Figure 6.

Preferential adherence of CEA‐expressing cancer cells to fibronectin is the result of a direct CEA N domain‐Fn interaction. CEA‐expressing (MC38.CEA) cells, but not the parental MC38 cells, preferentially adhere to immobilized Fn (Panels A and B). Cells (2.5 × 104) were dispensed to E‐plates wells pre‐coated with either 1 μg of Fn, BSA or in the absence of any immobilized protein. Cellular adhesion to Fn is reported as averaged ΔCell Index values (±SEM). C. Mapping the region of the CEA N domain responsible for Fn binding. Binding of His‐tagged rCEA segments spanning residues 1 to 290 of CEA to immobilized Fn was monitored by ELISA. The presence of bound His‐tagged proteins was detected using HRP‐coupled anti‐His monoclonal antibody (mAb His‐1; 1:5000). NS: not statistically significant; * significant when compared to control wells (P ≤ 0.05). Statistical analyses were performed using a Mann‐Whitney‐U‐test. D. Pull down of Fn by biotinylated CEA N108–115. Fn (5 μg) was incubated in the presence or absence of biotinylated heptapeptides (50 μg) and pulled down with magnetic Streptavidin beads. The presence of Fn was detected using anti‐Fn rabbit pAb (1:1000). The biotinylated 7‐mer peptides used were CEA N108‐115, FnBP, as well as the irrelevant P0 7‐mer. Presentation of soluble rCEA N domain significantly ameliorates the adherence of MC38.CEA cells to Fn‐coated surfaces (panels E and F). Blocking of cell surface CEA N domain through the addition of rCEA A3B3 (1 μM) results in a substantial reduction in adherence of MC38.CEA cells to Fn. NS: not statistically significant; * significant when compared to untreated cells (P ≤ 0.05). Statistically analyses were performed using a Student‐t‐test.