Impact of 5 fluorouracil chemotherapy on gut inflammation, functional parameters, and gut microbiota

Abstract

Emerging evidence suggests that gut microbiota may influence the response to chemotherapy. We sought to characterize the effects of 5 fluorouracil (5FU) chemotherapy on colon inflammation and functional measures in colorectal cancer (CRC) and to further determine whether gut microbiota can influence this response. 50 C57BL/6 were randomized into four groups; Control + Vehicle (n = 10), Control + 5FU (n = 10), AOM/DSS + Vehicle (n = 15), and AOM/DSS + 5FU (n = 15). CRC was induced chemically by a single 10 mg/kg injection of azoxymethane (AOM) followed by two cycles (2% and 1%) of dextran sodium sulfate (DSS). Mice were then treated with 3 cycles of vehicle or 5FU (cycle 1: 40 mg/kg, cycle 2 + 3: 20 mg/kg). Functional tests (grip strength and run-to-fatigue) were performed prior to 5FU treatment (baseline) and at the completion of the second cycle of 5FU. Following the third 5FU cycle, mice were euthanized and the colon was evaluated for expression of inflammatory genes using RT-qPCR and stool samples were profiled using 16S rRNA sequencing. A second experiment used fecal microbiota transplantation from 5FU treated mice to control mice (n = 10-15/group) to determine whether 5FU associated changes in the microbiota could influence functional measures and colon inflammation. 5FU reduced grip strength (p < 0.05) and caused a trending decrease in run-to-fatigue performance in cancer mice (p = 0.06). Select intestinal inflammatory genes were significantly elevated with 5FU treatment and this was further exacerbated with cancer (p < 0.05). Microbiota analysis revealed increased dissimilarity and alterations in bacterial taxonomy in 5FU and AOM/DSS-treated mice (p < 0.05). Fecal transplant from 5FU treated mice reduced functional performance (p < 0.05) and altered select colon inflammatory markers (p < 0.05). This study provides evidence of an effect of 5FU on inflammatory responses and functional measures in a mouse model of CRC and suggests that gut microbes may play a role in some, but not all, 5FU related perturbations.

Keywords: 5-Fluorouracil; Chemotherapy; Colorectal cancer; Fecal transplant; Inflammation; Microbiota; Toxicity.

Figure 1.. Study Design for experiment 1 and experiment 2.

A. Experiment 1 design and 5FU treatment regimen. B. Experiment 2 design, antibiotics treatment regimen and fecal transplantation.

Figure 2.. 5FU reduces tumor burden but decreases survival in AOM/DSS mice.

A. Gross body weight. B. Percent body weight change. C. Survival curve. D. Symptom score. * indicates statistical significance (p<0.05) for AOM/DSS + 5FU group vs. all groups, # indicates statistical signficiance (p<0.05) for Control + 5FU vs. Control + Vehicle from 3-way ANOVA. E-H. Liver, spleen, epididymal fat, and mesenteric fat, weights at euthanization. Groups not containing the same letters (a,b,ab) indicate statistical significance between groups (p<0.05) from two-way ANOVA, n=9–13/group. I. Total Polyp count. J. Polyp size. K. Dysplasia characterization. L. Representative H&E staining of distal colon specimens at 4X, insets are 20X. * indicates statistical significance (p<0.05) from students t-test, n = 9–13/group.

Figure 3.. Nonspecific toxicity of 5FU in distal ileum and liver tissue

A. Representative H&E staining of distal ileum specimens at 4X, insets are 20X. B. Measurement of villus length in small intestine. Groups not containing the same letters (a,b) indicate statistical significance between groups (p<0.05) from two-way ANOVA. C. Representative H&E staining of liver tissue specimens at 4X, insets are 20X. D. Histopathological analysis of liver inflammation. n = 9– 13/group.

Figure 4.. 5FU alters the immune profile of the colon.

A. qPCR analysis of TNFa, MCP-1, NOS2, IL6, IFNg, IL1b, IL10, IL4, FOXP3 genes. Data were normalized to vehicle treated controls and compared to two reference targets (TBP and H2AFV), which were evaluated for expression stability using GeNorm. Groups not containing the same letters (a,b,c,d) indicates statistical significance between groups (p<0.05) from two-way ANOVA, n = 9–13/group.

Figure 5.. 5FU affects functional measures of fatigue.

A. Relative grip strength measured in newtons/kilogram (N/Kg). B. Absolute grip strength measured in newtons (N). C. Percent performance change in run-to-fatigue test from pre-5FU treatment to post-5FU treatment. Groups not containing the same letters (a,b) indicates statistical significance between groups (p<0.05) from two-way ANOVA, n = 9–13/group.

Figure 6.. 5FU alters gut microbial profile.

A. Shannon plot of alpha-diversity in microbial communities. B. Total sequences per sample by Shannon rarefaction. C. Unweighted UniFrac Principle Coordinate Analysis (PCoA) of beta-diversity in microbial communities. D. Percent abundance of microbial phyla. E. Percent abundance of microbial class, n=5/group.

Figure 7.. Fecal Transplantation of 5FU altered microbiome does not alter body weight but does affect functional measures of fatigue.

A. Gross body weight. B. Percent body weight change. C-F. Liver, spleen, epididymal fat, and mesenteric fat, weights at euthanization. G. Relative grip strength measured in newtons/kilogram (N/Kg). H. Absolute grip strength measured in newtons (N). I. Characterization of performance changes in run-to-fatigue test. J. Percent performance change in run-to-fatigue test from pre-5FU treatment to post-5FU treatment. * indicates statistical significance between groups (p<0.05) from students t-test n=10– 15/group.

Figure 8.. 5FU alters macrophage population in the colon lamina propria and is replicated with fecal transplantation.

A. qPCR analysis of TNFa, MCP-1, NOS2, IL1b, IL10, IL4, Ly6G, and EMR1 genes in fecal transplantation mice. B,C. Representative H&E staining and CD68+ staining, respectively, in distal colons of fecal transplantation mice. Arrows indicate examples of positive staining. * indicates statistical significance between groups (p<0.05) from students t-test, n=10–15/group.