1H-NMR Based Metabolomics Technology Identifies Potential Serum Biomarkers of Colorectal Cancer Lung Metastasis in a Mouse Model

Figures & data

Figure 1 Representative ¹H-NMR spectra (δ9.5-δ0.5) of serum samples of different groups at 600 MHz Normal control; 7 days after administration; 14 days after administration; 21 days after administration.

Figure 2 Metabolomics analysis of serum samples of the normal and metastasis groups (A) PCA scores plot between the metastatic groups and normal control; (B) PLS-DA scores plot between the metastatic groups and normal control; (C) OPLS-DA scores plot between the metastatic groups and normal control; (D) The PLS-DA model was statistically verified by permutation analysis (200 times). R² represents explained variance, and Q² represents the predictive ability of the model.

Figure 3 Serum PR analysis of different metastasis groups and normal control group (A) OPLS-DA model was used to process score plots based on the normal group and each metastasis group; (B) statistical validation of PLS-DA model by permutation analysis (200 times). R² represents explained variance, and Q² represents the predictive ability of the model.

Table 1 Summary of Different Serum Metabolites Between Metastasis Groups and Normal Group

Metabolite	Multiplicity^a	Chemical Shift (ppm)	Normal vs 7 Day			Normal vs 14 Day			Normal vs 21 Day
			VIP^b	p-value^c	FC^d	VIP^b	p-value^c	FC^d	VIP^b	p-value^c	FC^d
VLDL: CH3-(CH2)n-	br	0.89	2.15	0.02077	1.55	2.08	0.01846	1.54	2.16	0.00495	2.35
3-Hydroxyisovalerate	s	1.271	2.80	0.00009	2.02	2.71	0.00006	2.02	2.56	0.00005	2.50
Acetate	s	1.93	1.99	0.01304	−2.15	1.90	0.00764	−2.47	1.82	0.00414	−3.04
Acetic acid	s	2.08	2.48	0.00061	2.52	2.47	0.00051	2.57	2.23	0.00002	3.36
Arginine	t	3.25	2.02	\	−1.17	2.09	0.04780	−1.17	2.12	0.00460	−1.40
Choline	s	3.2	2.65	0.00143	1.34	2.55	0.00035	1.75	1.91	\	−1.04
Creatine	s	3.04	1.98	0.01704	−2.10	1.94	0.01140	−2.29	1.78	0.00828	−2.56
	s	3.94	1.98	0.02991	−1.77	1.88	0.02319	−1.81	1.70	0.02442	−1.84
Glutamate	m	2.35	1.76	0.01567	−1.28	1.92	\	−1.19	2.13	0.02188	−1.36
	m (t)	3.77	1.86	0.01133	−1.49	1.69	0.00715	−1.57	1.85	0.01112	−1.52
Glutamine	m	2.45	1.74	0.01620	−1.36	1.79	\	−1.28	2.34	\	1.13
	m (t)	3.77	2.41	0.01133	−1.49	2.50	0.00715	−1.57	2.36	0.01112	−1.52
GPC (Glycerophosphochline)	s	3.23	2.02	0.03288	1.21	2.09	0.00222	1.36	2.12	\	1.23
Isoleucine	d	1.01	1.82	0.01051	−1.32	1.98	0.00477	−1.32	2.09	\	−1.12
Lactate	d	1.33	2.68	0.01447	1.36	2.53	0.00265	1.50	1.95	\	1.26
	q	4.11	2.73	0.00061	1.72	2.65	0.00050	1.72	2.39	\	1.39
Lipid,-CH=CH-	br	5.33	2.42	0.02136	2.04	2.28	0.02904	2.03	2.01	0.01274	3.13
Lysine	m	3.77	2.41	0.01133	−1.49	2.50	0.00715	−1.57	2.36	0.01112	−1.52
PC (Phosphochline)	s	3.21	2.65	0.00143	1.34	2.55	0.00035	1.75	1.91	\	−1.04
Phosphocreatine	s	3.04	1.98	0.01704	−2.10	1.94	0.01140	−2.29	1.78	0.00828	−2.56
	s	3.93	1.98	0.02991	−1.77	1.88	0.02319	−1.81	1.70	0.02442	−1.84
Scyllo-inositol	t	3.43	1.89	0.02075	−1.58	2.32	0.01328	−1.67	2.09	0.00797	−1.87
Methionine	s	2.13	1.60	\	−1.15	1.88	\	−1.22	2.09	0.01137	−1.35
Succinate	s	2.41	1.96	0.01650	−4.12	1.84	0.01430	−4.56	1.66	0.01110	−5.80
Taurine	t	3.27	2.02	\	−1.17	2.09	0.04780	−1.17	2.12	0.00460	−1.40
	t	3.43	1.89	0.02075	−1.58	2.32	0.01328	−1.67	2.09	0.00797	−1.87
Valine	d	1.05	1.38	0.00908	−1.34	2.01	0.00269	−1.37	2.10	\	−1.16

Notes: Multiplicity: ^aS singlet, d doublet, t triplet, q quartet, dd doublet of doublets, m multiplet. Variable: ^bImportance in the projection was obtained from OPLS-DA model with a threshold of 1.0. p-value: ^cObtained from Student's t-test. FC: ^dFold change (FC) between normal group and metastasis groups. Fold change with a positive value indicates a relatively higher concentration present in metastasis groups while a negative value means a relatively lower concentration as compared to the normal group. “\” represents this parameter is not statistically significant.

Figure 4 Box-and-whisker plots show differences between the normal control group and the metastatic groups. The Y-axis shows the relative abundance of the NMR signal (normalized to the total peak); median: horizontal line in the middle portion of the box; lower and upper quartile: bottom and top boundaries of boxes; whiskers, 5th and 95th percentiles; outliers: open circles; *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test.

Table 2 Pathway Enrichment

Pathways	Total Cmpd	Hits	Compound	Raw p	-Log p	FDR	Impact
Synthesis and degradation of ketone bodies	5	2	3-Hydroxybutanoate; Acetoacetate	6.74E-05	9.61	6.23E-04	0.60
Glycine, serine and threonine metabolism	34	8	Serine; Choline; Betaine; N,N-Dimethylglycine; Glycine; Threonine; Creatine; Pyruvate	2.33E-02	3.76	0.037	0.62
Pyruvate metabolism	22	4	Lactate; Pyruvate Acetate; Fumarate	7.75E-08	16.37	2.87E-06	0.35
Starch and sucrose metabolism	15	1	Glucose	2.95E-04	8.13	9.92E-04	0.33
Alanine, aspartate and glutamate metabolism	28	5	Alanine; Glutamate; Glutamine; Fumarate; Pyruvate	1.19E-04	9.04	8.81E-04	0.31
Aminoacyl-tRNA biosynthesis	48	12	Glutamine; Glycine; Serine; Methionine; Valine; Alanine; Lysine; Isoleucine; Leucine; Threonine; Tyrosine; Glutamate	6.68E-04	7.31	1.90E-03	0.17
Tyrosine metabolism	42	4	Fumarate; Pyruvate; Acetoacetate; Tyrosine	8.37E-04	7.09	2.21E-03	0.16
Glyoxylate and dicarboxylate metabolism	32	7	Serine; Glycine; Glutamate; Acetate; Pyruvate; Formate; Glutamine	2.70E-04	8.22	9.92E-04	0.15
Glycolysis/Gluconeogenesis	26	2	Pyruvate; Acetate	2.68E-04	8.22	9.92E-04	0.13
Cysteine and methionine metabolism	33	3	Serine; Methionine; Pyruvate	1.16E-03	6.76	2.69E-03	0.13
Pathways	Total Cmpd	Hits	Compound	Raw p	-log p	FDR	Impact
Butanoate metabolism	15	3	3-Hydroxybutanoate; Acetoacetate; Glutamate	6.71E-05	9.61	6.23E-04	0.11
Arginine and proline metabolism	38	4	Creatine; Glutamate; Phosphocreatine; Pyruvate	1.11E-06	13.71	2.06E-05	0.098
Glycerophospholipid metabolism	36	3	Choline phosphate; Choline; Phosphocholine	1.51E-04	8.80	9.32E-04	0.083
Citrate cycle (TCA cycle)	20	2	Pyruvate; Fumarate	1.14E-03	6.78	2.69E-03	0.076
Galactose metabolism	27	1	Glucose	2.95E-04	8.13	9.92E-04	0.035

Notes: *The Total is the total number of compounds in the pathway; the Hits is the actually matched number from the user uploaded data; the Raw p is the original p value calculated from the enrichment analysis; the FDR p is the p value adjusted using False Discovery Rate; the Impact is the pathway impact value calculated from pathway topology analysis.

Figure 5 Summary of pathway analysis by Metaboanalyst online. The further its location is from the (0,0), the more significant the pathway is.