Acutely increased extracellular pressure stimulates cancer cell adhesion to matrix proteins or endothelial cells via Src. We hypothesized that extracellular pressure might be chronically increased in rapidly growing tumors, and that such chronic pressure might similarly activate the metastatic potential of shed cancer cells via Src. We first measured interstitial pressure adjacent to and within human cancers undergoing percutaneous image-guided biopsy using a coaxial needle. Interstitial pressure in human cancers was on average 17 mmHg higher than interstitial pressure outside the tumors (n=11, p<0.05). Co26 murine colon cancer cells (105 cells/mL) were then exposed to ambient or 15 mmHg increased pressure for 30 minutes without or with the Src antagonist PP2 (20uM), and seeded into murine surgical wounds for 30 minutes before non-adherent cells were washed away and the wounds were closed. We measured time to palpable tumor and time to 100 mg tumor in 2 independent studies over 160 and 90 days. In the first study, pressure pretreatment of the cancer cells caused palpable tumors in 80.6% of mice vs. 47.2% for controls. (n=67, p<0.001) Time to 100 mg tumor paralleled time to palpable tumor. We then blocked Src in some cells with 20 uM PP2. Pressure again promoted tumor formation by vehicle control cells (67% vs 49% at ambient pressure, n=47, p<0.04). However, PP2 not only blocked the promotion of tumor formation by pressure, but reduced tumor formation by pressure-treated cells even below untreated ambient pressure controls (34% in the PP2 and pressure group vs. 54% in the untreated ambient pressure control, n=47, p<0.05). Because PP2 inhibits 9 Src-related kinases, we reduced Src by specific siRNA in human SW620 colon cancer cells to prove Src was the key target. Increased pressure for 48 hrs stimulated subsequent SW620 adhesion under ambient pressure (511 + 21 cells/HPF) compared to cells maintained for 48 hrs at ambient pressure (392 + 16 cells/HPF, n=42, P<0.0001). Maintaining the increased pressure during the adhesion assay further increased adhesion (595 + 30 cells/HPF, n=42, P<0.001). siRNA-reduction of Src by 67 + 8% (p<0.003) not only prevented stimulation of adhesion by 48 hrs of increased pressure but actually inhibited adhesion 46 + 7% vs. pressure-treated cells transfected with non-targeting control siRNA (n=60, p<0.01). Pressure-activated Src signaling may stimulate metastatic tumor formation in vivo, and mask a second counterregulatory pathway which inhibits adhesion. Src blockade may unmask this counterregulatory pathway and inhibit the stimulation of cancer cell metastatic potential by increased pressure within rapidly growing tumors.