The cancer chemotherapeutic 5-fluorouracil is a potent Fusobacterium nucleatum inhibitor and its activity is modified by intratumoral microbiota

Abstract

Fusobacterium nucleatum (Fn) is a dominant bacterial species in colorectal cancer (CRC) tissue that is associated with cancer progression and poorer patient prognosis. Following a small-molecule inhibitor screen of 1,846 bioactive compounds against a Fn CRC isolate, we find that 15% of inhibitors are antineoplastic agents including fluoropyrimidines. Validation of these findings reveals that 5-fluorouracil (5-FU), a first-line CRC chemotherapeutic, is a potent inhibitor of Fn CRC isolates. We also identify members of the intratumoral microbiota, including Escherichia coli, that are resistant to 5-FU. Further, CRC E. coli isolates can modify 5-FU and relieve 5-FU toxicity toward otherwise-sensitive Fn and human CRC epithelial cells. Lastly, we demonstrate that ex vivo patient CRC tumor microbiota undergo community disruption after 5-FU exposure and have the potential to deplete 5-FU levels, reducing local drug efficacy. Together, these observations argue for further investigation into the role of the CRC intratumoral microbiota in patient response to chemotherapy.

Keywords: 5-fluorouracil; CP: Cancer; CP: Microbiology; Fusobacterium nucleatum; cancer microbiome; chemotherapeutics; colorectal cancer; intratumoral microbiota.

Figure 1.. A bioactive library screen identifies several antineoplastic agents as Fn growth inhibitors

(A) Schematic describing the small-molecule screen workflow to determine Fn drug sensitivity. (B) Activity scores for n = 1,846 compounds on Fn viability, determined through ATP measurements. Inhibitors are compounds that are three standard deviations away from the neutral control (media alone). (C) A list of the antineoplastic agents found to be Fn inhibitors arranged by their respective activity scores. (D) Eight-point dose-response curves of Fn viability after 48 h exposure to 0.25–32 μM of the active metabolites for various chemotherapeutics used to treat metastatic colon cancer. The connecting line is a nonlinear regression of the log(inhibitor) versus response with a variable slope (four parameters). Error bars represent standard deviation (SD), n = 3 replicates. See also Figure S1 and Tables S1 and S2.

Figure 2.. Fn isolates are sensitive to physiological concentrations of 5-FU

(A) Heatmap depicting the concentrations of 5-FU where Fn isolates (n = 14) are 50% viable. The clinical isolates are grouped by the sampling source. The level of 5-FU in patient sera is labeled between 2.5 and 10 μM. (B) Eight-point dose-response curves of Fn viability for a single representative of each subspecies: animalis, polymorphum, vincentii, and nucleatum after exposure to 0.0375–4.8 μM 5-FU for 48 h. The connecting line is a nonlinear regression of the log(inhibitor) versus response with a variable slope (four parameters). Error bars represent SD, n = 3 biological replicates. See also Figure S2.

Figure 3.. E. coli isolates deplete 5-FU and reduces drug toxicity toward Fn and human CRC cells

(A) Thirteen-point dose-response curves of B. fragilis SB210, E. coli SB209, B. breve SB213, and P. micra SB214 viability after exposure to 0.0023–38.4 μM 5-FU for 48 h. The range of 5-FU in patient sera is labeled between 2.5 and 10 μM. The connecting lines are a nonlinear regression of the log(inhibitor) versus response with a variable slope (four parameters). The error bars represent SD, n = 3 biological replicates. (B) Measurement of 5-FU (4 μM) disappearance in the supernatant when exposed to the indicated bacterial strains or media alone for 0, 8, 24, and 48 h 100% is set to the media-alone condition at 0 h. Error bars represent SD, n = 3 biological replicates. (C) The viability of Fna SB010 when incubated in the conditioned supernatants of the indicated bacterial species for 48 h. The conditioned supernatants of the indicated bacterial species were incubated with 5-FU (4 μM) for 48 h (dark gray bars) or had fresh 5-FU (4 μM) added immediately prior to sterilization using a 0.2 μm filter (light gray bars). 100% is set to the Fn viability in the indicated bacterial supernatants alone for 48 h. Error bars represent SD, n = 3 biological replicates. (D) Relative growth of RKO CRC epithelial cells when incubated in conditioned supernatants of indicated bacterial species for 72 h. The conditioned supernatants were incubated with 5-FU (20 μM) for 48 h (dark gray bars) or had fresh 5-FU (20 μM) added immediately prior to sterilization using a 0.2 μm filter (light gray bars). This supernatant was diluted ¼ when added to the RKO culture, resulting anticipated 5 μM 5-FU. 0% growth is the confluency of the RKO cells in each condition at 0 h. 100% growth is the confluency of the RKO cells incubated with the indicated bacterial supernatant alone for 72 h. *p < 0.05, **p < 0.01, and ***p < 0.001 as determined by a two-sided Student’s t test. Error bars represent SD, n = 3 biological replicates. See also Figure S3 and Table S3.

Figure 4.. Patient-derived ex vivo CRC microbiota can deplete 5-FU and reduce chemotherapeutic toxicity

(A) Schematic depicting the workflow of patient tissue processing for bacterial community isolation and downstream 5-FU exposure for 48 h followed by metagenomic sequencing. (B) RNAscope-based fluorescence in situ hybridization (FISH) of tumor tissue from patients with CRC (n = 6 patients). Color key: eubacterial 16S rRNA (green) and DNA (blue). (C) Relative abundance of bacterial species greater than 1% in their respective tissue samples (n = 6 patients). The exposure to 5-FU is indicated for each community. (D) Measurement of 5-FU (4 μM) disappearance in the supernatant when exposed to the indicated ex vivo CRC bacterial communities or media alone for 0, 8, 24, and 48 h. Error bars represent standard deviation, n = 3 replicates. (E) Measurement of 5-FU (4 μM) disappearance in the supernatant when exposed to the indicated bacterial strains isolated from patient CRC_06 or media alone for 0, 24, and 48 h. **p < 0.01 and ***p < 0.001 as determined by a two-sided Student’s t test. Error bars represent standard deviation, n = 3 replicates. See also Figure S4 and Tables S4 and S5.