Evaluation of tyrosine receptor kinases in the interactions of head and neck squamous cell carcinoma cells and fibroblasts

Abstract

Objective: Despite treatment advancements, disease-free survival of head and neck squamous cell carcinoma (HNSCC) has not significantly improved. This may be a result of tumor-fibroblasts interactions providing protective pathways for oncogenic cells to resist therapy. Further understanding of these relationships in HNSCC may improve effectiveness of targeted therapies. In this article, we investigated the role of several receptor tyrosine kinases (RTKs) in the interactions between HNSCC cells and supporting cells (fibroblasts).

Materials and methods: HNSCC cell lines and human tumor samples were evaluated for FGFR1/2/3, and PDGF-beta expression levels. Cell lines (FADU, SCC1, OSC19, Cal27, SCC22A) were treated with a range of physiological concentrations of dovitinib and assessed for proliferation, cytotoxicity, and apoptosis. Mice bearing HNSCC xenografts were treated with dovitinib (20 mg/kg).

Results: Evaluation of HNSCC tumor specimens, cell lines and fibroblasts found variable expression of multiple RTKs (fibroblasts growth factor receptor, platelet derived growth factor receptor and vascular endothelial growth factor receptor) and their ligands, supporting previous theories of paracrine and autocrine signaling within the microenvironment. In a dose-dependent fashion, RTK inhibition reduced proliferation of HNSCC cell lines and fibroblast in vitro. When HNSCC cells were cocultured with fibroblasts, RTK inhibition resulted in a smaller reduction in the proliferation relative to untreated conditions. In vivo, RTK inhibition resulted in significant tumor regression and growth inhibition (p<0.05) and reduced the incidence of regional lymph node metastasis.

Conclusion: Effective treatment of HNSCC, therefore, may require inhibition of multiple RTKs in order to adequately inhibit the microenvironment's various signaling pathways.

Figure 1

Expression of FGFR, PDGFR-β, and VEGFR in human HNSCCs specimens. Tumor samples from 13 patients with HNSCC were stained by (A) immunohistochemistry or (B) immunofluorescence.

Figure 2

Orthotopic xenografts of HNSCC cell lines (A) SCC-1 and (B) OSC-19 were treated with dovitinib (20 mg/kg/day). In addition, the OSC-19 xenografts were treated with +/− radiation therapy. Marker, mean for triplicate; bars, SE. Statistical significance by unpaired t-test, *p<0.05.

Figure 3

Orthotopic OSC-19 xenografts were treated immediately following implantation with dovitinib (20 mg/kg/day). Tumor growth was followed for 17 days and imaged following systemic injection of labeled anti-EGFR antibody on day 17 (A). There was a significant stabilization of growth by day 8, which continued for the remainder of the study (B). Following treatment, tumors were harvested and histologically analyzed for proliferation by Ki67 staining (C). Lymph nodes were also harvested at the conclusion of the study and analyzed histologically for lymph node metastasis by H&E staining (D). Marker or columns, mean for triplicate; bars, SE. Statistical significance by unpaired t-test, *p<0.05.

Figure 4

Western blot analysis of receptor tyrosine kinases and their ligands in HNSCC cell lines and fibroblasts.

Figure 5

Dose-response curves for HNSCC cells and fibroblasts treated in vitro with dovitinib (0–1000nM). Boxes, mean for triplicate; bars, SE.

Figure 6

Cell proliferation of (A-C) HNSCC cells grown alone and cocultured with fibroblasts +/− dovitinib (100 nM). (D) Fibroblasts provide protection from proliferation inhibition by dovitinib. Columns, mean for triplicate; bars, SE.