Clinical significance of Fusobacterium nucleatum, epithelial–mesenchymal transition, and cancer stem cell markers in stage III/IV colorectal cancer patients

Figures & data

Table 1 Correlations between Fn level and clinicopathological parameters in stage III/IV CRC patients

Characteristics	Total	Fn level		p-value
		Low	High
Gender				0.705
Female	122	42	80
Male	158	51	107
Age				0.822
≤65 years	111	36	75
>65 years	169	57	112
Tumor location				0.579
Rectal	130	41	89
Colon	150	52	98
Tumor size				0.357
≤5 cm	214	68	146
>5 cm	66	25	41
Tumor differentiation				0.650
Poor	74	23	51
Well/moderate	206	70	136
Tumor invasion				0.015
T1–T2	96	41	55
T3–T4	184	52	132
Lymph node metastasis				0.008
N1	81	17	64
N2a	95	41	54
N2b	104	35	69
Distant metastasis				0.020
Absent	218	80	138
Present	62	13	49
Ki-67 expression				0.381
<30%	78	29	49
≥30%	202	64	138
Serum CEA level				0.274
≤5 ng/mL	99	37	62
>5 ng/mL	181	56	125
BMI				0.202
<18.5 kg/m^2	22	11	11
18.5–24.99 kg/m^2	178	58	120
≥25.0 kg/m^2	80	24	56

Abbreviations: Fn, Fusobacterium nucleatum; CRC, colorectal cancer; CEA, carcinoembryonic antigen; BMI, body mass index.

Figure 1 Fn level in tumor and adjacent normal tissues of stage III/IV CRC patients.

Notes: (A) Fn level is significantly higher in tumor tissues than in adjacent normal tissues of the whole cohort (tumor vs normal: 0.1092±0.2150 vs 0.0245±0.0553, n=280, p<0.001). (B) Fn level is significantly higher in tumor tissues than in adjacent normal tissues of stage III patients (tumor vs normal: 0.1043±0.2165 vs 0.0216±0.0450, n=218, p<0.001). (C) Fn level is significantly higher in tumor tissues than in adjacent normal tissues of stage IV patients (tumor vs normal: 0.1266±0.2106 vs 0.0348±0.0817, n=62, p=0.005). (D) ROC curve analysis determined the cutoff value of Fn level in tumor tissues to be 0.0282.
Abbreviations: Fn, Fusobacterium nucleatum; CRC, colorectal cancer; ROC, receiver operating characteristic.

Figure 2 Prognostic significance of Fn in stage III/IV CRC patients.

Note: CSS and DFS curves of stage III/IV CRC patients (A), stage IIIA patients (B), stage IIIB patients (C), stage IIIC patients (D), stage IV patients (E).
Abbreviations: Fn, Fusobacterium nucleatum; CRC, colorectal cancer; CSS, cancer-specific survival; DFS, disease-free survival.

Table 2 Univariate and multivariate analysis for prognostic factors in cancer-specific survival of stage III/IV CRC patients

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	p-value	HR	95% CI	p-value
Gender	0.835	0.594–1.175	0.300
Age	1.351	0.943–1.935	0.101
Tumor location	1.188	0.843–1.676	0.326
Tumor size	1.199	0.817–1.757	0.354
Tumor differentiation	0.731	0.497–1.074	0.110
Ki-67 positivity	0.914	0.629–1.328	0.638
Body mass index	0.806	0.597–1.087	0.158
Tumor invasion	1.595	1.097–2.319	0.015	1.341	0.920–1.955	0.127
Lymph node metastasis	1.426	1.136–1.789	0.002	1.430	1.158–1.766	0.001
Distant metastasis	3.507	2.425–5.071	<0.001	3.243	2.232–4.712	<0.001
Serum CEA level	1.466	1.006–2.136	0.046	1.515	1.038–2.212	0.031
Fn level	2.302	1.541–3.437	<0.001	2.222	1.483–3.329	<0.001

Abbreviations: Fn, Fusobacterium nucleatum; CRC, colorectal cancer; CEA, carcinoembryonic antigen; HR, hazard ratio; CI, confidence interval.

Table 3 Univariate and multivariate analysis for prognostic factors in disease-free survival of stage III/IV CRC patients

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	p-value	HR	95% CI	p-value
Gender	0.821	0.603–1.119	0.212
Age	1.092	0.796–1.498	0.585
Tumor location	1.149	0.842–1.568	0.381
Tumor size	1.132	0.796–1.609	0.490
Ki-67 positivity	0.998	0.708–1.408	0.993
Body mass index	0.871	0.665–1.143	0.319
Tumor differentiation	0.636	0.453–0.893	0.009	0.592	0.417–0.841	0.003
Tumor invasion	1.634	1.163–2.297	0.005	1.499	1.060–2.119	0.022
Lymph node metastasis	1.287	1.050–1.579	0.015	1.294	1.069–1.566	0.008
Distant metastasis	3.965	2.843–5.531	<0.001	3.914	2.788–5.495	<0.001
Serum CEA level	1.512	1.075–2.128	0.018	1.483	1.046–2.101	0.027
Fn level	2.133	1.496–3.041	<0.001	2.000	1.396–2.865	<0.001

Abbreviations: Fn, Fusobacterium nucleatum; CRC, colorectal cancer; CEA, carcinoembryonic antigen; HR, hazard ratio; CI, confidence interval.

Figure 3 Correlations between Fn level and chemotherapy benefits in stage III/IV patients.

Notes: (A) CSS and DFS of the whole cohort, (B) CSS and DFS of low Fn level group, and (C) CSS and DFS of high Fn level group stratified by chemotherapy reception. An interaction analysis indicates that patients with low Fn level benefit more from chemotherapy than those with high Fn level, in terms of DFS (CSS: p=0.134; DFS: p=0.048).
Abbreviations: Fn, Fusobacterium nucleatum; CRC, colorectal cancer; CSS, cancer-specific survival; DFS, disease-free survival.

Figure 4 Representative immunohistochemical staining images of EMT and CSC markers in CRC tissues.

Notes: High (left) and low (right) expression of E-cadherin (A), N-cadherin (B), Nanog (C), Oct-4 (D), Sox-2 (E). Magnification: ×200.
Abbreviations: CRC, colorectal cancer; EMT, epithelial-to-mesenchymal transition; CSC, cancer stem cell.

Figure 5 Prognostic significance of epithelial-to-mesenchymal transition and cancer stem cell markers in stage III/IV CRC patients.

Note: CSS and DFS curves stratified by E-cadherin expression (A), N-cadherin expression (B), Nanog expression (C), Oct-4 expression (D), Sox-2 expression (E).
Abbreviations: CRC, colorectal cancer; CSS, cancer-specific survival; DFS, disease-free survival.

Table 4 Correlations of Fn with EMT/CSC markers in stage III/IV CRC patients

Markers	N	Fn level		r	p-value
		Low	High
E-cadherin					<0.001
Low	167	36	131	−0.301
High	113	57	56
N-cadherin					<0.001
Low	139	71	68	0.377
High	141	22	119
Sox-2					0.078
Low	163	61	102	0.105
High	117	32	85
Oct-4					0.097
Low	155	58	97	0.099
High	125	35	90
Nanog					<0.001
Low	204	89	115	0.362
High	76	4	72

Abbreviations: Fn, Fusobacterium nucleatum; CRC, colorectal cancer; EMT, epithelial-to-mesenchymal transition; CSC, cancer stem cell.

Figure S1 The ROC curve analysis is used to determine the cutoff values of staining scores of epithelial–mesenchymal transition and cancer stem cell markers.

Notes: (A) E-cadherin; (B) N-cadherin; (C) Nanog; (D) Oct-4; (E) Sox-2.

Abbreviation: ROC, receiver operating characteristic.

Table S1 Correlations between epithelial-to-mesenchymal transition/cancer stem cell markers and clinicopathological characteristics

Characteristics	Total	E-cadherin		p-value	Total	N-cadherin		p-value	Total	Sox-2		p-value	Total	Oct-4		p-value	Total	Nanog		p-value
		Low	High			Low	High			Low	High			Low	High			Low	High
Gender				0.761				0.892				0.621				0.279				0.434
Female	122	74	48		122	60	62		122	69	53		122	72	50		122	86	36
Male	158	93	65		158	79	79		158	94	64		158	83	75		158	118	40
Age				0.960				0.607				0.878				0.892				0.559
≤65 years	111	66	45		111	53	58		111	64	47		111	62	49		111	83	28
>65 years	169	101	68		169	86	83		169	99	70		169	93	76		169	121	48
Tumor location				0.708				0.117				0.748				0.803				0.538
Rectal	130	76	54		130	58	72		130	77	53		130	73	57		130	97	33
Colon	150	91	59		150	81	69		150	86	64		150	82	68		150	107	43
Tumor size				0.297				0.233				0.904				0.664				0.731
≤5 cm	214	124	90		214	102	112		214	125	89		214	120	94		214	157	57
>5 cm	66	43	23		66	37	29		66	38	28		66	35	31		66	47	19
Tumor differentiation				0.555				0.311				0.422				0.992				0.134
Poor	74	42	32		74	33	41		74	46	28		74	41	33		74	49	25
Well/moderate	206	125	81		206	106	100		206	117	89		206	114	92		206	155	51
Tumor invasion				0.275				0.555				0.630				0.120				0.010
T1–T2	96	53	43		96	50	46		96	54	42		96	47	49		96	79	17
T3–T4	184	114	70		184	89	95		184	109	75		184	108	76		184	125	59
Lymph node metastasis				0.029				0.024				0.046				0.609				0.003
N1	81	39	42		81	31	50		81	43	38		81	45	36		81	59	22
N2a	95	58	37		95	56	39		95	65	30		95	56	39		95	80	15
N2b	104	70	34		104	52	52		104	55	49		104	54	50		104	65	39
Distant metastasis				0.039				0.012				0.396				<0.001				0.118
Absent	218	123	95		218	117	101		218	124	94		218	134	84		218	154	64
Present	62	44	18		62	22	40		62	39	23		62	21	41		62	50	12
Ki-67 positivity				0.042				0.382				0.483				0.394				0.041
<30%	78	54	24		78	42	36		78	48	30		78	40	38		78	50	28
≥30%	202	113	89		202	97	105		202	115	87		202	115	87		202	154	48
Serum CEA level				0.199				0.971				0.174				0.764				0.276
≤5 ng/mL	99	54	45		99	49	50		99	63	36		99	56	43		99	76	23
>5 ng/mL	181	113	68		181	90	91		181	100	81		181	99	82		181	128	53
BMI				0.472				0.887				0.367				0.394				0.166
<18.5 kg/m^2	22	12	10		22	12	10		22	14	8		22	11	11		22	13	9
18.5–24.99 kg/m^2	178	111	67		178	88	90		178	98	80		178	104	74		178	128	50
≥25.0 kg/m^2	80	44	36		80	39	41		80	51	29		80	40	40		80	63	17

Abbreviations: CEA, carcinoembryonic antigen; BMI, body mass index.