40130164

Table S1: ACC Patients' and tumour characteristics divided by H-scores of LEF-1, CTNNB1, GR and the lymphocyte infiltration (CD3, CD4, CD8, FoxP3).

Parameter	Immunohistochemistry cohort
Patients n	59
Age at diagnosis (range)	47 (19-75)
Sex
Female	37 (63 %)
Male	20 (34 %)
unknown	2 (3 %)
ENSAT Stage (I-IV)
I	4 (7 %)
	27 (46 %)
	15 (25 %)
IV	11 (19 %)
unknown	2 (3 %)
Hormone status
Inactive	14 (24 %)
GC	15 (26 %)
A	3 (5 %)
multiple	10 (17 %)
unknown	16 (28 %)
R-status (ENSAT I-III) 0 1	unknown
2
Ki-67 index n (%)
<10	17 (28 %)
≥10	29 (50 %)
unknown	13 (22 %)

Colon LEF-1

50 M

2 mm

50 UM

Figure S1. LEF-1 protein expression by immunohistochemistry in healthy colon tissue

Consecutive tissue slices were stained for LEF-1. LEF1 expression is clearly expressed predominantly in the goblet cells of the crypt with exclusive nuclear expression of the epithelial cells.

Figure S2, related to Figure 4 and 5. Correlation of GR and immune infiltration levels in the tumour. Protein expression levels of GR as well es CD3+ T cells (A), CD4 + T cells (B), CD8+ T cells (C), and FoxP3 + T cells (D) levels were divided into quartiles according to expression level and visualized in a stacked bar graph, respectively. The white colour is associated with low number of immune cells, the green is associated with intermediate number of immune cells and the lilac is associated with high levels of immune cells. Pearson correlation was used to show the linear relationship between the expression of GR (H-scores) and the immune cell infiltration (HPF), respectively. Testing for significance fisher's exact test was used.

A

CD3+ T cells

30

Number of samples [n]

p = 0.8050

CD3+ T cells (number/HPF)

150

Correlation of H scores

20

148-

R2=0.0107

146

144

100

10

50

0

0

low

intermediate

high

0

100

200

300

GR

GR

C

CD8+ T cells

30

Number of samples [n]

p = 0.8698

20

CD8+ T cells (number/HPF)

150-

Correlation of H scores R2=0.0089

140-

130

120

60

10

40-

20

0

0

low

intermediate

high

0

100

200

300

GR

GR

☐ low

☒ intermediate

☒ high

B

CD4+ T cells

30

p = 0.3439

Number of samples [n]

30-

Correlation of H scores

20

R2=0.0019

20

10.

CD4+ T cells (number/HPF)

10-

0

0

0

100

200

300

low

intermediate

high

GR

GR

D

FoxP3+ T cells

30

Number of samples [n]

p = 0.4645

FoxP3+ T cells (number/HPF)

30-

Correlation of H scores R2=0.0071

20

25

20-

15

10

10

6

4.

2.

0

0

low

intermediate

high

0

100

200

300

GR

GR

Figure S3, related to figure 6. Correlation of LEF-1 (A), CTNNB1 (B), and GR (C) with the hormone status, respectively. Hormone status of tumour (inactive, glucocorticoid excess (GC) or other (GCA, A, GCAO, multiple) were correlated and visualized as a stacked bar graph with the protein expression levels of LEF-1, CTNNB1 or GR, respectively. For statistical testing fisher's exact test was used.

A

LEF-1

CTNNB1

15

15

Number of samples [n]

p = 0.3562

Number of samples [n]

p = 0.4788

10

B

10

5

5

0

0

inactive GCA other

inactive GCA other

☐ low

☒ intermediate

☒ high

C

GR

10

Number of samples [n]

8

p = 1

6

4

2

0

inactive GCA other

Table S2: Numbers of T cell subtypes of CD3+, CD4+, CD8+, and FoxP3+ T cells and LEF-1 protein expression divided into tertiles .

CD3 LEF-1	low	intermediate	high
low	7	6	8
intermediate	8	5	6
high	6	9	7

CD4 LEF-1	low	intermediate	high
low	8	5	8
intermediate	9	5	5
high	5	9	8

CD8 LEF-1	low	intermediate	high
low	7	7	7
intermediate	8	7	4
high	6	5	11

FoxP3 LEF-1	low	intermediate	high
low	9	2	10
intermediate	7	2	10
high	8	5	9

Table S3: Numbers of T cell subtypes of CD3+, CD4+, CD8+, and FoxP3+ T cells and CTNNB1 protein expression divided into tertiles .

CD3 CTNNB1	low	intermediate	high
low	6	2	13
intermediate	7	4	5
high	8	14	3

CD4 CTNNB1	low	intermediate	high
low	8	3	10
intermediate	6	4	6
high	8	12	5

CD8 CTNNB1	low	intermediate	high
low	6	5	10
intermediate	7	5	4
high	8	9	8

FoxP3 CTNNB1	low	intermediate	high
low	9	1	11
intermediate	5	0	11
high	10	8	7

Table S4: Numbers of T cell subtypes of CD3+, CD4+, CD8+, and FoxP3+ T cells and CTNNB1 protein expression divided into tertiles.

CD3 GR	low	intermediate	high
low	8	6	5
intermediate	6	5	8
high	7	9	8

CD4 GR	low	intermediate	high
low	9	2	8
intermediate	5	4	10
high	8	1	15

CD8 GR	low	intermediate	high
low	8	5	6
intermediate	6	7	6
high	7	7	10

FoxP3 GR	low	intermediate	high
low	10	1	8
intermediate	5	4	10
high	9	4	11

A

300

GR expression (H-score)

200

100

0

0

50

100

150

200

250

LEF-1 expression (H-score)

B

Figure S4. Correlation of GR expression (H-score) with LEF-1 and CTNNB1 H-score, respectively. GR expression (H-score) was correlated with the protein expression levels of LEF-1 expression (R2=0.0006) and CTNNB1 (R2=0.0040), respectively. For statistical testing simple linear regression was used.

300

GR expression (H-score)

200

100

0

0

100

200

300

CTNNB1 expression (H-score)

LEF-1

Figure S5. Correlation of ENSAT stage with LEF-1 (low, intermediate, high). ENSAT stage was correlated and visualized as a stacked bar graph with the protein expression levels of LEF-1 divided into low, intermediate and high. For statistical testing fisher's exact test was used ..

30

p = 0.4013

Number of samples [n]

20

10

0

1

2

3

4

ENSAT stage

☐ low

☒ intermediate

☒ high

A

FKBP5 low vs. high

Figure S6: Expressional differences of FKBP5 at RNA level. Expression of 354 immune-related genes were measured and quantified by Nanostring nCounter from FFPE tissue to analyse expressional differences between FKBP5-high and -low tumours (A) as well es GC-secreting vs. hormonal inactive tumours (B-D). Significant genes (p<0.05-0.01) are marked in green and highly significant genes (p<0.01) are marked in purple. Analysis performed by Nanostring nSolver® and Prism. Adjusted p value was calculated by Benjamini-Yekutieli.

15

FKBP5

log10(p-value)

10

5

MMP9

SLC26A11

IL1R1

CD206

0

-3

-2

-1

0

1

2

3

Log2 fold change

B

C

0.7472

0.2020

2500

2500

normalized counts

2000

normalized counts

2000

1500

:

1500

:

1000

1000

500

500

0

GC

0

inactive

GCA inactive

D

0.3617

2500

normalized counts

2000

1500

:

1000

500

0

GC+GCA

inactive