Understanding factors affecting cell invasion influences the design ofengineered constructs for tissue regeneration. The objective of this workwas to investigate the effect of matrix stiffness on invasion of tumor cellsthrough a synthetic hydrogel with well-defined properties. A novel staracrylate-functionalized polyethylene glycol-co-lactide (SPELA) macromer wassynthesized to produce hydrogels with well-defined water content, elasticmodulus, degree of crosslinking and hydrophilicity. The hydrogel was formedby photo-polymerization of the macromer with or without integrin-bindingcell adhesive RGD peptide. Cell invasion experiments were carried out in atranswell with SPELA hydrogel as the invading matrix and 4T1 mouse breastcancer cells. The invading cells on the lower membrane side were countedwith an inverted fluorescent microscope. The concentration of SPELA macromerranged from 10-25 wt% and that of RGD ranged from 1x10-4 to 1x10-2 M. The shear modulus of the hydrogel varied from 200 Pato 25 kPa as the SPELA concentration increased from 10 to 25 wt%. Cellinvasion slightly increased with increasing RGD concentration. However, RGDconcentration >1% resulted in a significant decrease in cell migration.As the matrix stiffness increased from 0.15 to 0.4, 3, 5, 6, 14, and 25 kPathe invasion rate decreased from 18.0 to 5.5, 6, 5.7, 5.2, 1.5, and 1.0 cells/mm2/h, respectively. There was a sharp decrease ininvasion rate for matrix stiffness greater than 10 kPa. Results demonstratethat matrix stiffness plays a major role in invasion of tumor cell through agelatinous matrix.