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.
Highlights
Induction of invasion is the key property differentiating malignant from benign lesions
Two distinct carcinoma-associated fibroblasts (CAF) subtypes were identified in this study by transcriptome and secretome analysis: CAF-N with a gene expression pattern and secretory profile closer to normal oral fibroblasts (NOF), and CAF-D with a divergent transcriptome and secreting very high levels of TGF-b1
The CAF-N subtype included a high percentage of intrinsically motile CAFs, and the number of migratory cells could be increased in response to TGF-b1, whereas the CAF-D subtype included few motile cells and the motility phenotype was largely unchanged in response to TGF-b1
Summary
Induction of invasion is the key property differentiating malignant from benign lesions. There is convincing evidence that dysplasia-associated fibroblasts (DAF) and carcinoma-associated fibroblasts (CAF) differ from those associated with normal epithelium [4, 5], and that these adaptations have functional consequences for tumor progression and invasion [6, 7]. These carcinoma-promoting effects were attributed mainly to the subpopulation of myofibroblasts in the mixed CAF populations that had higher ability to secrete stimulative paracrine factors [3, 8, 9]
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