Using high-density oligonucleotide arrays, we measured expression of >12,000 genes in surgical excisions of invasive human squamous cell carcinomas (SCCs) versus site-matched control skin. This analysis defined >1,900 genes with altered expression in SCCs that were statistically different from controls. As SCCs are composed of epithelial cells, which are both hyperplastic and invasive, we sought to define gene sets associated with these biologic processes by comparing gene expression to psoriasis vulgaris, which is a condition of benign keratinocyte hyperplasia without invasiveness or pre-malignant potential. Through this analysis, we found genes that were commonly upregulated in both conditions and unique genes with increased expression in SCCs. Differential gene regulation in these two conditions was confirmed by real-time reverse transcription-PCR and immunohistochemistry. We found that benign hyperplasia is associated with upregulation of genes including DEFB4 (defensin B4), SERPINB3 (serine proteinase inhibitor, member 3), STAT1 (signal transducer and activator of transcription 1), K16 (keratin 16), CEACAMs (carcinoembryonic antigen-related cell adhesion molecules), and WNT 5A (wingless-type MMTV integration site family, member 5A). WNT receptor frizzled homolog 6 (FZD6) and prostaglandin-metabolizing enzyme hydroxyprostaglandin dehydrogenase were increased in SCC alone. Growth factor pleiotrophin (PTN) was expressed at higher levels in non-tumor-bearing skin adjacent to excised SCC. SCC was further characterized by upregulation of matrix metalloproteinases 1, 10, and 13, cathepsin L2, cystatin E/M as well as STAT3 and microseminoprotein, beta (MSMB), and downregulation of inducible nitric oxide synthase, granzyme B, CD8, and CD83. The current study defines a unique gene expression signature for cutaneous SCC in humans and suggests potential roles for WNT, FZD, and PTN in the pathogenesis of SCC.

Basal cell carcinoma (BCC), the most common human cancer, undergoes spontaneous regression in certain circumstances, which is potentially immune-mediated. To understand the immune response surrounding BCCs, we characterized the genomic, protein, and cellular microenvironment associated with BCC in comparison to normal skin. Our results demonstrated the following: (1) CD4+ CD25+ Foxp3+ surround epithelial tumor aggregates; (2) Immature dendritic cells (DCs) were abundant in the tumor microenvironment; (3) BCC showed increased expression of IL-4, IL-10, and CCL22 and increased expression of interferon-associated genes (IFI27, IRF1, IRF7, and G1P2) and IL-12/23, gene indicating a Th2 dominant microenvironment. Our findings suggest a dynamic state within the immune microenvironment associated with BCC. The finding of phenotypic T regs, in conjunction with immature DCs and Th2 cytokines, suggests an attenuated state of immunity to human BCC. In contrast, abundant CD8+ T cells, an interferon signal, and IL-12/23 suggest partial host antitumor response. A better understanding of these opposing forces within the immune microenvironment may facilitate development of more potent immune-based treatment for BCC and other human carcinomas.

In order to determine the phenotype and function of myeloid DCs from human cutaneous squamous cell carcinoma (SCC), we studied their surface marker expression and allo-stimulatory potential ex vivo. There were abundant CD11c+ myeloid DCs, as well as TNF and inducible nitric oxide synthase (iNOS)-producing DCs (TIP-DCs), in and around SCC tumor nests. Although myeloid DCs from SCC, adjacent non-tumor bearing skin, and normal skin, were phenotypically similar by flow cytometry, and there was a pronounced genomic signature of mature DCs in SCC, they showed different T cell stimulatory potential in an allo-MLR. Myeloid DCs from SCC were less potent stimulators of allogeneic T cell proliferation than DCs from non-tumor bearing skin. Culture with a DC-maturing cytokine cocktail (IL-1β, IL-6, TNF, and PGE2) enhanced stimulatory potential in DCs from non-tumor-bearing skin, while SCC associated DCs remained poor stimulators of T cell proliferation. The microenvironment associated with SCC showed expression of TGFβ, IL-10 and VEGF-A, factors capable of suppressing DC function. These findings indicate that CD11c+/HLA-DRhi DCs from SCC are mature, but are not potent stimulators of T cell proliferation compared with phenotypically similar DCs isolated from non-tumor-bearing skin. Identification of mechanisms responsible for suppression of tumor-associated DCs may provide insight into evasion of immunosurveillance by SCC.

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