosis and prognosis (97). These microorganisms correlated with clinical characteristics of OSCC, including histological grade and tumor stage. Intratumoral microorganisms could be used as diagnostic and prognostic biomarkers for OSCC. The association between oral microbiome and clinical parameters of OSCC necessitates further clinical studies. H. parainfluenzae might be negatively associated with the first-line treatment outcomes in NSCLC patients (98). Staphylococcus crista and Staphylococcus haemolyticus were related to longer progression-free survival (PFS). Serratia marcescens was associated with better OS while Corynebacterium jergeri and H. parainfluenzae were associated with poorer OS. It is still obscure how intratumoral microbiota influences the treatment and prognosis of lung cancer. A microbiome analysis on biopsy samples from different sites of patients with distinct primary tumors (breast, CRC and lung) showed that Acinetobacter, Burkholderia, Corynebacterium, Cutibacterium, Flavobacterium, Pelomonas, Rheinheimera, Sphingobium, Staphylococcus and Streptococcus were the ten most represented genera in metastatic tumor tissues (99). The richness of intratumoral microbiota was significantly related to OS and PFS in cancer patients. Further experiments to determine the clinical value of intratumoral microbiota are required. The relative abundance of intratumoral Pseudomonas was significantly higher in PLC patients with LTS than in those with STS (17). The level of Pseudomonas was positively correlated with clinical prognosis in PLC patients. This bacterium might represent a prognostic biomarker for PLC. Tumor microbial diversity was positively correlated with OS in patients with resected PDAC (53). The increased number of Pseudoxanthomonas, Saccharopolyspora and Streptomyces was associated with better clinical outcomes in PDAC patients. Another study showed that the composition and diversity of intratumoral microbiota were apparently distinct between long-term and short-term PDAC survivors (64). The high abundance of Megasphaera and Sphingomonas positively correlated with the prolonged OS. The high level of opportunistic pathogen Clostridium was related to shortened survival time. These dysregulated microbes might be promising prognostic biomarkers for PDAC. The high load of intratumoral F. nucleatum was markedly related to better OS, DFS and MFS in patients with anal squamous cell carcinoma (ASCC) (100). In addition, the association between intratumoral fungi and patient survival and therapy response in different cancer types was also documented (9). The oncogenic fungus, M. globosa, was correlated with shorter OS in breast cancer patients (49). The Phaeosphaeria genus was dramatically linked with shorter PFS in patients with ovarian cancer. The high amount of Cladosporium might be negatively associated with immunotherapy response in patients with metastatic melanoma. Thus, intratumoral fungi hold the promise as potential biomarkers and therapeutic targets in cancer. The clinical implication of intratumoral fungi remains to be detected in larger cohorts. Given a paucity of publicly-available fungal genomes, a great deal of investigative work is required to better understand the fungal functional repertoires in cancer.

In light of recent advances in the field of intratumor microbiome, microbiota-based therapies are considered to hold great application potential ( Figure 7 ). Many clinical trials exploring the therapeutic benefits of wild-type/modified microorganisms in cancer patients are now underway ( Table 3 ). A randomized, placebo-controlled, phase I dose-escalation trial in 26 patients with advanced PC indicated the safety, immunogenicity and tolerability of an oral T cell vaccine VXM01, which was composed of live attenuated Salmonella typhi carrying a eukaryotic expression plasmid encoding vascular endothelial growth factor receptor 2 (VEGFR2) (101). Another phase I clinical trial demonstrated that the VXM01 vaccine was efficacious in inducing VEGFR2-specific effector T cell responses and attenuating tumor perfusion in 30 patients with advanced PC (102).

CRS-207 is a live attenuated Listeria monocytogenes expressing mesothelin (103). In a multicenter, open-label phase Ib study, CRS-207 infusion in combined with chemotherapy decreased tumor size in 35 patients with malignant pleural mesothelioma (MPM). CRS-207 treatment induced marked changes in the local TME, as evidenced by elevated infiltration levels of T cells, DCs and NK cells, an increased ratio of CD8⁺ T cells to regulatory T cells (Tregs), and the transformation of M2-like macrophages into M1-like macrophages (103). Accordingly, CRS-207 combined with chemotherapy resulted in enhanced antitumor immunity and induced objective tumor responses in MPM patients. A multicenter, randomized, phase II trial involving 90 PC patients indicated that administration of CRS-207 plus cyclophosphamide (Cy)/the cancer vaccine GVAX exhibited favorable safety profile (104). The combined treatment promoted mesothelin-specific CD8 T cell responses and prolonged survival of PC patients. However, a randomized, controlled phase IIb clinical trial in patients with advanced PC showed that CRS-207 in combination with Cy/GVAX did not improve OS over standard chemotherapy (105). The engineered Listeria strain ADXS11-001, which expresses the HPV16-E7 fusion protein to target HPV-positive tumors, could activate antigen-specific T cell responses and lead to the increased serum level of CCL22 in patients with HPV-associated oropharyngeal cancer (106). The combination of ADXS11-001 with standard chemoradiation was proven to be well tolerable in patients with anal cancer (107). All nine patients showed complete clinical response, and eight patients (89%) exhibited no evidence of disease at a median follow-up of 42 months. ADXS31-142 is an attenuated L. monocytogenes-based immunotherapy targeting prostate-specific antigen (PSA) (108). An open-label phase I/II KEYNOTE-046 study including 50 patients with metastatic castration-resistant prostate cancer (mCRPC) preliminarily verified the safety and tolerability of ADXS31-142 combined with pembrolizumab. This combination treatment might improve OS in mCRPC patients with visceral metastasis, which warrants additional evaluation in future studies. ADXS31-164, a highly attenuated, recombinant L. monocytogenes expressing a chimeric human HER2/neu fusion protein, induced robust T cell immune responses, retarded tumor growth and extended OS in animal models (109, 111). ADXS31-164 might have significant translational relevance for patients with HER2/neu-positive cancers. An open-label, multicenter, phase I study demonstrated that a single intratumoral inoculation of nontoxic Clostridium novyi (C. novyi-NT) reduced the size of the injected tumor in nine patients with injectable, treatment-refractory solid tumors (41%) (110). C. novyi-NT administration induced a temporary systemic cytokine response and potentiated tumor-specific T cell responses. However, C. novyi-NT treatment might raise safety concerns including limb abscess, pathological fracture, rash and respiratory insufficiency. Thus, it is of great importance to assess the safety and therapeutic value of C. novyi-NT. Future studies with larger cohorts will be required to verify the protective effect of these antitumor microbiota therapies. An in-depth investigation of the immune alterations in the TME will contribute to uncovering the mechanisms of action of microbiota-centered therapies.