Methods: Two distinct FAK-depleted- and two distinct Src-depleted human foreskin fibroblasts (HFF) lines were generated, using lentiviral-mediated sh-RNA approach. FAK and Src knockdown was quantified by q-RT-PCR. Bioengineered human tissues were generated by stratification of layers of E-cadherin-suppressed SCC tumor cells on control-, sh-FAK- or sh-Src-HFF-populated collagen gels. Tissue morphology, tumor cell invasion, and Laminin 5 deposition were analyzed by H&E and IF staining. ELISA was used to quantify HGF levels in the growth medium of 2D cultures and 3D tissues.
Results: In 2D cultures while cell morphology was not altered, FAK/Src-depleted HFFs resulted in ~9-fold increase in HGF secretion compared to control-HFFs. Elevated HGF secretion was also observed in 3D tissues after seeding SCC tumor cells onto FAK- or Src-depleted HFF-populated collagen gels. In comparison to control tissues, tissues comprised of FAK/Src-depleted HFFs showed ~4-fold increase in invasion of small clusters of tumor cells into the underlying collagen. The increase in tumor cell invasion was associated with increased Laminin 5 secretion in the superficial layers of the epithelium, and degradation of Laminin 5 at the epithelial-stromal interface.
Conclusions: Our findings suggest a novel role for FAK and Src in regulating HGF secretion by quiescent fibroblasts in normal and precancerous human tissues. They demonstrate that FAK/Src-depleted fibroblasts can alter the tissue microenvironment, support the invasive behavior of E-cadherin suppressed SCC tumor cells, and potentially enhance the progression of a premalignant lesion to invasive carcinoma.