Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, with incidence rates reaching 19.5 per 100,000 individuals in 2020. The increase is attributed to dietary changes (high red meat, low fiber), sedentary lifestyles, and genetics. Current treatments, including surgery, radiotherapy, and chemotherapy, have limitations: surgery is unsuitable for advanced tumors, radiotherapy can cause complications, and chemotherapy affects healthy cells. This necessitates exploring novel therapeutic strategies, with probiotic therapy emerging as a promising alternative. Several probiotic strains have shown anti-cancer effects. *Lactobacillus reuteri* stimulates CD8+ T cell cytotoxicity, *Lactobacillus gallinarum* modulates the gut microbiota and produces indole-3-lactic acid, and *Streptococcus thermophilus* secretes β-galactosidase, all contributing to reduced tumorigenesis. However, the colonization efficiency of these probiotics is low (0.5-1%), highlighting the need for more effective strains. Probiotics' role in regulating intestinal microbiota and maintaining homeostasis is well-documented. *Pediococcus acidilactici* BT36 and butyrate-producing *Clostridium* spores wrapped in fructo-oligosaccharide demonstrate this through microbiota modulation. Fecal microbiota transplantation also shows promise in counteracting dysbiosis and inhibiting tumor proliferation. Oxidative stress, an imbalance in redox homeostasis, contributes to various diseases, including cancer. Antioxidants like Vitamin C (VC) show inhibitory effects on CRC growth, but the mechanisms aren't fully understood. CRC patients frequently exhibit gut microbial dysbiosis, characterized by increased opportunistic pathogens and harmful metabolites. This dysbiosis can exacerbate oxidative stress. For example, *E. faecalis* produces hydroxyl radicals, and deoxycholic acid is linked to oxidative damage. Cancer cells consistently experience oxidative stress. Therefore, probiotics, with their antioxidant and microbiota-modulating properties, may effectively combat CRC. "Jiangshui", a traditional fermented food, is rich in natural probiotics, and *Limosilactobacillus fermentum* GR-3, isolated from it, exhibits high antioxidant capacity and has shown promise in reducing uric acid levels. This study aimed to evaluate GR-3's effects on AOM/DSS-induced CRC in mice, examining intestinal barrier integrity, inflammation, oxidative stress, tumor apoptosis, and gut microbiome shifts.

Existing research highlights the potential of probiotic therapy in colorectal cancer (CRC) treatment. Studies have demonstrated the anti-cancer effects of various probiotic strains, including *Lactobacillus reuteri*, which stimulates the cytotoxicity of CD8+ T cells by secreting indole-3-aldehyde; *Lactobacillus gallinarum*, which modulates the gut microbiota and produces indole-3-lactic acid to inhibit intestinal tumorigenesis; and *Streptococcus thermophilus*, which inhibits colorectal tumorigenesis by secreting β-galactosidase. However, a significant limitation of these naturally occurring probiotics is their low colonization efficiency in the gut (0.5-1%), suggesting that their inherent capabilities might be insufficient for effective CRC treatment. The literature also emphasizes the crucial role of probiotics in regulating the intestinal microbiota and maintaining intestinal homeostasis. Studies have shown the efficacy of probiotics such as *Pediococcus acidilactici* BT36 in modulating gut microbiota to counteract chromium toxicity and butyrate-producing *Clostridium* spores in suppressing CRC by increasing short-chain fatty acids (SCFAs). Fecal microbiota transplantation has also been shown to counteract gut microbiome dysbiosis and inhibit tumor proliferation. The importance of redox status and oxidative stress in overall health and disease development, including CRC, is also well-established. Antioxidants play a critical role in maintaining redox homeostasis and preserving cellular function. Vitamin C (VC), for instance, has demonstrated inhibitory effects on CRC growth and proliferation, although the specific mechanisms remain to be fully elucidated. The role of gut microbiota dysbiosis and inflammation-induced oxidative stress in CRC progression is widely recognized, making it a key area of focus in the search for effective therapeutic strategies.

This study employed both in vitro and in vivo methods. Seven "Jiangshui"-derived probiotics were initially screened for their in vitro antioxidant capacity using DPPH free radical scavenging activity and total antioxidant capacity assays. *Limosilactobacillus fermentum* GR-3 exhibited the highest antioxidant capabilities and was selected for further investigation. In vitro co-culture experiments were conducted to assess the effects of GR-3 and a control strain (*P. acidilactici* GR-6) on the viability of human colon cancer cell lines (RKO and SW480) using CCK-8 assays and flow cytometry. Quantitative real-time PCR (qRT-PCR) was used to analyze the expression levels of apoptosis-related genes (p53, Bax, Bcl-2) and cell proliferation markers (NF-κB, β-catenin). Non-target metabolite analysis identified indole derivatives produced by GR-3 and GR-6. For in vivo studies, a colitis-associated colorectal cancer (CRC) mouse model was induced using azoxymethane (AOM) and dextran sulfate sodium (DSS). Mice were randomly assigned to six groups: control (CK), model (AOM/DSS only), GR-3, GR-6, 5-fluorouracil (5-FU), and vitamin C (VC). GR-3 and GR-6 were administered orally, 5-FU intraperitoneally, and VC orally. Body weight, fecal bleeding, stool consistency, and colon length were monitored weekly. After euthanasia, colon tissues were collected for histological analysis (H&E and Alcian blue staining), immunohistochemistry (Bax and TLR4), and qRT-PCR (apoptosis, proliferation, inflammation markers). Serum and fecal samples were analyzed for inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-4, IL-10), oxidative stress markers (MDA, GSH, SOD), and LPS levels. Intestinal permeability was assessed using FITC-dextran. Fecal samples were subjected to 16S rRNA sequencing and metagenomic shotgun sequencing to analyze gut microbiota composition and function. Untargeted metabolomic analysis (LC-MS) was performed on fecal samples to identify differential metabolites between the groups. Statistical analysis was performed using appropriate methods, including ANOVA, Kruskal-Wallis test, Kaplan-Meier analysis, and PLS-DA.

In vitro, *L. fermentum* GR-3 displayed the highest antioxidant capacity among seven tested probiotics and showed significant anti-cancer effects on SW480 cells, inducing apoptosis and reducing cell viability. GR-3 produced higher levels of IPA compared to the control strain GR-6. In the AOM/DSS-induced CRC mouse model, GR-3 treatment significantly improved body weight, reduced fecal bleeding and colon shortening, and decreased tumor incidence by 51.2% compared to the model group. GR-3 effectively reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and increased the levels of anti-inflammatory cytokines (IL-4, IL-10) in colon and serum. GR-3 also alleviated oxidative stress by reducing MDA levels and increasing GSH and SOD levels. It improved intestinal barrier integrity by decreasing serum LPS and FITC-dextran levels and upregulating the expression of MUC2, TFF3, ZO-1, and Occludin. 16S rRNA sequencing revealed that GR-3 significantly modulated the gut microbiota composition, increasing the abundance of beneficial bacteria such as *Alloprevotella* and *Lachnospiraceae NK4A136 group* while suppressing the expansion of *Bacteroides*. Metagenomic analysis further supported this finding by showing increased abundance of butyrate-producing species (*Muribaculum intestinale*) and decreased abundance of *Bacteroides fragilis*. GR-3 intervention upregulated genes involved in antioxidant activity (*xseA*, ALDH), butyrate synthesis (*bcd*), and vitamin biosynthesis while downregulating genes involved in harmful metabolic processes. Untargeted metabolomics revealed that GR-3 increased the levels of beneficial metabolites like SCFAs (acetate, propionate, butyrate), indole derivatives (ICA, IPA), Vitamin B12, and Vitamin D3 while reducing levels of harmful secondary bile acids (CA, DCA, LCA). Correlation analysis showed that DCA was negatively correlated with Vitamin B12, Vitamin D3, and calcitriol, while IPA was negatively correlated with DCA and L-tryptophan. These results suggest that GR-3's anti-tumor effects are mediated by its antioxidant properties, anti-inflammatory effects, improvement of the intestinal barrier, and modulation of the gut microbiome.

This study demonstrates the significant therapeutic potential of *Limosilactobacillus fermentum* GR-3 in mitigating colitis-associated tumorigenesis. The findings support the hypothesis that probiotics with strong antioxidant properties can combat CRC by reshaping the gut microbiome. GR-3's superior in vivo efficacy, despite not exhibiting the strongest in vitro anti-cancer effects compared to GR-6, emphasizes the importance of considering both in vitro and in vivo data when evaluating probiotic candidates for CRC therapy. GR-3's ability to colonize tumor tissues directly contributes to its effectiveness. The observed improvements in intestinal barrier function, reduction of inflammation, and modulation of gut microbiota composition, leading to increased beneficial metabolites and decreased harmful metabolites, highlight GR-3's multifaceted mechanisms of action. The observed positive correlations between beneficial metabolites (SCFAs, indole derivatives, vitamins) and the reduction of CRC markers further support the role of GR-3 in maintaining intestinal homeostasis and preventing tumor development. The negative correlation between DCA and beneficial metabolites also suggests a potential mechanism by which GR-3 reduces CRC risk by inhibiting *Bacteroides* proliferation. These results are consistent with the literature supporting the role of gut microbiota dysbiosis and inflammation-induced oxidative stress in CRC progression. The study provides valuable insights into the potential of utilizing probiotics with strong antioxidant properties as effective and safe therapeutic agents for CRC.

This study demonstrates the significant anti-tumor effects of *L. fermentum* GR-3 in an AOM/DSS-induced CRC mouse model. GR-3's efficacy is attributed to its ability to modulate the gut microbiota, enhance antioxidant capacity, reduce inflammation, and improve intestinal barrier integrity. The findings highlight the potential of GR-3 as a promising therapeutic agent for CRC prevention and treatment. Future research should focus on elucidating the precise molecular mechanisms underlying GR-3's effects and conducting human clinical trials to validate its clinical efficacy and safety. Further investigation into the interaction of GR-3 with specific immune cells and pathways would enhance our understanding of its mode of action.

The study was conducted using a mouse model, which may not perfectly replicate the complexities of human CRC. The sample size in some analyses could be considered relatively small. The long-term effects of GR-3 on CRC development and potential adverse effects require further investigation in larger-scale studies. The study focused primarily on the effects of GR-3 on the gut microbiota and metabolites; therefore further investigations are needed to fully understand the underlying molecular mechanisms.