M2-Type Macrophages Induce Tregs Generation by Activating the TGF-β/Smad Signalling Pathway to Promote Colorectal Cancer Development

Figures & data

Table 1 Clinical Characteristics of the 250 CRC Patients

Variables	n=250	Percent (%)
Age (years)
<65	105	42.00
≥65	145	58.00
Gender
Male	153	61.20
Female	97	38.80
TNM staging
I	37	14.80
II	108	43.20
III	77	30.80
IV	28	11.20
Histologic types
Tubular adenocarcinoma	232	92.80
Mucinous carcinoma	12	4.80
Other types	6	2.40

Figure 1 Representative IHC staining of CD163, Foxp3 and Smad3 in normal, paracancerous and CRC tissues (400× field). (A–C) The cell membrane and cytoplasm of M2-type macrophages are stained brown. (D–F) The nucleus of Tregs are stained brown. (G–I) The cell membrane and cytoplasm of Smad3+ cells are stained brown.

Figure 2 The expression levels of CD163, Foxp3 and Smad3 are different in normal, paracancerous and cancer tissues. (A) The number of CD163 in CRC tissues, their matched normal and paracancerous tissues. (B) The number of Foxp3 in CRC tissues, their matched normal and paracancerous tissues. (C) The staining indices of Smad3 in CRC tissues, their matched normal and paracancerous tissues.

Table 2 Relationship Between CD163 and Foxp3 and Clinicopathological Characteristics

Clinicopathological Characteristics	n (%) =250	CD163				Foxp3
		Low Expression	High Expression	χ^2	p-value	Low Expression	High Expression	χ^2	p-value
Age (years)				0.562	0.453			1.896	0.169
<65	105 (42)	29	76			28	77
≥65	145 (58)	34	111			28	117
Gender				2.649	0.104			2.166	0.141
Male	153 (61)	44	109			39	114
Female	97 (39)	19	78			17	80
Hospitalization time (days)				0.021	0.884			0.368	0.544
<16	125 (50)	31	94			30	95
≥16	125 (50)	32	93			26	99
Tumour maximum diameter (cm)				0.207	0.649			0.022	0.883
<5	145 (58)	35	110			32	113
≥5	105 (42)	28	77			24	81
BMI				2.557	0.279			1.076	0.584
<18.9	11 (4)	5	6			3	8
18.5–23.9	135 (54)	32	103			33	102
>23.9	104 (42)	26	78			20	84
Preoperative blood glucose				0.147	0.929			2.682	0.267
<3.89	4 (2)	1	3			1	3
3.89–6.11	186 (74)	48	138			37	149
>6.11	60 (24)	14	46			18	42
Preoperative Intestinal obstruction				0.681	0.409			0.501	0.479
Absent	225 (90)	55	170			49	176
Present	25 (10)	8	17			7	18
Preoperative fecal blood				0.255	0.614			0.069	0.794
Absent	102 (41)	24	78			22	80
Present	148 (59)	39	109			34	114
Anatomic tumour region				3.315	0.652			3.751	0.585
Cecum	8 (3)	2	6			2	6
Ascending colon	46 (18)	12	34			11	35
Transverse colon	3 (1)	0	3			0	3
Descending colon	10 (4)	4	6			2	8
Sigmoid	69 (28)	14	55			20	49
Rectum	114 (46)	31	83			21	93
Pathological type				2.119	0.145			0.379	0.538
Exogenous type	195 (78)	45	150			42	153
Ulcer type	55 (22)	18	37			14	41
Histologic types				2.007	0.367			0.160	0.923
Tubular adenocarcinoma	232 (93)	57	175			52	180
Mucinous carcinoma	12 (5)	5	7			3	9
Other types	6 (2)	1	5			1	5
Differentiation degree				0.641	0.726			1.668	0.434
Low	56 (22)	12	44			9	47
Moderate	177 (71)	46	131			43	134
High	17 (7)	5	12			4	13
Depth of infiltration				9.881	0.019			4.111	0.249
Infiltrate submucosa	10 (4)	5	5			4	6
Infiltrate muscular	34 (14)	14	20			9	25
Infiltrate the subserosal layer	151 (60)	34	117			35	116
Penetrating visceral peritoneum	55 (22)	10	45			8	47
Lymph node metastases				9.862	0.019			9.177	0.027
0	150 (60)	48	102			43	107
1–3	64 (25)	8	56			10	54
4–6	24 (10)	5	19			2	22
≥7	12 (5)	2	10			1	11
Distant metastases				3.510	0.061			2.477	0.115
Absent	222 (89)	60	162			53	169
Present	28 (11)	3	25			3	25
TNM staging				19.397	0.000			9.056	0.029
I	37 (15)	19	18			12	25
II	108 (43)	28	80			30	78
III	77 (30)	13	64			11	66
IV	28 (11)	3	25			3	25
Vascular invasion				4.993	0.026			4.278	0.039
Absent	188 (75)	54	134			48	140
Present	62 (25)	9	53			8	54
Nerve invasion				1.311	0.252			1.123	0.289
Absent	202 (81)	54	148			48	154
Present	48 (19)	9	39			8	40

Figure 3 Representative IHC staining of CD163, Foxp3 and Smad3 in different tumour TNM staging (400× field). (A–D) The expression of CD163 in CRC at stages I, II, III and IV. (E–H) The expression of Foxp3 in CRC at stages I, II, III and IV. (I–L) The expression levels of Smad3 in CRC at stages I, II, III and IV. (M–O) The changing trend of CD163, Foxp3 and Smad3 in CRC in different tumour TNM staging.

Table 3 Correlation Analysis of CD163 and Foxp3 Expression in Colorectal Cancer

		Foxp3
CD163	r	0.565
	p	0.000

Table 4 Correlation Analysis of Smad3 with CD163 and Foxp3 in Colorectal Cancer

		Smad3
CD163	r	0.657
	p	0.000
Foxp3	r	0.510
	p	0.000

Figure 4 Correlation analysis among CD163, Foxp3 and Smad3 in CRC. The linear correlation model is shown with a red line. Corresponding 95% confidence intervals (CI) are shown with the gray area. (A) Correlation between the number of CD163+ cells and Foxp3+ cells. (B) Correlation between the number of CD163+ cells and the staining indices of Smad3+ cells. (C) Correlation between the number of Foxp3+ cells and the staining indices of Smad3+ cells.

Table 5 Correlation Analysis of the Number of M2-Type Macrophages and Tregs with Preoperative CEA, CA19-9 and CA72-4

Tumour Markers	The Mean Number of M2-Type Macrophages		The Mean Number of Tregs
	r	p-value	r	p-value
Preoperative CEA (μg/L)	0.466	0.000	0.386	0.000
Preoperative CA19-9 (U/mL)	0.218	0.001	0.144	0.023
Preoperative CA72-4 (U/mL)	0.178	0.005	0.121	0.056