Figure 6. Activation of fibroblasts by sCEA upregulates the production and deposition of cellular Fn-EDA.

A. Activation of HNNFbs by either sCEA (200 ng/mL) or TGF-β (5pg/mL) specifically upregulate Fn-EDA expression. B. Addition of sCEA induces a dose-dependent upregulation of Fn-EDA expression in fibroblasts. Semi-confluent HNNFbs cultures were stimulated with sCEA for 24 hours followed by western blot analysis of Fn-EDA expression using mAb IST-9. C. Immunohistochemical (IHC) detection of increases in cellular Fn-EDA in liver biopsies from late stage CRC patients, but not in normal liver. D. Quantification of DAB (3,3’-diaminobenzidine) signals of cellular Fn-EDA in stained specimens taken from 23 late stage CRC patients with metastatic disease. DAB signals (defined as % DAB staining) were quantified using the Sedeen software image analysis package. E. Fn-EDA IHC DAB signals co-register with the membrane-bound CEA [mCEA] and α-SMA signals [fibroblasts] in liver biopsies from late stage CRC patients with metastatic disease. F. Enlargement of a representative hepatic tumor nodule highlighting the co-registration of DAB signals for mCEA together with α-SMA and Fn-EDA signals. G. Quantification of DAB signals of mCEA, α-SMA and cellular (EDA⁺) Fn in stained specimens taken from 23 colorectal cancer patients with metastatic disease. DAB signals were quantified using the Sedeen software image analysis package (Pathcore). Signals registered from the tumor-free as well as the tumor afflicted areas of the stained liver specimens were compared with signals quantified from liver specimens taken from healthy patients. NS; not statistically significant. * P < 0.05 when compared to signals registered from stained healthy liver specimens. ** P < 0.05 when compared to non-tumor areas within liver specimens. Statistical significance was determined using the Mann-Whitney test.