Combination of a pesco-vegetarian diet with non-steroidal anti-inflammatory drugs for colorectal cancer prevention: tumor suppression and gut microbiota modulation in Apc- mutated PIRC rats

Combination of a pesco-vegetarian diet with non-steroidal anti-inflammatory drugs for colorectal cancer prevention: tumor suppression and gut microbiota modulation in Apc- mutated PIRC rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Combination of a pesco-vegetarian diet with non-steroidal anti-inflammatory drugs for colorectal cancer prevention: tumor suppression and gut microbiota modulation in Apc- mutated PIRC rats Sofia Chioccioli, Niccolò Meriggi, Mariela Mejia Monroy, Sonia Renzi, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7216019/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 13 You are reading this latest preprint version Abstract Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide, with genetic predispositions such as FAP contributing significantly to early-onset disease. This study investigated the chemopreventive potential of two non-steroidal anti-inflammatory drugs (NSAIDs), acetylsalicylic acid (ASA) and sulindac (SU), in combination with a pesco-vegetarian diet (PVD), using Apc -mutated PIRC rats, a well-established model of CRC. Animals were treated over three months with two doses of ASA or a single dose of SU, and tumor burden and gut microbiota composition were assessed. Results confirmed the robust protective effect of the PVD diet in reducing the intestinal tumorigenesis, particularly in the colon, independent of pharmacological treatment. ASA treatment, especially at the higher dose, significantly reduced tumour incidence in both dietary groups, with additive effects seen in combination with PVD, while SU did not show a significant protective effect. Microbiota analysis revealed distinct shifts in bacterial composition associated with both dietary and pharmacological interventions. Notably, taxa such as Roseburia and Colidextribacter , previously linked to intestinal homeostasis and anti-inflammatory activity, were modulated by ASA and diet, suggesting a microbiome-mediated mechanism of chemoprevention. These findings underscore the independent and complementary roles of diet and pharmacological interventions in CRC prevention, and highlight the gut microbiota as a promising target for future personalised preventive strategies. Biological sciences/Cancer Biological sciences/Drug discovery Health sciences/Gastroenterology Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Introduction Colorectal cancer (CRC) is the second leading cause of cancer death worldwide (WHO, https://www.iarc.who.int/cancer-type/colorectal-cancer/ ). Besides genetic alterations, which drive the entire process of colon carcinogenesis, environmental factors, and dietary habits in particular, play a significant role in CRC development 1 . Accordingly, based on several epidemiological and experimental studies, the World Cancer Research Fund identified red and processed meat, as well as alcohol consumption, as risk factors, and whole grains and vegetables as protective (WCRF, https://wcrf.org/diet-activity-and-cancer/ ). Increased evidence suggests that the consumption of fish may reduce the CRC risk, due to the presence of long-chain ω-3 polyunsaturated fatty acids 2 , but there is not enough evidence to have a specific recommendation on eating fish. Recently, exploiting different models of colon carcinogenesis in vivo , we reported a strong protective effect of a pesco-vegetarian diet (PVD), which was able to reduce colon tumorigenesis in both chemically induced and spontaneous carcinogenesis in rats carrying a germ-line mutation in the Apc gene, a key genetic alteration in CRC development 3 . We also documented that this diet promotes the selection of specific bacterial taxa and metabolites that contribute to its protective benefits—an effect also observed when the diet-modulated fecal microbiota was transplanted into germ-free animals treated with carcinogens 3 . Data from clinical and preclinical studies also documents that CRC is affected by regular intake of non-steroidal anti-inflammatory drugs (NSAIDs) such as Aspirin (ASA), or Sulindac (SU), reducing the risk of developing CRC 4 – 6 . Accordingly, ASA has extensively reported to prevent CRC especially the sporadic type, i.e . not associated with specific genetic alterations 7 . For high-risk patients like those with FAP, a genetic syndrome (germ-line mutations in APC gene), leading to the development of hundreds to thousands of intestinal adenomas, which inevitably evolve to cancers, the evidence of a beneficial effect of ASA is not strong, and the available studies have yielded contradictory results 8 – 10 . As for SU, it has been reported to reduce colonic adenomas in FAP patients 11 and to be effective in experimental models of CRC 12 , 13 . Studies in humans and experimental animals also show that drugs can modify the gut microbiota composition, and, on the other hand, that an alteration of the microbiota composition could, directly or not, influences drug effect as documented by Zhao and colleagues 14 , showing that the effects of ASA in reducing CRC in mice are dependent on the composition of gut microbiota. Evidence also exists that variation in the microbiome composition is driven by variation in dietary habits, and we and others hypothesized that an important determinant of the diet-associated CRC risk is, in fact, the intestinal microbiome 3 , 15 , 16 . Given these considerations and the reported enhanced activity of a combination of two chemopreventive regimens reported in some studies 17 – 19 , we aimed to understand whether the strong preventive effect that we observed with the PVD diet 3 could be further strengthened by a combination of this same diet with two non-steroidal anti-inflammatory drugs (NSAIDs), namely ASA and SU. Furthermore, the co-administration of these drugs with a protective diet could, at least theoretically, allow a lowering of the dosages, thus limiting the unwanted side-effects often associated with their chronic use, which may limit their long-term administration in patients at risk. We used as experimental model PIRC (Polyposis in the Rat Colon) rats, carrying an Apc mutation which leads to the spontaneous formation of tumours in the colon and in the small intestine, and thus mimicking both FAP and CRC more closely than other Apc- based rodent models (i.e. Min mice), developing tumors mostly in the small intestine. While ASA has been shown to exert a preventive effect in chemically induced models of CRC in rats 6 , its effect in genetic models involving Apc mutation is less clear 20 , 21 . ASA has not been tested before in PIRC rats, while for SU, previous data from our group document that 320 ppm in the diet effectively decreases colon tumorigenesis, while lower doses were less active in the colon 12 . Thus, in the present study, we determined intestinal tumorigenesis in PIRC rats maintained on either a PVD diet or an AIN-76 based diet used as control diet (CTR) 3 , containing ASA at doses of 800 ppm or 1600 ppm 22 , or SU at a dose of 80 ppm, which were administered for three months. In addition, given the reported effect of drugs and diet on the intestinal microbiota we also studied the microbiota profiles in the different experimental groups. Materials and methods Animal housing, monitoring, and ethical approval PIRC rats (F344/NTac-Apc am1137 ) originated from the National Institutes of Health (NIH), Rat Resource and Research Center (RRRC) (University of Missouri, Columbia, MO, USA) were bred in CESAL (Housing Centre for Experimental Animals of the University of Florence, Italy) by mating heterozygous PIRC rats with Wild-Type F344. Pups, aged 3 weeks, were genotyped as previously described 23 . All animals were housed in ventilated cages (IVCs) under controlled environmental conditions (temperature, humidity, and a 12 h light/dark cycle). To promote animal welfare and reduce stress, each cage was enriched with wooden sticks to support the rats' natural gnawing behaviour, which is not satisfied by the powdered consistency of the experimental diets. Animals were monitored every two days during dietary administration for any signs of distress or adverse effects. Body weight was recorded weekly to evaluate general health status and identify potential treatment-related toxicity. At the end of the 12-week treatment period, animals were humanely euthanized by carbon dioxide (CO₂) inhalation. This method was selected in compliance with current ethical standards to minimize pain and suffering, and to allow timely collection of biological samples. All procedures involving animals were carried out in accordance with the European Directive 2010/63/EU and the Italian Legislative Decree 26/2014 on the protection of animals used for scientific purposes. The study complied with the ARRIVE guidelines, and all the experimental protocols were approved by the Italian Guidelines for Animal Care, DL 26/2014 under the authorization 496/2021-PR. Experimental design, dietary interventions, and pharmacological treatments Only male PIRC animals entered the experiment. Starting at one month of age, rats were fed for three months a control diet (CTR) or a pesco-vegetarian diet (PVD) using the low-calcium AIN76 diet as reference 3 . Diets were prepared in our laboratory using components in powder from Totofood (Laboratorio Dottori Piccioni, Milano, Italy) and stored at -20°C to avoid oxidation. Diets were administered in powder, which, in the case of the PVD group, included portions of cooked codfish pieces (Geloin, Florence, Italy) and lyophilised spinach (SAS Lyophilise.fr (Lorient, France)) as described in detail in De Filippo et al. 2024 3 . ASA (Bayer, Italy) and SU (Fisher Scientific) were added directly to both diets as detailed below. Animals were randomized to enter in the following groups: NT-CTR (n = 12) fed the CTR diet with no drug, ASA1-CTR (n = 12) fed the CTR supplemented with 800 ppm of ASA; ASA2-CTR (n = 12) fed the CTR supplemented with 1600 ppm of ASA; SU-CTR (n = 10) fed the CTR supplemented with 80 ppm of SU. NT-PVD (n = 12) fed the PVD diet with no drug; ASA1-PVD (n = 12) fed the PVD supplemented with 800 ppm of ASA; ASA2-PVD (n = 11) fed the PVD supplemented with 1600 ppm of ASA; SU-PVD (n = 10) fed the PVD supplemented with 80 ppm of SU. Assuming for rats a mean body weight of 300 g and about 12 g of daily diet consumption, a diet containing 800 ppm ASA provides 9.6 mg of ASA per rat/day, i.e. 32 mg/Kg body weight. Considering the different metabolic rate in humans and rats 24 , this dosing corresponds to roughly 5 mg/kg in humans, that is about 350 mg ASA/day in a 70 kg man or, in the case of 1600 ppm to 700 mg/day. Animals remained in the treatment for three months until the sacrifice when faeces were collected for microbiota analysis (see below). The entire colon and small intestine were longitudinally opened to enumerate the tumours in each experimental group as previously described 3 , 25 . Intestinal carcinogenesis evaluation At sacrifice, the entire intestine was washed with cold saline and longitudinally opened as described in Femia et al. 2015 12 . The number of tumors in the colon and small intestine was determined by macroscopic examination. The total number of tumors was recorded for the entire intestinal tract; however, tumor size assessment was restricted to the colon, in line with the study’s primary objective of evaluating colorectal cancer development. Mean tumor diameters and corresponding standard deviations for each experimental group was reported in the Results section. Evaluation of apoptosis in colonic normal mucosa and tumours Apoptosis was evaluated in histological longitudinal sections of the normal colon mucosa (n = 9 in CTR, n = 10 in CTR-ASA1 and PVD, n = 7 in CTR-ASA2 and CTR-SU, n = 11 in PVD-ASA1 and PVD-ASA2, n = 8 in PVD-SU) and tumours (n = 13 in CTR group, n = 9 in CTR-ASA1, n = 12 in CTR-ASA2, n = 10 in PVD, PVD-ASA1 and PVD-ASA2, n = 7 in CTR-SU and n = 8 in PVD-SU) as previously described 25 . PGE2 determination Circulating Prostaglandin E2 (PGE2) levels were determined in plasma obtained by centrifuging blood collected at the time of sacrifice in tubes containing sodium citrate as an anticoagulant. According to manufacturer instructions, PGE2 determination was assessed using the Prostaglandin E2 ELISA Kit - Monoclonal (Cayman Chemical Inc kit Ann Arbor, MI). DNA extraction and 16S rRNA (V3–V4) gene amplification and sequencing Total DNA extraction from faecal samples was carried out by using DNeasy PowerLyzer PowerSoil Kit (QIAGEN) following the manufacturer’s instructions, then quantified fluorometrically using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and stored at − 20°C until 16S (V3-V4) rRNA gene library preparation. The 16S (V3-V4) rRNA gene was amplified by using the primer pairs 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACNVGGGTWTCTAATCC-3′) with overhang Illumina adapters 3 . The barcoded libraries were balanced, pooled at equimolar concentrations, then sequenced using the Illumina MiSeq platform in paired-end mode (300x2). Amplicon sequence variants inference Primer pairs used for library preparation were removed by using cutadapt tool version 4.2 in paired-end mode 26 . The amplicon sequence variants (ASVs) inference from raw sequences was carried out using the DADA2 pipeline version 1.16 27 . Quality reads were improved using the “filterAndTrim” function, filtered reads based on an expected error threshold of 2 for both forward and reverse read pairs. Denoising step was performed using the “dada” function after error rate estimation using the “learnErrors” function. Denoised reads were merged (forward and reverse sequences with any mismatches and/or an overlap length shorter than 12 bp were removed). Chimeric sequences were removed using the “removeBimeraDenovo'' function. The taxonomic classification was carried out by using DECIPHER package version 2.30 28 against the latest version of the pre-formatted Silva small-subunit reference database (SSU version 138 available at: http://www2.decipher.codes/Downloads.html ). The dada2 pipeline showed adequate preservation of the number of reads after the filtering steps (Figure S4a). To further improve dataset quality, all variants not classified as Bacteria were removed with sequences classified as Archaea, chloroplasts, or mitochondria. After quality filtering, no differences in sequencing depth between dietary treatment groups were observed, excluding bias from sequencing depth (Figure S4b). After all quality steps, a total of 3’122’843 reads (median: 44’531.5) collapsed into a total of 1’993 different bacterial ASVs were obtained. After quality filtering, two samples (E153-38 and E153-39) reported 0 counts; thus, they were removed to avoid statistical bias and properly perform the downstream statistical analyses. Statistical analysis All statistical analyses were performed using the R software version 4.3.1 29 . Analysis of variance (Type III Anova) was fitted using a linear model by “lm” function on the model formula, which included both variables, diet and treatment , and their interaction. Dunnett's test for comparing all group combinations of diet and treatment groups against the NT-CTR group (designed as the control level) was performed using the “DunnettTest” function of “DescTools” package version 0.99.60 30 . Tukey HSD test between each group was performed by using “tukey_hsd” of “rstatix” package version 0.7.2 31 . Beta diversity analysis was performed by using “vegan” package version 2.6.10 32 . Differences in beta diversity were estimated using Bray-Curtis distance matrix after ASVs relative abundance transformation and singleton removal. Distances were reported by using principal coordinate analysis (PCoA) using the “cmdscale” function of “stats” package version 4.3.1 29 . The effect of diet and treatment on bacterial diversity was tested using permutational multivariate analysis of variance (also referred to as adonis PERMANOVA) by using the "adonis2" function of the “vegan” package version 2.6.8. The mean within and between group dissimilarities was assessed using the “meandist” function of the “vegan” package version 2.6.8. Environmental fitting was conducted on unconstrained ordination between ASVs abundance and tumour rate by principal component Analysis (PCA) based on Hellinger distance, and it was assessed after ASVs relative abundance transformation, singleton removal, and log + 1 scaling to reduce possible bias related to the different ASVs coverage. Environmental fitting analysis was carried out using the “envfit” function of the “vegan” package version 2.6.8 and the resulting p-value was adjusted using Bonferroni correction method. Significantly different ASVs between CTR and PVD groups within each treatment group were assessed using Wald's test (Benjamin–Hochberg correction for the P-values) implemented in the "DESeq" function of “DESeq2” package version 1.42.1 33 . Differences in genus-level relative abundances between different Aspirin ppm dosage (0, 800, and 1600) within each diet-related group were first assessed using Kruskal-Wallis rank sum test, then significant genus-level features (p < 0.05) were compared between each treatment group by using Wilcoxon test. Differences in genus-level relative abundances between each treatment group (NT, SU, ASA1, and ASA2) were assessed using the Wilcoxon test. The genus-level relative abundance transformation was carried out after singleton removal and after pruning ASVs present in less than 5% of samples. Figures were generated using “ggplot2” package version 3.5.2 34 and edited using the open-source graphics editor Inkscape ( http://inkscape.org/ ) to improve graphic rendering. Results Our study aims to investigate whether ASA and SU associated with PVD may enhance the protective activity of this diet against CRC. To this end, we treated Apc -mutated PIRC animals with two different doses of ASA and a single dose of SU over a three-month period. Chemopreventive activity was assessed by analyzing the number of intestinal tumors. The obtained data were integrated with 16S rRNA sequencing results to determine whether the chemopreventive effects could be attributed to modulation of the intestinal microbiota. PIRC rats aged one month were allocated to the different dietary groups (mean body weight at the beginning of the dietary treatment: mean 83.1 g (± 15.6 g; n = 91 rats). Dietary treatments were modulated in an isocaloric manner as described in De Filippo et al. 3 , therefore, at the end of the experimental period, we observed no differences in mean weight between the two dietary groups: (CTR: mean 320 g (sd 15), PVD: mean 324 g (sd 20)) (Anova, p = 0.26). Concerning the pharmacological treatment, in the CTR group diet, we did not observe differences among the different experimental groups; in the PVD group diet, while ASA treatment did not affect weight gain, SU was associated with a body weight slightly lower than the other groups (Table S2). The mean tumor diameter in the colon was comparable across the experimental groups and the following values were recorded (mean ± standard deviation): CTR-NT: 2.43 ± 1.02 mm; CTR-SU: 2.20 ± 1.02 mm; CTR-ASA1: 2.39 ± 1.33 mm; CTR-ASA2: 2.38 ± 1.05 mm; PVD-NT: 2.97 ± 1.34 mm; PVD-SU: 2.58 ± 1.00 mm; PVD-ASA1: 2.60 ± 1.16 mm; PVD-ASA2: 2.50 ± 1.24 mm. The PVD group determined a significant decrease in the total tumours and tumours in the colon tract regardless of the pharmacological treatment (Fig. 1 b). The pharmacological treatment effect was, however, evidenced only for the CTR group, showing a significant reduction in the number of total tumours in animals treated with ASA2 compared to both SU and NT groups (Fig. 1 b). The same treatment effect, i.e. reduction in the number of tumours in ASA2 compared to SU and NT in the CTR group, was also observed in the small intestine, although an effect of diet was not evident (Fig. 1 b). The effect of diet and pharmacological treatment, together with their interaction, in affecting the number of tumours per rat for each intestinal tract was assessed using type III Anova (Table S3). Both variables (diet and pharmacological treatment) influenced the onset of tumours observed in the colon tract and in all the intestinal tract (i.e. total tumours), whereas the number of tumours accounted for in the small intestine was influenced by the treatment only (Table S3). No significant interaction effect was highlighted, demonstrating that the two variables were effective in influencing the onset of tumours independently of each other (Table S3 and Figure S1 ). To better understand the significance of the values obtained, and due to the nested design of the experiment, we can also consider the group fed the control diet and without pharmacological treatment, i.e. the NT-CTR group, as the true control to which to compare each experimental group. Therefore, we compared the mean values of the number of tumours of each group (intended as every possible combination of levels within the diet and treatment variables) against the NT-CTR group, designated as control (Fig. 1 c and Table S4). Regarding the colon, we thus found that the groups NT-PVD, SU-PVD, ASA2-PVD, and ASA1-PVD had a statistically significant lower number of tumours than the NT-CTR group (Fig. 1 c). In the small intestine only the ASA2-CTR group showed significantly lower values than the NT-CTR group, while considering the entire intestinal tract (total: colon and small intestine), the two groups treated with ASA2 (ASA2-PVD and ASA2-CTR) had a significantly lower number of tumours than the NT-CTR group. As expected, the concentration of the circulating prostaglandin E2 was affected by the ASA supplementation in both concentrations (800 and 1600 ppm) compared to the non-treated group (Figure S2). The higher dose of ASA in both CTR and PVD diets resulted in a significant reduction in PGE2 levels compared to NT (Figure S2). The effect of diet and pharmacological intervention on the rate of apoptotic cells (i.e., apoptotic cells per crypt) in normal mucosa was assessed, and no significant effect was observed (Figure S3). We also evaluated the effect of diet and pharmacological treatment on the intestinal microbiota. The effect of diet and treatment on bacterial diversity (beta diversity) was assessed by multivariate analysis adonis PERMANOVA and depicted by multidimensional analysis (Fig. 2 a). In detail, the multidimensional analysis (PCoA based on Bray-Curtis distance in Fig. 2 a) showed that the diet was the most effective variable in the sample separation and this evidence was also corroborated by the adonis PERMANOVA (Fig. 2 a). To better highlight the effect of the pharmacological treatment, the same analyses were conducted separately by dividing the dataset according to the diet. The pharmacological treatment showed a similar effect in modulating bacterial diversity in both CTR and PVD datasets (R-squared in Fig. 2 b), however, specific differences between treatment levels within each diet-related group were highlighted as shown by the pairwise adonis PERMANOVA analysis (Figs. 2 d). The pairwise adonis PERMANOVA showed significant differences in bacterial diversity between ASA2 and SU in both diet-related groups and between ASA2 and NT in CTR dataset only (Fig. 2 d). Treatment was also effective in modulating bacterial alpha diversity in CTR dataset only (Shannon index in Fig. 2 c). Correlation between changes in bacterial diversity and tumour rate was assessed by environmental fitting analysis. The analysis showed a significant correlation between the number of colon tumours per rat and bacterial diversity; in particular, the microbial diversity in PVD groups showed an anticorrelated trend with the increase in the number of colon tumours (Fig. 3 a and Table S5). To better describe correlations between bacterial diversity and tumour rate, we also performed the environmental fitting analysis splitting the dataset according to each pharmacological treatment (Figure S5 and Table S6). The analysis showed that the correlation effect between diet and tumour rates was more evident in the SU and ASA2 groups, corroborating an anticorrelation trend between the PVD diet and the increase in the number of colon tumours (Figure S5). We assess the taxonomic variants significantly associated with the two diet groups within each treatment dataset using Wald's test (Wald's test of DESeq), identifying 111, 79, 129 and 129 ASVs differentially influenced by diet in the NT, SU, ASA1, and ASA2 treatment datasets respectively (All significant ASVs are reported in Table S7). We observed that diet selected specific ASVs in all four pharmacological treatments, highlighting a strong effect of diet in selecting a pattern of specific bacterial variants. In particular, diet was effective in selecting 21 different ASVs significantly associated with the same diet in all pharmacological treatments (Fig. 3 b). Most variants associated with the PVD diet (62.5%) were assigned to the Lachnospiraceae and Prevotellaceae families (ASV 75: Lachnospiraceae, ASV 353: Lachnospiraceae, ASV 348: Lachnospiraceae, ASV 3: Prevotellaceae, ASV 29: Lachnospiraceae, ASV 240: Lachnospiraceae, ASV 223: Lachnospiraceae, ASV 197: Lachnospiraceae UCG-001 , ASV 149: Lachnospiraceae, ASV 14: Prevotellaceae NK3B31 group ) (Fig. 3 b). Variation in the abundances of the main bacterial genera among different pharmacological treatments in the two different diet-related datasets was also evaluated (Relative abundances and significant comparisons are reported in Figure S6). Considering the significant effect of ASA on colon and intestinal tumours, we were interested in understanding the role of ASA treatment on bacterial abundances. Therefore, we evaluated the variations in relative abundances according to different ASA dosages, i.e. 0 (NT), 800 and 1600 ppm. ASA determined several variations of specific genera differently in the two dietary groups (Fig. 3 c and Table S8). We identified 12 different bacterial genera with significant change in relative abundance among different ASA dosages within different diet-related datasets. The different ASA dosages produced significant variations in these bacterial genera based on the diet Akkermansia , Anaerovorax , Barnesiella , Butyricimonas , and Oscillibacter were significantly affected by the treatment in the CTR diet only, whereas Blautia , Caproiciproducens , Coprococcus , Enterorhabdus , Roseburia , and Eubacterium xylanophilum group were significantly affected by the treatment in PVD diet only (Fig. 3 c). The bacterial genera Colidextribacter was affected by the ASA dosage in both diet-related datasets (Fig. 3 c). Discussion In this study, we investigated whether the chemopreventive efficacy of two NSAIDs, ASA and SU, could be enhanced when administered in combination with a pesco-vegetarian diet PVD. Using Apc -mutated PIRC rats, a well-established model for both CRC and FAP, we administered two different doses of ASA and one dose of SU over a three-month period and assessed both intestinal tumour burden and changes in gut microbiota composition. Our findings demonstrate that both dietary and pharmacological interventions significantly modulate tumour development in the intestinal tract. Notably, the PVD alone exerted a strong protective effect, consistent with our previous findings in other experimental models of carcinogenesis 3 . Animals fed the PVD exhibited a markedly lower number of tumours, especially in the colon, compared to CTR-fed animals. This reduction was statistically significant regardless of pharmacological treatment, further supporting epidemiological and experimental evidence linking high consumption of fish and vegetables to a reduced risk of CRC (WCRF, http://dietandcancerreport.org ). Regarding the drug treatments (two doses of ASA and one of SU), a significant effect was observed in the CTR group, where the number of total intestinal tumours in rats treated with the highest dose of ASA (CTR-ASA2) was significantly lower than in untreated controls (NT-CTR). In the PVD experimental groups, which received the same dosages of ASA, the protective effect of the higher dose was appreciable, especially in the colon, but statistical significance was not reached, likely due to the strong protective effect of the diet itself. Considering the group fed with a control diet and with no treatment (NT-CTR group), as a reference for the various comparisons, we found that the two groups treated with the higher dose of ASA (ASA2-PVD and ASA2-CTR) exhibited a significantly lower number of total tumours. These findings suggest that this ASA dosage is effective regardless of the diet, as further supported by the marked reduction in circulating PGE2 levels, particularly in the PVD group at both dosages (Figure S2). With respect to the lower ASA dose (800 ppm), a mild protective effect was observed in the CTR group, though it did not reach statistical significance. However, the analysis showed that ASA treatment influenced tumour development at least in the small intestine (Table S3). Taken together, these findings demonstrate that both the dietary treatment and pharmacological interventions independently modulates tumours development, with a more pronounced effect when ASA is administered alongside the protective PVD diet. Although several studies have shown that ASA intake reduces the risk of sporadic CRC, its effect of ASA in FAP remains less clear. For instance, Ishikawa et al. 35 reported a reduction in the number and size of colorectal polyps in FAP patients treated with low-dose ASA (100 mg/day). Conversely, other studies using higher doses (e.g. 600 mg/day in Burn et al. 8 showed only a trend toward reduced polyp load (number and size), without a significant reduction in polyp count. Similarly, data from genetic models of intestinal carcinogenesis, such as Apc -mutated Min mice, remain inconclusive. When a protective effect of ASA is observed in these models, it is typically limited only to the small intestine, with no significant impact on the colon 20 , 21 . ASA has not previously been tested in PIRC animals. The doses used in this study were based on a previous investigation that evaluated ASA in azoxymethane-treated rats, a model of sporadic CRC 36 . Considering the different metabolic rates between humans and rats, the equivalent doses in a 70-kg human would correspond to approximately 350 mg/day and 700 mg/day for ASA1 and ASA2, respectively—roughly equivalent to low and medium doses that have also been administered in FAP patients 8 , 35 . We hypothesized that the lowest dose of ASA (ASA1) administered with the CTR diet would lead to a modest reduction in colon tumorigenesis, while its combination with the PVD diet would result in a more pronounced protective effect. Indeed, we found a slight protective effect of ASA1 in the CTR group, and a stronger effect in the PVD group. However, as noted above, the strong protective effect of the PVD made it difficult to detect a statistically significant contribution from ASA1 beyond the effect of the diet alone. The highest dose of ASA (ASA2) significantly reduced total intestinal tumour burden, suggesting that in both diets this dosage of ASA is indeed effective. Our results also showed that SU, administered at a single dose, did not significantly reduce tumour development. In previous studies from our group 12 a strong protective effect was observed in PIRC rats treated with higher doses of SU (320 ppm), whereas animals treated with 80 ppm—the dose used in the present study—showed only a modest reduction in the number of total intestinal tumours. Unlike that previous study, in which treatment lasted eight months, the current study involved a shorter treatment period of three months, explaining the limited effect observed. Notably, the lack of efficacy for 80 ppm SU has also been reported in carcinogen-induced CRC models 37 , suggesting that higher doses are needed to afford protective effects against carcinogenesis. Given the protective effects observed with both dietary interventions and pharmacological treatment on the development of intestinal carcinogenesis, and according to previous findings, we sought to explore whether the gut microbiota could play a mediating or contributing role in modulating intestinal carcinogenesis. Our results indicate that the PVD diet plays a central role in reducing colon tumour burden, while drug treatment, particularly with the higher ASA dose (ASA2), further modulates tumour incidence. Moreover, gut microbiota analyses provide compelling evidence of diet- and drug-induced microbial shifts that may contribute to the observed chemopreventive effects. The significant impact of diet on bacterial diversity, as revealed by beta-diversity analyses and multivariate analyses, suggests that PVD creates a distinct microbial environment associated with reduced tumorigenesis. These findings align with growing evidence that microbiome composition influences CRC progression, with beneficial bacterial taxa potentially contributing to the observed protective effects 3 . In particular, the PVD promoted the enrichment of bacterial families known for their beneficial metabolic functions, such as Lachnospiraceae and Prevotellaceae, consistent with our previous observations 3 , regardless of the pharmacological treatment. These bacterial families have been implicated in maintaining intestinal homeostasis through the production of protective metabolites and the regulation of bile acid metabolism, thereby contributing to CRC prevention. The presence of these beneficial bacterial communities across all pharmacological treatment groups within the same dietary group confirms that diet exerts a primary role in shaping the gut microbiota composition, independently of drug administration. Therefore, while both pharmacological and dietary treatments influence gut microbial communities, they do so independently, without evidence of synergistic interaction, consistent with the lack of synergistic effects on tumour reduction. The effect of ASA treatment on microbiota composition was also evident, although less pronounced than that of diet. ASA selected for distinct bacterial genera in a dose- and diet-dependent manner. Specifically, Akkermansia , Anaerovorax , Barnesiella , Butyricimonas , and Oscillibacter were affected in the CTR dietary group, while Blautia , Caproiciproducens , Coprococcus , Enterorhabdus , Roseburia , and Eubacterium xylanophilum were modulated in the PVD group. In particular, Blautia has been associated with anti-inflammatory properties and improved intestinal barrier function 38–40 , supporting the hypothesis that pharmacological treatment may enhance ASA’s chemopreventive activity by promoting the growth of beneficial bacteria when combined with a protective diet. The abundance of Roseburia was also increased in the PVD group, reinforcing its potential protective role against CRC, as Kang et al. highlight that Roseburia intestinalis , a butyrate-producing gut probiotic often depleted in CRC patients, can suppress tumorigenesis and restore gut barrier function in CRC mouse models 41 Furthermore, the identification of Colidextribacter as a genus responsive to ASA across both dietary groups suggests a broader, diet-independent role for Aspirin in modulating the gut microbiota, with potential implications for developing targeted microbiota-based CRC prevention strategies. Overall, our results underscore the independent and significant contributions of both dietary and pharmacological interventions in preventing CRC. The observed gut microbiota changes point to a mechanistic link between diet composition, microbial diversity, and tumour suppression. Future studies should further explore the functional relevance of these microbial shifts, particularly in relation to metabolite production and immune modulation. A deeper understanding of these interactions may pave the way for integrated dietary and pharmacological approaches to optimize CRC prevention and therapy. Conclusions Our results demonstrate that both dietary and pharmacological interventions independently and significantly contribute to the prevention of colorectal cancer. The pesco-vegetarian diet (PVD) exerted a strong protective effect, associated with distinct shifts in gut microbiota composition, including the enrichment of beneficial bacterial features belonging to Lachnospiraceae and Prevotellaceae families. Among the taxa most notably associated with the PVD, the increased abundance of Roseburia supports its emerging role as a key microbial mediator of diet-induced protection against CRC. Aspirin treatment, especially at higher doses, also contributed to tumour reduction and modulated the gut microbiota in a dose- and diet-dependent manner. Interestingly, Colidextribacter was identified as a genus consistently responsive to ASA across both dietary contexts, suggesting a diet-independent microbial target of aspirin with potential relevance for microbiota-driven prevention strategies. Altogether, these findings point to a mechanistic link between dietary composition, microbial diversity, and suppression of intestinal tumorigenesis. The modulation of specific microbial taxa, such as Roseburia and Colidextribacter , may underlie part of the chemopreventive effects observed and offer new targets for intervention. Future studies should focus on characterizing the metabolic and immunological functions of these microbes and their interactions with dietary and pharmacological treatments. A deeper understanding of these dynamics could pave the way for integrated microbiota-informed strategies to optimize colorectal cancer prevention and therapy. Declarations Data availability and materials The raw sequences have been deposited to the European Nucleotide Archive (ENA) under the accession project code PRJEB88871. All results from statistical analyses were reported as figures and tables in the main text and supplementary file. Further information can be provided upon reasonable request to the corresponding author. Funding This project was funded by the following grants: (i) National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.3 - Call for tender No. 341 of 15 March 2022 of Italian Ministry of University and Research funded by the European Union - NextGenerationEU; Project code PE00000003, Concession Decree No. 1550 of 11 October 2022 adopted by the Italian Ministry of University and Research, Project title “ON Foods - Research and innovation network on food and nutrition Sustainability, Safety and Security - Working ON Foods”. (ii) European Union, NextGenerationEU, National Recovery and Resilience Plan, Mission 4 Component 2, Investment 1.5, THE (Tuscany Health Ecosystem), ECS00000017, CUP B83C22003920001. (iii) The Joint Programming Initiative a Healthy Diet for a Healthy Life-Intestinal Microbiomics (JPI HDHL-INTIMIC) Call for Joint Transnational Research Proposals on “Interrelation of the Intestinal Microbiome, Diet and Health” (reference number JTC-2017–7). (iv) HDHL INTIMIC-Knowledge Platform on food, diet, intestinal microbiomics, and human health (expression of interest no. 895). (v) University of Florence (Fondo ex 60%), Italy. Author information Sofia Chioccioli and Niccolò Meriggi have contributed equally as co-first authors. Authors and Affiliations Department of Neurosciences, Psychology, Drug Research and Child Health (NEUROFARBA), University of Florence, Florence, Italy. Sofia Chioccioli & Giovanna Caderni Institute of Agricultural Biology and Biotechnology (IBBA), National Research Council (CNR), Pisa, Italy. Niccolò Meriggi,Carlotta De Filippo & Mariela Mejia Monroy Department of Molecular and Developmental Medicine (DMMS), University of Siena, Siena, Italy. Mariela Mejia Monroy Department of Biology, University of Florence, Florence, Italy. Sonia Renzi & Benedetta Cerasuolo Contributions C.D.F. and G.C.: designed this study. S.C. and M.M.M.: sample management, DNA extraction and biometric data production. N.M.: data management and computational analyses. S.R. and B.C.: library preparation and sequencing. C.D.F., G.C., N.M., S.F. and M.M.M. wrote the manuscript. All authors reviewed the final version of the manuscript. Corresponding authors Correspondence to Carlotta De Filippo ( [email protected] ). Acknowledgements The authors acknowledge CeSAL (Centro Stabulazione Animali da Laboratorio) at University of Florence. Ethics declarations Competing interests The authors declare no competing interests. Ethical approval and consent to participate The experimental protocols were approved by the Italian Guidelines for Animal Care, DL 26/2014 under the authorization 496/2021-PR. References Keum, N. & Giovannucci, E. Global burden of colorectal cancer: emerging trends, risk factors and prevention strategies. Nat Rev Gastroenterol Hepatol . 16, 713-732 (2019). Caini, S. et al. 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Burn, J. et al. A randomized placebo-controlled prevention trial of aspirin and/or resistant starch in young people with familial adenomatous polyposis. Cancer Prev Res (Phila) . 4, 655-665 (2011). Ishikawa, H. et al. Chemoprevention with low-dose aspirin, mesalazine, or both in patients with familial adenomatous polyposis without previous colectomy (J-FAPP Study IV): a multicentre, double-blind, randomised, two-by-two factorial design trial. Lancet Gastroenterol Hepatol . 6, 474-481 (2021). Zaffaroni, G. et al. Updated European guidelines for clinical management of familial adenomatous polyposis (FAP), MUTYH-associated polyposis (MAP), gastric adenocarcinoma, proximal polyposis of the stomach (GAPPS) and other rare adenomatous polyposis syndromes: a joint EHTG-ESCP revision. Br J Surg . 11, znae070 (2024). Giardiello, F. M. et al. Treatment of colonic and rectal adenomas with sulindac in familial adenomatous polyposis. N Engl J Med . 328, 1313–1316 (1993). Femia, A. P., Soares, P. V., Luceri, C., Lodovici, M., Giannini, A. & Caderni, G. Sulindac, 3,3'-diindolylmethane and curcumin reduce carcinogenesis in the Pirc rat, an Apc-driven model of colon carcinogenesis. BMC cancer . 15, 611 (2015a). Davis, J. S. et al. Sulindac plus a phospholipid is effective for polyp reduction and safer than sulindac alone in a mouse model of colorectal cancer development. BMC Cancer . 20, 871 (2020). Zhao, R. et al. Aspirin reduces colorectal tumor development in mice and gut microbes reduce its bioavailability and chemopreventive effects. Gastroenterology , 159, 969–983.e4 (2020). Sofi, F. et al. Fecal microbiome as determinant of the effect of diet on colorectal cancer risk: comparison of meat-based versus pesco-vegetarian diets (the MeaTIc study). Trials . 20, 688 (2019). Abu-Ghazaleh, N., Chua W. J. & Gopalan V. Intestinal microbiota and its association with colon cancer and red/processed meat consumption. J Gastroenterol Hepatol . 36, 75-88 (2021). Reddy, B. S. et al. 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DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods . 13, 581-583 (2016). Wright, E.S. Using DECIPHER v2.0 to analyze big biological sequence data in R. The R Journal , 8, 352-359 (2016). R Core Team. R: A language and environment for statistical computing . Version 4.3.1 R Foundation for Statistical Computing, Vienna, Austria.https://www.R-project.org/ (2023). Signorell, A. DescTools: tools for descriptive statistics . R package version 0.99.58, https://CRAN.R-project.org/package=DescTools (2024). Kassambara, A. rstatix: Pipe-Friendly Framework for Basic Statistical Tests . R package version 0.7.2,https://CRAN.R-project.org/package=rstatix (2023). Oksanen, J. et al . vegan:Community Ecology Package . R package version 2.6–4. Comprehensive R archive network(CRAN). https://github.com/ vegandevs/vegan (2022). Love, M. I., Huber, W., & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol . 15, 1-21(2014). Wickham, H., Chang, W., & Wickham, M. H. ‘ggplot2’. Create elegant data visualisations using the grammar of graphics . Version 2, 1-189 (2016). Ishikawa, H. et al. Preventive effects of low-dose aspirin on colorectal adenoma growth in patients with familial adenomatous polyposis: double-blind, randomized clinical trial. Cancer Med . 2, 50-56 (2013). Mohammed, A. et al. Intermittent dosing regimens of aspirin and naproxen inhibit azoxymethane-induced colon adenoma progression to adenocarcinoma and invasive carcinoma. Cancer Prev Res (Phila). 12, 751–762 (2019). Agarwal, B. et al. Lovastatin augments sulindac-induced apoptosis in colon cancer cells and potentiates chemopreventive effects of sulindac. Gastroenterology . 117, 838-847 (1999). Mohebali, N., Ekat, K., Kreikemeyer, B., & Breitrück, A. Barrier protection and recovery effects of gut commensal bacteria on differentiated intestinal epithelial cells in vitro. Nutrients . 12, 2251 (2020). Nie, K. et al. Roseburia intestinalis: a beneficial gut organism from the discoveries in genus and species. Front Cell Infect Microbiol . 11, 757718 (2021). Holmberg, S. M. et al. The gut commensal Blautia maintains colonic mucus function under low-fiber consumption through secretion of short-chain fatty acids. Nat Commun. 15, 3502 (2024). Kang, X. et al. Roseburia intestinalis-generated butyrate boosts anti-PD-1 efficacy in colorectal cancer by activating cytotoxic CD8⁺ T cells. Gut 72 , 2112–2122 (2023 Additional Declarations No competing interests reported. Supplementary Files ChiocciolietalSupplementaryfile.docx TableS7.csv Table S7. Result’s table reporting the ASVs with related taxonomic assignment, significantly influenced by diet in each treatment -related dataset selected by using Wald’s test (Wald test of DESeq2). (see .csv file) Cite Share Download PDF Status: Published Journal Publication published 16 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 03 Oct, 2025 Reviews received at journal 30 Sep, 2025 Reviewers agreed at journal 29 Sep, 2025 Reviewers agreed at journal 28 Sep, 2025 Reviews received at journal 25 Sep, 2025 Reviewers agreed at journal 22 Sep, 2025 Reviews received at journal 19 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers agreed at journal 15 Sep, 2025 Reviewers invited by journal 13 Sep, 2025 Editor assigned by journal 11 Aug, 2025 Submission checks completed at journal 05 Aug, 2025 First submitted to journal 05 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":89184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Experimental design: one-month-old male rats were randomly allocated to CTR or PVD diets; within each dietary group, animals received: no pharmacological treatment (NT: not treated); SU: diet (CTR or PVD) supplemented with 80 ppm of SU; ASA1: diet (CTR or PVD) supplemented with 800 ppm of ASA; ASA2: diet (CTR or PVD) supplemented with 1600 ppm of ASA. Rats were sacrificed after three months of treatment, feces and samples were collected, and intestinal tumorigenesis was determined. \u003cstrong\u003eb\u003c/strong\u003e, Barplot reports the number of tumours per rat in the colon, small intestine, and the total number of intestinal tumours. The group comparisons are carried out between different pharmacological treatments within each dietary group and between dietary groups. The different pharmacological treatments are reported using a colour scheme, while dietary groups are reported on the x-axis labels. Significant pairs (Tukey HSD test) are reported using asterisks (*, p\u0026lt;0.05; **, p\u0026lt;0.01; ***, p\u0026lt;0.001). \u003cstrong\u003ec\u003c/strong\u003e, All treatment groups are compared against the non-treated control diet group (NT-CTR), designed as the control level by using Dunnett’s test. Points represent differences between the means of the groups (compared to NT-CTR) while error bars report the confidence intervals of mean differences for each group for each dataset, and statistically significant comparisons are highlighted using coloured points as reported by the colour scheme in the legend. The analysis is conducted and reported separately for each tissue type, with the number of tumours recorded accordingly.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7216019/v1/304fd095bcdd6754e281f7bd.png"},{"id":91711960,"identity":"59038964-d31c-464d-8180-b4b09b4ddc44","added_by":"auto","created_at":"2025-09-19 12:49:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":162463,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, PCoA (Bray-Curtis) performed on the entire dataset represents the sample distribution according to diet (shape pattern) and treatment (colour pattern). Results from adonis PERMANOVA (R-squared values and significance) are reported inside the panel and the confidence interval (0.95) is represented by the ellipses. \u003cstrong\u003eb\u003c/strong\u003e, PCoA (Bray-Curtis) performed on both CTR and PVD datasets separately. Treatment groups are represented by using the color scheme in the legend (see panel \u003cstrong\u003ea\u003c/strong\u003e), and solid empty circles represent centroids. \u003cstrong\u003ec\u003c/strong\u003e, box plots reported differences in alpha diversity metrics (Observed and Shannon index) in both CTR and PVD datasets. Significant differences between treatment groups tested using Wilcoxon test (Benjamini-Hochberg adjusted) are highlighted using asterisks (**, p\u0026lt;0.01). \u003cstrong\u003ed\u003c/strong\u003e, Point plot represents the average dissimilarity (point size) for each pairwise combination between pharmacological treatment groups and the R-squared values (colour gradient) from pairwise adonis PERMANOVA. Significant comparisons from pairwise adonis PERMANOVA are highlighted using blue circles around the points. Permutational multivariate analyses (adonis PERMANOVA and pairwise adonis PERMANOVA) are carried out setting a 1000 number of permutations.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7216019/v1/9595a68741aef1b91f6c446f.png"},{"id":91713562,"identity":"640c8384-0f43-4d91-aa2a-bd17e04e215a","added_by":"auto","created_at":"2025-09-19 12:57:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":207011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, PCA (Hellinger) reports the sample distributions produced by the overall variation in bacterial ASVs composition in relationships to the associated tumour rate associated with each intestinal tract (Colon: tumours in the colon tract, Sm. Intestine: tumours in the small intestine, Total: tumours in the entire intestinal tract). Samples are represented according to treatment (colour pattern) and diet (shape pattern) variables, while arrows indicate direction and magnitude of the linear relationship between tumour rate and the gradient of bacterial ASVs composition depicted by PCA (\u003cem\u003eenvfit\u003c/em\u003e function). The R-squared value of a significantly correlated variable is reported inside the panel, and significance is highlighted using asterisks (**, p\u0026lt;0.01).\u003cstrong\u003e b\u003c/strong\u003e, Bar plot reports the ASVs selected by Wald’s test (Wald’s test of DESeq2) in the same diet group (ASV with the same type of Log2 fold change variation, i.e. positive or negative, compared to the CTR diet in each treatment group) within each treatment group (colour pattern). Each ASV is reported with the related taxonomic assignment. \u003cstrong\u003ec\u003c/strong\u003e, Line chart reports the variation in genus-level relative abundance percentage between different ASA ppm dosage within CTR and PVD groups (colour pattern). Genera in which relative abundance showed significant variations (Kruskal-Wallis test) among different ASA dosages are highlighted using asterisks (*, p\u0026lt;0.05; **, p\u0026lt;0.01). Standard error is reported by using the error bar. Environmental fitting analysis is carried out setting a 1000 number of permutations.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7216019/v1/d0cd097c4018b84cf3569d95.png"},{"id":107350765,"identity":"3b48f128-c6a4-4214-8453-c1bfb81877a5","added_by":"auto","created_at":"2026-04-20 16:03:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":855455,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7216019/v1/26a07493-96d5-4e8d-a6d6-2d0b2e605d9a.pdf"},{"id":91711524,"identity":"d2b52c45-707e-4fa0-830a-c7ee9c8eac33","added_by":"auto","created_at":"2025-09-19 12:41:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":355921,"visible":true,"origin":"","legend":"","description":"","filename":"ChiocciolietalSupplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7216019/v1/6ef71d229035874845bee087.docx"},{"id":91711521,"identity":"8df00372-e46e-4bd5-aa00-fec38ed9c617","added_by":"auto","created_at":"2025-09-19 12:41:18","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":42598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S7\u003c/strong\u003e. Result’s table reporting the ASVs with related taxonomic assignment, significantly influenced by diet in each treatment -related dataset selected by using Wald’s test (Wald test of DESeq2).\u003c/p\u003e\n\u003cp\u003e(see .csv file)\u003c/p\u003e","description":"","filename":"TableS7.csv","url":"https://assets-eu.researchsquare.com/files/rs-7216019/v1/ca7e6dd39bf336adac7c239e.csv"}],"financialInterests":"No competing interests reported.","formattedTitle":"Combination of a pesco-vegetarian diet with non-steroidal anti-inflammatory drugs for colorectal cancer prevention: tumor suppression and gut microbiota modulation in Apc- mutated PIRC rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is the second leading cause of cancer death worldwide (WHO, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.iarc.who.int/cancer-type/colorectal-cancer/\u003c/span\u003e\u003cspan address=\"https://www.iarc.who.int/cancer-type/colorectal-cancer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Besides genetic alterations, which drive the entire process of colon carcinogenesis, environmental factors, and dietary habits in particular, play a significant role in CRC development\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Accordingly, based on several epidemiological and experimental studies, the World Cancer Research Fund identified red and processed meat, as well as alcohol consumption, as risk factors, and whole grains and vegetables as protective (WCRF, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wcrf.org/diet-activity-and-cancer/\u003c/span\u003e\u003cspan address=\"https://wcrf.org/diet-activity-and-cancer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Increased evidence suggests that the consumption of fish may reduce the CRC risk, due to the presence of long-chain ω-3 polyunsaturated fatty acids\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, but there is not enough evidence to have a specific recommendation on eating fish. Recently, exploiting different models of colon carcinogenesis \u003cem\u003ein vivo\u003c/em\u003e, we reported a strong protective effect of a pesco-vegetarian diet (PVD), which was able to reduce colon tumorigenesis in both chemically induced and spontaneous carcinogenesis in rats carrying a germ-line mutation in the \u003cem\u003eApc\u003c/em\u003e gene, a key genetic alteration in CRC development\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. We also documented that this diet promotes the selection of specific bacterial taxa and metabolites that contribute to its protective benefits\u0026mdash;an effect also observed when the diet-modulated fecal microbiota was transplanted into germ-free animals treated with carcinogens\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eData from clinical and preclinical studies also documents that CRC is affected by regular intake of non-steroidal anti-inflammatory drugs (NSAIDs) such as Aspirin (ASA), or Sulindac (SU), reducing the risk of developing CRC\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Accordingly, ASA has extensively reported to prevent CRC especially the sporadic type, \u003cem\u003ei.e\u003c/em\u003e. not associated with specific genetic alterations\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. For high-risk patients like those with FAP, a genetic syndrome (germ-line mutations in \u003cem\u003eAPC\u003c/em\u003e gene), leading to the development of hundreds to thousands of intestinal adenomas, which inevitably evolve to cancers, the evidence of a beneficial effect of ASA is not strong, and the available studies have yielded contradictory results\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. As for SU, it has been reported to reduce colonic adenomas in FAP patients\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and to be effective in experimental models of CRC\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eStudies in humans and experimental animals also show that drugs can modify the gut microbiota composition, and, on the other hand, that an alteration of the microbiota composition could, directly or not, influences drug effect as documented by Zhao and colleagues\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, showing that the effects of ASA in reducing CRC in mice are dependent on the composition of gut microbiota. Evidence also exists that variation in the microbiome composition is driven by variation in dietary habits, and we and others hypothesized that an important determinant of the diet-associated CRC risk is, in fact, the intestinal microbiome\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGiven these considerations and the reported enhanced activity of a combination of two chemopreventive regimens reported in some studies\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, we aimed to understand whether the strong preventive effect that we observed with the PVD diet\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e could be further strengthened by a combination of this same diet with two non-steroidal anti-inflammatory drugs (NSAIDs), namely ASA and SU. Furthermore, the co-administration of these drugs with a protective diet could, at least theoretically, allow a lowering of the dosages, thus limiting the unwanted side-effects often associated with their chronic use, which may limit their long-term administration in patients at risk.\u003c/p\u003e\u003cp\u003eWe used as experimental model PIRC (Polyposis in the Rat Colon) rats, carrying an \u003cem\u003eApc\u003c/em\u003e mutation which leads to the spontaneous formation of tumours in the colon and in the small intestine, and thus mimicking both FAP and CRC more closely than other \u003cem\u003eApc-\u003c/em\u003ebased rodent models (i.e. Min mice), developing tumors mostly in the small intestine. While ASA has been shown to exert a preventive effect in chemically induced models of CRC in rats\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, its effect in genetic models involving \u003cem\u003eApc\u003c/em\u003e mutation is less clear\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. ASA has not been tested before in PIRC rats, while for SU, previous data from our group document that 320 ppm in the diet effectively decreases colon tumorigenesis, while lower doses were less active in the colon\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThus, in the present study, we determined intestinal tumorigenesis in PIRC rats maintained on either a PVD diet or an AIN-76 based diet used as control diet (CTR)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, containing ASA at doses of 800 ppm or 1600 ppm\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, or SU at a dose of 80 ppm, which were administered for three months. In addition, given the reported effect of drugs and diet on the intestinal microbiota we also studied the microbiota profiles in the different experimental groups.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cem\u003eAnimal housing, monitoring, and ethical approval\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePIRC rats (F344/NTac-Apc\u003csup\u003eam1137\u003c/sup\u003e) originated from the National Institutes of Health (NIH), Rat Resource and Research Center (RRRC) (University of Missouri, Columbia, MO, USA) were bred in CESAL (Housing Centre for Experimental Animals of the University of Florence, Italy) by mating heterozygous PIRC rats with Wild-Type F344. Pups, aged 3 weeks, were genotyped as previously described\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAll animals were housed in ventilated cages (IVCs) under controlled environmental conditions (temperature, humidity, and a 12 h light/dark cycle). To promote animal welfare and reduce stress, each cage was enriched with wooden sticks to support the rats' natural gnawing behaviour, which is not satisfied by the powdered consistency of the experimental diets. Animals were monitored every two days during dietary administration for any signs of distress or adverse effects. Body weight was recorded weekly to evaluate general health status and identify potential treatment-related toxicity.\u003c/p\u003e\u003cp\u003eAt the end of the 12-week treatment period, animals were humanely euthanized by carbon dioxide (CO₂) inhalation. This method was selected in compliance with current ethical standards to minimize pain and suffering, and to allow timely collection of biological samples.\u003c/p\u003e\u003cp\u003e All procedures involving animals were carried out in accordance with the European Directive 2010/63/EU and the Italian Legislative Decree 26/2014 on the protection of animals used for scientific purposes. The study complied with the ARRIVE guidelines, and all the experimental protocols were approved by the Italian Guidelines for Animal Care, DL 26/2014 under the authorization 496/2021-PR.\u003c/p\u003e\u003cp\u003e\u003cem\u003eExperimental design, dietary interventions, and pharmacological treatments\u003c/em\u003e\u003c/p\u003e\u003cp\u003eOnly male PIRC animals entered the experiment. Starting at one month of age, rats were fed for three months a control diet (CTR) or a pesco-vegetarian diet (PVD) using the low-calcium AIN76 diet as reference\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Diets were prepared in our laboratory using components in powder from Totofood (Laboratorio Dottori Piccioni, Milano, Italy) and stored at -20\u0026deg;C to avoid oxidation. Diets were administered in powder, which, in the case of the PVD group, included portions of cooked codfish pieces (Geloin, Florence, Italy) and lyophilised spinach (SAS Lyophilise.fr (Lorient, France)) as described in detail in De Filippo et al. 2024\u003csup\u003e3\u003c/sup\u003e. ASA (Bayer, Italy) and SU (Fisher Scientific) were added directly to both diets as detailed below. Animals were randomized to enter in the following groups: NT-CTR (n\u0026thinsp;=\u0026thinsp;12) fed the CTR diet with no drug, ASA1-CTR (n\u0026thinsp;=\u0026thinsp;12) fed the CTR supplemented with 800 ppm of ASA; ASA2-CTR (n\u0026thinsp;=\u0026thinsp;12) fed the CTR supplemented with 1600 ppm of ASA; SU-CTR (n\u0026thinsp;=\u0026thinsp;10) fed the CTR supplemented with 80 ppm of SU. NT-PVD (n\u0026thinsp;=\u0026thinsp;12) fed the PVD diet with no drug; ASA1-PVD (n\u0026thinsp;=\u0026thinsp;12) fed the PVD supplemented with 800 ppm of ASA; ASA2-PVD (n\u0026thinsp;=\u0026thinsp;11) fed the PVD supplemented with 1600 ppm of ASA; SU-PVD (n\u0026thinsp;=\u0026thinsp;10) fed the PVD supplemented with 80 ppm of SU. Assuming for rats a mean body weight of 300 g and about 12 g of daily diet consumption, a diet containing 800 ppm ASA provides 9.6 mg of ASA per rat/day, \u003cem\u003ei.e.\u003c/em\u003e 32 mg/Kg body weight. Considering the different metabolic rate in humans and rats\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, this dosing corresponds to roughly 5 mg/kg in humans, that is about 350 mg ASA/day in a 70 kg man or, in the case of 1600 ppm to 700 mg/day.\u003c/p\u003e\u003cp\u003eAnimals remained in the treatment for three months until the sacrifice when faeces were collected for microbiota analysis (see below). The entire colon and small intestine were longitudinally opened to enumerate the tumours in each experimental group as previously described\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIntestinal carcinogenesis evaluation\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAt sacrifice, the entire intestine was washed with cold saline and longitudinally opened as described in Femia et al. 2015\u003csup\u003e12\u003c/sup\u003e. The number of tumors in the colon and small intestine was determined by macroscopic examination. The total number of tumors was recorded for the entire intestinal tract; however, tumor size assessment was restricted to the colon, in line with the study\u0026rsquo;s primary objective of evaluating colorectal cancer development. Mean tumor diameters and corresponding standard deviations for each experimental group was reported in the Results section.\u003c/p\u003e\u003cp\u003e\u003cem\u003eEvaluation of apoptosis in colonic normal mucosa and tumours\u003c/em\u003e\u003c/p\u003e\u003cp\u003eApoptosis was evaluated in histological longitudinal sections of the normal colon mucosa (n\u0026thinsp;=\u0026thinsp;9 in CTR, n\u0026thinsp;=\u0026thinsp;10 in CTR-ASA1 and PVD, n\u0026thinsp;=\u0026thinsp;7 in CTR-ASA2 and CTR-SU, n\u0026thinsp;=\u0026thinsp;11 in PVD-ASA1 and PVD-ASA2, n\u0026thinsp;=\u0026thinsp;8 in PVD-SU) and tumours (n\u0026thinsp;=\u0026thinsp;13 in CTR group, n\u0026thinsp;=\u0026thinsp;9 in CTR-ASA1, n\u0026thinsp;=\u0026thinsp;12 in CTR-ASA2, n\u0026thinsp;=\u0026thinsp;10 in PVD, PVD-ASA1 and PVD-ASA2, n\u0026thinsp;=\u0026thinsp;7 in CTR-SU and n\u0026thinsp;=\u0026thinsp;8 in PVD-SU) as previously described\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePGE2 determination\u003c/em\u003e\u003c/p\u003e\u003cp\u003eCirculating Prostaglandin E2 (PGE2) levels were determined in plasma obtained by centrifuging blood collected at the time of sacrifice in tubes containing sodium citrate as an anticoagulant. According to manufacturer instructions, PGE2 determination was assessed using the Prostaglandin E2 ELISA Kit - Monoclonal (Cayman Chemical Inc kit Ann Arbor, MI).\u003c/p\u003e\u003cp\u003e\u003cem\u003eDNA extraction and 16S rRNA (V3\u0026ndash;V4) gene amplification and sequencing\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTotal DNA extraction from faecal samples was carried out by using DNeasy PowerLyzer PowerSoil Kit (QIAGEN) following the manufacturer\u0026rsquo;s instructions, then quantified fluorometrically using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until 16S (V3-V4) rRNA gene library preparation. The 16S (V3-V4) rRNA gene was amplified by using the primer pairs 341F (5\u0026prime;-CCTACGGGNGGCWGCAG-3\u0026prime;) and 805R (5\u0026prime;-GACTACNVGGGTWTCTAATCC-3\u0026prime;) with overhang Illumina adapters\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The barcoded libraries were balanced, pooled at equimolar concentrations, then sequenced using the Illumina MiSeq platform in paired-end mode (300x2).\u003c/p\u003e\u003cp\u003e\u003cem\u003eAmplicon sequence variants inference\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePrimer pairs used for library preparation were removed by using cutadapt tool version 4.2 in paired-end mode\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The amplicon sequence variants (ASVs) inference from raw sequences was carried out using the DADA2 pipeline version 1.16\u003csup\u003e27\u003c/sup\u003e. Quality reads were improved using the \u0026ldquo;filterAndTrim\u0026rdquo; function, filtered reads based on an expected error threshold of 2 for both forward and reverse read pairs. Denoising step was performed using the \u0026ldquo;dada\u0026rdquo; function after error rate estimation using the \u0026ldquo;learnErrors\u0026rdquo; function. Denoised reads were merged (forward and reverse sequences with any mismatches and/or an overlap length shorter than 12 bp were removed). Chimeric sequences were removed using the \u0026ldquo;removeBimeraDenovo'' function. The taxonomic classification was carried out by using DECIPHER package version 2.30\u003csup\u003e28\u003c/sup\u003e against the latest version of the pre-formatted Silva small-subunit reference database (SSU version 138 available at: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www2.decipher.codes/Downloads.html\u003c/span\u003e\u003cspan address=\"http://www2.decipher.codes/Downloads.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The dada2 pipeline showed adequate preservation of the number of reads after the filtering steps (Figure S4a). To further improve dataset quality, all variants not classified as Bacteria were removed with sequences classified as Archaea, chloroplasts, or mitochondria. After quality filtering, no differences in sequencing depth between dietary treatment groups were observed, excluding bias from sequencing depth (Figure S4b). After all quality steps, a total of 3\u0026rsquo;122\u0026rsquo;843 reads (median: 44\u0026rsquo;531.5) collapsed into a total of 1\u0026rsquo;993 different bacterial ASVs were obtained. After quality filtering, two samples (E153-38 and E153-39) reported 0 counts; thus, they were removed to avoid statistical bias and properly perform the downstream statistical analyses.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were performed using the R software version 4.3.1\u003csup\u003e29\u003c/sup\u003e. Analysis of variance (Type III Anova) was fitted using a linear model by \u0026ldquo;lm\u0026rdquo; function on the model formula, which included both variables, \u003cem\u003ediet\u003c/em\u003e and \u003cem\u003etreatment\u003c/em\u003e, and their interaction. Dunnett's test for comparing all group combinations of \u003cem\u003ediet\u003c/em\u003e and \u003cem\u003etreatment\u003c/em\u003e groups against the NT-CTR group (designed as the control level) was performed using the \u0026ldquo;DunnettTest\u0026rdquo; function of \u0026ldquo;DescTools\u0026rdquo; package version 0.99.60\u003csup\u003e30\u003c/sup\u003e. Tukey HSD test between each group was performed by using \u0026ldquo;tukey_hsd\u0026rdquo; of \u0026ldquo;rstatix\u0026rdquo; package version 0.7.2\u003csup\u003e31\u003c/sup\u003e. Beta diversity analysis was performed by using \u0026ldquo;vegan\u0026rdquo; package version 2.6.10\u003csup\u003e32\u003c/sup\u003e. Differences in beta diversity were estimated using Bray-Curtis distance matrix after ASVs relative abundance transformation and singleton removal. Distances were reported by using principal coordinate analysis (PCoA) using the \u0026ldquo;cmdscale\u0026rdquo; function of \u0026ldquo;stats\u0026rdquo; package version 4.3.1\u003csup\u003e29\u003c/sup\u003e. The effect of \u003cem\u003ediet\u003c/em\u003e and \u003cem\u003etreatment\u003c/em\u003e on bacterial diversity was tested using permutational multivariate analysis of variance (also referred to as adonis PERMANOVA) by using the \"adonis2\" function of the \u0026ldquo;vegan\u0026rdquo; package version 2.6.8. The mean within and between group dissimilarities was assessed using the \u0026ldquo;meandist\u0026rdquo; function of the \u0026ldquo;vegan\u0026rdquo; package version 2.6.8. Environmental fitting was conducted on unconstrained ordination between ASVs abundance and tumour rate by principal component Analysis (PCA) based on Hellinger distance, and it was assessed after ASVs relative abundance transformation, singleton removal, and log\u0026thinsp;+\u0026thinsp;1 scaling to reduce possible bias related to the different ASVs coverage. Environmental fitting analysis was carried out using the \u0026ldquo;envfit\u0026rdquo; function of the \u0026ldquo;vegan\u0026rdquo; package version 2.6.8 and the resulting p-value was adjusted using Bonferroni correction method. Significantly different ASVs between CTR and PVD groups within each treatment group were assessed using Wald's test (Benjamin\u0026ndash;Hochberg correction for the P-values) implemented in the \"DESeq\" function of \u0026ldquo;DESeq2\u0026rdquo; package version 1.42.1\u003csup\u003e33\u003c/sup\u003e. Differences in genus-level relative abundances between different Aspirin ppm dosage (0, 800, and 1600) within each diet-related group were first assessed using Kruskal-Wallis rank sum test, then significant genus-level features (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were compared between each treatment group by using Wilcoxon test. Differences in genus-level relative abundances between each treatment group (NT, SU, ASA1, and ASA2) were assessed using the Wilcoxon test. The genus-level relative abundance transformation was carried out after singleton removal and after pruning ASVs present in less than 5% of samples. Figures were generated using \u0026ldquo;ggplot2\u0026rdquo; package version 3.5.2\u003csup\u003e34\u003c/sup\u003e and edited using the open-source graphics editor Inkscape (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://inkscape.org/\u003c/span\u003e\u003cspan address=\"http://inkscape.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to improve graphic rendering.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eOur study aims to investigate whether ASA and SU associated with PVD may enhance the protective activity of this diet against CRC. To this end, we treated \u003cem\u003eApc\u003c/em\u003e-mutated PIRC animals with two different doses of ASA and a single dose of SU over a three-month period. Chemopreventive activity was assessed by analyzing the number of intestinal tumors. The obtained data were integrated with 16S rRNA sequencing results to determine whether the chemopreventive effects could be attributed to modulation of the intestinal microbiota.\u003c/p\u003e\u003cp\u003ePIRC rats aged one month were allocated to the different dietary groups (mean body weight at the beginning of the dietary treatment: mean 83.1 g (\u0026plusmn;\u0026thinsp;15.6 g; n\u0026thinsp;=\u0026thinsp;91 rats). Dietary treatments were modulated in an isocaloric manner as described in De Filippo et al.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, therefore, at the end of the experimental period, we observed no differences in mean weight between the two dietary groups: (CTR: mean 320 g (sd 15), PVD: mean 324 g (sd 20)) (Anova, p\u0026thinsp;=\u0026thinsp;0.26). Concerning the pharmacological treatment, in the CTR group diet, we did not observe differences among the different experimental groups; in the PVD group diet, while ASA treatment did not affect weight gain, SU was associated with a body weight slightly lower than the other groups (Table S2).\u003c/p\u003e\u003cp\u003eThe mean tumor diameter in the colon was comparable across the experimental groups and the following values were recorded (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation): CTR-NT: 2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 mm; CTR-SU: 2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 mm; CTR-ASA1: 2.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33 mm; CTR-ASA2: 2.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05 mm; PVD-NT: 2.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34 mm; PVD-SU: 2.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 mm; PVD-ASA1: 2.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 mm; PVD-ASA2: 2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24 mm.\u003c/p\u003e\u003cp\u003eThe PVD group determined a significant decrease in the total tumours and tumours in the colon tract regardless of the pharmacological treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The pharmacological treatment effect was, however, evidenced only for the CTR group, showing a significant reduction in the number of total tumours in animals treated with ASA2 compared to both SU and NT groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The same treatment effect, i.e. reduction in the number of tumours in ASA2 compared to SU and NT in the CTR group, was also observed in the small intestine, although an effect of diet was not evident (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eThe effect of diet and pharmacological treatment, together with their interaction, in affecting the number of tumours per rat for each intestinal tract was assessed using type III Anova (Table S3). Both variables (diet and pharmacological treatment) influenced the onset of tumours observed in the colon tract and in all the intestinal tract (i.e. total tumours), whereas the number of tumours accounted for in the small intestine was influenced by the treatment only (Table S3). No significant interaction effect was highlighted, demonstrating that the two variables were effective in influencing the onset of tumours independently of each other (Table S3 and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo better understand the significance of the values obtained, and due to the nested design of the experiment, we can also consider the group fed the control diet and without pharmacological treatment, i.e. the NT-CTR group, as the true control to which to compare each experimental group. Therefore, we compared the mean values of the number of tumours of each group (intended as every possible combination of levels within the diet and treatment variables) against the NT-CTR group, designated as control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and Table S4). Regarding the colon, we thus found that the groups NT-PVD, SU-PVD, ASA2-PVD, and ASA1-PVD had a statistically significant lower number of tumours than the NT-CTR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). In the small intestine only the ASA2-CTR group showed significantly lower values than the NT-CTR group, while considering the entire intestinal tract (total: colon and small intestine), the two groups treated with ASA2 (ASA2-PVD and ASA2-CTR) had a significantly lower number of tumours than the NT-CTR group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs expected, the concentration of the circulating prostaglandin E2 was affected by the ASA supplementation in both concentrations (800 and 1600 ppm) compared to the non-treated group (Figure S2). The higher dose of ASA in both CTR and PVD diets resulted in a significant reduction in PGE2 levels compared to NT (Figure S2).\u003c/p\u003e\u003cp\u003eThe effect of diet and pharmacological intervention on the rate of apoptotic cells (i.e., apoptotic cells per crypt) in normal mucosa was assessed, and no significant effect was observed (Figure S3).\u003c/p\u003e\u003cp\u003eWe also evaluated the effect of diet and pharmacological treatment on the intestinal microbiota. The effect of diet and treatment on bacterial diversity (beta diversity) was assessed by multivariate analysis adonis PERMANOVA and depicted by multidimensional analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In detail, the multidimensional analysis (PCoA based on Bray-Curtis distance in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) showed that the diet was the most effective variable in the sample separation and this evidence was also corroborated by the adonis PERMANOVA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). To better highlight the effect of the pharmacological treatment, the same analyses were conducted separately by dividing the dataset according to the diet. The pharmacological treatment showed a similar effect in modulating bacterial diversity in both CTR and PVD datasets (R-squared in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), however, specific differences between treatment levels within each diet-related group were highlighted as shown by the pairwise adonis PERMANOVA analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The pairwise adonis PERMANOVA showed significant differences in bacterial diversity between ASA2 and SU in both diet-related groups and between ASA2 and NT in CTR dataset only (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Treatment was also effective in modulating bacterial alpha diversity in CTR dataset only (Shannon index in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCorrelation between changes in bacterial diversity and tumour rate was assessed by environmental fitting analysis. The analysis showed a significant correlation between the number of colon tumours per rat and bacterial diversity; in particular, the microbial diversity in PVD groups showed an anticorrelated trend with the increase in the number of colon tumours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Table S5). To better describe correlations between bacterial diversity and tumour rate, we also performed the environmental fitting analysis splitting the dataset according to each pharmacological treatment (Figure S5 and Table S6). The analysis showed that the correlation effect between diet and tumour rates was more evident in the SU and ASA2 groups, corroborating an anticorrelation trend between the PVD diet and the increase in the number of colon tumours (Figure S5).\u003c/p\u003e\u003cp\u003eWe assess the taxonomic variants significantly associated with the two diet groups within each treatment dataset using Wald's test (Wald's test of DESeq), identifying 111, 79, 129 and 129 ASVs differentially influenced by diet in the NT, SU, ASA1, and ASA2 treatment datasets respectively (All significant ASVs are reported in Table S7). We observed that diet selected specific ASVs in all four pharmacological treatments, highlighting a strong effect of diet in selecting a pattern of specific bacterial variants. In particular, diet was effective in selecting 21 different ASVs significantly associated with the same diet in all pharmacological treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Most variants associated with the PVD diet (62.5%) were assigned to the Lachnospiraceae and Prevotellaceae families (ASV 75: Lachnospiraceae, ASV 353: Lachnospiraceae, ASV 348: Lachnospiraceae, ASV 3: Prevotellaceae, ASV 29: Lachnospiraceae, ASV 240: Lachnospiraceae, ASV 223: Lachnospiraceae, ASV 197: \u003cem\u003eLachnospiraceae UCG-001\u003c/em\u003e, ASV 149: Lachnospiraceae, ASV 14: \u003cem\u003ePrevotellaceae NK3B31 group\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eVariation in the abundances of the main bacterial genera among different pharmacological treatments in the two different diet-related datasets was also evaluated (Relative abundances and significant comparisons are reported in Figure S6). Considering the significant effect of ASA on colon and intestinal tumours, we were interested in understanding the role of ASA treatment on bacterial abundances. Therefore, we evaluated the variations in relative abundances according to different ASA dosages, i.e. 0 (NT), 800 and 1600 ppm. ASA determined several variations of specific genera differently in the two dietary groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and Table S8). We identified 12 different bacterial genera with significant change in relative abundance among different ASA dosages within different diet-related datasets. The different ASA dosages produced significant variations in these bacterial genera based on the diet \u003cem\u003eAkkermansia\u003c/em\u003e, \u003cem\u003eAnaerovorax\u003c/em\u003e, \u003cem\u003eBarnesiella\u003c/em\u003e, \u003cem\u003eButyricimonas\u003c/em\u003e, and \u003cem\u003eOscillibacter\u003c/em\u003e were significantly affected by the treatment in the CTR diet only, whereas \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eCaproiciproducens\u003c/em\u003e, \u003cem\u003eCoprococcus\u003c/em\u003e, \u003cem\u003eEnterorhabdus\u003c/em\u003e, \u003cem\u003eRoseburia\u003c/em\u003e, and \u003cem\u003eEubacterium xylanophilum group\u003c/em\u003e were significantly affected by the treatment in PVD diet only (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The bacterial genera \u003cem\u003eColidextribacter\u003c/em\u003e was affected by the ASA dosage in both diet-related datasets (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated whether the chemopreventive efficacy of two NSAIDs, ASA and SU, could be enhanced when administered in combination with a pesco-vegetarian diet PVD. Using \u003cem\u003eApc\u003c/em\u003e-mutated PIRC rats, a well-established model for both CRC and FAP, we administered two different doses of ASA and one dose of SU over a three-month period and assessed both intestinal tumour burden and changes in gut microbiota composition. Our findings demonstrate that both dietary and pharmacological interventions significantly modulate tumour development in the intestinal tract. Notably, the PVD alone exerted a strong protective effect, consistent with our previous findings in other experimental models of carcinogenesis\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Animals fed the PVD exhibited a markedly lower number of tumours, especially in the colon, compared to CTR-fed animals. This reduction was statistically significant regardless of pharmacological treatment, further supporting epidemiological and experimental evidence linking high consumption of fish and vegetables to a reduced risk of CRC (WCRF, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dietandcancerreport.org\u003c/span\u003e\u003cspan address=\"http://dietandcancerreport.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRegarding the drug treatments (two doses of ASA and one of SU), a significant effect was observed in the CTR group, where the number of total intestinal tumours in rats treated with the highest dose of ASA (CTR-ASA2) was significantly lower than in untreated controls (NT-CTR). In the PVD experimental groups, which received the same dosages of ASA, the protective effect of the higher dose was appreciable, especially in the colon, but statistical significance was not reached, likely due to the strong protective effect of the diet itself. Considering the group fed with a control diet and with no treatment (NT-CTR group), as a reference for the various comparisons, we found that the two groups treated with the higher dose of ASA (ASA2-PVD and ASA2-CTR) exhibited a significantly lower number of total tumours. These findings suggest that this ASA dosage is effective regardless of the diet, as further supported by the marked reduction in circulating PGE2 levels, particularly in the PVD group at both dosages (Figure S2). With respect to the lower ASA dose (800 ppm), a mild protective effect was observed in the CTR group, though it did not reach statistical significance. However, the analysis showed that ASA treatment influenced tumour development at least in the small intestine (Table S3).\u003c/p\u003e\u003cp\u003eTaken together, these findings demonstrate that both the dietary treatment and pharmacological interventions independently modulates tumours development, with a more pronounced effect when ASA is administered alongside the protective PVD diet. Although several studies have shown that ASA intake reduces the risk of sporadic CRC, its effect of ASA in FAP remains less clear. For instance, Ishikawa et al.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e reported a reduction in the number and size of colorectal polyps in FAP patients treated with low-dose ASA (100 mg/day). Conversely, other studies using higher doses (e.g. 600 mg/day in Burn et al.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e showed only a trend toward reduced polyp load (number and size), without a significant reduction in polyp count. Similarly, data from genetic models of intestinal carcinogenesis, such as \u003cem\u003eApc\u003c/em\u003e-mutated Min mice, remain inconclusive. When a protective effect of ASA is observed in these models, it is typically limited only to the small intestine, with no significant impact on the colon\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eASA has not previously been tested in PIRC animals. The doses used in this study were based on a previous investigation that evaluated ASA in azoxymethane-treated rats, a model of sporadic CRC\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Considering the different metabolic rates between humans and rats, the equivalent doses in a 70-kg human would correspond to approximately 350 mg/day and 700 mg/day for ASA1 and ASA2, respectively\u0026mdash;roughly equivalent to low and medium doses that have also been administered in FAP patients\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe hypothesized that the lowest dose of ASA (ASA1) administered with the CTR diet would lead to a modest reduction in colon tumorigenesis, while its combination with the PVD diet would result in a more pronounced protective effect. Indeed, we found a slight protective effect of ASA1 in the CTR group, and a stronger effect in the PVD group. However, as noted above, the strong protective effect of the PVD made it difficult to detect a statistically significant contribution from ASA1 beyond the effect of the diet alone. The highest dose of ASA (ASA2) significantly reduced total intestinal tumour burden, suggesting that in both diets this dosage of ASA is indeed effective.\u003c/p\u003e\u003cp\u003eOur results also showed that SU, administered at a single dose, did not significantly reduce tumour development. In previous studies from our group\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e a strong protective effect was observed in PIRC rats treated with higher doses of SU (320 ppm), whereas animals treated with 80 ppm\u0026mdash;the dose used in the present study\u0026mdash;showed only a modest reduction in the number of total intestinal tumours. Unlike that previous study, in which treatment lasted eight months, the current study involved a shorter treatment period of three months, explaining the limited effect observed. Notably, the lack of efficacy for 80 ppm SU has also been reported in carcinogen-induced CRC models\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, suggesting that higher doses are needed to afford protective effects against carcinogenesis.\u003c/p\u003e\u003cp\u003eGiven the protective effects observed with both dietary interventions and pharmacological treatment on the development of intestinal carcinogenesis, and according to previous findings, we sought to explore whether the gut microbiota could play a mediating or contributing role in modulating intestinal carcinogenesis.\u003c/p\u003e\u003cp\u003eOur results indicate that the PVD diet plays a central role in reducing colon tumour burden, while drug treatment, particularly with the higher ASA dose (ASA2), further modulates tumour incidence. Moreover, gut microbiota analyses provide compelling evidence of diet- and drug-induced microbial shifts that may contribute to the observed chemopreventive effects.\u003c/p\u003e\u003cp\u003eThe significant impact of diet on bacterial diversity, as revealed by beta-diversity analyses and multivariate analyses, suggests that PVD creates a distinct microbial environment associated with reduced tumorigenesis. These findings align with growing evidence that microbiome composition influences CRC progression, with beneficial bacterial taxa potentially contributing to the observed protective effects\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In particular, the PVD promoted the enrichment of bacterial families known for their beneficial metabolic functions, such as Lachnospiraceae and Prevotellaceae, consistent with our previous observations\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, regardless of the pharmacological treatment. These bacterial families have been implicated in maintaining intestinal homeostasis through the production of protective metabolites and the regulation of bile acid metabolism, thereby contributing to CRC prevention. The presence of these beneficial bacterial communities across all pharmacological treatment groups within the same dietary group confirms that diet exerts a primary role in shaping the gut microbiota composition, independently of drug administration.\u003c/p\u003e\u003cp\u003eTherefore, while both pharmacological and dietary treatments influence gut microbial communities, they do so independently, without evidence of synergistic interaction, consistent with the lack of synergistic effects on tumour reduction. The effect of ASA treatment on microbiota composition was also evident, although less pronounced than that of diet. ASA selected for distinct bacterial genera in a dose- and diet-dependent manner. Specifically, \u003cem\u003eAkkermansia\u003c/em\u003e, \u003cem\u003eAnaerovorax\u003c/em\u003e, \u003cem\u003eBarnesiella\u003c/em\u003e, \u003cem\u003eButyricimonas\u003c/em\u003e, and \u003cem\u003eOscillibacter\u003c/em\u003e were affected in the CTR dietary group, while \u003cem\u003eBlautia\u003c/em\u003e, \u003cem\u003eCaproiciproducens\u003c/em\u003e, \u003cem\u003eCoprococcus\u003c/em\u003e, \u003cem\u003eEnterorhabdus\u003c/em\u003e, \u003cem\u003eRoseburia\u003c/em\u003e, and \u003cem\u003eEubacterium xylanophilum\u003c/em\u003e were modulated in the PVD group. In particular, \u003cem\u003eBlautia\u003c/em\u003e has been associated with anti-inflammatory properties and improved intestinal barrier function\u003csup\u003e38\u0026ndash;40\u003c/sup\u003e, supporting the hypothesis that pharmacological treatment may enhance ASA\u0026rsquo;s chemopreventive activity by promoting the growth of beneficial bacteria when combined with a protective diet.\u003c/p\u003e\u003cp\u003eThe abundance of \u003cem\u003eRoseburia\u003c/em\u003e was also increased in the PVD group, reinforcing its potential protective role against CRC, as Kang et al. highlight that \u003cem\u003eRoseburia intestinalis\u003c/em\u003e, a butyrate-producing gut probiotic often depleted in CRC patients, can suppress tumorigenesis and restore gut barrier function in CRC mouse models\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e Furthermore, the identification of \u003cem\u003eColidextribacter\u003c/em\u003e as a genus responsive to ASA across both dietary groups suggests a broader, diet-independent role for Aspirin in modulating the gut microbiota, with potential implications for developing targeted microbiota-based CRC prevention strategies.\u003c/p\u003e\u003cp\u003eOverall, our results underscore the independent and significant contributions of both dietary and pharmacological interventions in preventing CRC. The observed gut microbiota changes point to a mechanistic link between diet composition, microbial diversity, and tumour suppression. Future studies should further explore the functional relevance of these microbial shifts, particularly in relation to metabolite production and immune modulation. A deeper understanding of these interactions may pave the way for integrated dietary and pharmacological approaches to optimize CRC prevention and therapy.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results demonstrate that both dietary and pharmacological interventions independently and significantly contribute to the prevention of colorectal cancer. The pesco-vegetarian diet (PVD) exerted a strong protective effect, associated with distinct shifts in gut microbiota composition, including the enrichment of beneficial bacterial features belonging to Lachnospiraceae and Prevotellaceae families. Among the taxa most notably associated with the PVD, the increased abundance of \u003cem\u003eRoseburia\u003c/em\u003e supports its emerging role as a key microbial mediator of diet-induced protection against CRC.\u003c/p\u003e\u003cp\u003eAspirin treatment, especially at higher doses, also contributed to tumour reduction and modulated the gut microbiota in a dose- and diet-dependent manner. Interestingly, \u003cem\u003eColidextribacter\u003c/em\u003e was identified as a genus consistently responsive to ASA across both dietary contexts, suggesting a diet-independent microbial target of aspirin with potential relevance for microbiota-driven prevention strategies.\u003c/p\u003e\u003cp\u003eAltogether, these findings point to a mechanistic link between dietary composition, microbial diversity, and suppression of intestinal tumorigenesis. The modulation of specific microbial taxa, such as \u003cem\u003eRoseburia\u003c/em\u003e and \u003cem\u003eColidextribacter\u003c/em\u003e, may underlie part of the chemopreventive effects observed and offer new targets for intervention. Future studies should focus on characterizing the metabolic and immunological functions of these microbes and their interactions with dietary and pharmacological treatments. A deeper understanding of these dynamics could pave the way for integrated microbiota-informed strategies to optimize colorectal cancer prevention and therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequences have been deposited to the European Nucleotide Archive (ENA) under the accession project code PRJEB88871. All results from statistical analyses were reported as figures and tables in the main text and supplementary file. Further information can be provided upon reasonable request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was funded by the following grants:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(i) National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.3 - Call for tender No. 341 of 15 March 2022 of Italian Ministry of University and Research funded by the European Union - NextGenerationEU; Project code PE00000003, Concession Decree No. 1550 of 11 October 2022 adopted by the Italian Ministry of University and Research, Project title “ON Foods - Research and innovation network on food and nutrition Sustainability, Safety and Security - Working ON Foods”.\u003c/p\u003e\n\u003cp\u003e(ii) European Union, NextGenerationEU, National Recovery and Resilience Plan, Mission 4 Component 2, Investment 1.5, THE (Tuscany Health Ecosystem), ECS00000017, CUP B83C22003920001.\u003c/p\u003e\n\u003cp\u003e(iii) The Joint Programming Initiative a Healthy Diet for a Healthy Life-Intestinal Microbiomics (JPI HDHL-INTIMIC) Call for Joint Transnational Research Proposals on “Interrelation of the Intestinal Microbiome, Diet and Health” (reference number JTC-2017–7).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(iv) HDHL INTIMIC-Knowledge Platform on food, diet, intestinal microbiomics, and human health (expression of interest no. 895).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(v) University of Florence (Fondo ex 60%), Italy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSofia Chioccioli and Niccolò Meriggi have contributed equally as co-first authors.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors and Affiliations\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Neurosciences, Psychology, Drug Research and Child Health (NEUROFARBA), University of Florence, Florence, Italy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSofia Chioccioli \u0026amp; Giovanna Caderni\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitute of Agricultural Biology and Biotechnology (IBBA), National Research Council (CNR), Pisa, Italy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNiccolò Meriggi,Carlotta De Filippo \u0026amp;\u0026nbsp;Mariela Mejia Monroy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Molecular and Developmental Medicine (DMMS), University of Siena, Siena, Italy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMariela Mejia Monroy\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Biology, University of Florence, Florence, Italy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSonia Renzi \u0026amp; Benedetta Cerasuolo\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eContributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eC.D.F. and G.C.: designed this study. S.C. and M.M.M.: sample management, DNA extraction and biometric data production. N.M.: data management and computational analyses. S.R. and B.C.: library preparation and sequencing. C.D.F., G.C., N.M., S.F. and M.M.M. wrote the manuscript. All authors reviewed the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCorresponding authors\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Carlotta De Filippo ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge CeSAL (Centro Stabulazione Animali da Laboratorio) at University of Florence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthical approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocols were approved by the Italian Guidelines for Animal Care, DL 26/2014 under the authorization 496/2021-PR.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKeum, N. \u0026amp; Giovannucci, E. 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Roseburia intestinalis-generated butyrate boosts anti-PD-1 efficacy in colorectal cancer by activating cytotoxic CD8⁺ T cells. \u003cem\u003eGut\u003c/em\u003e\u003cstrong\u003e72\u003c/strong\u003e, 2112\u0026ndash;2122 (2023\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7216019/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7216019/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eColorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide, with genetic predispositions such as FAP contributing significantly to early-onset disease. This study investigated the chemopreventive potential of two non-steroidal anti-inflammatory drugs (NSAIDs), acetylsalicylic acid (ASA) and sulindac (SU), in combination with a pesco-vegetarian diet (PVD), using \u003cem\u003eApc\u003c/em\u003e-mutated PIRC rats, a well-established model of CRC. Animals were treated over three months with two doses of ASA or a single dose of SU, and tumor burden and gut microbiota composition were assessed. Results confirmed the robust protective effect of the PVD diet in reducing the intestinal tumorigenesis, particularly in the colon, independent of pharmacological treatment. ASA treatment, especially at the higher dose, significantly reduced tumour incidence in both dietary groups, with additive effects seen in combination with PVD, while SU did not show a significant protective effect. Microbiota analysis revealed distinct shifts in bacterial composition associated with both dietary and pharmacological interventions. Notably, taxa such as \u003cem\u003eRoseburia\u003c/em\u003e and \u003cem\u003eColidextribacter\u003c/em\u003e, previously linked to intestinal homeostasis and anti-inflammatory activity, were modulated by ASA and diet, suggesting a microbiome-mediated mechanism of chemoprevention. These findings underscore the independent and complementary roles of diet and pharmacological interventions in CRC prevention, and highlight the gut microbiota as a promising target for future personalised preventive strategies.\u003c/p\u003e","manuscriptTitle":"Combination of a pesco-vegetarian diet with non-steroidal anti-inflammatory drugs for colorectal cancer prevention: tumor suppression and gut microbiota modulation in Apc- mutated PIRC rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-19 12:41:14","doi":"10.21203/rs.3.rs-7216019/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-03T05:17:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T02:40:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171755351473557957395429372691310907562","date":"2025-09-29T17:55:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21670430249439568227243392098965431392","date":"2025-09-28T18:49:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-25T15:17:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82847206416397249102039261499868203972","date":"2025-09-23T01:40:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-19T19:02:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161524332136275874443438298632226327402","date":"2025-09-15T14:22:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164169250978090185273091228594549271264","date":"2025-09-15T10:33:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-13T15:53:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-11T05:09:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-05T16:59:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-05T14:28:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e7197ceb-c6f5-42e0-a1a8-1be934c2a594","owner":[],"postedDate":"September 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":54966152,"name":"Biological sciences/Cancer"},{"id":54966153,"name":"Biological sciences/Drug discovery"},{"id":54966154,"name":"Health sciences/Gastroenterology"},{"id":54966155,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-20T16:01:31+00:00","versionOfRecord":{"articleIdentity":"rs-7216019","link":"https://doi.org/10.1038/s41598-026-48074-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-16 15:57:07","publishedOnDateReadable":"April 16th, 2026"},"versionCreatedAt":"2025-09-19 12:41:14","video":"","vorDoi":"10.1038/s41598-026-48074-5","vorDoiUrl":"https://doi.org/10.1038/s41598-026-48074-5","workflowStages":[]},"version":"v1","identity":"rs-7216019","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7216019","identity":"rs-7216019","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}