Head and neck cancers are characterized by a vigorous desmoplastic response, but the contribution of stromal-derived growth factors to the tumor microenvironment is poorly understood. We evaluated the expression of stromal growth factor expression in head and neck squamous cell carcinoma (HNSCC) in normal and tumor-associated stromal cells. Stromal tissue was isolated from epithelial cells with laser capture microdissection (LCMD) and analyzed by cDNA array for the expression of TGFalpha, TGF-beta1, HGF, PDGF-alpha, IGFII, bFGF, aFGF, VEGFC, and VEGF. Primary fibroblasts were isolated in vitro from HNSCC tumors, adjacent histologically normal mucosa, and skin in vitro. Fibroblast populations were assessed for TGF-beta1 expression by ELISA and luciferase reporter assay to assess protein expression. We identified TGF-beta1 and IGFII overexpression in normal and tumor-associated stromal cells; however, only TGF-beta1 was significantly overexpressed (3.4-fold) in tumor-associated stroma. Assessment of carcinoma-associated fibroblasts (CAFs), normal dermal fibroblasts (NDFs), and normal mucosal fibroblasts (NMFs) in propagated fibroblasts demonstrated persistently elevated levels of TGF-beta1 in CAFs compared to NMF and NDF populations. Elevated levels of TGF-beta1 were identified in the stromal compartment of HNSCC tumors compared to normal mucosa by immunohistochemical analysis. These results suggest that TGF-beta1 mRNA and protein is specifically upregulated in CAFs in vitro and in vivo.

To elucidate tumor-stromal interactions during tumor invasion by assessing the expression of proteolytic enzymes by carcinoma-associated fibroblasts (CAFs) in vivo using complementary DNA (cDNA) array analysis. Methods: Tumor-associated stroma was isolated from tumor and adjacent mucosal specimens of the same patient by laser capture microdissection, and the messenger RNA (mRNA) was assessed by cDNA microarray specific for proteolytic enzymes and their inhibitors. Protein overexpression was then analyzed by immunoblotting of primary fibroblast isolates derived from skin, mucosa, and tumor specimens. Results: Array analysis of 4 tumor and 4 adjacent mu-cosal samples demonstrated significant (2.6-fold) overexpression of membrane type 1 matrix metalloproteinase (MT1-MMP) but not of serine proteases or other matrix metalloproteinases. Analysis of normal dermal fibroblasts, normal mucosal fibroblasts, and CAFs similarly demonstrated up-regulation of MT1-MMP. Conclusions: These results suggest that MT1-MMP mRNA is specifically up-regulated in CAFs in vivo whereas MT1-MMP protein is specifically up-regulated in CAFs in vitro. Known to induce tumor cell invasion when expressed in tumor cells, CAF expression of MT1-MMP may be important in the stromal response to tumor cells that characterizes the desmoplastic reaction.

Background: The murine orthotopic oral cavity tumor model allows evaluation of tumor growth and invasion. Currently, serial measurements of tissue growth are difficult to obtain since invasive procedures or animal sacrifice is necessary to evaluate tumor size. High-resolution ultrasound was evaluated as a noninvasive method to monitor tumor size in vivo. Methods: Sixteen immunodeficient mice, age 9 weeks, were injected transcervically with a human squamous cell carcinoma cell line into the tongue, and tumor volume was assessed by high-frequency ultrasound at 11 days. The animals were subsequently sacrificed and the tumors processed for histology. Tumor size was then calculated by caliper measurement in two dimensions. Results: Tumor dimensions obtained using ultrasound were found to significantly correlate with the histologic measurements (Spearman coefficient 0.90, p < 0.0001). Tumor dimensions were on average larger using ultrasound versus histologic measurements, although this was not significantly different than zero (95% confidence interval –13.96 to 62.37 mm²). Conclusions: High-resolution ultrasound accurately measures tumor volume in the murine orthotopic oral cavity tumor model without sacrifice.

Objective:To determine whether acellular human dermis is degraded by matrix metalloproteinases (MMPs), a large class of matrix-degrading enzymes. Methods:The degradation of acellular human dermis specimens was evaluated in vitro. Wild-type murine fibroblasts with a broad-spectrum MMP inhibitor, GM6001, and MMP-2-deficient fibroblasts were placed on the basement membrane and dermal surfaces of acellular human dermis. Matrix degradation and fibroblast infiltration into the matrix were assessed after a 20-day incubation period. Results:The basement membrane thickness of the specimens cultured with wild-type fibroblasts was significantly less than that of specimens cultured with GM6001 (PϽ.001), and the infiltration of fibroblasts into the der-mal surface was limited by the addition of GM6001 (P=.002). To determine whether MMP-2 was involved in this in vitro phenotype, MMP-2-deficient fibroblasts were assessed in comparison with wild-type fibroblasts. Wildtype fibroblasts degraded the basement membrane surface (PϽ.001) and infiltrated the dermal surface (P=.003) more efficiently than did MMP-2-deficient fibroblasts. Conclusions:The results from our in vitro experiments suggest that MMPs and specifically MMP-2 may play an important role in the resorption of acellular human dermis. Addition of MMP inhibitors to implanted dermal matrices may slow fibroblast infiltration and improve their longevity in vivo.

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