The Barrier–Microbiota–Inflammation Axis in Colorectal Cancer: Mechanisms and Emerging Diagnostic & Therapeutic Strategies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors The paper is - first of all- very hard to read. It is a fully narrative review, with very little data and just ideas following one another. There are no tables with data extracted from literature or any other experimental proof for the theories discussed. The paper presents no ground-breaking theories. Several of the discussed phenomena have been extensively studied, but also heavily discussed in other review papers. In that case, what is its novelty? Finally, several sections only hypothesize on the connection of a certain phenomenon with CRC, without an experimental proof. This means that they should not be presented or discussed as literature facts. Specific comments

1. Please correct Graphical abstract. In the graph, what "Intestinal" refers to? The phrase "The disruption of the intestinal barrier and concomitant alterations in gut microbiota lead to dysbiosis" is not correct (alterations in gut microbiota are the dysbiosis).
2. Chapter "2.2. Mechanisms Linking Intestinal Barrier Disruption to CRC Development" totally neglects intrinsic / intracellular mechanisms underlying CRC.
3. Lines 288-290" "Beyond metabolites, the impact of gut microbial taxa extends to local immune regulation, particularly the balance between Th17 and Treg cells, which is crucial for shaping the CRC tumor microenvironment and facilitating immune evasion [65].". What is EXACTLY the role in microbiota / dysbiosis in immune dysregulation? The paragraph is very vague on this issue.
4. Chapter 4 repeats many ideas discussed earlier in the manuscript. There is no detailed description on how inflammation changes microbial composition in the gut.
5. Chapter 5.1. (Biomarkers) mentions 4 bacterial species at first, but later ignores to mention which species are taken into consideration.
6. Lines 448-477 - how these interventions actually influence microbial status and / or gut barrier state and function. 

Author Response

Reviewer #1:

1. Please correct Graphical abstract. In the graph, what "Intestinal" refers to? The phrase "The disruption of the intestinal barrier and concomitant alterations in gut microbiota lead to dysbiosis" is not correct (alterations in gut microbiota are the dysbiosis).

Our response:

We thank the reviewer for this insightful comment. In response to the feedback, we have made the following revisions to the graphical abstract:

Clarified the term "Intestinal" by changing it to "Intestinal epithelium", which better reflects the specific context of intestinal barrier disruption depicted in the figure.

Revised the wording regarding microbiota changes: The phrase "alterations in gut microbiota lead to dysbiosis" has been updated to "dysbiosis in the gut microbiota", as dysbiosis itself represents the alteration in the gut microbiota.

Additionally, we have ensured that the graphical abstract more clearly illustrates the complex mechanisms linking intestinal barrier disruption, microbial factors, inflammation, and colorectal cancer (CRC) progression. The updated diagram now clearly reflects these relationships in a more accurate and visually cohesive manner.

We believe these changes significantly improve the clarity and accuracy of the graphical abstract and align it more closely with the manuscript content. As your suggestions, the graphical abstract in our revised manuscript Cancers-4104399.R1 has been revised as follows:

 

Graphical abstract

The disruption of the intestinal barrier and dysbiosis in the gut microbiota impair mucosal repair mechanisms. This microbial imbalance and persistent chronic inflammation drive a self-perpetuating cycle: impaired barrier function allows for microbial invasion, triggering an inflammatory cascade that further exacerbates the initial damage. As the condition progresses, this cycle of inflammation and barrier dysfunction ultimately contributes to the development of colorectal cancer.

 

2. Chapter "2.2. Mechanisms Linking Intestinal Barrier Disruption to CRC Development" totally neglects intrinsic / intracellular mechanisms underlying CRC.

Our response:

We thank the reviewer for this insightful comment. We agree that in the original version of Section 2.2, the discussion primarily focused on barrier disruption–associated extrinsic mechanisms, such as microbial translocation, luminal metabolites, and inflammation, while the intrinsic and intracellular mechanisms underlying colorectal carcinogenesis were not sufficiently explicit.

To address this concern, we have revised Section 2.2 by adding clarifying and integrative statements to better highlight epithelial cell–intrinsic mechanisms within the existing framework. Specifically, we now explicitly describe how intestinal barrier impairment facilitates intracellular oncogenic signaling in epithelial cells, including activation of NF-κB, STAT3, and Wnt/β-catenin pathways, induction of epithelial–mesenchymal transition (EMT), and accumulation of oxidative stress–induced DNA damage. We further clarify that microbial products such as lipopolysaccharide (LPS) and secondary bile acids act not only as luminal or inflammatory factors, but also as upstream triggers of intracellular signaling cascades that directly influence epithelial cell fate and genomic stability. Importantly, these additions emphasize that intestinal barrier disruption should not be regarded solely as an extrinsic event, but rather as an upstream initiator that converges with intrinsic epithelial vulnerabilities, forming an integrated extrinsic–intrinsic carcinogenic axis. These targeted revisions enhance the mechanistic depth and conceptual clarity of Section 2.2, while preserving the original structure and focus of the manuscript. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

The compromise of the intestinal barrier act as both a contributor and a consequence of early CRC-associated dysbiosis, establishing a feed-forward loop in which microbial products and metabolites progressively erode junctional integrity and mucus structure [33,34]. Recent research suggests a high-fat diet (HFD) can directly alter the intestinal epithelial structure by reshaping the microbiota, leading to dysbiosis and the accumulation of carcinogenic metabolites [35]. Mouse models confirm that HFD or high sucrose diets downregulate tight junction protein expression, increase intestinal permeability, dramatically increase LPS levels, and promote the development of large and more numerous colorectal tumors [36]. Beyond diet, potent antibiotics can severely perturb microbial homeostasis, favoring the expansion of pathobionts, thinning the mucus layer, and thereby enhancing bacterial adhesion, transcytosis, and invasion. Furthermore, inflammatory bowel disease (IBD) induces chronic inflammation that extensively damages mucosal architecture and the intestinal barrier, which significantly increases CRC risk [37]. Clinical epidemiologic meta-analyses suggest that IBD patients face a substantially increased relative risk of CRC after a disease duration of 10 years or more [38]. Beyond serving as a permissive inflammatory environment, chronic barrier damage in these contexts also primes epithelial cells for intracellular oncogenic signaling, thereby lowering the threshold for malignant transformation. [The 1^st paragraph in the 2.2]

The breach of the intestinal barrier allows luminal contents to infiltrate the lamina propria, where they directly interact with intestinal stem cells or immune cells to initiate pro-carcinogenic processes. For example, in vitro studies that LPS triggers the secretion of pro-inflammatory cytokines, downregulates tight junction proteins, promotes epithelial cell migration, and induces epithelial-to-mesenchymal transition (EMT). Importantly, these effects are mediated through intracellular activation of TLR4-dependent NF-κB and STAT3 signaling pathways, directly linking barrier-derived stimuli to epithelial cell reprogramming [39,40]. Similarly, deoxycholic acid (DCA) exhibits direct cytotoxicity toward colonic epithelial cells, induces reactive oxygen species (ROS) production, causes deoxyribonucleic acid (DNA) damage, and increases mutation rates in key genes such as p53 [41]. At the intracellular level, DCA-induced oxidative stress and DNA damage promote genomic instability and facilitate mutations in key tumor suppressor genes, reinforcing its role as a direct epithelial carcinogen rather than a purely luminal toxin [42]. Both molecules also enhance the permeation and accumulation of other carcinogens by upregulating permeability-associated channels like pIgR, thereby establishing a pathological cycle of chronic leakage, persistent stimulation, and genomic instability. [The 2^nd paragraph in the 2.2]

Following barrier disruption, multiple epithelial cell–intrinsic oncogenic signaling pathways are activated, forming a complex molecular network that drives carcinogenesis. These pathways represent core intracellular mechanisms of CRC initiation, indicating that barrier failure actively engages epithelial oncogenic programs rather than acting solely through secondary inflammation. For example, the Wnt/β-catenin signaling pathway, a well-established mediator of CRC, is coactivated by LPS and DCA, leading to nuclear translocation of β-catenin and upregulation of oncogenes such as Myc and cyclin D1 [43]. Concurrently, inflammatory signaling via NF-κB and signal transducer and activator of transcription 3 (STAT3) is rapidly engaged through the TLR4-LPS signaling pathway, promoting anti-apoptotic effects, proliferation, and immune suppression—all critical for tumor progression [44]. Significant crosstalk exists among these pathways; NF-κB activity enhances STAT3 expression, while stabilized β-catenin signaling promotes to form a malignant regulatory network characterized by inflammation-induced proliferation and immunosuppression [45]. Collectively, these insights reposition the intestinal barrier from a passive victim to an active upstream initiator of CRC. Compromise of barrier integrity enables molecular permeation and triggers powerful carcinogenic amplification cascades. Consequently, therapeutic strategies aimed at preserving or restoring barrier function could form the cornerstones of primary CRC prevention. [The 3^rd paragraph in the 2.2]

 

3. Lines 288-290" "Beyond metabolites, the impact of gut microbial taxa extends to local immune regulation, particularly the balance between Th17 and Treg cells, which is crucial for shaping the CRC tumor microenvironment and facilitating immune evasion [65].". What is EXACTLY the role in microbiota / dysbiosis in immune dysregulation? The paragraph is very vague on this issue.

Our response:

We thank the reviewer for this important comment. We agree that the original wording in Lines 288–290 was overly general and did not sufficiently specify how gut microbiota dysbiosis mechanistically contributes to immune dysregulation in colorectal cancer.

To address this issue, we have revised this paragraph to provide a more explicit mechanistic explanation of microbiota-driven immune modulation. Specifically, we now clarify that CRC-associated dysbiosis promotes Th17/Treg imbalance through defined microbial and metabolic pathways. Revisions clarify the causal role of gut microbiota dysbiosis in shaping immune imbalance, rather than describing it as a correlative phenomenon. We believe this improves the mechanistic precision of the section and adequately addresses the reviewer’s concern. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

Beyond metabolites, the impact of gut microbial taxa extends to local immune regulation, particularly the balance between Th17 and Treg cells, which is crucial for shaping the CRC tumor microenvironment and facilitating immune evasion [96]. CRC-associated dysbiosis contributes to this imbalance through defined mechanisms, including the enrichment of pro-inflammatory pathobionts that promote IL-6/IL-23–driven Th17 polarization, as well as the depletion of short-chain fatty acid–producing bacteria that normally support Treg differentiation and immune tolerance [97-99]. Together, these microbiota-driven alterations sustain chronic inflammatory signaling and impair antitumor immune surveillance within the colorectal tumor microenvironment. Under homeostatic conditions, a balance between Tregs and Th17 cell responses maintains immune tolerance while allowing immune surveillance of the tumor. In CRC, this balance is disrupted, with an expansion of Th17 cells and a loss of Treg cell number and function, creating a chronic pro-tumor inflammatory state at tumor site [100]. Systematic immune profiling links greater Th17 cell infiltration in CRC with higher recurrence and poorer survival, whereas the prognostic value of Treg infiltration appears to be context-dependent [101]. Furthermore, certain gut microbiota and their metabolites can drive the polarization of T-cells away from a Treg phenotype and toward a pro-inflammatory Th17 pathway. Metabolites that induce Treg differentiation are often depleted in CRC-associated dysbiosis, disrupting networks that induce immunosuppression. Clinical observations note a paradoxical increase in Th17 frequency after curative chemotherapy, and it is hypothesized that Th17 may contribute to immune exhaustion by recruiting immunosuppressive myeloid-derived suppressor cells and sustaining chronic STAT3 and NF-κB pathways activation [102]. [The 6^th paragraph in the 3.2]

 

4. Chapter 4 repeats many ideas discussed earlier in the manuscript. There is no detailed description on how inflammation changes microbial composition in the gut.

Our response:

We thank the reviewer for this constructive comment. We acknowledge that certain core concepts related to the barrier–microbiota–inflammation axis are revisited in Chapter 4. This partial overlap was intended to maintain conceptual continuity; however, we agree that the original version of Chapter 4 did not sufficiently distinguish its mechanistic focus from earlier sections. To address this concern, we have revised Chapter 4 to more clearly emphasize the reverse directionality, namely how chronic intestinal inflammation actively reshapes gut microbial composition, rather than reiterating how dysbiosis initiates inflammation. Specifically, we now elaborate on inflammation-driven alterations in the gut environment, including cytokine-mediated changes in epithelial antimicrobial peptide secretion, oxygen and nitrate availability, bile acid metabolism, and mucosal barrier integrity, all of which create selective pressures favoring facultative anaerobes and pro-inflammatory pathobionts.

These additions clarify that Chapter 4 focuses on inflammation as an active driver of microbial remodeling, thereby complementing—rather than duplicating—the earlier sections that emphasize barrier dysfunction and dysbiosis as upstream events. We believe this revision improves the logical flow of the manuscript and strengthens the bidirectional framework of the barrier–microbiota–inflammation axis. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

A critical feedback loop perpetuates this process: NF-kB, activated by LPS, not only exacerbates intestinal barrier dysfunction but also sustains persistent inflammation and dysbiosis. Inflammatory signaling profoundly alters the gut ecological niche by increasing epithelial oxygenation, nitrate availability, and antimicrobial peptide release, which preferentially supports the expansion of facultative anaerobic bacteria such as Proteobacteria while suppressing obligate anaerobic commensals. In parallel, inflammation-driven changes in bile acid composition and mucin structure further disrupt microbial metabolic balance, promoting the persistence of pro-inflammatory pathobionts [111-113]. Importantly, current evidence suggests that gut microbiota alterations in this cycle function both as a contributor of barrier dysfunction and inflammation, rather than as a unidirectional initiating factor [70]. This creates a self-reinforcing cycle wherein barrier damage, microbial translocation, and inflammatory amplification are continuously maintained [114,115]. These mechanistic insights reveal promising intervention points. Blocking TLR/NLR signaling, modulating the microbiota with probiotics, or using drugs to restore barrier integrity represent viable strategies to interrupt early cascade events for CRC prevention [116]. Clinically, biomarkers of barrier integrity—such as elevated anti-flagellin antibodies, serum zonulin or DAO levels, and a metabolite profile characterized by low SCFAs and high deoxycholate—correlate with activation of TLR4/MyD88/NLRP3 pathways and associate with greater tumor burden upon transfer of dysbiotic microbiota into gnotobiotic or carcinogen-primed mice [117-121]. This LPS–TLR4–MyD88–NF-κB axis therefore represents a central inflammatory signaling node within the barrier–microbiota–inflammation network and will not be re-described in detail in subsequent sections. [The 3^rd paragraph in the 4.2]

 

5. Chapter 5.1. (Biomarkers) mentions 4 bacterial species at first, but later ignores to mention which species are taken into consideration.

Our response:

We thank the reviewer for pointing out this issue. We agree that in the original version of Section 5.1, several representative bacterial species were explicitly mentioned at the beginning, whereas later descriptions referred to microbial biomarkers in a more general manner, which may cause ambiguity. To improve clarity and consistency, we have revised Section 5.1 to explicitly restate representative bacterial species when discussing microbial biomarker panels. In particular, we now consistently specify commonly reported CRC-associated taxa, including Fusobacterium nucleatum, Parvimonas micra, Bacteroides fragilis, and Escherichia coli, when referring to microbial signatures used in diagnostic models. These revisions ensure that the discussion of microbial biomarkers remains concrete and traceable throughout the section. We believe this clarification improves the readability and coherence of Section 5.1 and adequately addresses the reviewer’s concern. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

Microbial signatures, typically derived from CRC-associated taxa such as Fusobacterium nucleatum, Parvimonas micra, Bacteroides fragilis, and Escherichia coli, represent a promising biomarker category. Enrichment of species such as F. nucleatum, B. fragilis, and P. micra in early CRC enables non-invasive stool screening via quantitative polymerase chain reaction (qPCR) [124]. A systematic review in 2023 demonstrated that random forest models incorporating any four species achieved AUC values of 0.86 to 0.94, outperforming established single markers like FIT and Septin 9 (AUC > 0.74) [125]. In blood, circulating microbial DNA (cfbDNA) has emerged as a novel liquid biopsy, offering an alternative for individuals reluctant or unable to undergo colonoscopy or stool testing. Metabolomics, which measure a type of host-microbiota interaction, also presents strong diagnostic potential. One multicenter study linked eight fecal metabolites to CRC, achieving an AUC of 0.94 for discriminating CRC from healthy controls and 0.92 for distinguishing CRC from adenomas, surpassing existing fecal DNA-based products [126]. [The 2^nd paragraph in the 5.1]

 

6. Lines 448-477 - how these interventions actually influence microbial status and / or gut barrier state and function.

Our response:

We thank the reviewer for this valuable comment. We agree that in the original version of Lines 448–477, the discussion primarily summarized potential intervention strategies without sufficiently clarifying how these approaches mechanistically influence gut microbial composition and/or intestinal barrier state and function. To address this concern, we have revised this section to explicitly link each intervention strategy to its corresponding effects on microbial ecology and barrier integrity. Specifically, we now clarify how dietary fibers and prebiotics reshape microbial composition by selectively enriching short-chain fatty acid–producing bacteria and restoring SCFA-mediated epithelial barrier support; how barrier-protective agents (e.g., zinc supplementation and GLP-2 analogs) enhance tight junction protein expression, mucus integrity, and epithelial regeneration; and how microbiota-targeted interventions such as probiotics and fecal microbiota transplantation (FMT) re-establish microbial diversity while indirectly improving barrier function and reducing inflammatory signaling. These additions provide a clearer mechanistic framework connecting early interventions with their effects on microbial status and intestinal barrier function, thereby strengthening the translational relevance of this section. We believe that these revisions adequately address the reviewer’s concern. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

These barrier-protective agents primarily act on the host epithelium by enhancing tight junction protein expression, supporting goblet cell function, and accelerating epithelial renewal, thereby indirectly stabilizing the microbial niche and limiting dysbiosis-driven inflammation [142]. Currently, there is a scarcity of large randomized controlled trial (RCT) demonstrating that early interventions can modify precancer precursors or epithelial dysfunctions driven by intestinal permeability. Consequently, further investigation is required before progressing to more definitive large-scale trials. [The 2^nd paragraph in the 5.2]

Nutritional strategies also play a key role in maintaining microbiome homeostasis. Dietary fibers act as natural prebiotics, fermented by specific butyrate-producing bacteria into SCFA—primarily butyrate—which in turn inhibit histone deacetylase and promote tumor cell apoptosis. Mechanistically, dietary fibers act as selective substrates for beneficial commensals, particularly short-chain fatty acid–producing bacteria, leading to increased luminal SCFA levels that reinforce epithelial tight junctions, promote mucus production, and suppress low-grade inflammation [143]. A 2024 Canadian translational study transplanted fecal microbiota from CRC patients into germ-free mice and then administered various dietary fibers. This intervention significantly altered microbial community structure, increased SCFA production, and improved healing of surgical intestinal anastomoses, suggesting a potential application in optimizing gut health for oncology surgery [144]. [The 3^rd paragraph in the 5.2]

FMT, while established in IBD, is also being explored in CRC. By restoring microbial diversity and reducing the dominance of pro-inflammatory pathobionts, microbiota-targeted interventions such as probiotics and fecal microbiota transplantation also contribute to secondary improvements in barrier integrity and immune homeostasis, highlighting the bidirectional relationship between microbial composition and epithelial function [145]. A systematic review published in 2023 with 23 identified RCTs evaluated FMT’s effects in patients undergoing CRC surgery, chemoradiotherapy, or risk polypectomy, finding that it can restore microbiota diversity, reduce pro-inflammatory taxa, and help reset immune homeostasis [146]. Although not yet a first-line treatment for CRC, FMT represents a promising platform for pre- and post-surgical interventions, with future potential lying in personalized donor matching and targeted application for high-risk individuals. [The 4^th paragraph in the 5.2]

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Despite extensive research, colorectal cancer remains one of the most common cancer diagnoses worldwide. Dysbiosis is considered a significant factor involved in the carcinogenesis and progression of colorectal cancer through its effects on immune responses, intestinal barrier integrity, and microbial metabolite profiles. In this context, this manuscript focuses on an important area of ​​research with a clinical impact. Although the manuscript is well written, I have the following suggestion for authors to improve the quality of the manuscript:

- Please explain all abbreviations used in Figures 1 and 2 in their legends.

- Please replace “gobbler cells” with “goblet cells” in line 130.

- An additional figure related to the main mechanisms linking intestinal barrier disruption to CRC development will highlight this subchapter of the manuscript.

- The link between dysbiosis and CMS subtypes of CRC in colorectal cancer is incompletely described (CMS4 subtype is often enriched with Fusobacterium nucleatum, while CMS1 may have different microbiota profiles).

- Please include a brief description of the role of dysregulation of sIgA secretion in the context of intestinal dysbiosis in CRC.

- Streptococcus gallolyticus has also been detected to be involved in colorectal cancer carcinogenesis. Please add supplementary data.

- Microbiota modulation (probiotics/prebiotics), faecal transplantation and diet are also novel therapeutic strategies in CRC patients, which are incompletely described in the manuscript.

- Please revise the text at lines 374-378 (...a study from 2022... and two references - 72 and 73). The same suggestion for lines 471-474 (Numerous longitudinal studies and meta-analyses.... and only one reference - 93).

- Please expand the data on the potential role of glucagon-like peptide-2 (GLP-2) analogues in intestinal barrier recovery in clinical trials.

- Please add a reference to line 128. The same suggestion also applies to lines 137, 141, 157, 188, 225, 228, 240, 328, 345, 407, 410, 438, 446, 471, 477, 480, 485 and 514.

Author Response

Reviewer #2:

1. Please explain all abbreviations used in Figures 1 and 2 in their legends.

Our response:

We thank the reviewer for this comment. We agree that all abbreviations used in Figures 1 and 2 should be clearly defined in their legends. Accordingly, we have revised the figure legends to explicitly explain all abbreviations appearing in each figure. We believe this improves clarity and readability for the readers. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

 

Figure 1. The CRC Gut Axis: Barrier Disruption, Gut Dysbiosis & Inflammation Cascade. This diagram illustrates the vicious cycle wherein intestinal barrier compromise drives gut dysbiosis, which in turn fuels chronic inflammation, thereby generating a pro-carcinogenic microenvironment. Current intervention strategies focus on these interconnected processes; however, clinical translation remains challenging due to issue of standardization, system bias, and the requisite for extensive cohort validation. Abbreviations: CRC, colorectal cancer; TJ, tight junction; LPS, lipopolysaccharide; SCFAs, short-chain fatty acids; NF-κB, nuclear factor kappa B; STAT3, signal transducer and activator of transcription 3; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation primary response 88.

 

Figure 3. Strategies for Early Diagnosis & Intervention in CRC. This figure outlines advanced strategies for CRC. Early diagnosis integrates serum biomarkers, microbial signatures, metabolomics, and AI models for high-accuracy detection. Early intervention focuses on barrier protection, microbiota modulation, and anti-inflammation. The integration of these elements aims to advance AI-driven precision medicine, pending resolution of challenges in standardization and clinical validation. Abbreviations: FIT, fecal immunochemical test; FMT, fecal microbiota transplantation; SCFAs, short-chain fatty acids; AI, artificial intelligence; CRC, colorectal cancer.

2. Please replace “gobbler cells” with “goblet cells” in line 130.

Our response:

We thank the reviewer for pointing this out. We have corrected the term “gobbler cells” to “goblet cells” in line 130 as follows:

Deficiency in either occludin or ZO-1 alone may not critically disrupt barrier integrity under homeostatic conditions; however, their combined loss under inflammatory or neoplastic conditions leads to severe barrier failure, creating a hazardous vulnerability[17]. The mucus layer, primarily composed of the gel-forming protein mucin2 (MUC2) secreted by goblet cells, prevents direct contact between microbes and epithelial cells. Reduced or aberrantly glycosylated MUC2 is associated with enhanced microbial penetration, inflammation and carcinogenesis [18,19]. Similarly, the mucosal immune system, through mechanisms such as polymeric immunoglobulin receptor (pIgR)-mediated transport of polyclonal immunoglobulin A (IgA), helps manage microbial antigens. [In the 1^st paragraph of section 2.1]

 

3. An additional figure related to the main mechanisms linking intestinal barrier disruption to CRC development will highlight this subchapter of the manuscript.

Our response:

We thank the reviewer for the helpful suggestion. In response to this comment, we have included an additional figure (Figure 2) that visually summarizes the key mechanisms linking intestinal barrier disruption to colorectal cancer (CRC) development. The figure highlights the contribution of microbial factors (e.g., LPS) and their role in intestinal permeability, immune activation, and carcinogenic signaling pathways (such as Wnt/β-catenin and NF-κB). We believe this figure strengthens the manuscript by offering a clear, visual representation of the processes discussed in the subchapter on the barrier-microbiota-inflammation axis in CRC. As your suggestions, the added Figure 2 in our revised manuscript Cancers-4104399.R1 has been revised as follows:

Figure 2. Key mechanisms linking intestinal barrier disruption to colorectal cancer development. Disruption of the intestinal barrier leads to increased intestinal permeability, microbial translocation, and activation of inflammatory pathways. Microbial factors, such as LPS, drive inflammation through TLR4–NF-κB signaling, contributing to carcinogenic signaling via pathways like Wnt/β-catenin. This cascade ultimately increases the risk of CRC development.

4. The link between dysbiosis and CMS subtypes of CRC in colorectal cancer is incompletely described (CMS4 subtype is often enriched with Fusobacterium nucleatum, while CMS1 may have different microbiota profiles).

Our response:

We thank the reviewer for this insightful comment. We agree that the association between gut microbiota dysbiosis and consensus molecular subtypes (CMS) of colorectal cancer was incompletely described in the original manuscript.

To address this issue, we have revised the relevant section to more explicitly link representative microbial features with specific CMS subtypes. In particular, we now highlight that CMS4 tumors are frequently enriched with pro-inflammatory pathobionts such as Fusobacterium nucleatum, consistent with their mesenchymal, inflammatory, and immunosuppressive microenvironment, whereas CMS1 tumors exhibit distinct microbiota profiles that are more closely associated with immune activation and microsatellite instability. We further clarify that microbiota–CMS associations are subtype-enriched rather than exclusive, and likely reflect bidirectional interactions between tumor biology, immune contexture, and the intestinal microbial niche.These additions improve the mechanistic clarity of the dysbiosis–CMS relationship while avoiding oversimplification, and better align the discussion with current subtype-specific CRC frameworks. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

Collectively, these findings demonstrate that CRC-associated gut dysbiosis and functional imbalance are consistent across geographical, ethnic and dietary backgrounds. This suggests that microbial dysbiosis is not merely an associated mark of tumorigenesis but may play an essential role in driving the disease. With ongoing advances in multi-omic technologies and artificial intelligence, microbiota-derived signatures hold promise as predictive, non-invasive tools for early CRC detection and risk stratification, potentially extending to other poorly defined disease conditions. Emerging evidence further suggests that gut microbiota dysbiosis is differentially associated with consensus molecular subtypes (CMS) of colorectal cancer [73,74]. CMS4 tumors, characterized by mesenchymal features, stromal activation, and chronic inflammatory signaling, are frequently enriched with pro-inflammatory pathobionts such as Fusobacterium nucleatum, consistent with their immunosuppressive and invasive tumor microenvironment [75,76]. In contrast, CMS1 tumors, which are typically microsatellite instability–high and immune-reactive, exhibit distinct microbiota profiles that differ from CMS4 and may reflect alternative microbiota–immune interactions [77]. These observations indicate that dysbiosis–CMS associations are subtype-enriched rather than mutually exclusive, highlighting the bidirectional interplay between microbial composition, immune contexture, and tumor molecular phenotype. [The last part of 3.1]

 

5. Please include a brief description of the role of dysregulation of sIgA secretion in the context of intestinal dysbiosis in CRC.

Our response:

We thank the reviewer for this helpful suggestion. We agree that secretory immunoglobulin A (sIgA) plays an important role in maintaining intestinal microbial homeostasis and that its dysregulation is relevant to intestinal dysbiosis in colorectal cancer. To address this point, we have added a brief description in the manuscript to clarify how impaired sIgA secretion contributes to dysbiosis in CRC. Specifically, we now describe that reduced or dysfunctional sIgA-mediated immune exclusion weakens mucosal control of microbial adherence and spatial organization, thereby facilitating microbial overgrowth, barrier penetration, and chronic inflammatory signaling. These additions integrate sIgA dysregulation into the barrier–microbiota–inflammation framework of CRC without expanding the scope beyond a concise mechanistic description. We believe this revision adequately addresses the reviewer’s comment. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

The intestinal barrier serves as the first interface between the host and the external environment. Beyond mere physical separation, it is a multifunctional system comprising the epithelial cell layer, the mucus layer, and the mucosal immune system, which together perform diverse and dynamic regulatory roles. A key component is the epithelial tight junctions (TJ), which govern selective paracellular permeability. Occludin, a transmembrane protein, acts primarily as a sealing element, while the junctional protein ZO-1 stabilizes the TJ and links them to the actin cytoskeleton to mediate stress responses [16]. Deficiency in either occludin or ZO-1 alone may not critically disrupt barrier integrity under homeostatic conditions; however, their combined loss under inflammatory or neoplastic conditions leads to severe barrier failure, creating a hazardous vulnerability[17]. The mucus layer, primarily composed of the gel-forming protein mucin2 (MUC2) secreted by goblet cells, prevents direct contact between microbes and epithelial cells. Reduced or aberrantly glycosylated MUC2 is associated with enhanced microbial penetration, inflammation and carcinogenesis [18,19]. Similarly, the mucosal immune system, through mechanisms such as polymeric immunoglobulin receptor (pIgR)-mediated transport of polyclonal immunoglobulin A (IgA), helps manage microbial antigens. Secretory IgA (sIgA) represents a critical immune barrier component that maintains microbial homeostasis by limiting bacterial adherence to the epithelium and shaping microbial spatial organization through immune exclusion [20]. Dysregulation of pIgR-mediated IgA transcytosis or reduced sIgA coating capacity compromises this control, allowing excessive microbial–epithelial contact and promoting dysbiosis-associated inflammation [21]. In the context of CRC, impaired sIgA function has been associated with altered microbial composition and increased mucosal permeability, thereby reinforcing the barrier–microbiota–inflammation axis [22]. Impaired IgA transport can lead to pathogenic dysbiosis, heightened inflammation, and increased cancer risk [23]. [The 1^st paragraph in the 2.1]

 

6. Streptococcus gallolyticus has also been detected to be involved in colorectal cancer carcinogenesis. Please add supplementary data.

Our response:

We thank the reviewer for this valuable comment. We agree that Streptococcus gallolyticus has been implicated in colorectal cancer carcinogenesis and represents an important CRC-associated bacterial species. To address this point, we have added Streptococcus gallolyticus to the discussion of CRC-associated microbiota and provided supplementary material summarizing key evidence supporting its involvement in colorectal carcinogenesis. Specifically, we have included a supplementary table that compiles representative studies describing the association of S. gallolyticus with CRC, including detection methods, proposed mechanisms, and clinical relevance. This addition complements the main text without altering the overall structure of the manuscript.

We believe that the inclusion of this supplementary information strengthens the completeness of the microbiota–CRC discussion and adequately addresses the reviewer’s comment. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

Across discovery and validation cohorts, patients with early-stage CRC and advanced adenomas consistently exhibit a remodelled gut microbiome, characterized by a 20-30% reduction in α-diversity, consistent β-diversity separation, and co-abundance networks that shift toward a Fusobacterium-centeric structure while losing short-chain fatty acid (SCFA)-producing modules [53,54]. Both mucosal and fecal samples show enrichment of oral-origin taxa (e.g., F. nucleatum, Parvimonas micra (P. micra), enterotoxigenic Bacteroides fragilis (ETBF)) and depletion of butyrate producers (e.g., Faecalibacterium prausnitzii (F. prausnitzii), Roseburia, Eubacterium rectale (E. rectale), Ruminococcus spp.) [55]. Metagenomic analyses reveal decreased microbial capacity for SCFA biosynthesis, with concurrent enrichment in pathways involved in bile-acid deconjugation, 7α-dehydroxylation, LPS biosynthesis, and oxidative-stress, and improved disease classification by strain-level/single nucleotide variant (SNV) features [53]. In addition to these taxa, Streptococcus gallolyticus has also been repeatedly associated with colorectal cancer and implicated in tumorigenesis through inflammation-related and host–microbe interaction mechanisms (summarized in Table 1). [The 1^st paragraph in the 3.1]

 

Table 1. Representative evidence linking Streptococcus gallolyticus to colorectal cancer.

Study	Sample type	Detection method	Key findings	Proposed mechanism	Reference
Abdulamir et al. (2011)	CRC tissue / blood	PCR, culture	Higher prevalence of Streptococcus gallolyticus (formerly S. bovis) in CRC patients	Chronic inflammation and bacterial translocation contribute to carcinogenesis	[56]
Butt et al. (2017)	Serum antibodies	Serology (multiplex)	Elevated anti-S. gallolyticus pilus protein antibodies (Gallo2178/2179) in CRC patients	Immune-mediated association; antibody response as biomarker for CRC risk	[57]
Boleij et al. (2011)	Tumor tissue	qPCR, sequencing	Enrichment of S. gallolyticus DNA and antigen expression in tumor vs. normal tissues	Disruption of epithelial barrier and induction of inflammatory signaling pathways	[58]

 

7. Microbiota modulation (probiotics/prebiotics), faecal transplantation and diet are also novel therapeutic strategies in CRC patients, which are incompletely described in the manuscript.

Our response:

We thank the reviewer for this constructive comment. We agree that microbiota modulation strategies, including probiotics/prebiotics, fecal microbiota transplantation (FMT), and dietary interventions, represent emerging and potentially novel therapeutic approaches in colorectal cancer, and that their roles were not sufficiently elaborated in the original manuscript. To address this concern, we have revised the relevant section to provide a more comprehensive yet concise description of these strategies in the context of CRC. Specifically, we now clarify how probiotics and prebiotics modulate microbial composition and metabolic outputs to attenuate inflammation and support barrier function; how FMT restores microbial diversity and ecological stability, with potential implications for tumor-associated inflammation and treatment responsiveness; and how dietary interventions reshape microbial metabolites and host–microbe interactions that influence tumor progression. We also emphasize that these approaches are currently considered adjunctive or exploratory strategies in CRC management, rather than established standalone therapies.

These additions improve the translational relevance of the manuscript while maintaining its review-oriented scope. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

Beyond their preventive potential, microbiota-targeted approaches are increasingly explored as novel therapeutic strategies in CRC patients. Probiotics and prebiotics aim to restore microbial balance by enriching beneficial commensals and enhancing the production of anti-inflammatory metabolites, thereby indirectly modulating tumor-associated inflammation and epithelial barrier function [98]. Fecal microbiota transplantation (FMT), although still experimental in CRC, has shown promise in re-establishing microbial diversity and reshaping the intestinal microenvironment, particularly in the context of inflammation and treatment responsiveness [147]. Dietary interventions further influence these processes by altering microbial substrates and metabolic outputs, highlighting the interconnected role of diet–microbiota–host interactions in CRC progression and therapy [148]. [The 5^th paragraph in the 5.2]

 

8. Please revise the text at lines 374-378 (...a study from 2022... and two references - 72 and 73). The same suggestion for lines 471-474 (Numerous longitudinal studies and meta-analyses.... and only one reference - 93).

Our response:

We thank the reviewer for this helpful comment. We agree that the wording in lines 374–378 and 471–474 overstated the breadth of evidence relative to the number of references cited.

To address this concern, we have revised the text to better align the strength of the statements with the supporting references. Specifically, we have rephrased the descriptions in lines 374–378 to refer more explicitly to representative studies rather than implying broader consensus. Similarly, the wording in lines 471–474 has been adjusted to avoid suggesting multiple longitudinal studies and meta-analyses when only a single reference was cited. These revisions improve the accuracy and balance of the manuscript without altering its overall conclusions. We believe that these changes adequately address the reviewer’s concern. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

Although chronic inflammation can arise from prolonged exposure to dietary factors or dysbiosis, direct evidence from recent studies demonstrated that the transplanting fecal microbiota from CRC patients into germ-free and IL-10⁻/⁻ mice enhanced gut permeability, activated TLR4/MyD88 and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammatory pathways, and increased tumor burden [108,109]. Importantly, different bacteria exhibit distinct carcinogenic potentials despite potentially similar capacities to induce inflammation. For example, colonization with E. coli NC101 significantly increases more colorectal tumors than E. faecalis, emphasizing the specificity of microbial genomics in cancer promotion and/or suppression [110]. [The 2^nd paragraph in the 4.2]

Substantial evidence implicates chronic inflammation as a key contributor of CRC development and progression, making it a compelling target for prevention and early intervention. Aspirin is one of the most well-established anti-inflammatory agents for CRC chemoprevention. It inhibits the cyclooxygenase-2 (COX-2), thereby reducing prostaglandin E2 (PGE2) production and modulating downstream IL-6/NF-κB/STAT3 anti-inflammatory signaling [149]. Available longitudinal evidence suggests that dietary interventions may influence CRC risk [146]. In addition, evidence suggests that aspirin may offer a particular survival benefit for patients whose tumors harbor a PIK3CA mutation [150]. [The 1^st paragraph in the 5.3]

 

9. Please expand the data on the potential role of glucagon-like peptide-2 (GLP-2) analogues in intestinal barrier recovery in clinical trials.

Our response:

We thank the reviewer for this constructive suggestion. We agree that the clinical evidence supporting glucagon-like peptide-2 (GLP-2) analogues in intestinal barrier recovery was insufficiently detailed in the original manuscript. To address this point, we have expanded the relevant paragraph by adding clinical trial data and clinically relevant endpoints of GLP-2 therapy. Specifically, we now summarize that GLP-2 analogues (e.g., teduglutide) have demonstrated clinically meaningful improvements in intestinal function in randomized and prospective studies, including reduced parenteral support requirements and enhanced intestinal absorptive capacity, together with evidence of mucosal structural recovery (e.g., increased villus height/crypt depth) and barrier-related molecular/functional changes (e.g., modulation of tight junction–associated markers and intestinal permeability readouts). We also briefly update the ongoing development of longer-acting GLP-2 analogues (e.g., apraglutide and glepaglutide) in clinical trials and clarify that most current human evidence for barrier recovery is derived from short bowel syndrome–associated intestinal failure, while broader CRC-specific applications remain investigational. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

With growing validation of the "barrier-microbiota-inflammation" axis in CRC, clinical research is increasingly focused on upstream interventions that are barrier-protective and microbiota-modulatory. These strategies, which include prebiotics and dedicated barrier-strengthening agents, are anticipated to form the cornerstone of prevention in high-risk or early screened cohorts. For example, zinc supplementation has been shown to enhance the expression of tight junction proteins including ZO-1 and occludin, and to reduce barrier-related inflammation by inhibiting the NF-κB related pathway [135]. A small prospective intervention trial found that zinc alpha-ketoacid nutritional formulations discouraged intestinal permeability in IBD patients, as measured inversely to serum barrier injury markers, such as DAO and D-lactate (P < 0.05), promoting the authors to suggest a potential role in CRC risk reduction [136]. Similarly, glucagon-like peptide-2 (GLP-2) analogs like teduglutide, known for supporting mucosal repair in short bowel syndrome, have been shown to stimulate MUC2 secretion from goblet cells, sustain mucous layer intactness, and promote epithelial proliferation and barrier recovery in preclinical and ongoing human studies [137]. In human studies—most robustly in short bowel syndrome–associated intestinal failure—GLP-2 therapy (e.g., teduglutide) has demonstrated clinically meaningful improvements in intestinal function, including significant reductions in parenteral support requirements and enhanced fluid and nutrient absorption, as shown in randomized controlled trials and prospective clinical studies [138,139]. These functional benefits are accompanied by evidence of mucosal structural recovery, such as increased villus height and crypt depth, reflecting enhanced epithelial regenerative capacity. Importantly, emerging clinical biomarker studies further suggest that GLP-2 treatment can modulate barrier-related molecular programs, including the expression of tight junction–associated proteins, and improve functional readouts of intestinal permeability in patients with intestinal failure [140]. In addition, longer-acting GLP-2 analogues, such as apraglutide and glepaglutide, are currently under active clinical development, aiming to provide sustained intestinal adaptation with less frequent dosing and improved patient compliance [141]. While these clinical data support GLP-2 analogues as promising agents for intestinal barrier recovery, their application in CRC settings should currently be considered adjunctive and investigational, pending dedicated clinical trials addressing CRC-specific endpoints and patient populations. [The 1^st paragraph in the 5.2]

 

10. Please add a reference to line 128. The same suggestion also applies to lines 137, 141, 157, 188, 225, 228, 240, 328, 345, 407, 410, 438, 446, 471, 477, 480, 485 and 514.

Our response:

We thank the reviewer for this careful and constructive comment. We agree that the statements at lines 128, 137, 141, 157, 188, 225, 228, 240, 328, 345, 407, 410, 438, 446, 471, 477, 480, 485, and 514 required additional literature support.

To address this concern, we have systematically reviewed the manuscript and added appropriate references at all the indicated locations to support the corresponding statements. These newly added citations include recent experimental, clinical, and review studies relevant to intestinal barrier function, microbiota dysbiosis, inflammation, and colorectal cancer. We believe that these revisions improve the rigor, transparency, and completeness of the manuscript. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

References

17. Zhang, Y.; Zhang, J.; Xia, Y.; Sun, J. Bacterial translocation and barrier dysfunction enhance colonic tumorigenesis. Neoplasia 2023, 35, 100847, doi:https://doi.org/10.1016/j.neo.2022.100847.
18. Deng, F.; Wu, Z.; Zou, F.; Wang, S.; Wang, X. The Hippo–YAP/TAZ Signaling Pathway in Intestinal Self-Renewal and Regeneration After Injury. 2022, Volume 10 - 2022, doi:10.3389/fcell.2022.894737.
19. Kayama, H.; Takeda, K. Regulation of intestinal epithelial homeostasis by mesenchymal cells. Inflammation and Regeneration 2024, 44, 42, doi:10.1186/s41232-024-00355-0.
20. Shao, X.; Liu, L.; Zhou, Y.; Zhong, K.; Gu, J.; Hu, T.; Yao, Y.; Zhou, C.; Chen, W. High-fat diet promotes colitis-associated tumorigenesis by altering gut microbial butyrate metabolism. International Journal of Biological Sciences 2023, 19, 5004-5019, doi:10.7150/ijbs.86717.
21. Tang, F.; Cao, F.; Lu, C.; He, X.; Weng, L.; Sun, L. Dvl2 facilitates the coordination of NF-κB and Wnt signaling to promote colitis-associated colorectal progression. 2022, 113, 565-575, doi:https://doi.org/10.1111/cas.15206.
22. Zhou, Y.; Xu, H.; Xu, J.; Guo, X.; Zhao, H.; Chen, Y.; Zhou, Y.; Nie, Y. F. prausnitzii and its supernatant increase SCFAs-producing bacteria to restore gut dysbiosis in TNBS-induced colitis. AMB Express 2021, 11, 33, doi:10.1186/s13568-021-01197-6.
23. Osman, M.A.; Neoh, H.-m.; Ab Mutalib, N.-S.; Chin, S.-F.; Mazlan, L.; Raja Ali, R.A.; Zakaria, A.D.; Ngiu, C.S.; Ang, M.Y.; Jamal, R. Parvimonas micra, Peptostreptococcus stomatis, Fusobacterium nucleatum and Akkermansia muciniphila as a four-bacteria biomarker panel of colorectal cancer. Scientific Reports 2021, 11, 2925, doi:10.1038/s41598-021-82465-0.
24. Zhang, H.; Wu, J.; Ji, D.; Liu, Y.; Lu, S.; Lin, Z.; Chen, T.; Ao, L. Microbiome analysis reveals universal diagnostic biomarkers for colorectal cancer across populations and technologies. 2022, Volume 13 - 2022, doi:10.3389/fmicb.2022.1005201.
25. Hirano, T.; Hirayama, D.; Wagatsuma, K.; Yamakawa, T.; Yokoyama, Y.; Nakase, H. Immunological Mechanisms in Inflammation-Associated Colon Carcinogenesis. 2020, 21, 3062.
26. Catalano, M.; Mini, E.; Nobili, S.; Vascotto, I.A.; Ravizza, D.; Amorosi, A.; Tonelli, F.; Roviello, F.; Roviello, G.; Nesi, G.J.W.J.o.G.O. Ulcerative colitis and colorectal cancer: Pathogenic insights and precision strategies for prevention and treatment. 2025, 17, 108514, doi:10.4251/wjgo.v17.i10.108514.
27. Zwezerijnen-Jiwa, F.H.; Sivov, H.; Paizs, P.; Zafeiropoulou, K.; Kinross, J. A systematic review of microbiome-derived biomarkers for early colorectal cancer detection. Neoplasia 2023, 36, 100868, doi:10.1016/j.neo.2022.100868.
28. Rye, M.S.; Garrett, K.L.; Holt, R.A.; Platell, C.F.; McCoy, M.J. Fusobacterium nucleatum and Bacteroides fragilis detection in colorectal tumours: Optimal target site and correlation with total bacterial load. PLOS ONE 2022, 17, e0262416, doi:10.1371/journal.pone.0262416.
29. Hu, Y.; Zhang, W.; Yang, K.; Lin, X.; Liu, H.-C.; Odle, J.; See, M.T.; Cui, X.; Li, T.; Wang, S.; et al. Dietary Zn proteinate with moderate chelation strength alleviates heat stress-induced intestinal barrier function damage by promoting expression of tight junction proteins via the A20/NF-κB p65/MMP-2 pathway in the jejunum of broilers. Journal of Animal Science and Biotechnology 2024, 15, 115, doi:10.1186/s40104-024-01075-8.
30. Xie, F.-F.; Xu, L.-B.; Zhu, H.; Yu, X.-Q.; Deng, L.-Y.; Qin, H.-Z.; Lin, S. Serum Metabolomics and NF-κB Pathway Analysis Revealed the Antipyretic Mechanism of Ellagic Acid on LPS-Induced Fever in Rabbits. 2024, 14, 407.
31. Li, P.; Huang, D. Targeting the JAK-STAT pathway in colorectal cancer: mechanisms, clinical implications, and therapeutic potential. 2024, Volume 12 - 2024, doi:10.3389/fcell.2024.1507621.
32. Li, X.; Wang, Z.; Zhang, S.; Yao, Q.; Chen, W.; Liu, F. Ruxolitinib induces apoptosis of human colorectal cancer cells by downregulating the JAK1/2‑STAT1‑Mcl‑1 axis. Oncol Lett 2021, 21, 352, doi:10.3892/ol.2021.12613.
33. Wang, D.; Yu, W.; Lian, J.; Wu, Q.; Liu, S.; Yang, L.; Li, F.; Huang, L.; Chen, X.; Zhang, Z.; et al. Th17 cells inhibit CD8+ T cell migration by systematically downregulating CXCR3 expression via IL-17A/STAT3 in advanced-stage colorectal cancer patients. Journal of Hematology & Oncology 2020, 13, 68, doi:10.1186/s13045-020-00897-z.
34. Byrd, D.A.; Gomez, M.F.; Hogue, S.R.; Burns, J.R.; Smith, N.; Sampson, J.; Loftfield, E.; Wolf, P.G.; Wan, Y.; Warner, A.; et al. Associations of the Colon Tissue Microbiome and Circulating Bile Acids With Colorectal Adenoma Among Average-Risk Women. 2025, 14, e71048, doi:https://doi.org/10.1002/cam4.71048.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript provides an integrated overview of how intestinal barrier disruption, microbial dysbiosis, and chronic inflammation cooperate from very early stages to drive colorectal cancer development. The authors summarise the key mechanisms, emerging biomarkers, and potential therapeutic interventions targeting this axis. The aim of the manuscript is particularly noteworthly, as the authors propose a unified framework for these processes and present them as a single, clinically actionable system for early diagnosis and prevention.

However, it should be noted that the manuscript adopts a predominantly descriptive narrative approach, with very limited critical examination of the robustness of its claims. At several points, it relies somewhat heavily on direct causal assertions despite the underlying data being correlational or preclinical, without consistently providing the analytical context needed to support them. This occasionally leads to a blending of different levels of evidence and to an insufficient acknowledgment of the limitations of the references cited.

Some examples of deficiencies that could be highlighted include:

Lines 87–89: there are paragraphs containing incorrect statements such as: “Shift in the chemical barrier, such as elevated microbiota-derived secondary bile acids and short-chain fatty acids, further suppress antitumor immunity and foster epithelial stress and low-grade inflammation [10,11].” The cited references do not support this claim (Ref 10 refers only to bile acids), and the assertion is not accurate with respect to SCFAs; see also Fig. 1.

Although a formal structure is outlined, the narrative integration of the intestinal barrier–microbiota–inflammation axis is inconsistent; for instance, inflammation is described with limited integration into the rest of the axis, rather than as another core component of the proposed narrative framework.

Regarding the stated objective of addressing “emerging diagnostic strategies,” clarity is lacking. The manuscript focuses heavily on biomarkers but not on other types of operational strategies, which are presented in a poorly structured manner. It would be advisable for the authors to include, for example, a comparative table with specific interventions, potentially adding sensitivity/specificity values where applicable.

With respect to methodology, although the manuscript appears to be a narrative review, the authors do not report any search strategy, making it impossible to assess the transparency, comprehensiveness, or reproducibility of the information presented. This may also explain why results from animal studies, in vivo experiments, and observational studies are mixed together without indicating their relative evidentiary weight, which can lead to confusion.

More critical discussion is needed. The dual role of SCFAs is mentioned but not addressed; the well‑known geographic variability is not discussed; nor are the biases inherent to metagenomic studies, or the key question of whether microbiota changes are a cause, a consequence, or both. Concepts are repeated—for example, the LPS–TLR4–MyD88–NF‑κB axis is explained several times in different sections with little synthesis.

In several paragraphs, the language used suggests strong causality (e.g., “drivers,” “dismantles,” “firmly establishes”) when the cited references provide mainly associative or preclinical evidence.

Finally, the manuscript does not include any limitations of this narrative description (as already mentioned above such as biases or heterogeneity of the referenced studies; results  data mixed from animal, in vitro, human studies;  the lack of clinical trials, etc.).

Some typographical errors in the text include: initation (initiation), elebated (elevated), microhome (microbiome).

Some errors in the figures:  Tihgt junctions, EARLY DIAGIOSIS, CACSADE, Bionrakers, Microhome, Accids.

Author Response

Reviewer #3:

1. Lines 87–89: there are paragraphs containing incorrect statements such as: “Shift in the chemical barrier, such as elevated microbiota-derived secondary bile acids and short-chain fatty acids, further suppress antitumor immunity and foster epithelial stress and low-grade inflammation [10,11].” The cited references do not support this claim (Ref 10 refers only to bile acids), and the assertion is not accurate with respect to SCFAs; see also Fig. 1.

Our response:

We thank the reviewer for carefully pointing out this issue. We agree that the statement in lines 87–89 was inaccurate and insufficiently supported by the cited references. Specifically, the original wording incorrectly grouped microbiota-derived secondary bile acids and short-chain fatty acids (SCFAs) together as immunosuppressive and pro-inflammatory factors.

To address this concern, we have revised the text to clearly distinguish the divergent roles of these metabolites. The revised version now specifies that elevated secondary bile acids are associated with epithelial stress, immune suppression, and pro-tumorigenic inflammation, whereas SCFAs—particularly butyrate—are generally recognized for their barrier-protective and immunoregulatory functions. We have also corrected the citations to ensure that the references accurately support the revised statements and aligned the text with the conceptual framework presented in Figure 1. We believe this correction improves the scientific accuracy and internal consistency of the manuscript and adequately addresses the reviewer’s concern. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

 

Figure 1. The CRC Gut Axis: Barrier Disruption, Gut Dysbiosis & Inflammation Cascade. This diagram illustrates the vicious cycle wherein intestinal barrier compromise drives gut dysbiosis, which in turn fuels chronic inflammation, thereby generating a pro-carcinogenic microenvironment. Current intervention strategies focus on these interconnected processes; however, clinical translation remains challenging due to issue of standardization, system bias, and the requisite for extensive cohort validation. Abbreviations: CRC, colorectal cancer; TJ, tight junction; LPS, lipopolysaccharide; SCFAs, short-chain fatty acids; NF-κB, nuclear factor kappa B; STAT3, signal transducer and activator of transcription 3; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation primary response 88.

Accumulating evidence indicates that intestinal barrier impairment and gut microbiota remodeling are detectable at the adenoma and early cancer stages rather than solely in advanced carcinomas [5,6]. Large-scale metagenomic analyses have consistently shown reproducible diversity shifts and enrichment of CRC–associated bacteria, such as Fusobacterium nucleatum (F. nucleatum) and pks-positive Escherichia coli (E. coli) [7]. Host-side changes include structural barrier defects—thinning of the mucus layer, loss of goblet-cell function, and downregulation of tight-junction proteins like zonula occludens-1 (ZO-1) and occludin—which collectively increase intestinal permeability and promote microbial translocation [8,9]. Shifts in the chemical barrier, such as elevated microbiota-derived secondary bile acids, have been associated with suppression of antitumor immunity, epithelial stress, and low-grade inflammation [10,11]. In contrast, short-chain fatty acids (SCFAs), particularly butyrate, are generally considered barrier-protective metabolites that support epithelial homeostasis and immune regulation [12]. These alterations activate pro-tumor inflammatory signaling, including lipopolysaccharide (LPS)-driven toll-like receptor 4 (TLR4)/ myeloid differentiation primary response 88 (MyD88)/nuclear factor-κB (NF-κB) (TLR4–MyD88–NF-κB) and Fusobacterium adhesins-mediated Wnt–β-catenin pathways [9,13]. This mechanistic axis not only offers novel biomarkers in stool and blood but also informs targeted strategies aimed at restoring barrier integrity and microbial balance [7]. [The 2^nd paragraph in the Introduction]

2. Although a formal structure is outlined, the narrative integration of the intestinal barrier–microbiota–inflammation axis is inconsistent; for instance, inflammation is described with limited integration into the rest of the axis, rather than as another core component of the proposed narrative framework.

Our response:

We thank the reviewer for this thoughtful and constructive comment. We agree that although the manuscript outlines a formal barrier–microbiota–inflammation framework, the narrative integration of inflammation as a core component of this axis was not sufficiently consistent throughout the original text.

To address this concern, we have revised the manuscript to more explicitly and consistently integrate inflammation into the barrier–microbiota–inflammation axis across relevant sections. Rather than describing inflammation as a downstream or isolated consequence, we now emphasize its bidirectional role as both an effector and an amplifier that actively interacts with intestinal barrier dysfunction and microbial dysbiosis. Specifically, we have strengthened cross-references between sections to clarify how inflammatory signaling both arises from and feeds back into barrier impairment and microbiota remodeling, thereby reinforcing the axis as a dynamic and interdependent framework.

These revisions improve the narrative coherence of the manuscript and ensure that inflammation is presented as an integral and continuous component of the proposed conceptual axis, rather than as a separate thematic element. We believe this improves the overall clarity and conceptual integrity of the review. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

While current screening primarily relies on colonoscopy and fecal immunochemical testing (FIT), meta-analytic estimates confirm that FIT has limited sensitivity for advanced adenomas and variable performance in programmatic settings [14]. Recent work demonstrates that combining FIT with blood/stool-based biomarkers and age can improve diagnostic accuracy and resource allocation [15]. In this context, this review focuses on the earliest window of tumorigenesis—adenoma and early colorectal cancer—and integrates microbial alterations with mechanical and chemical barrier disruptions, mapping these changes to inflammation nodes that are actionable for diagnosis and intervention. We first address barrier disruption, then microbiome changes with sample-type and stage stratification, followed by the inflammatory cascade and its feedback loops, and finally discuss diagnostic markers and interventions aligned to each node. Importantly, within this framework, inflammation is not treated as a downstream consequence alone, but as a core and dynamic component that both results from and actively reinforces intestinal barrier dysfunction and microbial dysbiosis. To integrate these interrelated processes, we propose a conceptual framework in which intestinal barrier disruption, gut microbiota dysbiosis, and chronic inflammation form a self-reinforcing axis that drives colorectal carcinogenesis, as illustrated in Figure 1. [The 3^rd paragraph in the Introduction]

Chronic intestinal inflammation is well-extablished independent risk factor for CRC, substantially elevating cancer risk in patients with IBD, including ulcerative colitis (UC) and Crohn's disease (CD). Epidemiologic studies have shown that patients with UC and CD face an approximately 2.4-fold increased relative risk (RR) of CRC, which escalates with longer disease duration and greater extent of lesions [103]. This risk can be amplified to 5-10 times that of the general population in specific high-risk subgroups, such as patients with disease lasting over 8 years, those with co-morbid primary sclerosing cholangitis, or individuals with a positive family history of CRC [104]. Consequently, endoscopic surveillance is recommended in clinical guidelines for these groups. While earlier sections focus on how intestinal barrier disruption and microbial dysbiosis initiate inflammatory signaling, this section emphasizes the reciprocal process by which sustained intestinal inflammation actively remodels gut microbial composition. Chronic inflammatory signaling alters the intestinal microenvironment, thereby imposing selective pressures that reshape microbial ecology and reinforce dysbiosis. [The 1^st paragraph in the 4.1]

Collectively, these findings underscore inflammation as a core component of the barrier–microbiota–inflammation axis, dynamically linking epithelial dysfunction and microbial dysbiosis to sustained tumor-promoting conditions in colorectal cancer. [The 4^th paragraph in the 4.2]

3. Regarding the stated objective of addressing “emerging diagnostic strategies,” clarity is lacking. The manuscript focuses heavily on biomarkers but not on other types of operational strategies, which are presented in a poorly structured manner. It would be advisable for the authors to include, for example, a comparative table with specific interventions, potentially adding sensitivity/specificity values where applicable.

Our response:

We thank the reviewer for this constructive and important comment. We agree that, in the original version, the concept of “emerging diagnostic strategies” was not sufficiently explicit and that the presentation leaned heavily toward individual biomarkers without a clear operational or comparative framework. This may have reduced the translational clarity of the diagnostic section.

To address this concern, we have substantially revised the manuscript to (i) clarify what we define as “diagnostic strategies”, extending beyond single biomarkers to include integrated, operational screening approaches, and (ii) improve structural organization and comparability across strategies.

Importantly, we have added a new comparative summary table that systematically contrasts representative emerging diagnostic strategies, including their sample type, biological basis, clinical application scenario, and reported diagnostic performance metrics (e.g., sensitivity, specificity, or AUC, where available). This table is intended to provide readers with a concise, practice-oriented overview rather than a purely descriptive biomarker list. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

Importantly, emerging diagnostic strategies should be understood not as isolated biomarkers but as operational frameworks that integrate biological signals into actionable screening or triage pathways. As summarized in Table 2, current approaches range from single-marker assays to composite panels and multimodal algorithms that combine microbiota-derived features, barrier integrity markers, metabolites, and clinical variables. These integrated strategies consistently outperform single-marker tests and are better aligned with real-world screening needs, particularly in FIT-based population programs and early-onset CRC risk stratification. [The 5^th paragraph in the 5.1]

 

Table 2. Emerging diagnostic strategies targeting the barrier–microbiota–inflammation axis in colorectal cancer

Diagnostic strategy	Sample type	Core biological basis	Representative markers / models	Intended clinical use	Diagnostic performance*	References
Single microbial biomarker assays	Stool	CRC-associated microbial enrichment	Fusobacterium nucleatum, Peptostreptococcus anaerobius	Non-invasive CRC screening	Fn: AUC ~0.82; combined markers improve detection	[130]
Barrier integrity–related serum markers / host response	Blood	Host immune response to microbiota; epithelial barrier dysfunction	Antibodies against Fn, anti-microbial serology	Adjunct early detection	Enhanced discrimination when combined	[131]
Stool metabolic & microbial dysbiosis profiling	Stool	Dysregulated microbial metabolites & taxa	Multi-taxa microbial panels	Early adenoma & CRC detection	Microbiome AUC ~0.80–0.89	[123]
Microbiota + FIT integration	Stool (FIT + microbes)	Occult bleeding complemented by microbial signals	FIT + Fusobacterium quantification	Improved FIT screening	Sensitivity 92.3%, specificity ~93% for CRC (PMC)	[132]
Machine learning microbiome models	Stool	Gut microbial community patterns with AI	Random forest & RF microbial risk score models	Early CRC & adenoma classification	AUC ~0.82–0.90	[133]
AI-ML gut microbiome prediction tools	Stool (multi-cohort)	Species-level profiles + ML	e.g., CRCpred (XGBoost)	Population screening enhancement	AUC ~0.90–0.91	[134]

 

4. With respect to methodology, although the manuscript appears to be a narrative review, the authors do not report any search strategy, making it impossible to assess the transparency, comprehensiveness, or reproducibility of the information presented. This may also explain why results from animal studies, in vivo experiments, and observational studies are mixed together without indicating their relative evidentiary weight, which can lead to confusion.

Our response:

We thank the reviewer for this comment. We acknowledge that the original manuscript did not explicitly describe the literature search process, which may have reduced transparency.

To address this, we have clarified that this work is a narrative review and have now added a brief description of the literature search strategy, including the main databases searched, key terms, and the temporal scope. This information is provided to enhance transparency, while recognizing that the manuscript is not intended to be a systematic review or meta-analysis.

In addition, we have revised the text to more clearly distinguish between preclinical (in vitro and animal) studies and human observational or clinical evidence, particularly when discussing mechanistic insights versus diagnostic or translational relevance. This clarification is intended to prevent overinterpretation and to guide readers regarding the relative evidentiary weight of different study types. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

This narrative review is based on literature retrieved from PubMed, Web of Science, and Scopus, using combinations of keywords related to colorectal cancer, intestinal barrier function, gut microbiota, and inflammation. Priority was given to recent and representative studies with mechanistic relevance or translational significance. The aim was to integrate key findings rather than to provide a systematic or exhaustive synthesis. [The 4^th paragraph in the Introduction]

5. More critical discussion is needed. The dual role of SCFAs is mentioned but not addressed; the well known geographic variability is not discussed; nor are the biases inherent to metagenomic studies, or the key question of whether microbiota changes are a cause, a consequence, or both. Concepts are repeated—for example, the LPS–TLR4–MyD88–NF κB axis is explained several times in different sections with little synthesis.

Our response:

We thank the reviewer for this insightful comment and agree that the original version of the manuscript was overly descriptive in parts and did not sufficiently emphasize critical discussion and conceptual synthesis.

To address this concern, we revised the manuscript by integrating concise critical clarifications directly into the relevant sections, rather than adding a separate discussion module. Specifically, the discussion of short-chain fatty acids (SCFAs) was expanded to emphasize their context- and stage-dependent effects, highlighting that SCFAs may exert distinct biological roles depending on epithelial metabolic state and disease stage, rather than being uniformly protective. In addition, we now explicitly acknowledge geographic and population-level variability in gut microbiota composition, noting its implications for the generalizability of microbiota-based biomarkers.

We also added brief clarifying statements regarding methodological biases inherent to metagenomic studies, including variability in sample type, sequencing platforms, and confounding clinical factors, to caution against overinterpretation of cross-study associations. Importantly, the manuscript now explicitly addresses the unresolved issue of causality versus consequence, clarifying that current evidence supports a bidirectional and self-reinforcing relationship between microbiota alterations, barrier dysfunction, and inflammation, rather than a unidirectional causal model.

Finally, to reduce redundancy and improve synthesis, repeated descriptions of inflammatory signaling pathways—particularly the LPS–TLR4–MyD88–NF-κB axis—were consolidated into a single core mechanistic framework, with subsequent sections referring back to this pathway instead of re-describing it in detail. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

In CRC-prone contexts, mechanical barrier failure becomes quantifiable, featuring reduced junctional continuity of ZO-1/occludin with cytoplasmic relocalization, thinning of the inner MUC2 layer, and glycan abnormalities [26,27]. These structural defects correlate with elevated biomarkers of permeability—such as serum LPS, anti-LPS/flagellin antibodies, diamine oxidase (DAO), and fecal/serum zonulin—which can be measured alongside TJ proteins fragments in translational studies [28,29]. Mechanistically, signaling pathways such as TLR4→MyD88→NF-κB/myosin light chain kinase (MLCK) promote TJ disassembly and paracellular leak, while disruption of E-cadherin further impairs adherens junctions, facilitating microbial translocation [30-32]. This LPS–TLR4–MyD88–NF-κB pathway represents a central inflammatory signaling axis within the barrier–microbiota–inflammation network and will not be re-described in detail in subsequent sections. [The 3^rd paragraph in the 2.1]

Notably, the composition and baseline abundance of CRC-associated microbial taxa exhibit substantial geographic and population-level variability [68]. Large-scale cohort studies have demonstrated that the prevalence of organisms such as Fusobacterium nucleatum differs markedly across regions, dietary patterns, and ethnic backgrounds [61,69]. This variability limits the universal applicability of fixed microbial signatures and suggests that diagnostic performance is often cohort-dependent [70,71]. Consequently, microbiota-based biomarkers should be interpreted within specific population contexts and require regional calibration and external validation before broad clinical implementation [72]. [The 4^th paragraph in the 3.1]

While SCFAs, particularly butyrate, are generally regarded as protective metabolites that support epithelial integrity and anti-inflammatory signaling, their biological effects are increasingly recognized as context-dependent rather than uniformly beneficial [93]. In normal colonic epithelium, SCFAs serve as a primary energy source and reinforce barrier function and immune tolerance. However, in transformed or metabolically reprogrammed epithelial cells, especially those exhibiting a glycolytic phenotype, intracellular accumulation of butyrate may exert distinct epigenetic and proliferative effects [94]. Experimental studies therefore suggest that the impact of SCFAs depends on local concentration, epithelial metabolic state, and disease stage, highlighting the need for caution when extrapolating their protective role across different phases of colorectal carcinogenesis [95]. [The 5^th paragraph in the 3.2]

A critical feedback loop perpetuates this process: NF-kB, activated by LPS, not only exacerbates intestinal barrier dysfunction but also sustains persistent inflammation and dysbiosis. Inflammatory signaling profoundly alters the gut ecological niche by increasing epithelial oxygenation, nitrate availability, and antimicrobial peptide release, which preferentially supports the expansion of facultative anaerobic bacteria such as Proteobacteria while suppressing obligate anaerobic commensals. In parallel, inflammation-driven changes in bile acid composition and mucin structure further disrupt microbial metabolic balance, promoting the persistence of pro-inflammatory pathobionts [111-113]. Importantly, current evidence suggests that gut microbiota alterations in this cycle function both as a contributor of barrier dysfunction and inflammation, rather than as a unidirectional initiating factor [70]. This creates a self-reinforcing cycle wherein barrier damage, microbial translocation, and inflammatory amplification are continuously maintained [114,115]. These mechanistic insights reveal promising intervention points. Blocking TLR/NLR signaling, modulating the microbiota with probiotics, or using drugs to restore barrier integrity represent viable strategies to interrupt early cascade events for CRC prevention [116]. Clinically, biomarkers of barrier integrity—such as elevated anti-flagellin antibodies, serum zonulin or DAO levels, and a metabolite profile characterized by low SCFAs and high deoxycholate—correlate with activation of TLR4/MyD88/NLRP3 pathways and associate with greater tumor burden upon transfer of dysbiotic microbiota into gnotobiotic or carcinogen-primed mice [117-121]. This LPS–TLR4–MyD88–NF-κB axis therefore represents a central inflammatory signaling node within the barrier–microbiota–inflammation network and will not be re-described in detail in subsequent sections. [The 3^rd paragraph in the 4.2]

In addition to standardization challenges, microbiome-based studies are subject to methodological biases that complicate cross-study comparison and interpretation. Variability in sample type (stool versus mucosal tissue), sequencing approaches (16S rRNA versus shotgun metagenomics), bioinformatic pipelines, and reference databases can substantially influence reported microbial profiles [160,161]. Moreover, confounding factors such as diet, medication use, bowel preparation, and comorbidities are often incompletely controlled [162,163]. These limitations underscore that microbiota–CRC associations are probabilistic rather than deterministic and should be interpreted with caution, particularly when extrapolating findings across platforms or populations [164]. [The 3^rd paragraph in the 6.1]

6. In several paragraphs, the language used suggests strong causality (e.g., “drivers,” “dismantles,” “firmly establishes”) when the cited references provide mainly associative or preclinical evidence.

Our response:

We thank the reviewer for this important comment and agree that some wording in the original manuscript may have implied stronger causality than is warranted by the available evidence.

To address this concern, we have carefully revised the manuscript to align causal language with the strength and nature of the supporting evidence. Specifically, terms suggesting definitive causation (e.g., “drivers,” “dismantles,” “firmly establishes”) were systematically replaced with more appropriately qualified expressions such as “is associated with,” “contributes to,” “is implicated in,” or “supports a mechanistic link,” particularly when conclusions were based on observational human studies or preclinical models.

In addition, where mechanistic insights were derived primarily from animal or in vitro studies, the text now explicitly reflects their preclinical nature, avoiding extrapolation beyond the evidence base. Stronger causal language is retained only in contexts where multiple lines of evidence converge or where conclusions are clearly limited to experimental models.

We believe these revisions improve conceptual precision and prevent overinterpretation, while preserving the integrative and translational perspective of the review.

7. Finally, the manuscript does not include any limitations of this narrative description (as already mentioned above such as biases or heterogeneity of the referenced studies; results data mixed from animal, in vitro, human studies; the lack of clinical trials, etc.).

Our response:

We thank the reviewer for this important comment and agree that the limitations of this narrative review were not sufficiently articulated in the original manuscript.

To address this issue, we have added a concise limitations paragraph to the manuscript that explicitly acknowledges the inherent constraints of this narrative synthesis. Specifically, we now discuss (i) the heterogeneity and potential biases of the referenced studies, including differences in study design, populations, and analytical platforms; (ii) the integration of evidence across in vitro, animal, and human studies, which provides mechanistic insight but limits direct clinical inference; and (iii) the current scarcity of large-scale prospective clinical trials, particularly those validating microbiota- or barrier-based biomarkers for clinical implementation.

These limitations are now clearly stated to contextualize the conclusions of the review and to avoid overgeneralization. We believe that explicitly acknowledging these constraints improves the transparency and interpretability of the manuscript while preserving its integrative and hypothesis-generating purpose. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

Beyond standardization, translational research on the barrier–microbiota–inflammation axis faces inherent methodological limitations. It should also be acknowledged that these constraints reflect inherent limitations of the present narrative review, which integrates evidence from heterogeneous in vitro, animal, and human studies rather than from uniformly designed clinical trials. As such, the conclusions drawn here are intended to be integrative and hypothesis-generating, rather than definitive or predictive at the clinical level. Many mechanistic findings are derived from animal models or small-scale human observational studies, lacking validation in large, prospective clinical studies. While animal studies consistently demonstrate the tumor-suppressive effects of SCFAs, for instance, the considerable variability in human diet, host metabolism, and gut microbiota composition has precluded a consensus on their predictive value for CRC risk or their utility in existing screening programs [165]. [The 1^st paragraph in the 6.2]

 

8. Some typographical errors in the text include: initation (initiation), elebated (elevated), microhome (microbiome). Some errors in the figures: Tihgt junctions, EARLY DIAGIOSIS, CACSADE, Bionrakers, Microhome, Accids.

Our response:

We thank the reviewer for carefully identifying these typographical errors. All the reported spelling mistakes in both the main text and figures have been corrected accordingly.

Specifically, errors in the text (e.g., initation, elebated, microhome) were corrected to their proper forms, and all typographical and labeling errors in the figures (e.g., Tihgt junctions, EARLY DIAGIOSIS, CACSADE, Bionrakers, Microhome, Accids) have been revised to ensure accuracy and consistency.

In addition, the entire manuscript and all figures were carefully rechecked to correct any remaining typographical or formatting issues. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

 

Graphical abstract

The disruption of the intestinal barrier and dysbiosis in the gut microbiota impair mucosal repair mechanisms. This microbial imbalance and persistent chronic inflammation drive a self-perpetuating cycle: impaired barrier function allows for microbial invasion, triggering an inflammatory cascade that further exacerbates the initial damage. As the condition progresses, this cycle of inflammation and barrier dysfunction ultimately contributes to the development of colorectal cancer.

 

Figure 1. The CRC Gut Axis: Barrier Disruption, Gut Dysbiosis & Inflammation Cascade. This diagram illustrates the vicious cycle wherein intestinal barrier compromise drives gut dysbiosis, which in turn fuels chronic inflammation, thereby generating a pro-carcinogenic microenvironment. Current intervention strategies focus on these interconnected processes; however, clinical translation remains challenging due to issue of standardization, system bias, and the requisite for extensive cohort validation. Abbreviations: CRC, colorectal cancer; TJ, tight junction; LPS, lipopolysaccharide; SCFAs, short-chain fatty acids; NF-κB, nuclear factor kappa B; STAT3, signal transducer and activator of transcription 3; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation primary response 88.

Figure 2. Key mechanisms linking intestinal barrier disruption to colorectal cancer development. Disruption of the intestinal barrier leads to increased intestinal permeability, microbial translocation, and activation of inflammatory pathways. Microbial factors, such as LPS, drive inflammation through TLR4–NF-κB signaling, contributing to carcinogenic signaling via pathways like Wnt/β-catenin. This cascade ultimately increases the risk of CRC development.

 

Figure 3. Strategies for Early Diagnosis & Intervention in CRC. This figure outlines advanced strategies for CRC. Early diagnosis integrates serum biomarkers, microbial signatures, metabolomics, and AI models for high-accuracy detection. Early intervention focuses on barrier protection, microbiota modulation, and anti-inflammation. The integration of these elements aims to advance AI-driven precision medicine, pending resolution of challenges in standardization and clinical validation. Abbreviations: FIT, fecal immunochemical test; FMT, fecal microbiota transplantation; SCFAs, short-chain fatty acids; AI, artificial intelligence; CRC, colorectal cancer.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript "The Barrier-Microbiota-Inflammation Axis in Colorectal Cancer: Mechanisms and Emerging Diagnostic & Therapeutic Strategies" represents an extensive non-systematic review on the role of intestinal microorganisms, integrity of epithelial barrier and inflammation in the pathogenesis of colorectal cancer. Considering the high frequency of colorectal carcinoma, the topic is important; it also corresponds to the scope of the journal "Cancers" and the section "Cancer Causes, Screening and Diagnosis".

Authors propose that the described biological processes and markers could be useful to identify novel diagnostic markers as well as targets for future treatment and/or prevention strategies. Thus, global scientific community could benefit from this publication.

Considering the weaknesses of the submitted manuscript, a more critical assessment of the compiled facts would be beneficial. Regarding different cancer models, it would be necessary to distinguish more clearly between the carcinogenesis on the background of long-standing inflammatory bowel disease (IBD) and other sporadic cases. While inflammation is a recognized driver in IBD-associated carcinogenesis, the morphological evidence of inflammation is more limited, regarding the early and precancerous events in the remaining burden of human colorectal carcinoma. 

Finally, I would like to thank the authors for their work input. 

Comments on the Quality of English Language

Please, correct the few minor misprints, e.g. "cacsade" in Figure 1; "elebated" (line 145) etc., throughout the manuscript. Please, indicate the type of your article (above the title). 

Author Response

Reviewer #4:

1. Considering the weaknesses of the submitted manuscript, a more critical assessment of the compiled facts would be beneficial. Regarding different cancer models, it would be necessary to distinguish more clearly between the carcinogenesis on the background of long-standing inflammatory bowel disease (IBD) and other sporadic cases. While inflammation is a recognized driver in IBD-associated carcinogenesis, the morphological evidence of inflammation is more limited, regarding the early and precancerous events in the remaining burden of human colorectal carcinoma.

Our response:

We thank the reviewer for this important and nuanced comment. We agree that the original manuscript did not sufficiently distinguish between IBD-associated colorectal carcinogenesis and sporadic colorectal cancer, particularly with respect to the strength and nature of inflammatory evidence across disease contexts.

To address this concern, we revised the manuscript to explicitly differentiate inflammation-driven carcinogenesis in long-standing IBD from the more subtle, context-dependent inflammatory involvement observed in sporadic CRC. Specifically, the text now clarifies that chronic, histologically evident inflammation represents a well-established driver in IBD-associated carcinogenesis, whereas in sporadic CRC, inflammatory signaling is often low-grade, localized, or molecularly inferred, rather than accompanied by overt morphological inflammation during early or precancerous stages.

In addition, we have strengthened the critical assessment by avoiding direct extrapolation from IBD models or inflammation-driven animal studies to sporadic CRC. Where inflammatory mechanisms are discussed in the context of sporadic disease, they are now framed as microenvironmental or barrier-associated inflammatory cues, rather than classical chronic colitis–like inflammation. This distinction improves conceptual precision and prevents overgeneralization across cancer models. As your suggestions, the corresponding text in our revised manuscript Cancers-4104399.R1 has been revised as follows:

The molecular mechanisms linking inflammation to CRC primarily involve canonical signaling pathways, predominantly NF-κB, STAT3, and the interleukin (IL)-16/IL-17 axis. Notably, much of the strongest causal evidence for inflammation-driven tumor initiation comes from colitis-associated models and IBD settings, whereas in sporadic CRC these pathways are more often implicated as tumor-promoting programs within the local microenvironment rather than as manifestations of overt chronic colitis. Inflammatory cells within the gut release cytokines such as IL-6, tumor necrosis factor-α (TNF-α), and IL-17, which activate the STAT3 and NF-κB pathways in target cells. This activation promotes the expression of anti-apoptotic genes, proliferation-inducing factors, and angiogenic molecules, thereby converting the intestinal microenvironment from a state of inflammation to one that is pro-carcinogenic [105]. IL-17, produced by Th17 cells, γδT cells, and innate lymphoid cells, serves as a critical bridge between chronic inflammation and CRC. Its persistent activation of STAT3 signaling in epithelial cells drives hyperproliferation and the accumulation of DNA damage [106]. Azoxymethane/dextran sodium sulfate-induced rodent models show significant increases in IL-6 and IL-17F levels alongside STAT3/NF-κB pathway activation, while pharmacological inhibition of STAT3 reduces tumor progression [107]. Mechanistic studies further reveal that inflammation reprograms not only epithelial cells but also immune cells, such as tumor-associated macrophages (TAMs) and dendritic cells, toward tumor-promoting phenotypes, thereby amplifying the cytokine cascades. The coordinated activation of STAT3 and NF-κB thus constitutes a central signaling hub that drives CRC pathogenesis, particularly in the context of IBD [23]. [The 2^nd paragraph in the 4.1]

Intestinal barrier dysfunction and gut microbiota dysbiosis act synergistically to initiate the inflammatory cascade, a relationship robustly validated in animal models. However, it should be noted that many experimental systems that demonstrate a strong inflammation-driven initiation component are colitis-associated or inflammation-amplified models, whereas in early sporadic CRC the morphological evidence of overt inflammation is often more limited and inflammatory involvement may be low-grade or locally confined. Under physiological conditions, tight junction proteins and the mucosal layer prevent bacterial components from translocating across the intestinal epithelia. When the intestinal barrier is compromised, microbial products and metabolites gain access to the lamina propria [9]. There, they are recognized by host pattern recognition receptors, triggering downstream signaling cascades, such as those involving MyD88, NF-κB, and mitogen-activated protein kinases (MAPK) that initiate robust cytokine production [108]. [The 1^st paragraph in the 4.2]

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

No further comments

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has improved with more details and clarifications. I recommend this manuscript for publication in its present form. 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have made a great deal of effort to improve their work on the integrated overview of how intestinal barrier disruption, microbial dysbiosis, and chronic inflammation cooperate from very early stages to drive colorectal cancer development. In this new version, the authors have correctly addressed the concerns, expanded the aspects for which further explanation was requested, and satisfactorily corrected the issues and mistakes indicated, resulting in a considerable improvement of the manuscript. I consider that it can now be accepted for publication.