Gut commensal Alistipes as a potential pathogenic factor in colorectal cancer

doi:10.21203/rs.3.rs-4911038/v1

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Gut commensal Alistipes as a potential pathogenic factor in colorectal cancer

https://doi.org/10.21203/rs.3.rs-4911038/v1

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Although previous research has shown that inflammation is associated with development of colorectal cancer (CRC), questions remain about whether inflammatory factor-secreting bacteria play a crucial role in CRC development. The potential role of gut microbiota in secreting inflammatory factors involved in the development of CRC among Chinese patients was explored in this study. 16S rRNA sequencing was utilized to evaluate the distinct microbial characteristics between patients with CRC and colorectal adenoma. Serum levels of inflammatory factors and the expression level of LRG1 tissue protein were studied. The correlation between gut microbiota and inflammatory signaling was analyzed to explore potential molecular mechanisms of CRC. Compared with the adenoma group, CRC patients exhibit distinct pathologies. Moreover, elevated levels of CEA, erythrocytes and haemoglobin in the blood of CRC patients were found. In addition, CRC patients have significantly higher levels of TNF-α, IL-6, IL-10, LRG1 and TGF-β1 when compared with adenoma patients. Spearman correlation analysis revealed that LRG1 was positively related to IL-6 and TNF-α, respectively. The correlation analysis result of TGF-β1 was consistent with the above. Furthermore, we found that the relative abundance of gut commensal Alistipes was significantly elevated in CRC patients. Moreover, a positive correlation between Alistipes and inflammatory signaling was also found. The results suggest that gut commensal Alistipes is a key bacterium with pro-inflammatory properties in the development of CRC. TNF-α and IL-6 associated with Alistipes might activate LRG1-TGF-β1 signaling which contributed to the pathogenesis of CRC.

Colorectal cancer

Gut microbiota

Alistipes

Pro-inflammatory cytokines

Globally, colorectal cancer (CRC) ranks the third in cancer incidence (10%) and is the second most common cause of cancer-related mortality (9.4%). The incidence of CRC, however, varies, with the highest number of cases reported in Asia (52.3%), followed by Europe (26.9%) and North America (9.3%)[1]. With the rising trend of mortality from CRC in Asia, there's a critical demand to discover the underlying mechanism for more efficient strategies to treat CRC, including early identification and prevention.

Recent research reveals that the development of CRC is a complex process involving multiple mechanisms. A large number of studies have shown a correlation between inflammation and tumors[2]. Research has also shown that an imbalance in gut microbiota can lead to CRC[3, 4]. However, little is known about the underlying mechanisms linking microorganisms, inflammation, and colorectal carcinogenesis. Certain cytokines, i.e., tumor necrosis factor (TNF-α) and interleukin(IL)-6, are known for regulating inflammation which can result in CRC development[5, 6]. Transforming growth factor-beta (TGF-β) from monocyte macrophages, platelets and glial cells, also regulate cell proliferation, migration, differentiation and immuno-inflammatory responses, which can further enhance carcinogenesis. Among several subtypes of TGF-β, TGF-β1 is the most widely distributed and active in cells, and coordinates in the majority of biological functions[7, 8]. TGF-β1 is a key factor in the induction of epithelial-mesenchymal transition (EMT) in tumor cells. Previous studies have revealed that LRG1 can promote TGF-β1-induced EMT process and angiogenesis in tumors[9].

Human leucine-abundant α-2-glycoprotein-1 (LRG1) is another potential indicator of CRC activity[10, 11]. Ladd and colleagues reported higher levels of LRG1 in CRC samples taken from female patients, even prior to diagnosis[12, 13]. Further, a study showed that the levels of mRNA for LRG1 and itself are significantly higher in CRC patients[10]. Study indicates that IL-6 plays a role in vascular instability by activating LRG1 transcription via STAT3 phosphorylation[14]. The activation of LRG1 is triggered by IL-1β and TNF-a, leading to the activation of NF-kB, which plays an important role in immune response[15]. Additionally, blocking IL-6 signaling in human lung endothelial cells with the antiIL6 receptor antibody Tocilizumab markedly decreased LRG1 expression. These studies suggest that LRG1 can be induced by certain cytokines.

Colorectal adenoma is a benign tumor of the colon. It is regarded as a precancerous lesion and is a common disease especially among elders. Study shows that 95% of CRC is composed of adenocarcinoma that progresses from adenomatous polyp or adenoma[16]. A large number of microorganisms in colon are involved in maintaining the homeostasis of colon's internal environment. Disruption of this homeostasis can lead to gut microbiota dysbiosis, which can potentially cause the development of CRC. The most common types of intestinal bacteria are Bacteroidetes and Firmicutes in healthy people, while Clostridium nucleatum and Bacteroides fragilis are involved in the development of CRC[17]. Research has shown that probiotics such as Lactobacillus and Bifidobacterium are significantly reduced, while carcinogenic bacteria such as Enterobacteriaceae and Puribacterium were significantly increased in CRC patients[18]. Further, stage III-IV rectal cancer patients have a distinct accumulation of strains of Parabacteria, Parabacteroides, Alistipes, and Ruminococcus[19, 20]. The presence of microbiota is associated with the onset and advancement of CRC through its impact on intestinal inflammation related to tumors[21, 22]. Another study suggested that oral gavage with A. onderdonkii kh33 would increase the levels of macrophage inflammatory protein 1 alpha and 2 (MIP-1α, MIP-2) and IL-17A/F before CRC surgery, while oral oral gavage with P. goldsteinii kh35 inhibited the production of those three cytokines[23]. Research has shown that Alistipes indistinctus can promote the maturation of mast cells by stimulating IL-6 production which may modulate local immune response. The abundance of these gut bacteria may suggest a possible cause in the progression of CRC.

Currently, most researches mainly focus on dietary structure and gut microbiota classification for patients with CRC. The molecular mechanism of CRC development is still mostly unknown, and the inflammatory factor-secreting bacteria that are present in the gut have not yet been fully identified. The underlying mechanisms also require further experimental exploration. Thus, the focus of this research is to study among CRC patients, what bacteria are involved in the development of CRC through inflammatory factors. This will help us understand the underlying mechanisms and further search for potential therapeutic targets.

2.1 Patients and sample collection

A total of 20 CRC patients, including 12 men and 8 women (aged 42 to 69 years), were operated on at the Anhui No.2 Provincial People’s Hospital from January 2023 to December 2023. In the adenoma group, there were 13 men and 7 women (aged 46 to 69 years). We collected intestinal mucosal tissue and serum samples of patients with colorectal adenoma and CRC. The diagnoses in all patients were made histologically with biopsied samples. Patients were excluded if they received neoadjuvant treatment before tissue harvesting, or had been treated with antibiotics for a minimum of four weeks before sampling. We also examined fecal samples from patients who were included. These fecal specimens were gathered 3 ~ 14 days prior to bowel cleanse of colonoscopy. Intact fresh feces were collected in sterile boxes and stored at -80°C on arrival to the laboratory. We obtained informed consent from all patients involved in this study. We followed regulations according to the Declaration of Helsinki and received approval from Medical Ethics Committee of Anhui No. 2 Provincial People’s Hospital. (No. R2023-012, China).

2.2 Reagents and materials

Rabbit anti-human LRG1 (13224-1-AP) and TGF-β1 (21898-AP) were purchased from Proteintech, China. Immunohistochemical kits were purchased from Beijing Zhongshan Jinqiao Company, China. Enzyme-linked immunosorbent assay (ELISA) kits (human TNF-α, human IL-6 and human IL-10) were purchased from Jianglai Biotechnology, China.

2.3 ELISA measurement of cytokines

Blood samples were used to extract blood serum. The blood samples were allowed to clot at 25℃ for 30 min before centrifugation (1500g, 4℃, 15 min), following which the serum was collected and stored at -80℃ until further analysis. The quantities of various cytokines, including human TNF-α, IL-6, and IL-10, were measured with ELISA kit, adhering to the guidelines provided by the manufacturer.

2.4 Fecal microbial community analysis

DNA quality and concentration measurements were conducted using 1.0% agarose gel electrophoresis and NanoDrop® ND-2000 spectrophotometer (Thermo Scientific Inc., USA). We preserved them at -80℃ before additional application. We extracted microbial genome and conducted bioinformatic analysis from collected feces[24]. Amplification of the bacterial 16S rRNA within V3-V4 areas was achieved using primers 336F and 806R. Sequencing was completed using the standard protocols of Majorbio BioPharm Technology Co., Ltd. (Shanghai, China).

2.5 Immunohistochemical staining

Tissue segments embedded in paraffin underwent dewaxing, rehydration, immersion in citric acid, followed by incubation in a microwave setup for antigen recovery. Tissue samples were examined using primary antibodies including rabbit anti-LRG1 (1:200) and rabbit anti-TGF-β1 (1:200). Sections of the colon underwent incubation using secondary antibodies. The technique of immunohistochemistry was carried out with an immunohistochemistry kit. Hematoxylin was used to stain the cell nuclei for a duration of 30 s.

Differentiation of the tissues was performed by mixing hydrochloric acid in ethanol for 30 s, followed by a 15 min water rinse and dehydration using aqueous alcohol and xylene. The immunohistochemical images were visualized by a microscope (Olympus, Japan).

2.6 Statistical analysis

We compared two groups (patients with adenoma/CRC) with t-test by calculating mean and standard deviation. Chi-Square and Fischer exact test were used to compare qualitative data between groups. The significance of bacterial percent abundance (with a threshold > 0.05% in at least one group) between groups was determined by Wilcoxon rank test. *P < 0.05 was considered to indicate statistically significant differences. Every statistical evaluation was calculated with SPSS (SPSS, Chicago, IL, USA). We used Graph Pad Prism 8.0 (Graph Pad Software, USA) to faciliate the graphical handling.

3.1 Clinical pathological differences between patients with CRC and colorectal adenoma

A total of 20 patients with colorectal adenoma (mean patient age was 57.25 ± 7.23 years) and 20 patients with CRC (mean patient age was 57.97 ± 8.12 years) were included. There was no statistical significance among the two groups with respect to sex and age (P > 0.05).

Figure 1A shows the histological differences between adenoma and CRC. The pathological manifestations of tumor cells in the colonic tissues of CRC patients include a solid striated arrangement, occasional glandular structures, infiltrative growth, and penetration into the muscularis propria of the mucosa, along with mucus components visible within the cytoplasm. These features align with the typical pathological characteristics of CRC. Carcinoembryonic antigen (CEA) is used clinically as a tumor marker for CRC. Hemoglobin and erythrocytes are also used to monitor CRC progression since CRC can cause intestinal hemorrhaging.

From our study, we found that among CRC patients, CEA is significantly higher, while erythrocyte count and hemoglobin levels are significantly lower when comparing to adenoma group (P < 0.05) (Fig. 1B-D). TNF-α (P < 0.001), IL-6 (P < 0.001) and IL-10 (P < 0.01) were significantly elevated in CRC patients (Fig. 1E-G).

3.2 Positive correlation between pro-inflammatory cytokines and LRG1 protein expression

The expression of LRG1 and TGF-β1 in CRC is significantly higher than in patients with adenomas (P < 0.01) (Fig. 2). Under immunohistochemical staining, LRG1 is highly expressed in CRC tissues, suggesting that LRG1 is involved in the progression of CRC.

Figure 3 depicts the correlation analysis of pro-inflammatory cytokines with the expression of LRG1 and TGF-β1. LRG1 is positively correlated with IL-6 (r = 0.846, P < 0.001); TGF-β1 is positively correlated with IL-6 (r = 0.496, P < 0.05); LRG1 is positively correlated with TNF-α (r = 0.734, P < 0.001); TGF-β1 is positively correlated with TNF-α (r = 0.529, P < 0.05).

3.3 Distinct microbial characteristics in patients with CRC and colorectal adenoma

We performed 16S rRNA sequencing to assess the composition of gut microbiota of CRC. The Venn diagram reveals two sets of partly overlapping operational taxonomic units (OTUs), suggesting a difference between OTUs in CRC and adenoma patients. There were 421 and 777 distinct OTUs in adenoma and CRC patients, respectively, while the number of OTUs common to both samples was 750 (Fig. 4A). CRC patients exhibited a greater number of OTUs compared to the adenoma controls. Consequently, these two groups possessed unique OTUs. The phylogenetic tree categorized the gut microbiota from two groups into two principal branches (Fig. 4B). Our results show that despite the significant differences between the two groups, we still observed mostly the same microbial community patterns in individual groups. Subsequently, we conducted weighted UniFrac PCoA evaluation to depict the comparative resemblance in the gut microbiota composition. In addition, PC1 and PC2 scores showed significant differences in flora composition between the 2 groups (R = 0.3327, P < 0.01) (Fig. 4C). PCoA analysis shows that adenoma and CRC clusters are visibly distinct. We performed species composition analyses to further explore the distribution of gut microbiota. The histograms illustrating the gut microbiota community structure showed the microbial species and their relative abundance at the genus level (Fig. 4D). At the genus level, the top 10 genera at the taxonomic level for both sample groups were Blautia, Escherichia- Shigella, Streptococcus, Bacteroides, Bifidobacterium, Faecalibacterium, Ruminococcus_torques_group, Akkermansia, Prevotella and Collinsella. The proportions of Blautia and Streptococcus in the CRC patients are less compared to the adenoma control. The proportions are significant for Blautia (P < 0.01) and Streptococcus (P < 0.05) (Fig. 4E). However, the proportions of Alistipes, Peptostreptococcus, and Porphyromonas in CRC patients are higher than those in adenoma group (P < 0.05) (Fig. 4E).

We further performed linear discriminant analysis (LDA) on the effect size (LefSe) to identify the primary biomarkers and predominant microbiota between both groups. As shown in Fig. 4F, the abundance of Oscillospiracea, Alistipes, Rikenellaceae, Christensenellaceae, Christensenellales, Porphyromonas, Porphyromonadaceae and Peptostreptococcus is the highest in the CRC patients. Nonetheless, the percentage of Lachnospiraceae was found to be greater in patients with adenoma than those with CRC. Our findings suggest that alterations in these microbial communities may be associated with the onset and development of CRC.

3.4 Correlation between Alistipes and inflammatory signaling factors

The redundancy analysis/canonical correlation analysis (RDA/CCA) analysis mainly reflects the relationship between bacterial microbiota and clinical inflammatory factors. A subset of clinical factors was obtained by using maximum correlation coefficient. We performed CCA analyses according to sample species distribution tables for clinical factors. Inflammatory factors we selected for this study included TNF-α, IL-6, IL-10, LRG1 and TGF-β1.

Analysis of the RDA/CAA environmental factors reveals that differences in inflammatory signaling were tightly associated with gut microbiota. This analysis further identified that TNF-α, IL-6, LRG1, and TGF-β1 exerted the most significant influence on tumor gut microbiota, with IL-10 following in importance (Fig. 5A).

Correlation between microbial classification and clinical inflammatory factors reveals that Alistipes is positively related to TNF-α (r = 0.5035, P < 0.05), IL-6 (r = 0.4921, P < 0.05), LRG1 (r = 0.4989, P < 0.05) and TGF-β1 (r = 0.4987, P < 0.05); Porphyromonas is positively related to TNF-α (r = 0.4305, P < 0.05), IL-6 (r = 0.4307, P < 0.05), LRG1 (r = 0.4305, P < 0.05) and TGF-β1 (r = 0.4323, P < 0.05) (Fig. 5B). Comparatively, Blautia is negatively associated with TNF-α (r = -0.5432, P < 0.01), IL-6(r = -0.5320, P < 0.01), LRG1 (r = -0.5191, P < 0.01) and TGF-β1 (r = -0.5159, P < 0.01); Butyricoccus is negatively associated with TNF-α (r= -0.5694, P < 0.01), IL-6 (r = -0.5732, P < 0.01), LRG1 (r = -0.5740, P < 0.01) and TGF-β1 (r = -0.5910, P < 0.01) (Fig. 5B). Besides, our results show that Alistipes has the strongest effect on inflammatory signaling.

A variety of factors affect the development of CRC and the underlying mechanism remains incompletely understood. Here, we profiled the fecal gut microbiota, the serum, and the tissue protein of CRC patients and compared them with those of precancerous colorectal adenoma patients. There are distinct differences in the serological and cytological presentations between the two. CEA is significantly higher, while erythrocyte count and hemoglobin levels are significantly lower when compared to the adenoma group. CRC patients have significantly higher levels of TNF-α, IL-6, IL-10, LRG1 and TGF-β1 when compared with adenoma patients. Spearman correlation analysis revealed that LRG1 was positively related to IL-6 and TNF-α, respectively. The correlation analysis result of TGF-β1 was consistent with the above. Furthermore, we found that the relative abundance of gut commensal Alistipes was significantly elevated in CRC patients. Moreover, a positive correlation between Alistipes and inflammatory signaling was also found. Our results suggest that gut commensal Alistipes is a key bacterium with pro-inflammatory properties in the development of CRC. TNF-α and IL-6 associated with Alistipes might activate LRG1-TGF-β1 signaling which contributed to the pathogenesis of CRC.

The inflammatory reactions ultimately lead to the occurrence of colon cancer. In the study of CRC, several aspects of inflammation are involved, including inflammatory factors, the development of immune cells, and the activation of related signaling pathways. Previous studies have suggested that upregulation of mucosal pro-inflammatory cytokines is associated with the development of CRC[18]. Consistent with previous studies, we confirmed the elevation of pro-inflammatory cytokines in patients with CRC compared to adenoma. In our study, all three cytokines, TNF-α, IL-6, and IL-10, were significantly elevated in CRC patients. Although IL-10 is a crucial anti-inflammatory cytokine, it may act as an inflammation-inducing factor in cancer patients. Li et al. [25] showed that IL-10 and IL-18 are highly expressed in the serum of CRC patients, making IL-10 and IL-18 useful as indicators to determine the prognosis of CRC patients. Consistent with these findings, our results indicated elevated IL-10 levels in CRC patients.

LRG1 in the context of development of inflammatory diseases and tumors, mainly through TGF-β1 signaling, has attracted the attention of researchers and has achieved important advances. Research indicates that the TGF-β1 signaling is the key factor for CRC. LRG1, as a preliminary controller of the TGF-β1 signaling pathway, likely plays a crucial role in driving the angiogenic transition in TGF-β1 signaling. However, the upstream mechanisms by which LRG1 is induced under disease conditions are not well defined by current research. There is also evidence that shows pro-inflammatory cytokines such as IL-6 and TNF-α could induce LRG1, which is a glycoprotein that is mainly produced by liver[26–28]. Consistent with previous studies, we found higher levels of LRG1 and TGF-β1 in CRC tissues compared to adenoma tissues. In this study, there were significant differences in the levels of inflammatory factors (TNF-α, IL-6 and IL-10), LRG1 proteins and TGF-β1 signaling between the two groups. Spearman correlation analysis revealed that LRG1 was positively related to IL-6 and TNF-α, respectively. The correlation analysis result of TGF-β1 was consistent with the above. This points to the crucial role that inflammatory signaling factors play in the pathogenesis of CRC.

Previous studies have shown that microbial species potentially promoting cancer, such as Fusobacterium nucleatum and Bacteroides fragilis, have been linked to the onset and progression of CRC by means of inducing inflammation, compromising the intestinal barrier, causing mucosal tissue damage, facilitating the accumulation of somatic oncogenic mutations, and altering the microenvironment[29]. Changes in some of these gut microbes may be associated with the development of CRC. The Venn diagram from 16S rRNA analysis shows that OTU from patients with CRC and adenoma are not completely overlapped, suggesting a discrepancy between these two groups. The number of OTUs is also higher in the CRC patients than in adenoma patients. CRC-associated gut microbiota exhibits increased species richness, reduced presence of potentially protective species, and elevated levels of precarcinogenic species, as indicated by prior research[14]. Consistent with previous research, these results suggest that increased diversity may not be a sign of a healthy gut, but rather the excessive growth of harmful bacteria, which further promotes the development of adenomas and cancers. In β-diversity parameters, PCoA analysis shows that adenoma and CRC clusters are visibly distinct. This indicates a significant difference in the composition of the gut microbial community between the two groups.

Then, we employed bar charts depicting community composition to examine variations in the community structure between the two groups. Our findings revealed that the top 10 genera at the taxonomic level for both sample groups. Although these bacterial species are commonly found in the two groups, fluctuations in their quantities could be linked to the onset of CRC. We performed species difference analyses and LDA on the effect size LefSe to identify the primary biomarkers and predominant microbiota between both groups. Our data suggest that the abundance of Alistipes is highest in CRC patients, and that Alistipes might be the key bacterium promoting CRC development. Elevated abundance of Alistipes may contribute to CRC progression.

Alistipes, a gram-negative bacterium belonging to the phylum Bacteroidetes, has been found to potentially offer protection against specific diseases, such as liver fibrosis and cardiovascular disease[30]. Conversely, research has also implicated Alistipes as a pathogen in CRC and as a factor linked to depression[30]. In addition, experimental findings reveal that Alistipes significantly triggers inflammation and cancer development in mouse model[31]. However, there are fewer studies on the mechanisms through which Alistipes promotes the development and progression of CRC.

Previous research also reveals that Alistipes may enhance the growth and development of mast cells through the activation of IL-6 synthesis[32]. Alistipes promote right-sided tumorigenesis through the IL-6/STAT3 pathway. Iida and colleagues suggested that the binding of the pro-inflammatory gram-negative bacterium A. shahii to TLR4 triggers TNF production[30]. Researchers suggested a direct link between Alistipes and the function of TLR4-priming/TNF generation[33]. These studies suggest that Alistipes is associated with pro-inflammatory processes. Further studies are required to explore the precise functions and underlying mechanisms of Alistipes in CRC.

We further examined the relationship between Alistipes and different inflammatory signaling factors (TNF-α, IL-6, IL-10, LRG1 and TGF-β1), in order to delineate the signaling pathways that may be involved. Analysis of the RDA/CAA environmental factors reveals that differences in inflammatory signaling factors were tightly associated with gut microbiota. Additionally, our results indicate that TNF-α, IL-6, LRG1, and TGF-β1 have a more significant impact on altering the bacterial composition within tumor tissue. Correlation between microbial classification and clinical inflammatory factors reveals that Alistipes has the strongest effect on inflammatory signaling. These results suggest that gut commensal Alistipes is a key bacterium with pro-inflammatory properties in the development of CRC.

Our investigation focuses on the interplay between LRG1 and Alistipes in disease, as well as their possible roles in the onset and progression of CRC. In the present study, we confirm that the inflammatory response is exacerbated in individuals with CRC, as indicated by elevated TNF-α and IL-6 levels detected in their serum. Gut commensal Alistipes is a key bacterium with pro-inflammatory properties in the development of CRC. TNF-α and IL-6 associated with Alistipes might activate LRG1-TGF-β1 signaling which contributed to the pathogenesis of CRC. Consequently, treatment of gut microbiota may be able to avert the rise of rectal carcinomas and improve treatment effectiveness. Our findings suggest possible pathogenic influence from Alistipes among CRC patients and propose targeting Alistipes for future therapeutic intervention.

Author contributionsJF and GL: conceptualization, designed the experiment, writing-original draft preparation and funding acquisition. XL, LC, SC and BR: collected the materials and conducted quantitative data analysis. YL and CT: methodology, investigation and data curation. SS: formal analysis and software. LJ: supervision, conceptualization, writing-review & editing and funding acquisition. All authors read and approved the final manuscript.

FundingThis research was funded by Anhui Medical University Research Foundation (Jingjing Fu, grant No. 2022xkj124), the Key Program of Natural Science Research of Anhui Provincial Education Department (Lei Jiang, grant No. 2023AH053381) and the Wu Jieping Medical Foundation (Lei Jiang, grant No. 320.6750.2022-20-30)

Data availabilityThe data presented in this study are available in this article and the Supplementary Materials.

Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki and approved by the Medical Ethic Committee of Anhui No.2 Provincial People’s Hospital (No. R2023-012, China). The written informed consent was obtained from each patient.

Consent for publication All authors approved the content and submission.

Competing interests The authors declare that they have no competing interests.

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Gut commensal Alistipes as a potential pathogenic factor in colorectal cancer

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Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 Patients and sample collection

2.2 Reagents and materials

2.3 ELISA measurement of cytokines

2.4 Fecal microbial community analysis

2.5 Immunohistochemical staining

2.6 Statistical analysis

3 Result

3.1 Clinical pathological differences between patients with CRC and colorectal adenoma

3.2 Positive correlation between pro-inflammatory cytokines and LRG1 protein expression

3.3 Distinct microbial characteristics in patients with CRC and colorectal adenoma

3.4 Correlation between Alistipes and inflammatory signaling factors

4 Discussion

5 Conclusion

Declarations

References

Additional Declarations

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