Follicle-forming cat thyroid cell lines synthesizing extracellular matrix and basal membrane components: a new tool for the study of thyroidal morphogenesis

Interactions between follicular epithelial cells and extracellular matrix ( ECM) are supposed to play an important Pole in the development and maintena nce of thyroid tissue architecture. In the present study we have therefore investigated the synthesis of ECM components by a feline thyroid cell line which is able to form follicle-like structures in vitro, and also in v-ras- transfected and control-transfected sublines. Transfections were performed by lipofection with pZSR (viral Harvey ras gene; neo) and pSV2-neo (control , neo only) plasmids. We have adapted a semisolid culture system composed e xclusively of polymerized alginate and therefore devoid of ECM components. Feline cells embedded in alginate gels as single cells and cultured for up to 90 days formed cell clusters within 10 days. Follicle-like structures we re formed in the original cell Lines and also in the v-ras- and control-tra nsfected cells. Differences in proliferation rates were observed, the v-ras -transfected cells growing up to two to three times faster than the non-tra nsfected cells. Immunostaining was done using rabbit first antibodies direc ted against mouse collagen IV, human fibronectin, laminin (tumor Engelbreth -Holm-Swarm laminin), perlecan and other ECM components. For comparison, im munostaining was also performed on cryosections of nodular goiters of six h yperthyroid cats. The cell lines and their transfected clones stained stron gly positive for collagen IV and fibronectin, and positively but less stron gly for laminin and perlecan. The cat goiter tissue stained positively for collagen IV, laminin, perlecan, and fibronectin, and positive staining for S-laminin (containing the beta 2-chain) was seen in blood vessel walls in t his tissue. In conclusion, cat cell lines grow three-dimensionally in alginate beads ov er several weeks, they form follicle-like structures and express the same E CM components as the native cat goiter tissue. Transfection with v-ras does increase proliferation rate, but does not fundamentally alter formation of follicle-like structures and ECM expression Alginate gel culture is a prom ising new tool for the study of follicular morphogenesis, polarity, the exp ression pattern of ECM components and of the interaction between thyrocytes and ECM. It avoids interference caused by gels composed of ECM components.