Supplementary Data
Materials and Methods
ELISA techniques
IL-8, VEGF-A and P-selectin levels were quantified using corresponding human DuoSet
ELISA (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions.
Release of VWF was quantified with a sandwich ELISA using a polyclonal rabbit anti-VWF
antibody (Dako, Hamburg, Germany) and a polyclonal rabbit peroxidase-labeled anti-human
VWF antibody (Dako, Hamburg, Germany). A standard curve was generated using Standard
Human Plasma (Behring, Marburg, Germany) with a defined VWF content (2nd International
Standard 87/718, National Institute for Biological Standards and Control, London).
In vitro Angiogenesis Assay
For in vitro angiogenesis studies, the ibidi tube formation assay (ibidi, Germany) was used
according to the instructions of the manufacturer utilizing ibidi μ-slides. Briefly, 10,000
HUVECs were suspended in 25 µl culture medium supplemented with 25µl tumor cell
conditioned medium. The cell suspension (50μl) was applied to the Matrigel-coated (BD
Biosciences, USA) well of each μ-slide and incubated in a humidified chamber at 37°C. Tube
formation was recorded 4 hours after cell seeding. The total tube length was quantified with
ImageJ software.
RNA preparation and qRT-PCR
Total RNA from UCB cells was extracted using the RNeasy mini kit (Qiagen, Hilden,
Germany) and cDNA was synthesized from 1µg of total RNA per sample with the Reverse
Transcription System from Promega according to the manufacturers’ protocols. To quantify
mRNA transcript levels, quantitative real-time polymerase chain reaction (qRT-PCR) was
performed applying the GoTaq® qPCR Master Mix (Promega, Heidelberg, Germany). The
following primer pairs were used: E-selectin ligand 1 (
ESL-1): sense 5’-GAG TGC AAA AAG
CGC CTC AA-3’, antisense 5’-CTT GGT GAT CCG TCC ACA CA-3′;
protease activated receptor 1 (PAR-1): sense 5’-CCT GCT TCA GTC TGT GCG G-3′, antisense 5’-CTG GTC
AAA TAT CCG GAG GCA-3′; prothrombin: sense 5’-TGT GAA CAT CAC CCG GTC
AG-3′, antisense 5’-ATT CAC ACT GGA GCC TTC GG-3′; selectin P ligand-1 (
SELPLG-1): sense
5’-GGC CAG TGG TCT AGA AGG AG-3′, antisense 5’-CAG AGG AGT GGT GTC AGT
GC-3′; TF: sense 5’-CCC AAA CCC GTC AAT CAA GTC-3′, antisense 5’-CCA AGT ACG
TCT GCT TCA CAT-3′; PDPN: sense 5’-AGG TGT CAG CTC TGC TCT TC-3′, antisense
5’-CCA CCA GAG TTG TCA AGC CA-3′. Gene expression was normalized to ß-actin
mRNA levels for each sample (primer pair AGA AAA TCT GGC ACC ACA CC-3′ and
5′-CCA TCT CTT GCT CGA AGT CC-3′).
Supplementary Figure 1: Schematic overview of the potential cross talk between bladder cancer cells, the endothelium and the coagulation system. Bladder cancer cells may expose several membrane anchored proteins potentially mediating the binding to the endothelium and/or platelets (e.g. selectin P ligand-1 (SELPLG), PDPN) and/or inducing the plasmatic coagulation via e.g. TF. Moreover, tumor cells are known to secrete factors promoting the procoagulant activation of platelets and endothelial cells (e.g. VEGF-A). Especially the secretion of VWF from endothelial cells has been associated to tumor progression in different entities.
Supplementary Figure 2: Expression of procoagulant factors and ability of T24 cells to produce thrombin. (A) Expression of candidate genes in RT4, T24, RT112 and UROtsa cells which are associated with the direct binding of cancer cells to platelets and endothelial cells (selectin P ligand-1 (SELPLG), E-selectin ligand 1 (ESL-1)) or with the cross-talk between cancer cells and the plasmatic coagulation (protease activated receptor 1 (PAR-1), prothrombin). Data were normalized to ß-Actin and mRNA expression is shown on a logarithmic scale. Data are presented as mean ± SD, n=3. (B) Thrombin generation assay to measure the ability of T24 cell extracts to convert plasmatic prothrombin into thrombin. The involvement of TF was confirmed by blocking T24 cell derived TF with a blocking antibody (+ anti TF, 10µg/ml). The specificity of the assay was tested by adding the thrombin inhibitor hirudin (+ hirudin, 10U/ml). The red dashed line indicates the plasma baseline. Data are presented as mean ± SD, n=3. * P < 0.05, student’s t test. (C) Surface expression of TF and TM on UCB cells was measured by flow cytometry.
Supplementary Figure 3: UCB cells trigger angiogenesis. (A) In vitro Matrigel angiogenesis assays were performed with HUVECs grown in 50% culture medium supplemented with 50% tumor cell supernatant. Total tube length was quantified after 4h and normalized to non-conditioned medium (control). Representative microscopic images show tube formation after 4h of incubation time with the control or RT4 supernatant. Data are presented as mean ± SD, n=3. * P < 0.05, student’s t test. (B) Representative immunofluorescence of HUVECs treated with medium conditioned by RT4, T24 cell or control. VWF was stained in green, CD31 was stained in red, and nuclei were stained in blue. Scale bars correspond to 50µm.
Supplementary Figure 4: VWF mediated blood vessel occlusion in peritumoral tissue. Representative microscopic images of bladder tissue (healthy control tissue, low grade and high grade tumor tissue) in which VWF was stained by immunohistochemistry. Scale bars correspond to 100 µm.
Supplementary Table 1. Correlation of the overall patients’ survival with the expression of the indicated genes.
Overall
survival living deceased
P-value MMP9 1141 6087 0.002 VEGF-A 5845 4573 0.004 PAR-1 609 736 0.004 ADAMTS13 108 79 0.008 ENTPD1 958 1126 0.01 HPSE 75 89 0.01 PAI-1 5556 7178 0.04 uPAR 1148 1284 0.04 VWF 2583 2955 0.08 TF 1668 1736 0.1 PROCR 657 571 0.1 TFPI 673 772 0.3 PDPN 1001 981 0.4 P-selectin 118 123 0.4 FII 22 17 0.4 TIMP1 9279 10224 0.6 THBD 2616 2313 0.9
Supplementary Video 1. Supernatants of UROtsa cells failed to induce endothelial activation. Calcein blue labeled HUVECs (blue) were perfused with calcein red-orange labeled platelets (red) resuspended in cell culture medium conditioned by UROtsa cells.
Supplementary Video 2. Supernatants of RT4 cells induced endothelial activation. Calcein blue labeled HUVECs (blue) were perfused with calcein red-orange labeled platelets (red) resuspended in cell culture medium conditioned by RT4 cells.
