Background
Methods
Patients and tissue specimens
Immunohistochemistry
Evaluation of immunoreactivity
Cell cultures and preparation of TAMs
Quantitative real-time PCR
Western blotting
Immunofluorescence microscopy
Wound-healing and invasion assays
Statistical analysis
Results
CD68-positive macrophages and CD163-positive macrophages in oral squamous cell carcinoma and peritumoral tissue
Variable | No. | CD68+ macrophages counts in tumor nest | CD68+ macrophages counts in tumor stroma | ||||
---|---|---|---|---|---|---|---|
Low(≤6/HPF) | High(>6/HPF) |
P
| Low(≤8/HPF) | High(>8/HPF) |
P
| ||
Sex |
0.176
|
0.060
| |||||
Male |
74
|
34
|
40
|
30
|
44
| ||
Female |
53
|
18
|
35
|
13
|
40
| ||
Age (years) |
0.773
|
0.927
| |||||
≤50 |
26
|
10
|
16
|
9
|
17
| ||
>50 |
101
|
42
|
59
|
34
|
67
| ||
Tumor location |
0.034
|
0.346
| |||||
Tongue |
52
|
29
|
23
|
20
|
32
| ||
Gingiva |
26
|
7
|
19
|
8
|
18
| ||
Buccal mucosa |
31
|
8
|
23
|
6
|
25
| ||
Lip |
4
|
3
|
1
|
2
|
2
| ||
Floor of the mouth |
2
|
1
|
1
|
1
|
1
| ||
Others |
12
|
4
|
8
|
6
|
6
| ||
Tumor size |
0.665
|
0.369
| |||||
T1 |
21
|
9
|
12
|
7
|
14
| ||
T2 |
68
|
28
|
40
|
21
|
47
| ||
T3 |
26
|
12
|
14
|
8
|
18
| ||
T4 |
12
|
3
|
9
|
7
|
5
| ||
Lymph node metastasis |
0.850
|
0.321
| |||||
N0 |
72
|
30
|
42
|
27
|
45
| ||
N (+) |
55
|
22
|
33
|
16
|
39
| ||
Metastasis |
0.793
|
0.627
| |||||
M0 |
125
|
51
|
74
|
42
|
83
| ||
M (+) |
2
|
1
|
1
|
1
|
1
| ||
Clinical stage |
0.262
|
0.699
| |||||
I |
12
|
5
|
7
|
4
|
8
| ||
II |
42
|
19
|
23
|
17
|
25
| ||
III |
34
|
16
|
18
|
8
|
26
| ||
IV |
39
|
12
|
27
|
14
|
25
| ||
Pathological grade |
0.135
|
0.296
| |||||
I |
76
|
34
|
42
|
24
|
52
| ||
II |
42
|
18
|
24
|
13
|
29
| ||
III |
9
|
0
|
9
|
6
|
3
| ||
Recurrence |
0.111
|
0.259
| |||||
No |
83
|
39
|
44
|
25
|
58
| ||
Yes |
42
|
13
|
29
|
18
|
24
| ||
Lost |
2
|
0
|
2
|
0
|
2
| ||
Follow-up |
0.005
|
0.218
| |||||
Live |
91
|
45
|
46
|
27
|
64
| ||
Dead |
33
|
7
|
26
|
15
|
18
| ||
Lost |
3
|
0
|
3
|
1
|
2
|
Variable | No. | CD163+ macrophages counts in tumor nest | CD163+ macrophages counts in tumor stroma | ||||
---|---|---|---|---|---|---|---|
Low(≤6/HPF) | High(>6/HPF) |
P
| Low(≤8/HPF) | High(>8/HPF) |
P
| ||
Sex |
0.037
|
0.846
| |||||
Male |
74
|
36
|
38
|
32
|
42
| ||
Female |
53
|
16
|
37
|
22
|
31
| ||
Age (years) |
0.874
|
0.674
| |||||
≤50 |
26
|
11
|
15
|
12
|
14
| ||
>50 |
101
|
41
|
60
|
42
|
59
| ||
Tumor location |
0.389
|
0.057
| |||||
Tongue |
52
|
24
|
28
|
22
|
30
| ||
Gingiva |
26
|
9
|
17
|
17
|
9
| ||
Buccal mucosa |
31
|
9
|
22
|
7
|
24
| ||
Lip |
4
|
3
|
1
|
2
|
2
| ||
Floor of the mouth |
2
|
1
|
1
|
1
|
1
| ||
Others |
12
|
6
|
6
|
5
|
7
| ||
Tumor size |
0.921
|
0.335
| |||||
T1 |
21
|
7
|
14
|
7
|
14
| ||
T2 |
68
|
31
|
37
|
30
|
38
| ||
T3 |
26
|
8
|
18
|
9
|
17
| ||
T4 |
12
|
6
|
6
|
8
|
4
| ||
Lymph node metastasis |
0.200
|
0.889
| |||||
N0 |
72
|
33
|
39
|
31
|
41
| ||
N (+) |
55
|
19
|
36
|
23
|
32
| ||
Metastasis |
0.235
|
0.829
| |||||
M0 |
125
|
52
|
73
|
53
|
72
| ||
M (+) |
2
|
0
|
2
|
1
|
1
| ||
Clinical stage |
0.059
|
0.725
| |||||
I |
12
|
5
|
7
|
4
|
8
| ||
II |
42
|
21
|
21
|
21
|
21
| ||
III |
34
|
16
|
18
|
13
|
21
| ||
IV |
39
|
10
|
29
|
16
|
23
| ||
Pathological grade |
0.211
|
0.469
| |||||
I |
76
|
35
|
41
|
35
|
41
| ||
II |
42
|
13
|
29
|
14
|
28
| ||
III |
9
|
4
|
5
|
5
|
4
| ||
Recurrence |
0.22
|
0.01
| |||||
No |
83
|
38
|
45
|
28
|
55
| ||
Yes |
42
|
13
|
29
|
24
|
18
| ||
Lost |
2
|
1
|
1
|
2
|
0
| ||
Follow-up |
0.04
|
0.040
| |||||
Live |
91
|
43
|
48
|
34
|
57
| ||
Dead |
33
|
8
|
25
|
17
|
16
| ||
Lost |
3
|
1
|
2
|
3
|
0
|
Variable | No. | CD68+ macrophages counts | CD163+ macrophages counts | ||||
High(>6/HPF) | Low(≤6/HPF) |
P
| High(>6/HPF) | Low(≤6/HPF) |
P
| ||
0.000
|
0.000
| ||||||
Normal |
23
|
0
|
23
|
0
|
23
| ||
Tumor |
127
|
75
|
52
|
75
|
52
| ||
Relationship between CD68+, CD163+ macrophages counts in tumor stroma and peritumoral stroma | |||||||
Variable | No. | CD68+ macrophages counts | CD163+ macrophages counts | ||||
High(>8/HPF) | Low(≤8/HPF) |
P
| High(>8/HPF) | Low(≤8/HPF) |
P
| ||
0.374
|
0.000
| ||||||
Normal |
23
|
12
|
11
|
0
|
23
| ||
Tumor |
127
|
84
|
43
|
73
|
54
|
Expression of CD68 and CD163 in oral squamous cell carcinoma patients
Variable | No. | CD68 | CD163 | ||||||
---|---|---|---|---|---|---|---|---|---|
N | L | H | P | N | L | H | P | ||
Sex |
0.081
|
0.008
| |||||||
Male |
74
|
15
|
19
|
40
|
35
|
25
|
14
| ||
Female |
53
|
6
|
8
|
39
|
13
|
18
|
22
| ||
Age (years) |
0.668
|
0.642
| |||||||
≤50 |
26
|
5
|
4
|
17
|
8
|
9
|
9
| ||
>50 |
101
|
16
|
23
|
62
|
40
|
34
|
27
| ||
Tumor location |
0.324
|
0.405
| |||||||
Tongue |
52
|
10
|
12
|
30
|
20
|
17
|
15
| ||
Gingiva |
26
|
3
|
2
|
21
|
8
|
11
|
7
| ||
Buccal mucosa |
31
|
3
|
8
|
20
|
11
|
9
|
11
| ||
Lip |
4
|
2
|
1
|
1
|
4
|
0
|
0
| ||
Floor of the mouth |
2
|
0
|
1
|
1
|
0
|
1
|
1
| ||
Others |
12
|
3
|
3
|
6
|
5
|
5
|
2
| ||
Tumor size |
0.419
|
0.124
| |||||||
T1 |
21
|
5
|
3
|
13
|
9
|
4
|
8
| ||
T2 |
68
|
9
|
14
|
45
|
23
|
24
|
21
| ||
T3 |
26
|
4
|
5
|
17
|
14
|
7
|
5
| ||
T4 |
12
|
3
|
5
|
4
|
2
|
8
|
2
| ||
Lymph node metastasis |
0.044
|
0.098
| |||||||
N0 |
72
|
15
|
19
|
38
|
33
|
22
|
17
| ||
N (+) |
55
|
6
|
8
|
41
|
15
|
21
|
19
| ||
Metastasis |
0.542
|
0.535
| |||||||
M0 |
125
|
21
|
27
|
77
|
48
|
42
|
35
| ||
M (+) |
2
|
0
|
0
|
2
|
0
|
1
|
1
| ||
Clinical stage |
0.564
|
0.114
| |||||||
I |
12
|
4
|
2
|
6
|
7
|
2
|
3
| ||
II |
42
|
7
|
10
|
25
|
17
|
14
|
11
| ||
III |
34
|
4
|
7
|
23
|
14
|
9
|
11
| ||
IV |
39
|
6
|
8
|
25
|
10
|
18
|
11
| ||
Pathological grade |
0.986
|
0.537
| |||||||
I |
76
|
12
|
17
|
47
|
31
|
25
|
20
| ||
II |
42
|
8
|
6
|
28
|
15
|
16
|
11
| ||
III |
9
|
1
|
4
|
4
|
2
|
2
|
5
| ||
Recurrence |
0.870
|
0.01
| |||||||
No |
83
|
14
|
16
|
53
|
35
|
32
|
16
| ||
Yes |
42
|
7
|
11
|
24
|
13
|
10
|
19
| ||
Lost |
2
|
0
|
0
|
2
|
0
|
1
|
1
| ||
Follow-up |
0.560
|
0
| |||||||
Live |
91
|
16
|
17
|
58
|
41
|
33
|
17
| ||
Dead |
33
|
5
|
10
|
18
|
7
|
9
|
17
| ||
Lost |
3
|
0
|
0
|
3
|
0
|
1
|
2
|
Expression of E-cadherin and vimentin in oral squamous cell carcinoma patients
Variable | No. | E-cadherin | Vimentin | ||||||
---|---|---|---|---|---|---|---|---|---|
N | L | H | P | N | L | H | P | ||
Sex |
0.990
|
0.205
| |||||||
Male |
74
|
16
|
21
|
37
|
37
|
22
|
15
| ||
Female |
53
|
12
|
15
|
26
|
19
|
17
|
17
| ||
Age (years) |
0.257
|
0.383
| |||||||
≤50 |
26
|
7
|
4
|
15
|
12
|
10
|
4
| ||
>50 |
101
|
21
|
32
|
48
|
44
|
29
|
28
| ||
Tumor location |
0.444
|
0.345
| |||||||
Tongue |
52
|
13
|
12
|
27
|
26
|
16
|
10
| ||
Gingiva |
26
|
3
|
6
|
17
|
12
|
10
|
4
| ||
Buccal mucosa |
31
|
9
|
11
|
11
|
10
|
8
|
13
| ||
Lip |
4
|
1
|
1
|
2
|
1
|
2
|
1
| ||
Floor of the mouth |
2
|
1
|
1
|
0
|
2
|
0
|
0
| ||
Others |
12
|
1
|
5
|
6
|
5
|
3
|
4
| ||
Tumor size |
0.848
|
0.819
| |||||||
T1 |
21
|
3
|
7
|
11
|
10
|
6
|
5
| ||
T2 |
68
|
18
|
19
|
31
|
27
|
24
|
17
| ||
T3 |
26
|
5
|
7
|
14
|
12
|
5
|
9
| ||
T4 |
12
|
2
|
3
|
7
|
7
|
4
|
1
| ||
Lymph node metastasis |
0.150
|
0.010
| |||||||
N0 |
72
|
20
|
17
|
35
|
40
|
19
|
13
| ||
N (+) |
55
|
8
|
19
|
28
|
16
|
20
|
19
| ||
Metastasis |
0.678
|
0.050
| |||||||
M0 |
125
|
28
|
35
|
62
|
56
|
39
|
30
| ||
M (+) |
2
|
0
|
1
|
1
|
0
|
0
|
2
| ||
Clinical stage |
0.071
|
0.046
| |||||||
I |
12
|
3
|
1
|
8
|
9
|
2
|
1
| ||
II |
42
|
15
|
10
|
17
|
21
|
12
|
9
| ||
III |
34
|
4
|
12
|
18
|
13
|
10
|
11
| ||
IV |
39
|
6
|
13
|
20
|
13
|
15
|
11
| ||
Pathological grade |
0.441
|
0.001
| |||||||
I |
76
|
16
|
19
|
41
|
40
|
25
|
11
| ||
II |
42
|
11
|
13
|
18
|
15
|
13
|
14
| ||
III |
9
|
1
|
4
|
4
|
1
|
1
|
7
| ||
Recurrence |
0.922
|
0.05
| |||||||
No |
83
|
18
|
24
|
41
|
43
|
24
|
16
| ||
Yes |
42
|
9
|
12
|
21
|
13
|
14
|
15
| ||
Lost |
2
|
1
|
0
|
1
|
0
|
1
|
1
| ||
Follow-up |
0.747
|
0.014
| |||||||
Live |
91
|
18
|
27
|
46
|
47
|
27
|
17
| ||
Dead |
33
|
9
|
9
|
15
|
9
|
11
|
13
| ||
Lost |
3
|
1
|
0
|
2
|
0
|
1
|
2
|
Variable | No. | E-cadherin | Vimentin | ||||||
---|---|---|---|---|---|---|---|---|---|
N | L | H | P | N | L | H | P | ||
Sex |
0.554
|
0.024
| |||||||
Male |
74
|
20
|
28
|
26
|
37
|
18
|
19
| ||
Female |
53
|
15
|
24
|
14
|
19
|
8
|
26
| ||
Age (years) |
0.988
|
0.209
| |||||||
≤50 |
26
|
7
|
11
|
8
|
12
|
8
|
6
| ||
>50 |
101
|
28
|
41
|
32
|
44
|
18
|
39
| ||
Tumor location |
0.123
|
0.508
| |||||||
Tongue |
52
|
15
|
21
|
16
|
26
|
9
|
17
| ||
Gingiva |
26
|
6
|
6
|
14
|
12
|
7
|
7
| ||
Buccal mucosa |
31
|
11
|
15
|
5
|
10
|
6
|
15
| ||
Lip |
4
|
1
|
1
|
2
|
1
|
2
|
1
| ||
Floor of the mouth |
2
|
1
|
1
|
0
|
2
|
0
|
0
| ||
Others |
12
|
1
|
8
|
3
|
5
|
2
|
5
| ||
Tumor size |
0.970
|
0.818
| |||||||
T1 |
21
|
5
|
8
|
8
|
10
|
4
|
7
| ||
T2 |
68
|
21
|
28
|
19
|
27
|
16
|
25
| ||
T3 |
26
|
6
|
13
|
7
|
12
|
4
|
10
| ||
T4 |
12
|
3
|
3
|
6
|
7
|
2
|
3
| ||
Lymph node metastasis |
0.236
|
0.003
| |||||||
N0 |
72
|
23
|
25
|
24
|
40
|
15
|
17
| ||
N (+) |
55
|
12
|
27
|
16
|
16
|
11
|
28
| ||
Metastasis |
0.665
|
0.159
| |||||||
M0 |
125
|
35
|
51
|
39
|
56
|
26
|
43
| ||
M (+) |
2
|
0
|
1
|
1
|
0
|
0
|
2
| ||
Clinical stage |
0.236
|
0.043
| |||||||
I |
12
|
3
|
3
|
6
|
9
|
2
|
1
| ||
II |
42
|
17
|
13
|
12
|
21
|
8
|
13
| ||
III |
34
|
5
|
19
|
10
|
13
|
6
|
15
| ||
IV |
39
|
10
|
17
|
12
|
13
|
10
|
16
| ||
Pathological grade |
0.659
|
0.007
| |||||||
I |
76
|
20
|
29
|
27
|
40
|
16
|
20
| ||
II |
42
|
13
|
20
|
9
|
15
|
10
|
17
| ||
III |
9
|
2
|
3
|
4
|
1
|
0
|
8
| ||
Recurrence |
0.460
|
0.01
| |||||||
No |
83
|
22
|
32
|
29
|
43
|
19
|
21
| ||
Yes |
42
|
12
|
20
|
10
|
13
|
6
|
23
| ||
Lost |
2
|
1
|
0
|
1
|
0
|
1
|
1
| ||
Follow-up |
0.301
|
0.001
| |||||||
Live |
91
|
22
|
39
|
30
|
47
|
21
|
23
| ||
Dead |
33
|
12
|
13
|
8
|
9
|
4
|
20
| ||
Lost |
3
|
1
|
0
|
2
|
0
|
1
|
2
|
Association between the expression of CD163, E-cadherin and vimentin
Variable | CD163 |
---|---|
P
| |
E-cadherin
| |
In tumor |
0.040
|
At tumor invasive front |
0.038
|
Vimentin
| |
In tumor |
0.000
|
At tumor invasive front |
0.000
|
Survival analysis
covariate |
P-value | Risk ratio | 95 % CI |
---|---|---|---|
Sex (male, female) | 0.4 | 1.35 | (0.655, 2.043) |
Age (≤50 or younger,>50) | 0.13 | 2.49 | (1.300, 3.680) |
Tumor location Post1 | 0.75 | 1.16 | (0.244, 2.082) |
Tumor location Post2 | 0. 448 | 1.38 | (0.551, 2.201) |
Tumor location Post3 | 0.98 | 0 | (−1242.048, 1242.048) |
Tumor location Post4 | 0.99 | 0 | (−1596.148, 1596.148) |
Tumor location Post5 | 0.77 | 0.8 | (−0.692, 2.288) |
Tumor size (T1-T4) | 0.45 | 1.18 | (0.757, 1.595) |
Nodal metastasis (N0, N(+)) | 0.44 | 1.31 | (0.618, 2.006) |
Metastasis (M0, M(+)) | 0.69 | 0.05 | (0.760, 1.470) |
Clinical stage (I, II, III, IV) | 0.55 | 1.12 | (1.146, 2.200) |
Pathological grade | 0.06 | 1.67 | (0.244, 2.082) |
CD68+ macrophages counts in tumor nest (Low, High) | 0.01 | 3.08 | (2.236, 3.914) |
CD68+ macrophages counts in tumor stroma (Low, High) | 0.3 | 0.69 | (−0.010, 1.390) |
CD163+ macrophages counts in tumor nest (Low, High) | 0.02 | 2.83 | (1.991, 3.669) |
CD163+ macrophages counts in tumor stroma (Low, High) | 0.48 | 0.78 | (0.085, 1.473) |
CD68 expression in tumor (Negative, Low, High) | 0.97 | 1.01 | (0.555, 1.465) |
CD163 expression in tumor (Negative, Low, High) | 0 | 2.2 | (1.735, 2.667) |
E-cadherin expression in tumor (Negative, Low, High) | 0.61 | 0.89 | (0.465, 1.323) |
E-cadherin expression at the invasive front of tumor (Negative, Low, High) | 0.19 | 0.74 | (0.280, 1.1937) |
Vimentin expression in tumor (Negative, Low, High) | 0.01 | 1.79 | (1.373, 2.207) |
Vimentin expression at the invasive front of tumor (Negative, Low, High) | 0 | 1.88 | (1.474, 2.295) |