S100A4 expression is increased in tumor-associated stroma but is not correlated with S100A4 levels in the tumor cells. Sections of normal tongue (A) and skin (B) tissue from BALB/C nude mice were immunohistochemically stained for S100A4. Representative areas of the samples are presented. S100A4 staining is shown in brown, and nuclei are stained blue. C: S100A4 mRNA levels in tumor-associated stroma in tongue and skin were analyzed by RT-PCR. Expression levels in stroma close to the tumors of HSC-4, Ishikawa (ISH), and UT-SCC-12A (black bars; n = 3) were quantified relative to the expression levels in stroma further away (>0.5 mm) from the tumors (white bars; n = 3). All expression levels were normalized to the expression of the reference genes Ppia and Nono. Error bars represent SEM. D: Immunoblots of cell lysates from SCC9, SCC9/A4, and SCC9/EGFP cells probed with anti-S100A4 and anti–β-actin. Immunohistochemical S100A4 staining of tongue tumors of the SCC9/A4 cell line with sparse stromal staining (E) and more pronounced stromal staining (F) and of the SCC9 cell line with strong stromal staining (G). Representative areas of three parallel tumors are presented; S100A4 staining is shown in brown, and nuclei are stained blue. t, tumor area; and s, stromal area.