MP4 file - 752K, 1. Time-lapse microscopy of CAF1 plated onto collagen I showing the diversity of CAF movement, with a predominance of motile cells with longer tracks. Images were captured every 10 mins and analysed using IMARIS software. The centre of each CAF is marked by a grey square. Migration was visualized by a track that changes colour in time as shown by the scale lower right corner. Examples of highly motile CAF are shown in the left quadrants and less motile CAF at the junction of the upper and lower left quadrants. Quantification of the migration is shown in supplementary Table ST1.
ARTICLE ABSTRACT
Heterogeneity of carcinoma-associated fibroblasts (CAF) has long been recognized, but the functional significance remains poorly understood. Here, we report the distinction of two CAF subtypes in oral squamous cell carcinoma (OSCC) that have differential tumor-promoting capability, one with a transcriptome and secretome closer to normal fibroblasts (CAF-N) and the other with a more divergent expression pattern (CAF-D). Both subtypes supported higher tumor incidence in nonobese diabetic/severe combined immunodeficient (NOD/SCID) Ilγ2(null) mice and deeper invasion of malignant keratinocytes than normal or dysplasia-associated fibroblasts, but CAF-N was more efficient than CAF-D in enhancing tumor incidence. CAF-N included more intrinsically motile fibroblasts maintained by high autocrine production of hyaluronan. Inhibiting CAF-N migration by blocking hyaluronan synthesis or chain elongation impaired invasion of adjacent OSCC cells, pinpointing fibroblast motility as an essential mechanism in this process. In contrast, CAF-D harbored fewer motile fibroblasts but synthesized higher TGF-β1 levels. TGF-β1 did not stimulate CAF-D migration but enhanced invasion and expression of epithelial–mesenchymal transition (EMT) markers in malignant keratinocytes. Inhibiting TGF-β1 in three-dimensional cultures containing CAF-D impaired keratinocyte invasion, suggesting TGF-β1–induced EMT mediates CAF-D–induced carcinoma cell invasion. TGF-β1–pretreated normal fibroblasts also induced invasive properties in transformed oral keratinocytes, indicating that TGF-β1–synthesizing fibroblasts, as well as hyaluronan-synthesizing fibroblasts, are critical for carcinoma invasion. Taken together, these results discern two subtypes of CAF that promote OSCC cell invasion via different mechanisms. Cancer Res; 73(13); 3888–901. ©2013 AACR.
