UCSF

Studies of the Carcinoma-Associated Fibroblast in Human Oral Squamous Cell Carcinoma

S. Thaddeus Connelly

Abstract

Our understanding of the development and progression of oral squamous cell carcinoma (Oral SCC) remains limited. This is reflected by the fact that oral cancer is associated with one of the poorest 5-year survival rates of any malignancy in the body. It is becoming increasingly evident that the underlying stroma has an important role to play in tumorogenesis. Our studies focus on the central controller of the stroma, the fibroblast and its tumor-associated counterpart, the carcinoma-associated fibroblast (CAF). The molecular and functional characteristics of CAFs associated with Oral SCC remain incomplete. Thus, we studied the influence of the stroma on the tumor microenvironment through a histologic evaluation of cancerous and pre-cancerous lesions. Additionally, we assessed the molecular response that CAFs and fibroblasts display to TGFβ or TGFβ-mediated stimulation by direct measurement of expression levels of downstream genes and in an organotypic co-culture system, respectively. Lastly, we used expression profiling, immunofluorescence and functional assays of CAFs and normal fibroblasts (NFs) in an attempt to classify a CAF specific expression signature, CAF subsets or cellular origin and to identify CAF genes or gene pathways that might contribute to proliferation, migration and invasion. The results demonstrate that the stromal lymphocytic infiltrate increases with lesion severity of pre-cancers with the most in Oral SCC, indicating a causal relationship. Fibroblast response to TGFβ or TGFβ-mediated stimulation was varied based on anatomic site and this heterogeneity was further appreciated in the results of the immunofluorescence and expression profiling, however these studies confirmed that αSMA is a robust CAF marker. Despite the heterogeneity in the CAF/NF expression profiles, a set of genes was found and validated to be differentially expressed between gingiva CAFs and NFs and tongue and gingival fibroblasts in general. Finally, analysis of fibroblast groups defined through functional assays reveal differential gene expression between fibroblasts that are most or least inhibitory to keratinocyte/tumor cell line proliferation and those that increase or decrease invasion/migration. SFRP2 from the proliferation assay and DKK1 from the invasion/migration assay are both differentially expressed negative modulators of WNT signaling, indicating a potentially important pathway in the reciprocal tumor/stroma evolutionary relationship.