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1 Supplementary Figure S1 Cancer cells in vitro Pretreatment with Control IgG (18h) Pretreatment with anti-u-par (18h) Acid Wash/Pretreatment with Control IgG (18h) Acid Wash/Pretreatment with anti-u-par (18h) Adhe esion (numbe er of cells) D Omental Culture Matrigel
2 Supplementary Figure S2 Cancer cells in vitro Proliferation Rate of Pr roliferation (Fold-chang ge) Control IgG anti-u-par
3 Supplementary Figure S3 CaOV3 tumor in vivo A B Relative mrna expression ACTG2/ hgapd DH +hgus Relative mrna expression ACTG2/ hgapd DH +hgus CaOV3 Cancer cells in vitro Control IgG anti-u-par Control IgG anti-u-par
4 Supplemental Methods Acid wash. To dissociate endogenously produced upa from upar, the CaOV3, HeyA8, and SKOV3ip1 ovarian cancer cells cells and/or the 3D omental culture were subjected to a mild acid wash. The cells were treated in a in phosphate-buffered saline (PBS) at 4 C with: 1 mm HEPES ph 7.4 for 2 minutes, 5 mm glycine-hcl and 1 mm NaCl ph 3. for 2 minutes, and.5 M HEPES and.1 M NaCl, ph 7.5 for 2 minutes. The cells were washed three times with full growth media, incubated with antibodies for 18 hours, and adhesion assay performed to Matrigel and 3D omental culture as previously described in Materials and Methods Section. Proliferation. CaOV3, HeyA8, and SKOV3ip1 ovarian cancer cells (2x1 4 ) were treated with control mouse IgG or u-par antibody (ATN-658) for 4 days in serum-free media. The proliferation of ovarian cancer cells was measured using a fluorescence dye (CyQuant Molecular Probes, Eugene, OR) as reported previously [1]. i
5 Supplemental Figure Legends Fig. S1. Anti-u-PAR treatment of in vitro acid-washed ovarian cancer cells reduces cell adhesion. Cells were acid-washed to remove all u-par bound upa and pretreated for 18 hours with an u-par antibody, and adhesion after 3 minutes to the 3D omental culture or Matrigel was measured. Fig. S2. Anti-u-PAR treatment has no effect on ovarian cancer cell proliferation. SKOV3ip1, HeyA8 and CaOV3 cells were plated in complete medium with either 2ug/ml control IgG or u-par antibody and assayed at 72 hours. Each bar is the mean +/- standard deviation of n=5, and is representative of three independent experiments. No significance is found when a student s t-test is used to compare control to u-par antibody treatment in each graph. Fig. S3. Treatment with u-par antibody decreases ACTG2 expression and production in vivo and in vitro. Anti-u-PAR treatment significantly reduced ACTG2 mrna (real-time RT-PCR) levels in xenograft ovarian cancer tumors (A) and in ovarian cancer cells cultured on plastic (B). Bar graph show results (n=5, in vitro) representative of three independent experiments and shows results of three animals (in vivo) +/- standard deviation. A student s t-test compares control to u-par antibody treatment in each graph. indicates p<.1. ii
6 Reference List [1] Kaur S, Kenny HA, Jagadeeswaran S, et β3-integrin expression on tumor cells inhibits tumor progression, reduces metastasis, and is associated with a favorable prognosis in patients with ovarian cancer. Am J Pathol 29;175: iii
7 Supplementary Table S1. Summary of current literature reporting u-par positive ovarian cancer tumors. Authors Journal Year Reference Brief Description of Paper Method used to Detect u-par Number of Patients with Ovarian Cancer u-par Positive Patients Casslen et Schmalfeldt B et Chambers S. et Tecimer C et Eur J of Can Can Res Int J of Gyn Can Int J of Gyn Can Investigated the binding of 125 I- labelled upa to cell membranes of ovarian tumors Investigated the upa, u-par, PAI-1 and PAI-2 concentrations in primary tumors and tumor-infiltrated omentum and retroperitoneal lymph nodes of ovarian cancer patients Investigated the prognostic value of upa or u-par expression in ovarian cancer epithelium and stroma and relationship of upa/u-par and CSF- 1/CSF-1 receptor expression Investigated the prognostic of upa, u- PAR and PAI-1 levels in extracts of ovarian cancer tissues Receptor assayscatchard analysis (I 125 -upa) Immunohistochemistry n=1 n=33 FIGO III-IV omental metastases and primary tumors n=13; FIGO I and II (18 primary + 4 metastatic tumors); FIGO III and IV (85 primary and 77 metastatic tumors) n=43 1/1 (1%) 33/33 (1%) 33/13 (32%); stage I+II: primary 6/18 (33%), metastatic 2/4 (5%); stage III and IV: primary 27/85 (32%), metastatic 17/77 (22%) 41/43 (95%) median 4.7 (.-71.) ; mean 7.3+/ Borgfeldt C et Borgfeldt C et Int J Can Int J Can Investigated mrna levels for upa, u- PAR, and PAI-1 in serous ovarian tumors Investigated the prognostic value of upa and u-par in homogenates of ovarian tumors RT-PCR n=1 n= 67; 62 primary and 5 metastatic tumors (51 invasive) 1/1 (1%) primary 49/62 (79%) and metastatic 5/5 (1%) Mabrouk RA et Clin Biochem Investigated the role of u-par and c- erbb-2 in breast and ovarian cancer n= 2 primary tumors and benign tissue primary 13/2 (65%), benign /2 (%) Wang L et Kenny HA et Gyn Oncol Current Study exact numbers not published NA Investigated the distribution of upa and u-par in epithelial ovarian cancer patients, primary vs metastasis, and relationship of upa/u-par and MMP expression. Investigated the distribution of u-par in ovarian cancer patients (all origins) and primary vs metastasis Immunohistochemistry Immunohistochemistry n=1 primary and n=3 metastatic tumors n=162 FIGO I-IV :n=8 subtype serous papillary; primary (n=77) and metastatic (n=77) primary 88/1 (88%), metastatic 27/3 (9%) 149/162 (92%): primary 71/77 (92.2%) metastatic 67/77 (9%) sub-type serous papillary
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