Supplementary Table 1: Details of the chosen assays for Real Time-RT-PCR analysis. Use the hyperlinks at the Assay-ID numbers to access the homepage of Applied Biosystems with all details of the respective assay.
ELISA measurement of growth factors in tissue homogenates A small part of frozen tissue (approximately 3x3x3 mm) was homogenized in PBS with protease inhibitor (Complete Mini Protease Inhibitor Cocktail Tablets; Roche, Basel, Switzerland). After 2 min centrifugation with 14 000g at 4°C the supernatant (without topping fat layer) was used for ELISA measurement. FGF-2, PDGF-AA, VEGF-D and CXCL12 were determined with Quantikine ELISAs (R&D Systems; Abingdon, United Kingdom), VEGF-A and ANG1 were measured using DuoSet ELISA Kits (R&D Systems) according to the manufacturer’s instructions. The amounts of the respective factors were calculated in relation to the amount of total protein in the homogenates as measured by a protein quantification assay (BCA Protein Assay Kit; Pierce, Rockford, USA).
Supplementary Figures
Figure S1. Schematic diagram of the areas of tissue sampling.
Breast cancer tissue was obtained from the middle ring of the tumor in a macroscopically vital looking area of the tumor by the pathologist directly after surgical removal of the tumor. Additionally, tumor adjacent tissue (distance to tumor 1-2 cm) was collected.
10
ANG-1 10
1
0.1
ANG-2
*
***
10
1
FGF-2
***
1
0.1
HGF
*** relative expression
***
relative expression
***
relative expression
relative expression
10
1
0.01
0.1
Tumor adj acent
Tumor
Healthy tissue
0.01
Tumor adj acent
Tumor
Healthy tissue
10
***
***
PDGF-A
0.1
Tumor adj acent
Tumor
Healthy tissue
Tumor adj acent
Tumor
Healthy tissue
10
PDGF-B
*
**
PDGF-C
**
10
***
***
PDGF-D
1
relative expression
1
relative expression
relative expression
relative expression
10
1
1
0.1
0.1 0.1
Tumor adj acent
Tumor
Healthy tissue
100
Tumor
Healthy tissue
Tumor adj acent
10
VEGF-A
1
Healthy tissue
Tumor adj acent
1
VEGF-C
*** relative expression
10
Tumor
Tumor
Healthy tissue
10
VEGF-B
*** relative expression
relative expression
0.01
Tumor adj acent
*
10
relative expression
0.1
1
***
***
VEGF-D
1
0.1
0.01
0.001 0.1
0.1
Tumor adj acent
Tumor
Healthy tissue
0.1
Tumor adj acent
Tumor
Healthy tissue
Tumor adj acent
Tumor
Healthy tissue
Tumor adj acent
Tumor
Healthy tissue
20
***
**
CXCL1 10
***
***
10
CXCL2
***
***
CXCL3
15
***
***
CXCL5
10
1
0.1
relative expression
1
relative expression
relative expression
relative expression
10 1
0.1
0.1
5 1.0 0.8 0.6 0.4
0.01
0.2 0.01
0.01
0.001
Tumor adj acent
Tumor
Healthy tissue
Tumor adj acent
Tumor
Tumor adj acent
25 20
***
**
CXCL6
100
***
0.0
Healthy tissue
***
Healthy tissue
10
CXCL4
Tumor adj acent
Tumor
TSP-1
*
15
Healthy tissue
TSP-2
**
*
5 5 4 3 2
10
relative expression
10
1
relative expression
10
relative expression
relative expression
Tumor
1
1
0.1
1 0.01
0
*
Healthy tissue
***
CXCL9
1
0.1
0.1
Tumor adj acent
10
relative expression
relative expression
10
Tumor
Tumor
***
Healthy tissue
***
0.1
Tumor adj acent
Tumor
Healthy tissue
***
***
CXCL11
1
0.1
0.001
0.001
0.001
Healthy tissue
0.01
0.01
0.01
Tumor
10
CXCL10
1
0.1
Tumor adj acent
relative expression
Tumor adj acent
Tumor adj acent
Tumor
Healthy tissue
Tumor adj acent
Tumor
Healthy tissue
Tumor adj acent
Tumor
Healthy tissue
Figure S2. Expression of angiogenic, antiangiogenic and lymphangiogenic growth factors and chemokines in primary breast cancer The mRNA expression of angiogenic, antiangiogenic and lymphangiogenic growth factors and chemokines was quantified by Real Time RT-PCR in breast cancer tissues, tumor adjacent tissues and healthy breast tissues and normalized to the mean expression of four different housekeeping genes. Relative expression was calculated with the ΔΔCt method. The horizontal line indicates the median of each group. * p < 0.05, ** p < 0.01 and *** p < 0.001.
100
10
VEGF-B
10
1
** 1
0.1
Normal
RCC
RCC 100
0.1
** 1
0.1
Normal
RCC
ANG-2 relative expression
1
RCC
PDGF-D relative expression
relative expression
Normal
10
PDGF-C
**
1
0.1
Normal
10
HGF
* relative expression
relative expression
relative expression
**
VEGF-A
10
1
0.1
Normal
RCC
Normal
RCC
Supplementary Figure S3. Expression of angiogenic growth factors in clear cell renal carcinoma The mRNA expression of angiogenic growth factors was quantified by Real Time RT-PCR in clear cell renal carcinoma samples (RCC) and normal kidney tissues and normalized to the mean expression of four different housekeeping genes. Relative expression was calculated with the ΔΔCt method and normalized to the calibrator of the breast cancer samples. The horizontal line indicates the median of each group. * p < 0.05 and ** p < 0.01 with two-sided paired t-test.
0.01
0.1
0.01
Tumor adjacent
Tumor
10
1
Tumor adjacent
Tumor
Tumor adjacent
Tumor
1 10
*
ANG-1
1
0.1
VEGF-D (ng/mg total protein)
0.1
1
PDGF-AA
100
Angiopoietin-1 (ng/mg total protein)
1
FGF-2
PGDF-AA (pg/mg total protein)
VEGF-A
FGF basic (ng/mg total protein)
VEGF-A (ng/mg total protein)
10
**
VEGF-D
0.1
0.01
0.001
Tumor adjacent
Tumor
Tumor adjacent
Tumor
Supplementary Figure S4. Quantification of angiogenic and lymphangiogenic growth factors on protein level The protein amount of angiogenic and lymphangiogenic factors were determined by ELISA in homogenates of breast cancer tissues and tumor adjacent tissues of the same donors and normalized to the amount of total protein as determined by a protein quantification assay. The horizontal line indicates the median of each group. * p < 0.05 and ** p < 0.01.
10
10
1
1
10
VEGF-R3
10
***
*** relative expression
***
NRP-1
***
***
1
NRP-2
** relative expression
VEGF-R2
*** relative expression
relative expression
10
relative expression
VEGF-R1
1
1
0.1 0.1 0.1
Healthy tissue
0.1
Tumor adjacent
Tumor
Healthy tissue
Tumor adjacent
10
***
1
PDGF-Rβ
***
*** relative expression
***
0.1
Tumor adjacent
Tumor
Healthy tissue
Tumor adjacent
1
Tumor
Healthy tissue
10
PDGF-Rα
relative expression
relative expression
***
Healthy tissue
10
TIE-2 10
Tumor
***
***
1
CXCR2
100
CXCR3
*** relative expression
Tumor
relative expression
Tumor adjacent
10
1
1
0.1
0.1 0.1
0.1 0.1
Tumor adjacent
Tumor
Healthy tissue
Tumor adjacent
Tumor
Healthy tissue
0.01
Tumor adjacent
Tumor
Healthy tissue
Tumor adjacent
Tumor
Healthy tissue
Tumor adjacent
Tumor
Healthy tissue
Supplementary Figure S5. Expression of receptors for angiogenic and lymphangiogenic factors in primary breast cancer The mRNA expression of angiogenic and lymphangiogenic receptors was quantified by Real Time RT-PCR in breast cancer tissues, tumor adjacent tissues and healthy breast tissues and normalized to the mean expression of four different housekeeping genes. Relative expression was calculated with the ΔΔCt method. The horizontal line indicates the median of each group. ** p < 0.01 and *** p < 0.001.
10
10
***
**
***
10
***
***
***
10
***
***
1
0.1
relative expression
0.1
relative expression
relative expression
relative expression
1 1
0.1
0.01
1
0.1
0.001
CD146 0.01
VE-Cadherin 0.01
Tumor adjacent
Tumor
Healthy tissue
LYVE1
0.01
PROX1
0.0001
Tumor adjacent
Tumor
Healthy tissue
Tumor adjacent
Tumor
Healthy tissue
Tumor adjacent
Tumor
Healthy tissue
Supplementary Figure S6. Expression of markers for endothelial and lymphatic endothelial cells in primary breast cancer The mRNA expression of markers for endothelial cells (CD146, VE-Cadherin) and lymphatic endothelial cells (LYVE1, PROX1) was quantified by Real Time RT-PCR in breast cancer tissues, tumor adjacent tissues and healthy breast tissues and normalized to the mean expression of four different housekeeping genes. Relative expression was calculated with the ΔΔCt method. The horizontal line indicates the median of each group. ** p < 0.01 and *** p < 0.001.
1
*
1
LYVE-1
p = 0.09
relative expression
0.1
10
VE-Cadherin
relative expression
CD146
relative expression
relative expression
1
10
p = 0.05
0.1
0.01
PROX-1
1
0.1
0.1 0.001
0.01
Grade 1
Grade 3
Grade 1
Grade 3
0.01
Grade 1
Grade 3
Grade 1
Grade 3
Supplementary Figure S7. Comparison of expression levels of markers for endothelial and lymphatic endothelial cells in primary breast cancer Relative expression data of markers for endothelial and lymphatic endothelial cells were analyzed according to the histopathologic grading of the primary breast tumors. The horizontal line indicates the median of each group. * p < 0.05.
*
* relative expression
relative expression
1
0.1
1
CD146 0.01
VE-Cadherin 0.1
Normal
RCC
Normal
RCC
Supplementary Figure S8. Expression of markers for endothelial cells in clear cell renal carcinoma The mRNA expression of markers for endothelial cells was quantified by Real Time RT-PCR in clear cell renal carcinoma samples (RCC) and normal kidney tissues and normalized to the mean expression of four different housekeeping genes. Relative expression was calculated with the ΔΔCt method and normalized to the calibrator of the breast cancer samples. The horizontal line indicates the median of each group. * p < 0.05 with two-sided paired t-test.
