Akkermansia Muciniphila Potentiates the Antitumor Efficacy of FOLFOX in Colon Cancer

FOLFOX (oxaliplatin, fluorouracil and calcium folinate) is the first-line chemotherapy regimen for colon cancer therapy in the clinic. It provides superior efficacy than oxaliplatin alone, but the underlying mechanism remains unclear. In the present study, pharmacomicrobiomics integrated with metabolomics was conducted to uncover the role of the gut microbiome behind this. First, in vivo study demonstrated that FOLFOX exhibited better efficacy than oxaliplatin alone in colon cancer animal models. Second, 16S rDNA gene sequencing analysis showed that the abundance of Akkermansia muciniphila (A. muciniphila) remarkably increased in the FOLFOX treated individuals and positively correlated with the therapeutic effect. Third, further exploration confirmed A. muciniphila colonization significantly enhanced the anti-cancer efficacy of FOLFOX. Last, metabolomics analysis suggested dipeptides containing branched-chain amino acid (BCAA) might be responsible for gut bacteria mediated FOLFOX efficacy. In conclusion, our study revealed the key role of A. muciniphila in mediating FOLFOX efficacy, and manipulating A. muciniphila might serve as a novel strategy for colon cancer therapy.


Sample preparation
For GC-MS analysis, about 20 mg fecal samples were homogenized with precooled saline (4 μL/mg). Methanol was added to the fecal homogenate (4:1, μL/μL), vortex mixed for 15 min and centrifuged twice (4 °C, 12000 rpm, 10 min). Then, 80 μL of the supernatant was transferred into a brown glass vial and added with 25 μL methoxyamine hydrochloride (10 mg/mL in pyridine). The mixture was incubated at 37 °C for 90 min and vacuum dried at 50 °C for 2 h (Labconco CentriVap® , Kansas, USA). Lastly, 120 μL of MSTFA was added to the dried extracts and incubated at 37 °C for 2 h, the supernatant was collected for GC-MS analysis.

GC-MS.
Column temperature was started at 70 °C (0 to 2 min), followed by an increase from 70 to 320 °C at the rate of 10 °C/min (2 to 27 min) and maintained at 320 °C (27 to 29 min). The flow rate of carrier gas (Helium) was 1.0 mL/min. Injection volume was 1μL and the split ratio was 30:1. The temperatures of the injector, transfer line and ion source was 250, 250, and 200 °C, respectively. Electron impact mode (EI, 70 eV) using full scan mode (from m/z 45 to 600) was carried out for data acquisition.

LC-MS.
The column temperature was 40 °C. The mobile phase consists of (A) 0.1% formic acid in water and (B) acetonitrile. The elution program (flow rate: 0.4 mL/min) was as follows: mobile phase A was decreased from 95 to 5% from 0 to 20 min, and maintained at 5% A for 3 min, then brought back to 95% A and maintained from 23 to Electrospray ionization (ESI) source in both positive and negative modes with full scan mode (from m/z 100 to 1000) was carried out. Injection volume was 5μL. TOF analyzer detector voltage was 1.80 kV. The interface voltage was 4.5 kV for positive ion mode and -3.5 kV for negative ion mode.

Quality control and differential metabolites screening
To check the robustness of the non-target metabolomics workflow, quality control samples (QC) were prepared by pooling equal aliquot of each sample, processed together with actual samples, and injected every six samples in the analytical sequence. Orthogonal partial least-squares-discriminant analysis (OPLS-DA) models were constructed to explore the differences between model and treatment groups. The variable importance in projection (VIP) generated from OPLS-DA models and p values from non-parametric Mann-Whitney U test (SPSS 19.0, Chicago, USA) were used to determine whether a feature is significantly different between the two groups.
Only the features meet the requirements of VIP > 1 and p < 0.05 were considered for metabolite annotation.

Metabolite annotation
Identification of the metabolite detected by GC-MS was performed by comparing the mass spectra with those available in National Institute of Standards and Technology (NIST 11) library (with similarity ≥ 80%), and they were further confirmed by retention time, accurate mass and mass spectra with available standard compounds in the laboratory.     The p-values<0.05 were considered statistically significant, *p<0.05, **p<0.01.