Retinoic acid modulates the radiosensitivity of head-and-neck squamous carcinoma cells grown in collagen gel.
OBJECTIVE
Collagen gels are increasingly regarded as reliable scaffolds for studying cells in vitro, displaying the same three-dimensional network of collagen fibers as encountered in vivo. As a contribution to therapeutic control of head-and-neck cancer, we grew HSCO86 cells in collagen gel and assessed their behavior in the presence of retinoic acid (RA) and radiation.


METHODS
The malignant epithelial cell line HSCO86 was isolated from a postirradiation human oropharyngeal squamous carcinoma; it was EGFR-negative by immunocytochemical criteria. The cells were embedded in hydrated collagen I at a density of 10(6) cells/mL, and on Days 8, 10, and 12 of culture, they were treated with 10(-5) M retinoic acid. Radiation was administered using two different schedules: simultaneously with RA in three daily doses totaling 10 Gy, or with a single dose of 8 Gy on Day 29 of culture, after the effects of RA had taken place. Cell proliferation was evaluated by the MTT assay, whereas morphometric characteristics were detected in the cultured gels directly or in the gels after they were fixed and stained with hematoxylin.


RESULTS
Contrary to growth in monolayer, where HSCO86 cells displayed a high proliferation rate, in collagen gel only a tiny fraction of the cells, usually less than 0.02%, survived the environmental stress; these cells spontaneously organized themselves into clonal multicellular spheroids growing up to 0.8 mm in diameter. After exposure to 10(-5) M retinoic acid, cell proliferation first declined and then, about 15 days after treatment, it started to increase to a level far above that in the control group. This surge in proliferation was ascribed to the appearance of numerous fibroblast-like cells at the edge of the spheroids. These cells, called HSCO-F, were the result of epithelial-to-mesenchymal conversion. When the gels were disaggregated by collagenase, and the cells were seeded in monolayer, HSCO-F cells reversed their morphology into parental HSCO86 cells. Treatment of collagen gels with 10 Gy, fractionated in three daily doses, did not substantially affect the growth of HSCO86 spheroids. However, when radiation was given simultaneously with RA, cell growth was significantly inhibited, both in terms of cell proliferation and size of spheroids (p < 0.0001 vs. untreated controls). This synergism applied mainly to parental HSCO86 cells, because no significant damage was induced by radiation on the HSCO-F cells previously generated by treatment with RA.


CONCLUSIONS
Differences in the radiosensitivity of HSCO86 and HSCO-F cells are surprising in view of their common origin; this suggests a scenario in which, to overcome a microenvironmental stress, head-and-neck carcinoma cells can temporarily shift from an epithelial to a mesenchymal phenotype. In particular, morphologic and functional data suggested that HSCO-F cells were transformed into vascular endothelial cells whose characteristics included the following: (1) distinctive expression of Factor VIII and beta(1)-integrin, not detected in parental HSCO86 cells; (2) active migration in the collagen network by extruded pseudopodia, frequently appearing as colonies of filamentous cells aligned along the radial axis of the spheroids; and (3) efficient contraction of floating collagen gels. The implication of our study is that head-and-neck carcinomas may respond to RA treatment by selecting cell populations both resistant to radiation and capable of migrating inside the connective tissue, mimicking the behavior of vascular capillaries.
Rossi
Lorenzo
L
Laboratory of Comparative Oncology, University of Genoa, Genoa, Italy. lorenzo.rossi@istage.it
Corvò
Renzo
R
D002455:Cell Division; D003094:Collagen / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000473:pathology; D004847:Epithelial Cells; D005782:Gels / Q000494:pharmacology*; D006258:Head and Neck Neoplasms / Q000532:radiotherapy*; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology; D007150:Immunohistochemistry; D010641:Phenotype; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D014212:Tretinoin / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D005782:Gels; D013778:Tetrazolium Salts; D013844:Thiazoles; D014212:Tretinoin; D006820:Hyaluronic Acid; D003094:Collagen; C022616:thiazolyl blue