Supplementary Figure Legend

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1 Supplementary Figure Legend Supplementary Figure S1. Effects of MMP-1 silencing on HEp3-hi/diss cell proliferation in 2D and 3D culture conditions. (A) Downregulation of MMP-1 expression in HEp3-hi/diss cells by sirna interference. Western blot analysis of MMP-1 was performed on CM from HEp3-hi/diss cells silenced by transient transfection with control or MMP-1 sirna constructs. Position of 50-kDa MMP-1 is indicated on the right. (B) Effects of MMP-1 deficiency on proliferation potential of HEp3-hi/diss cells in 2D cultures. HEp3-hi/diss cells were treated with control or MMP-1-specific sirna and plated at 1x103 cells per well. At the indicated time points, the cells were detached and counted. Data are expressed as means determined from triplicate counts. Presented is one of two independent experiments. (C) Effects of MMP-1 deficiency on proliferation potential of HEp3-hi/diss cells in 3D culture conditions. HEp3-hi/diss cells, treated with control or MMP-1-specific sirna, were mixed with neutralized type I collagen. Polymerized collagen gels were overlaid with D-10 and the number of cells in gels was determined on day 8 of incubation. Data are from a

2 representative experiment performed in triplicate. *, P<0.05; two-tailed Student s t-test. Supplementary Figure S2. HEp3-hi/diss tissue colonization does not depend on MMP-1 expression. (A) Analysis of vascular arrest and colonization in an experimental metastasis model. The assay was performed as described in Fig. 1B using HEp3-hi/diss cells transfected with control (Ctrl) or MMP-1-specific shrna and sirna constructs. Two hrs later, the CAM tissue was harvested from 5 to 10 embryos per variant and the number of human cells was quantified by Alu-qPCR to determine the levels of cell arrest (left panel). The levels of tumor cell colonization in the CAM (middle panel) and liver (right panel) were analyzed 5 days after cell inoculations. Colonization data from two independent experiments, each employing from 9 to 18 embryos per variant, were analyzed as percentage of control group (100%). Bars are means ± SEM from pooled data. ***, P <0.0001; two-tailed Student s t test. (B) Fluorescence microscopy analysis of CAM tissue. The cells were treated with control and MMP-1-specific sirnas, pre-labeled with CellTracker Green and then inoculated i.v.

3 into day 12 embryos. Five days after cell injections, the CAM vasculature was highlighted in red with Rhodamine-conjugated LCA. The portions of the CAM were analyzed without fixation in fluorescent microscope. Bar, 50 μm. Supplementary Figure S3. Effects of MMP-1 downregulation on HEp3-hi/diss cell adhesion and invasion. (A) MMP-1 deficiency does not affect adhesion to the ECM proteins in vitro. HEp3- hi/diss cells transfected with control or MMP-1 sirna were plated at into wells pre-coated with type I collagen, fibronectin or Matrigel and allowed to adhere for 30 minutes. The levels of adhesion were determined in comparison to control group (100%). Bars, means ± SEM determined from the data from 2 independent experiments performed in triplicate. (B) MMP-1 deficiency does not affect Transwell invasion in vitro. To create a matrix barrier, the upper sides of Transwells were coated with neutralized type I collagen (left and middle graphs) or Matrigel (right graph). Invasion was induced by 5% FCS in 0.5 ml of DMEM placed into the lower chamber. Where indicated, 5 μm of GM6001 was added to both the upper and lower chambers. The HEp3-hi/diss cells treated with control or MMP-1 sirnas were allowed to invade matrix barriers for 48 hours, after which time the transmigrated cells were counted. Data are presented as percentage of control (100%)

4 determined for each individual Transwell. Bars are means ± SEM from pooled data of two individual experiments performed in triplicate. *, P<0.05; two-tailed Student s t-test. Supplementary Figure S4. Inhibition of vascular permeability in HEp3-hi/diss microtumors by the PAR1 antagonist RWJ HEp3-hi/diss cells were grafted on the CAM of day 10 ex ovo embryos to generate topical microtumors (1x105 cells per microtumor; 4-5 microtumors per embryo). Developing individual microtumors were treated with 20 μl of the PAR1 inhibitor RWJ (applied as 10 μm or 50 μm solution) or vehicle control (1% DMSO in PBS) on days 2 and 4 after cell grafting. On day 5, the embryos were injected i.v. first with TITCconjugated 155-kDa dextran, followed in 45 min by inoculation of FITC-conjugated 2,000-kDa dextran. Individual microtumors were excised from the CAM and lysed in 0.3 ml mripa buffer. The red and green fluorescence was measured in the tumor lysates at 492/516 and 557/576 nm, respectively. The ratio of the red fluorescence signal (permeable dextran exudation) to the green fluorescence signal (volume of perfusable vasculature) was calculated to determine the relative permeability of tumor vasculature. Data are from one representative experiment

5 employing 6-7 embryos per treatment condition. The effects of treatments with RWJ at 10 μm (RWJ-10) and 50 μm (RWJ-50) are calculated as fold differences compared to the vehicle control (assigned as 1.0). Data are means ± SEM. ***, P< Supplementary Figure S5. Inhibition of HEp3-hi/diss intravasation by a PAR1 antagonist. HEp3-hi/diss cells were grafted on the CAM of day 10 ex ovo embryos to generate topical microtumors (1x105 cells per microtumor; 4-5 microtumors per embryo). Developing microtumors were each treated with 20 μl of vehicle control (1% DMSO in PBS) or 5 μm PAR1 inhibitor SCH79797 applied on days 1, 3 and 5 after cell grafting. On day 6, the portions of the CAM distal to location of microtumors were excised and processed for AluqPCR analysis. The actual numbers of human tumor cells were determined using a standard curve. The pooled data are from 3 independent experiments, each involving from 5 to 7 embryos per treatment variant. The data (means±sem) are expressed as a fold difference in intravasated cell numbers compared to the vehicle control (1.0). *, P<0.05, twotailed Mann- Whitney test.

6 Supplementary Figure S6. MMP-1 produced by the human HNSCC Detroit 562 concomitantly regulates vascular permeability and intravasation. (A) Downregulation of MMP-1 expression in Detroit 562 cells by sirna interference. Kinetic western blot analysis of MMP-1 was performed on 20-μl CM samples collected at the indicated time points after transient transfection of Detroit 562 cells with control (Ctrl) or MMP-1 sirna constructs. CM from Hep3-hi/diss cells (20 μl) was run to allow for comparison of relative levels of MMP-1 production. Position of mol. wt. markers in kda is indicated on the left. Position of 52-kDa MMP-1 is indicated on the right. (B and C) MMP-1 silencing concomitantly reduces the levels of Detroit 562 intravasation and vascular permeability. Detroit cells transfected with control or MMP-1- specific sirnas were grafted on the top the CAM to generate topical microtumors. After 6 days of incubation, the levels of intravasation to the CAM (B) were quantified by AluqPCR in the CAM samples harvested away from microtumors. To determine the levels of vascular permeability (C), the embryos were injected i.v. with the permeable low mol. wt. TRITCdextran, incubated for 45 min, and additionally inoculated with the non-permeable high mol. wt. FITC-dextran. Immediately, the individual microtumors were excised from the CAM and lysed (3 microtumors per sample). The levels of red and green fluorescence were measured at 590 and 560 nm, respectively. Permeability of intratumoral vasculature in individual tumors

7 is presented as ratio of dextran exudation (red fluorescence) to total volume of perfusable vasculature (green fluorescence). In (B) and (C), the data are means ± SEM of fold differences between control and MMP-1-silenced conditions. ** and ***, P<0.01 and <0.0001, respectively; two-tailed Student s t-test.