Dynamic interplay of intratumoral microbiota within the colorectal cancer microenvironment

Colorectal cancer (CRC) is a complex malignancy, influenced not only by cancer cells but also by the tumor microenvironment (TME). Within the TME, emerging evidence highlights the presence and functional roles of diverse microbial entities, referred to as intratumoral microbiota. The distribution of these microbiota exhibits significant heterogeneity and engages in dynamic interactions with tumor cells, forming a unique ecosystem. Certain bacterial strains distinctly influence the TME of CRC, affecting the characteristics and progression of the tumor. This review summarizes the intricate roles of intratumoral microbiota within CRC's TME, emphasizing their importance in the disease's development and progression, and discuss the opportunities and challenges in the field.

tions between immune cells, extracellular matrix (ECM) components, blood vessels, and signaling molecules, culminating in a specialized niche that fosters cancer cell growth. 3The comprehension of the TME's pivotal role in cancer biology has undergone significant evolution.This has catalyzed the emergence of innovative therapeutic approaches directed at manipulating this dynamic microenvironment. 4Nonetheless, a comprehensive elucidation of the TME's multifaceted components and its potential repercussions on tumorigenesis remains a challenge.
Recent advancements in high-throughput sequencing technologies 5 and metagenomics 6 have revolutionized our understanding of the human microbiome, revealing that microorganisms inhabit diverse human niches.Particularly in CRC, a dynamic microbial ecosystem has been identified within the tumor tissue, paving the way for novel perspectives on tumor progression and its interplay with resident microorganisms.The potential ramifications of this intricate interplay between the TME and the intratumoral microbiota in CRC cannot be overstated, as it could redefine diagnostic and therapeutic modalities.
Understanding the specificity of intratumoral microbiota in CRC has profound implications for developing innovative diagnostic and therapeutic strategies.Targeting the intratumoral microbiota within the TME or supplementing bacteria with antitumor effects holds promise in advancing personalized medicine, tailoring therapeutic interventions based on individual patients' unique microbial profiles.
This review summarizes the characteristics of intratumoral microbiota in CRC and the interplay between the TME and intratumoral microbiota, aiming to enhance our understanding of CRC pathogenesis and highlight potential avenues for innovative diagnostic and therapeutic strategies in CRC treatment.

CHARACTERISTICS OF TME IN CRC
Tumors should not be viewed as isolated entities.Rather, they represent intricate and ever-evolving ecosystems.This complexity is particularly evident in CRC.The TME of CRC can be likened to a garden, with tumor cells as aggressive weeds.Just as weeds require nourishing soil to thrive, CRC cells heavily depend on the diverse milieu of the TME, composed of cancer cells, stromal cells, the ECM, and a vast array of immune cells. 7 standout feature of the CRC TME is its pronounced infiltration by effector memory lymphocytes, 8 predominantly T cells, which suggests a rapid immune response to threats. 9,10This contrasts with some cancers, such as melanoma, where the T-cell profile might initially be diverse but becomes dominated by "exhausted" T cells with inhibitory receptors such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4(CTLA-4). 11In CRC, tumor-associated macrophages often exhibit a tumor-promoting M2 phenotype, 12 while myeloid-derived suppressor cells (MDSCs) further dampen antitumor immunity and bolster tumor growth. 13enturing deeper into the TME, the stromal landscape in CRC stands out.The matrix is densely populated with cancer-associated fibroblasts (CAFs), 14 which actively contribute to tumor progression by secreting cytokines, remodeling the ECM, and promoting angiogenesis.While CAFs can be found in many tumor types, their interaction and cross-talk with CRC cells are distinctly enhanced, leading to an environment conducive for tumor growth and metastasis.Furthermore, the vascular architecture in CRC, supported by pericytes and vascular endothelial cells, displays particular adaptations in response to the tumor's microenvironmental needs.Particularly, vascular endothelial growth factor, found abundantly in CRC samples, 15 plays a central role in angiogenesis, which can significantly influence patient outcomes. 16The ECM in CRC, comprising various proteins, glycoproteins, and proteoglycans, has a unique composition and density that can sway cellular behavior, migration, and resistance to treatment.Furthermore, the TME is a hotbed for cytokines, 17 with CRC notably producing high levels of interleukin-6 (IL-6) and transforming growth factor-β, which fuel tumor growth while suppressing immune responses.
A unique dimension of CRC's TME is its intimate relationship with the gut microbiota. 18Given the intestine's role in human health, including its vast microbial community, its interaction with CRC is profound.Certain bacterial species, notably Fusobacterium nucleatum, have been implicated in CRC progression, either bolstering or inhibiting the disease through various mechanisms, such as immune modulation or the production of genotoxic metabolites.This microbiota interplay, influenced by diet and lifestyle, adds complexity to the CRC's TME, making it a nexus of internal and external factors.
To sum it up, while some common patterns in TME across various cancers emerge, the immune landscape of CRC, shaped by genetic, epigenetic, environmental, and stochastic factors, stands distinct.This complexity underscores the importance of understanding CRC's TME in devising therapeutic strategies, echoing the sentiment of managing both the "weeds" and their nurturing environment to control CRC's progression effectively.

INTRATUMORAL MICROBIOTA, AN INTEGRAL AND INDISPENSABLE COMPONENT OF THE TME
The microbiome is increasingly recognized as an integral component of the TME. 19Contrary to past beliefs that tumor tissues were sterile, recent studies have unveiled the existence of intratumoral microbiota in at least 33 significant cancer types, including CRC, 20 as intratumoral microbiota exhibit tumor-type specificity, and each subtype harbors a distinct bacterial community with specific metabolic functions. 20,21This intertwining of microbiota within the tumor has given rise to the concept of the "tumor microbiome microenvironment." CRC tissues are notable for their higher bacterial and fungal load in comparison to healthy tissue, with particular bacterial strains enriched within the tumor. 22he intratumoral microbiota in CRC can be attributed to three primary sources. 23,24First, the mucosal barrier of the colon, which naturally houses diverse microorganisms, becomes a conduit for these microbes during instances of mucosal damage caused by tumor development.This is further complicated by the heterogeneous nature of gut microbiota across different segments of the colon, which can influence the types and prevalence of bacteria within the TME. 25 The distinction in microbial composition between proximal and distal tumors accentuates the role of mucosal pathways in bacterial infiltration. 18djacent to the CRC tissues, the surrounding normal tissue too displays similar bacterial composition, suggesting a second pathway for microbial entry.These bacteria possibly exploit the immunosuppressive and hypoxic conditions prevalent in tumor areas to establish a stronghold.Such a condition, arising due to the altered cellular metabolism and immunological responses within the CRC TME, tends to create a favorable environment for these microorganisms, allowing them to thrive and interact dynamically with the host cells.
A third mode of bacterial access is through the bloodstream, influenced by various factors such as oral or gut microbial imbalance.Escherichia coli, after compromising the gut's vascular integrity, can circulate through the bloodstream, anchoring itself in distant organs like the liver.Such colonization can be instrumental in setting up the stage for metastasis. 26hile CRC-associated bacteria mainly reside within structures such as tumor and immune cells, this does not necessarily indicate direct bacterial invasion.These microbial interactions are multifaceted, often involving cellular transport processes.However, direct bacterial infiltration from the bloodstream to tumor sites remains a possibility. 27n conclusion, the TME in CRC is significantly shaped by its intratumoral microbiota.The vast microbial diversity within the colon indicates that microbiota profiles may vary among CRC subtypes.This variance could influence tumor development, host interactions, and treatment outcomes, underscoring the importance of understanding these microbial dynamics in CRC's pathophysiology (Figure 1).

Heterogeneity of the TME of CRC
The TME of CRC exhibits a remarkable degree of heterogeneity, reflecting the intricately diverse and multifaceted nature of their components. 23Delving into the role of intratumoral microbiota in CRC demands a deep understanding of its unique TME landscape.
Research spearheaded by Nejman's team provided insights into the complex realm of the intratumoral microbiome across various tumor types, identifying unique microbiome compositions. 20While multiple studies have discussed the intratumoral microbiota in different tumors (Figure 2), a particular emphasis on gastrointestinal cancers, such as CRC, has been observed. 28Specifically, Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria, and Actinobacteria have been identified as the dominant bacterial phyla within CRC, a composition distinct from normal colorectal tissues. 28ithin the CRC TME, the heterogeneity of the microbial community becomes even more pronounced.A study by Bullman's team using spatial transcriptomics identified distinct patterns of bacterial distribution within CRC 29 (Figure 3).They found that bacterial clusters within the TME often correlated with regions of highly mutated cancer cells, especially those with diminished wild-type p53 protein expression.This suggests that intratumoral microbiota may play a role in modulating cancer cell behavior in CRC.Furthermore, areas with a high bacterial load within the TME of CRC displayed characteristics such as reduced cellular proliferation potential and elevated MAPK/ERK signaling pathway activity, indicating a potential interplay between bacteria and tumor cell signaling pathways.The TME of CRC not only varies from other cancer types but also exhibits regional differences within the same tumor, highlighting the intricate nature of its microbial ecosystem.A pressing question remains: do changes in the TME result from bacterial colonization, or do specific bacterial populations naturally gravitate toward particular niches within the CRC TME? 29

The cause of heterogeneity of TME of CRC
The TME of CRC plays a pivotal role in shaping the colonization and behavior of microbial communities within the tumor.While current spatial transcriptome has proved that there is heterogeneity in intratumoral microbiota in CRC, the exact distribution of bacterial species within the CRC TME remains to be elucidated.Existing research shows that the microbiome composition within breast cancer tissues shows a notable shift from anaerobic to facultative anaerobic bacteria with a rise in aerobic bacteria during lung metastasis, which can be attributed to the lung's enhanced oxygenation.This differential bacterial distribution suggests varied oxygen gradients and living conditions in tumor tissues.Such findings provide insights for envisioning the potential microbiome distribution within CRC (Figure 4).

Oxygen gradients
The varying oxygen gradients within the CRC's TME, influenced by factors such as tumor vascularity and cellular growth rates, create distinct niches.The innermost regions of CRC, often hypoxic due to limited blood supply, offer a conducive environment for anaerobic bacteria like F. nucleatum. 30Studies revealed the overabundance of F. nucleatum in CRC tissues compared to healthy ones, 31,32 emphasizing its potential role in cancer progression.On the other hand, the peripheries of the tumor, which tend to be better oxygenated owing to proximity to blood vessels, might support the proliferation of aerobic microbes, such as E. coli and Enterococcus species.

pH degree
Acidity is a fundamental characteristic of the TME, providing an energy source that fuels the malignant progression of CRC. 33The Warburg effect, 34 a metabolic shift in cancer cells, leads to increased production of lactic acid as a byproduct of anaerobic glycolysis, contributing to the acidic conditions observed in areas closer to the tumor core.Various factors, such as cancer cell metabolism, tissue hypoxia, and immune response, contribute to the alterations in pH of TME.This acidic milieu tends to favor acidophilic bacteria.For example, a genus such as Lactobacillus, which is known to thrive in acidic environments, might find the TME more hospitable.

Nutritional conditions
In the nutritional battlefield of the TME, CRC cells voraciously consume glucose to sustain their rapid growth.This altered nutrient matrix, consequently, limits the bacterial communities.Bacteria such as Bacteroides, which can metabolize alternative energy sources when glucose is scarce, might have an edge in such environments. 35s Han et al. pointed out in 2019, specific strains of Bacteroides have a mutualistic relationship with CRC cells, benefiting from the metabolites the tumor cells produce.Fusobacteria possess unique metabolic adaptations that give them an advantage in the tumor environment.Unlike Enterobacteriaceae, they do not rely on glucose but instead utilize amino acids and peptides for nourishment.

Inflammatory environment
Chronic inflammation, endemic to CRC, profoundly influences both tumor progression and intratumoral microbiota distribution, arising from events such as accelerated tumor cell growth and the influx of immune cells.This heightened inflammatory milieu not only nurtures the tumor's advancement but also dictates the settlement patterns of Heterogeneity of intratumor microbiota in different tumors.(A) Lung tumors.There are several bacteria that influence the progress and metastasis of lung cancers.These bacteria perform their roles using different pathways.(B) Gastrointestinal tumors.The composition and function of microbiota in gastrointestinal tumors is complex.Reproduced with permission. 23Copyright 2023, John Wiley and Sons.
specific microbial communities.The presence of various inflammatory signals can steer certain bacteria toward particular niches within the CRC landscape, such as IL-8 and tumor necrosis factor-alpha.These mediators act as chemoattractants, influencing the recruitment and distribution of specific bacterial species, such as F. nucleatum and E. coli.
It is evident that microbial colonization within tumors is influenced by multiple factors, such as nutrient availability, oxygen gradients, and pH levels, leading to the observed spatial microbial diversity.Recognizing the TME's heterogeneity in CRC is essential for understanding its intricate influence on microbial distribution.Clinically, these insights have profound implications for CRC treatment.Tailoring therapeutic approaches based on indi-vidual TME and microbial profiles can potentially enhance treatment efficacy.Furthermore, specific microbial signatures could offer predictive insights into CRC progression and outcomes.Overall, a comprehensive understanding of CRC's TME not only elucidates underlying tumor mechanisms but also holds promise for advancing clinical applications.

HOW CAN INTRATUMORAL MICROBIOTA INFLUENCE THE TME OF CRC
It is not only the TME that influences the activity and dynamics of intratumoral microbiota; these microorganisms, in turn, exert profound impacts on the TME, creating a complex interplay and balance.

DNA damage
Research has highlighted the crucial role tumoral microbiota play in the genesis of CRC, primarily through DNA damage.Studies by Dejea et al. disclosed a heightened presence of enterotoxigenic B. fragilis and E. coli in the colonic mucosa of patients diagnosed with familial adenomatous polyposis. 36Further corroborating this, experimental data from mice studies illustrated amplified DNA damage when colonized with both aforementioned bacterial strains.E. coli, in particular, not only possess the capability to directly modulate tumor cell behavior, 37 but The mutual selection between tumor microenvironment (TME) and intratumoral microbiota leads to heterogeneity of tumors.Aerobic bacteria are mainly concentrated on the surface of tumors and around blood vessels.Acidic and anaerobic bacteria are mainly concentrated in the core of tumor.Image created with BioRender.com.
its genetic makeup also reveals a fascinating mechanism by which it might promote carcinogenesis.Specifically, a region within its genome, termed polyketide synthase, is responsible for the synthesis of colibactin. 38This compound is implicated in initiating DNA damage in host cells. 39Such genetic perturbations can subsequently lead to genomic instability, fostering pathogenic mutations. 40his series of events underscores the profound influence of E. coli and, by extension, intratumoral microbiota in the evolution of CRC via DNA damage pathways.

Modulation of immune responses
Bacterial strains, especially in the context of the TME, are fundamental in orchestrating immune responses.A notable exemplar is B. fragilis, known for producing a molecule termed polysaccharide A (PSA).This particular molecule could create an immunosuppressive environment, which, in turn, is favorable for CRC progression. 41It is F. nucleatum that also prominently shapes the immunological landscape in CRC.Elevated levels of F. nucleatum correlate with diminished CD3+ T-cell infiltration, indicating its potent immunomodulatory role.Interestingly, the tumor subtype predominantly associated with Fusobacterium showcases an abundant influx of tumorinfiltrating lymphocytes. 42This surge in immune cells underscores the intricate relationship between F. nucleatum and the host response, facilitating the progression of colorectal neoplasian. 43Such findings accentuate the imperative of understanding and potentially regulating the TME, especially concerning the role of F. nucleatum 44 However, F. nucleatum's influence is not merely passive or limited to its abundance.It actively shapes the CRC TME through a myriad of mechanisms.By driving inflammation and fine-tuning immune reactions, F. nucleatum creates an environment that amplifies CRC cells proliferation and invasion.Additionally, it plays a role in the initiation of processes such as the epithelial-mesenchymal transition (EMT) and augments resistance to chemotherapy. 45,46he enrichment of this bacterium may inadvertently offer growth advantages to CRC cells, particularly as it can coax tumor-promoting reactions from myeloid immune cells.Moreover, F. nucleatum has been found to impede antitumor T-cell-mediated adaptive immunity, further emphasizing its role in CRC's progression.

Production of virulence factors
The complex landscape of CRC is markedly influenced by the myriad of metabolites and virulence factors produced by the resident microbes.At the forefront of this interaction is F. nucleatum, which employs a virulence factor, FadA, to specifically bind to E-cadherin, 47 through activating the Wnt/β-catenin signaling in intestinal epithelial cells. 48This adhesion is complemented by its ability to secrete virulence factors that notably inhibit T-cell activity, degrading antitumor T-cell-mediated adaptive immunity. 49,50Similarly, enterotoxigenic B. fragilis (ETBF) secretes fragilysin, a potent toxin with dual actions.While it can disrupt cell-cell interactions-thereby promoting tumor proliferation. 51B. fragilis utilize fragilysin for its cytotoxic effects on CRC cells, potentially hindering tumor expansion.Moreover, this bacterium synthesizes a range of virulence factors, with enterotoxins and PSA standing out.PSA, in particular, shifts the immune dynamics by bolstering the pro-inflammatory T-helper 17 cell population, furthering CRC progression. 52This action is mediated through the IL-17-dependent signaling cascade, leading to the activation of pathways like nuclear factor-κB (NF-κB) and Wnt. 53The culmination of these pathways shapes an inflammatory environment in the gut. 54However, B. fragilis showcases its paradoxical nature by also boosting the release of anti-inflammatory cytokines, such as IL-10, potentially counteracting inflammation and constraining tumor growth.

Persistent inflammatory reactions
Chronic inflammation stands as a pivotal trigger in the genesis of CRC.A multitude of bacterial agents, with F. nucleatum leading the charge, have been identified as key orchestrators of these persistent inflammatory cascades.Notably, F. nucleatum interacts with toll-like receptors (TLR) within the TME, activating the TLR4/MYD88/NF-κB signaling pathway.This pathway's activation creates a pro-inflammatory milieu conducive to CRC cells survival, while simultaneously inhibiting apoptosis. 55This forms a positive feedback cycle that induces pro-inflammatory responses and promotes progression of CRC.In line with this, F. nucleatum further escalates its influence by boosting miR-21 expression within CRC cells.This leads to the suppression of RAS protein activator like 1 (RASA1), thus setting in motion the intrinsic RAS signaling pathway.The activation of this pathway culminates in an increased transcription of genes closely associated with cellular proliferation. 56This mechanism underscores the concept that bacterial-induced inflammation is not a transient event, but rather, a consistent driver of tumor progression.Specific strains of B. fragilis and certain E. coli are known to trigger pro-inflammatory responses.These responses result in the recruitment of immune cells, including neutrophils and MDSCs, to the tumor site. 57Depending on the TME, these immune cells may either promote or inhibit tumor progression, influenced by their interactions with the bacteria and the host.Depending on the TME, these immune cells may either promote or inhibit tumor progression, influenced by their interactions with the bacteria and the host.

Metastasis and recurrence
Recent discoveries have illuminated the role of microbes in the metastasis and recurrence of CRC.Strikingly similar strains of Fusobacterium have been identified in both primary CRC and its metastatic derivatives, 58 suggesting that these bacteria might accompany CRC cells during their metastatic migration to distant sites.This propensity for metastasis can be linked to Fusobacteria's exceptional adhesive and invasive capabilities toward epithelial cells. 43,44n tandem with these observations, recent clinical studies have highlighted a significant correlation between F. nucleatum levels and the prognosis of CRC patients. 45,59articularly, noteworthy is the finding that patients with persistent F. nucleatum post-neoadjuvant chemoradiotherapy are at an increased risk of recurrence.Conversely, the elimination of the bacterium after treatment is associated with favorable immune resurgence, epitomized by a marked upsurge in cytotoxic CD8+ T cells.Such insights accentuate the cruciality of closely monitoring and potentially targeting F. nucleatum as a part of comprehensive CRC therapeutic strategies.
Understanding the interplay between intratumoral microbiota and CRC's TME offers avenues for innovative therapeutic interventions.Recognizing each microbe's unique influence and the resultant cascade of cellular events is imperative for devising comprehensive strategies to combat CRC.

TARGETING INTRATUMORAL MICROBIOTA FOR CRC DIAGNOSIS AND TREATMENT
The intricate relationship between specific bacteria and the TME of CRC has unveiled a new dimension in cancer research.As researchers delve into the impact of these bacteria on CRC's complex biological landscape, they also uncover opportunities to utilize this knowledge for innovative diagnostic and therapeutic strategies.The identification of these specific bacteria and their interactions within the TME also provides a foundation for refining diagnostic precision and developing targeted treatments.By bridging the gap between understanding the role of bacteria in the TME and harnessing this knowledge for clinical applications, the prospect of effectively targeting intratumoral microbiota for improved CRC diagnosis and treatment becomes increasingly promising.

Biomarkers for CRC diagnosis and prognosis
Interestingly, a number of investigations have underscored the potential of F. nucleatum in enhancing the early diagnosis of CRC. 60,61Studies indicated that combining fecal F. nucleatum with immunochemical tests can augment the identification of advanced colorectal adenoma and carcinoma. 62Remarkably, the relative abundance of fecal F. nucleatum was found to be 132 times higher in the CRC group than in the control group.Diagnostic tests using fecal F. nucleatum alone produced sensitivity and specificity rates of 80.2% and 80.7%, respectively.Sensitivity further improved when fecal F. nucleatum was combined with FIT and other fecal bacteria. 63eyond F. nucleatum, metagenomic analysis has brought other potential markers to the forefront.The discovery of butyryl-CoA dehydrogenase in CRC fecal microbiomes serves as an exemplar.Its presence was noticeably elevated in CRC patients, drawing attention to its potential as a novel diagnostic tool. 64Additionally, a groundbreaking study conducted across several medical centers revealed that serum anti-Fn-IgA levels could be a predictor of early-stage CRC, with a specificity rate surpassing 85%.Furthermore, the intratumoral microbiota is emerging as a potent source of biomarkers for CRC diagnosis and prognosis due to its role in tumorigenesis and progression.Several investigations have shown that the amount of F. nucleatum DNA in tissue has a positive correlation with CRC-specific mortality, suggesting its potential as an adverse prognostic biomarker. 65An evident association was also observed between the prevalence of F. nuclea-tum and lymph node metastasis in CRC cases.Poore et al. have highlighted the presence of distinct tumor microbiomes in the bloodstream, hinting at their potential as blood-based diagnostic tools. 66If these findings are corroborated through extensive clinical sampling, the tumor microbiome could emerge as a revolutionary microbial marker, enabling precise prognosis and perhaps revealing new CRC subtypes.
However, while the promise of the intratumoral microbiota as biomarkers is undeniable, challenges persist.The intricacies of the microbiome, individual variations, and distinctions between cancer types highlight the need for standardizing and validating these biomarkers.To solidify microbial biomarkers for clinical use, comprehensive longitudinal studies combined with multi-omics methodologies are imperative.

Therapy targeting intratumoral microbiota in CRC
The intratumoral microbiota has emerged as a focal point in the advancement of cancer therapy, particularly since the identification of its presence in solid tumors.This microbial community within tumors can significantly influence cancer progression and treatment outcomes, making it a potential therapeutic target. 67ntriguingly, F. nucleatum is sensitive to several antibiotics, such as metronidazole and clindamycin.In patientderived xenograft models of CRC with an abundance of F. nucleatum, treatment with metronidazole led to reduced tumor sizes, demonstrating the potential of antimicrobial agents in eliminating intratumoral microbiota.Similarly, cefoxitin has exhibited effectiveness in eradicating ETBF colonies in mice and decreasing IL-17A levels in the colon. 68n innovative approach to the modulation of intratumoral microbiota was observed in a study by Cai's team (Figure 5). 69Administering antibiotic mixtures (ATBx) through the tail vein effectively eliminated the intratumoral microbiota while preserving the gut microbiota.Interestingly, when ATBx was administered through gavage into mice, it had the ability to eradicate both intestinal microbiota and intracellular bacteria.Such findings suggest that the route of antibiotic administration can differentially affect the microbiota landscapes in various body sites, offering a method for targeted microbiota manipulation in CRC.Moreover, numerous antibiotics have been extensively used in clinical settings, with their safety and tolerability well validated.They effectively treat various infectious diseases, and their associated side effects and adverse reactions are widely studied and understood.As a result, antibiotic therapy emerges as the swiftest, safest, F I G U R E 5 Various administration strategies of antibiotics and the influences on the gut and tumor microbiota.Reproduced with permission. 69Copyright 2022, Elsevier.and most clinically feasible approach to target the tumor microbiome.
Furthermore, drugs traditionally not considered antibiotics, such as metformin, have shown potential in targeting F. nucleatum. 70A seminal study documented metformin's ability to counteract F. nucleatum-induced tumorigenicity, providing a fresh perspective on repurposing existing drugs for CRC therapy 70 .Zerumbone, the primary compound found in Zingiber zerumbet, displays antibacterial, antiinflammatory, and antitumor attributes. 71Notably, it effectively reduces ETBF-induced intestinal inflammationlinked CRC by influencing pathways such as IL-17, βcatenin, Stat3, and NF-κB.
Another promising avenue is phage therapy.Bacteriophages, or phages, exhibit specificity in infecting and lysing bacteria. 72,73Zheng et al. isolated a phage strain capable of targeting F. nucleatum.To amplify its therapeutic potential, they conceived a hybrid nanosystem combining phages with irinotecan and dextran nanoparticles.This approach not only effectively eliminated F. nucleatum but also improved chemoresponse, showcasing the potential of combining traditional and novel treatments.

Probiotic therapy
Within the TME, a spectrum of bacteria has been identified, including those commonly recognized as probiotics, such as Lactobacillus spp., Bifidobacterium spp., Faecalibacterium spp., Roseburia spp., Clostridium spp., and other species of Lachnospiraceae family.However, their preva-lence within the TME is notably lower compared to that in feces and the intestinal microbiota. 74,75espite their limited abundance and the exploration in the TME is still in the early stages, these probiotics work: from reinforcing gut barrier integrity, normalizing intestinal microflora, and curbing inflammation, to directly mitigating tumor initiation and growth. 76otably, an intriguing correlation has been observed between the abundance of Bifidobacteria and the presence of signet ring cells in CRC tissue, indicating a potential role for Bifidobacteria within the TME in influencing tumor differentiation during development. 77Clinical evidence further attests to the promising role of probiotics in CRC management.In a notable trial, post-CRC surgery patients were administered a concoction of Bifidobacterium animalis subsp.lactis, Lactobacillus casei, and Lactobacillus plantarum.The results were encouraging, with significant enhancements in beneficial bacterial populations and concurrent reductions in CRC-associated bacterial strains. 78nother intervention using L. plantarum, Lactobacillus acidophilus, and Bifidobacterium longum documented similar positive shifts in microbial diversity, strengthening the mucosal barrier in CRC patients. 79Intriguingly, specific strains of F. nucleatum could engage in horizontal gene transfer, revealing a tight-knit relationship with other gut microbes. 50,80This is evident in the therapeutic effects of microecologic products 81 that mitigate the virulence of genera by overseeing the complete microbial composition and restraining the acquisition of virulence genes by Fusobacterium.For instance, probiotic intervention in CRC patients resulted in a reduction of Fusobacterium and an increase in microbial diversity, suggesting that microecologic products could benefit CRC patients by suppressing CRC-associated genera. 82robiotics boost the breakdown of dietary fiber through fermentation, leading to increased concentrations of potential anticancer agents.These agents, including SCFAs, phenols, and conjugated linoleic acids, exhibit therapeutic benefits against CRC. 83,84Consistent consumption of probiotic bacteria can lead to a reduction in pathogen within the gut, so that lowering the carcinogenic compounds. 83n conclusion, probiotics' foray into TME investigations hints at their substantial promise in both CRC prevention and treatment.Their potential therapeutic benefits underscore the importance of delving deeper into their efficacy, safety, and precise mechanisms in CRC management.As research advances, probiotics could well emerge as an exciting frontier in CRC therapeutics.

Bacterial engineering
The enhanced survival of bacteria within tumors opens up possibilities for bacterial engineering, which holds the potential to produce localized therapeutics in specific regions of the body, thereby increasing the efficacy of certain drugs through local transformation. 85,86his approach involves the targeted modification of live pathogenic bacteria to create unique anticancer vectors that can effectively treat tumor-like tissues in a personalized manner. 87llustratively, E. coli Nissle 1917 has been bio-engineered.This engineered bacterium effectively inhabited tumor sites and transformed nitrogen-hydrogen compounds within the TME into L-arginine.L-arginine, a pivotal factor for bolstering antitumor T-cell reactions, showcased the potential of this modified bacterium for potential application in cancer therapy. 88To sum up, the progression and utilization of engineered bacteria constitute a significant stride in the realm of cancer treatment advancement (Figure 6).

CHALLENGES IN EXPLORING INTRATUMORAL MICROBIOTA IN CRC
Examining the intratumoral microbiota in CRC presents formidable challenges, stemming from the intricacies of the TME and the interplay between microbiota and tumor dynamics.
Contrary to tumors located in sterile regions, securing uncontaminated samples that genuinely represent the intratumoral microbiota of CRC is notably arduous.CRC's intimate anatomical juxtaposition 89,90 and its symbiotic relationship 91 with intestinal microbiota exacerbate the risk of contamination.
Despite the burgeoning literature on CRC's intratumoral microbiota, comprehensive studies isolating microbiota devoid of gut biome influences are limited, and findings often diverge.For instance, Shah et al. employed minimally invasive colonoscopy biopsies, 92 while Wang et al. scrutinized the intratumoral microbiota of surgically excised CRC samples. 93Additionally, Liu et al. utilized the innovative honeycomb biopsy sampling approach to discern differences between tumor tissue and mucosal microbiota. 94Nonetheless, these methods still grapple with contamination risks, with inevitable external microbiota from the colon jeopardizing the fidelity of the results.
Exogenous contamination further compounds the issue.The introduction of foreign microbes during sample handling, combined with the inherently low microbial biomass of tumor samples, heightens the potential for DNA contaminants from reagents and lab apparatus.Such contamination introduces confounding variables into sequencing datasets, challenging the delineation of authentic microbial signatures.
In summation, the pursuit to elucidate the intratumoral microbiota in CRC is laden with complexities.Elements such as the tumor's anatomical positioning, its symbiotic relationship with the gut microbiota, and inherent sampling pitfalls underscore the imperative for advanced methodologies and analytical rigor.Addressing these intricacies is pivotal for a comprehensive understanding of microbiota's nuanced role in CRC pathophysiology.

CONCLUSION AND FUTURE PERSPECTIVES
The microbiota within tumors exhibits tumor specificity, and this review specifically focuses on its relevance to CRC.Our comprehensive exploration encompassed the characteristics of the TME of CRC and highlighted the emerging role of intratumoral microbiota as a novel TME participant.Our investigation delved into the heterogeneous distribution of intratumoral microbiota and emphasized the mutual selection processes between the TME and intratumoral microbiota, where the TME provides nutrients and supports immune escape for microorganisms, while intratumoral microbiota, in turn, influence the TME through immune regulation, metabolic products, and induction of inflammation, contributing to the observed heterogeneity.Within the TME of CRC, specific bacterial strains were identified to exert distinct effects on tumor behavior, including promoting inflammation, modulating immune responses, enhancing tumor cell proliferation and invasion, inducing EMT, and facilitating chemotherapy resistance.Notably, the specificity of intratumoral microbiota in CRC was evident, with particular bacterial strains exhibiting unique abundance and roles.Additionally, we analyzed the possibility of reduced abundance of probiotics in the TME, and proposed the potential of supplementing probiotics in the intestine to affect probiotics within tumors.
The potential diagnostic significance of targeting intratumoral microbiota within the TME was emphasized.Microorganisms within the tumor may secrete specific bacterial products, such as SCAFs or bacterial extracellular vesicles carrying components from parent bacteria. 95tecting these specific bacterial components in circulation may offer new opportunities for early diagnosis of CRC.However, factors such as dietary habits, cultural variations, environmental pollution, and antibiotic use can influence gut microbiota composition and affect the reproducibility of gut microbiota research.Therefore, tumor microbiota selected from the TME may provide more stable and reliable evidence for predicting the prognosis of CRC.
The intravenous injection of ATBx, as found by Shang's team, could target the removal of intratumoral microbiota without affecting intestinal microbiota.If validated through extensive experimentation, this approach could become a safe and commonly used auxiliary antitumor method.Personalized treatments based on modulating specific bacterial strains and microbiota composition hold promise for addressing the heterogeneity of the TME in different CRC patients.Such interventions may offer tailored immunotherapy and precision drug treatments for CRC patients.
Despite the belief in the existence of microorganisms within tumors, questions about their origin and exact function persist.Characterizing tumor microbiota has been challenging due to its relatively low abundance, uncertain cultivation methods, 96 severe host genome contamination, 97 and the risk of external contamination during tissue treatment or reagent introduction. 98CRC, in particular, pose challenges as their anatomical proximity to the colon results in lower microbial abundance within the tumor and potential contamination from the intestinal wall.Developing new sampling methods is imperative to obtain a more reliable overall landscape of the microbiome within CRC.
Despite progress, the exact mechanisms underlying the role of intratumoral microbiota in CRC remain incompletely understood.Future research should focus on exploring the complex interactions between intratumoral microbiota, TME, tumor development, and treatment responses.Additionally, clinical studies and validation of targeted approaches for manipulating intratumoral microbiota are essential to ensure safety and efficacy.
In conclusion, intratumoral microbiota as a novel component of the TME of CRC, plays a vital role in CRC development and treatment.Continued research and application of intratumoral microbiota provide exciting prospects for potential diagnostic and therapeutic applications in CRC, offering new hope and personalized treatment options for patients.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

F I G U R E 1
Sources of intratumor microbiota of CRC.(1) Mucosal: microbes invade the tumor site due to mucosal destruction during tumorigenesis.(2) Normal adjacent tissues (NATs): NAT is a potential source of intratumor microbiota.(3) Hematogenous spread: intratumor microbes enter tumor sites from oral cavity, gut, lung, and other sites through the bloodstream.Image created with BioRend.com.

F
I G U R E 3 10× Visium spatial transcriptomics detected a specimen of CRC.(A) Spatial distribution of total bacterial reads and total UMI transcripts throughout the tumor tissue from human CRC specimens.(B) Pie chart of the top 10 most dominant bacterial genera.(C) RNAscope-FISH imaging showing the distribution of bacteria across the tumor tissue.(D) Spatial distribution of Fusobacterium, Bacteroides, and Leptotrichia UMIs detected in CRC.(E) RNAscope-CISH images show the distribution of Fusobacterium nucleatum (dark red) and other bacterial communities in the tumor tissue; a sequential immunohistochemistry image shows the distribution of CD45+ (red) and PanCK+ (green) cells to identify the immune and epithelial compartments, respectively, in the tumor tissue.UMI: unique molecular identifier; FISH: fluorescence in situ hybridization; CISH: chromogenic in situ hybridization.Reproduced with permission. 29Copyright 2022, Springer Nature.

F I G U R E 6
The intratumor microbiota can span the entire process of early patient diagnosis, precise treatment, and prognosis prediction for colorectal cancer (CRC).Image created with BioRender.com.