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33
8.
Tables
Table 1. Primer sequence and conditions used for the real-time RT-PCR analysis
34
Table 2. Relationship between MAFB+ cells density and the Clinicopathological
features in Nodal involvement-negative (n = 60) and nodal involvement-positive (n
= 60) groups of patients with non-metastatic lung adenocarcinoma.
Nodal
involvement
(N-)
(n = 60)
Nodal
involvement
(N+)
(n = 60)
40
34
20
26
Male
27
29
Female
33
31
Never
23
22
Former or current
37
38
I (n = 57)
57
II+III (n = 63)
60
Low
24
Mid+High
Cancer Recurrence
36
54
Positive
13
42
Negative
47
18
Ly(–)
52
19
Ly(+)
41
V(–)
42
12
V(+)
18
48
PL(–)
43
24
PL(+)
17
36
Variables
P value of
Fisher's exact test
Age (yr.)
<70
≥70
Gender
0.3479
0.8549
Smoking history
Clinical Stage
<0.0001
MAFB positive cells density
0.0003
<0.0001
Lymphatic permeation
<0.0001
Vessel invasion
<0.0001
Pleural infiltration
0.0009
The correlations were evaluated by Fisher’s exact test, *p < 0.05, **p < 0.001
35
Table 3. Correlation between MAFB+ cell density and the clinicopathological factors
in non-metastatic lung adenocarcinoma.
Variables
Low-MAFB+ (≤0.005)
Mid-MAFB+ (0.006–0.016)
No of Case n = 30 (25%)
No of Case n = 59 (49%)
<70
≥70
20 (67%)
10 (33%)
Male
Female
15 (50%)
15 (50%)
Never
Former or current
12 (40%)
18 (60%)
I (n = 57)
II + III (n = 63)
24 (42%) ***
6 (10%) ***
Negative
Positive
22 (73%) *
8 (27%) *
Negative (N−)
Positive (N+)
24 (80%) ***
6 (20%) ***
Ly(–)
Ly(+)
26 (87%) ***
4 (13%) ***
V(–)
V(+)
23 (77%) ***
7 (23%) ***
PL(–)
PL(+)
23 (77%) *
7 (23%) *
Age (yr)
39 (66%)
20 (34%)
Gender
21 (36%) *
38 (64%) *
Smoking history
25 (42%)
34 (58%)
Clinical Stage
30 (53%)
29 (46%)
Cancer Recurrence
33 (56%)
26 (44%)
Nodal involvement
32 (54%)
27 (46%)
Lymphatic permeation
34 (58%)
25 (42%)
Vessel invasion
26 (44%)
33 (56%)
Pleural infiltration
31 (53%)
28 (47%)
High-MAFB+ (≥0.017)
P value of
Fisher’s
No of Case n = 31 (26%)
exact test
15 (48%)
16 (52%)
0.2249
20 (65%) *
11 (35%) *
0.0302
8 (26%)
23 (74%)
0.3046
3 (5%) ***
28 (44%) ***
<0.001
10 (32%) *
21 (68%) *
0.006
4 (13%) ***
27 (87%) ***
<0.0001
12 (39%) **
19 (61%) **
<0.001
5 (16%) ***
26 (84%) ***
<0.0001
13 (42%)
18 (58%)
0.019
According to density of cells expressing MAFB, 120 lung adenocarcinoma patients
with stages I, II, and III were grouped into low-MAFB+, mid-MAFB+, and highMAFB+ cell density groups. Correlation between MAFB expression and clinical
factors among groups was recorded and statistically analyzed using Fisher’s exact
test, * p < 0.05, ** p < 0.001, *** p < 0.0001.
36
Table 4. Univariate analysis of disease-free survival and overall survival in nonmetastatic lung adenocarcinoma stages.
Univariate Analysis
Disease-Free Survival
Hr (95% Ci)
p Value
MAFB (low versus
mid)
2.998 (0.9882–
0.0828
12.95)
MAFB (low versus
high)
7.423 (2.393–
0.0018
32.44)
Gender (male versus 1.856 (0.9236–
0.0861
female)
3.847)
Smoking (Yes versus
No)
3.115 (1.369–
0.0122
8.376)
Age (<70 versus ≥70)
1.010 (0.9697–
0.6397
1.058)
Pstage (I versus II + III)
7.500 (3.194–
<0.0001
20.72)
Nodal involvement
(Yes versus No)
6.497 (2.880–
<0.0001
16.71)
Lymphatic invasion
(Yes versus No)
3.072 (1.522–
6.398)
Vessel invasion (Yes
versus No)
5.444 (2.377–
0.0002
14.71)
0.002
Pleural infiltration (Yes 2.740 (1.350–
0.0062
versus No)
5.803)
Multivariate Analysis
Disease-Free
Overall Survival
Overall Survival
Survival
Hr (95% Ci) p Value Hr (95% Ci) p Value Hr (95% Ci) p Value
3.039
1.665
1.966 (0.5985–
(1.001–
0.079
(0.5008– 0.4468
0.3086
8.859)
13.13)
7.565)
8.105
1.773
2.001 (0.5587–
(2.620–
0.0011 (0.4873– 0.4230
0.3263
9.646)
35.36)
8.669)
1.826
(0.9081–
0.095
3.790)
0.3208
3.106
3.117
2.989 (1.231–
0.0123
(1290–
0.0182
0.0235
(1.368–
8.437)
8.724)
8.390)
1.013
(0.9715–
0.5578
1.062)
8.464
(3.614–
<0.0001
23.24)
7.723
4.382
5.304 (1.845–
(3.411–
<0.0001 (1.555– 0.0078
0.0032
17.12)
19.85)
13.88)
3.294
1.049
1.094 (0.4918–
(1.626–
0.0011 (0.4812– 0.9064
0.8285
2.522)
6.887)
2.384)
4.875
1.843
1.370 (0.4730–
(2.136–
0.0005 (0.6468– 0.2753
0.5786
4.458)
13.14)
5.939)
2.644
1.228
1.216 (0.5498–
(1.306–
0.008
(0.5665– 0.6108
0.6363
2.805)
5.585)
2.793)
Statistically significant differences between groups were determined using Cox
proportional hazard model (p < 0.05). HR, hazard ratio; CI, confidence interval.
37
1.
Figures
Figure 1. Classification of Lung cancers.
Created with BioRender.com
Lung cancers are classified into small cell and non-small cell types, with the latter
group including adenocarcinoma, squamous cell carcinoma and large cell
undifferentiated carcinoma.
38
Figure 2. Generating Mafb-GFP expressing mice.
Created with BioRender.com
Homologous recombination strategy for GFP reporter gene knock-in to the Mafb
locus (A) Mafb targeting vector construct (B) heterozygous (+/−Neo) mice were
crossed to Ayu1-Cre mice to remove the Neo cassette.
39
Figure 3. Morphometrical quantification of MAFB-positive macrophages in
human lung cancer.
100 μm
100 μm
100 μm
100 μm
100 μm
100 μm
40
Immunohistochemical analysis of MAFB expression in lung adenocarcinoma
showing metastasis potential. The left panel shows (A) Lung adenocarcinoma
samples with negative metastasis, (B) the calculation of total tissue area in yellow
color, and (C) the number of MAFB positive area in red color. The right panel shows
(D) Lung adenocarcinomas samples with positive metastasis, (E) the calculation of
total tissue area in yellow color, and (F) the number of MAFB positive area in red
color. Scale bar 100 μm.
41
Figure 4. Stages of Lung cancers according to TNM staging system.
There are 4 stages of Lung cancer according to TNM staging (which accounts for T;
primary tumor size, N; nodal involvement, and M; metastasis).
42
Figure 5. Mafbgfp/+ in bright or dark field, and Immunofluorescence staining.
(A) Whole-mount observation of LLC tumor harvested from both WT and Mafbgfp/+
in bright or dark field 2 weeks after s/c injection and (B) immunohistochemical
analysis of LLC tumors using anti-F4/80 and anti-GFP antibodies to check merging
expression of Mafbgfp/+ within F4/80+ cells.
43
Figure 6. Clustering strategy of myeloid series.
(A) 0-33 clusters were analyzed (B) myeloid series (Cluster 4, 5, 6, 25, 30 and 33)
were extracted by using myeloid makers, LYZ, MARCO, CD68, FCGR3A.
44
Figure 7. Single-cell RNA sequencing (scRNA-seq) analysis obtained from 44
patients with treatment-naive lung adenocarcinoma.
45
Single-cell RNA raw data included normal lung tissue (n = 11), tumor tissue (stage
I and III, n = 7), and advanced tumor tissue (stage IV, n = 4). Raw data were
downloaded and processed using sctransform function in Seurat (v3). (A) Identified
17 clusters of the myeloid population. (B) monocytes (Mo), alveolar macrophages
(AM), macrophages lineage (ML), and dendritic cells (DC) cluster distribution in
normal lung tissues, tumors, and advanced tumors. (C,D) CD68 expression pattern
in normal lung tissues, tumor, and advanced tumor. (E,F) CD204 expression pattern
in normal lung tissues, tumor, and advanced tumor. (G,H) MAFB expression pattern
in normal lung tissues, tumor, and advanced tumor. (I) Heatmap analysis of the
expression of MAFB, CD68, and CD204 in AM. (J) Heatmap analysis of the
expression of MAFB, CD68, and CD204 in monocytes. (K) Heatmap analysis of the
expression of MAFB, CD68, and CD204 in macrophage lineage.
46
Figure 8. Analysis of myeloid cluster.
(A) A heatmap of marker genes Expression for Alveolar macrophage, Macrophage
lineage and Monocyte. (B) UMAP of the macrophage marker CCR2 expression in
normal lung, tumor, and advanced Tumor. (C) A heatmap of other TAM markers,
CD206, CD163 pattern of expression.
47
Figure 9. Grouping non-metastatic lung adenocarcinoma tissue according to
MAFB+ cells density.
50 μm
48
50 μm
(A) Representative data of immunohistochemical analysis of human lung
adenocarcinomas with anti-human MAFB. (B) MAFB-positive area relative to
tissue area (MAFB/tissue area) was morphometrically quantified. Tissue samples
were grouped into top 25% (high-MAFB+ group, MAFB expression area/tissue area
= 0–0.005 (n = 30)), 25–50% (mid-MAFB+ group, MAFB expression area/tissue
area = 0.006–0.016 (n = 59)), and bottom 25% (low-MAFB+ group, MAFB
expression area/tissue area = 0.017–0.121 (n = 31)). (C) MAFB expression in three
groups was tested for correlation to tumor sizes. Data are presented as means ± SEM;
data is considered significant at * p < 0.05, ** p< 0.01.
49
Figure 10. OS and DFS of low-, mid-, and high- in MAFB+ cells Kaplan–Meier
analysis.
(A) overall survival and (B) disease-free survival of the three groups: low-MAFB+,
mid-MAFB+, and high-MAFB+. Difference in survival was compared using log-rank
test. Pearson correlation analysis was performed between MAFB expression and (C)
survival time (R score: −0.366, p = 0.000043) and (D) disease-free survival (R score:
−0.378, p = 0.000023). Data are presented as means ± SEM; data is considered
significant at * p < 0.05; ** p < 0.01.
50
Figure 11. Kaplan–Meier analysis for OS and DFS of patients with or without
nodal involvement.
(A) Overall survival of patients with or without nodal involvement. (B) Disease free
survival with or without nodal involvement,**p<0.001.
51
Figure 12. Smoking Index and tumor diameter correlation.
Smoking Index and tumor diameter were not corelated with MAFB expression
Pearson Correlation analysis was performed between MAFB expression and
Smoking index (R score: -0.043, p = 0.6)
52
Figure 13. Overall survival (OS) and Disease-free survival (DFS) of patients of
all, male, and female smokers and non-smokers.
(A) Overall survival (OS) and Disease-free survival (DFS) of patients of smokers
and non-smokers.
53
(B) OS and DFS of female patients of smokers and non-smokers. (C) OS and DFS
of male patients of smokers and non-smokers. data is considered significant at * p <
0.05; ** p < 0.01.
54
Figure 14. OS and DFS analysis in smokers and non-smoker patients
Kaplan–Meier analysis of (A) the overall survival and disease-free survival of the
low-MAFB+, mid-MAFB+, and high-MAFB+ in the total smoking and non-smoking
patients; (B) the overall survival and disease-free survival of the low-MAFB+, midMAFB+, and high-MAFB+ in female smoker and non-smoker groups; and
55
(C) the overall survival and disease-free survival of the low-MAFB+, mid-MAFB+,
and high-MAFB+ in male smoker and non-smoker groups. Difference in survival
was compared using log-rank test. *, p < 0.05; **, p < 0.01.
56
Graphical abstract
Created with BioRender.com
57
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