Fig. 4.

Defining a cancer stem cell signature for SCCs. (A) Hierarchical cluster analysis of Affymetrix 430 2.0 array data indicates that, whether CD34⁺ or CD34⁻, SCC CSCs are more similar to each other than they are to wild-type SCs from either epidermis or HFs. Note that core markers of CD34⁺ HF-SCs, including Nfatc1, Lhx2, vitamin D receptor (VdR), and Lgr5, are strikingly reduced or absent in CSCs. Other adult SC markers [Lrig1, PR domain containing 1, with ZNF domain (Prdm1), and Sox9] are not uniformly elevated in CSCs. (B) Differential expression analyses indicate key distinctions between CSCs and wild-type SCs. Shown are fold changes (log₂ scale) of previously described HF-SC markers between α6β1^hiCD34^hi and α6β1^hiCD34^lo cells of wild-type, TβRII^KO, dKO, and FAK^KO SCCs and wild-type skin epithelium. Note that HF-SC marker genes other than CD34 are not differentially expressed in these two CSC populations. Bars indicate the average of two independent experiments. HF-SCs (α6^hiβ1^hiCD34^hi) vs. epidermis (α6^hiβ1^hiCD34^neg) serves as a positive control. (C) Venn diagram revealing a core CSC signature of 742 genes that are up-regulated by twofold or more (P < 0.05) in all CSCs irrespective of their genotype and whether compared with wild-type HF-SCs or epidermal progenitors. Functional hierarchical cluster analysis indicates enrichment for processes that are commonly deregulated in cancers. (D) Selected sets of skin genes that are elevated (red) or abrogated (blue) by more than twofold in CSCs compared with wild-type-SCs. (E–K) Immunofluorescence microscopy (E–I) and immunoblot analyses (K) validate the CSC signature and define the CSC niche at the tumor–stroma interface. Validation was performed on independent primary SCCs that developed in wild-type mice. (Scale bars: 20 μm.) FACS analysis in J shows that surface marker CD44, up-regulated in CSCs and localized at the tumor–stroma interface (I), is in the integrin-high population.