Wenyue Zhang

Fibroblast-derived MT1-MMP promotes tumor progression in vitro and in vivo

BackgroundIdentification of fibroblast derived factors in tumor progression has the potential to provide novel molecular targets for modulating tumor cell growth and metastasis. Multiple matrix metalloproteases (MMPs) are expressed by both mesenchymal and epithelial cells within head and neck squamous cell carcinomas (HNSCCs), but the relative importance of these enzymes and the cell source is the subject of controversy.MethodsThe invasive potential of HNSCC tumor cells were assessed in vitro atop type I collagen gels in coculture with wild-type (WT), MMP-2 null, MMP-9 null or MT1-MMP null fibroblasts. A floor of mouth mouse model of HNSCC was used to assess in vivo growth after co-injection of FaDu tumor cells with MMP null fibroblasts.ResultsHere we report changes in tumor phenotype when FaDu HNSCCs cells are cocultured with WT, MMP-2 null, MMP-9 null or MT1-MMP null fibroblasts in vitro and in vivo. WT, MMP-2 null and MMP-9 null fibroblasts, but not MT1-MMP null fibroblasts, spontaneously invaded into type I collagen gels. WT fibroblasts stimulated FaDu tumor cell invasion in coculture. This invasive phenotype was unaffected by combination with MMP-9 null fibroblasts, reduced with MMP-2 null fibroblasts (50%) and abrogated in MT1-MMP null fibroblasts. Co-injection of FaDu tumor cells with fibroblasts in an orthotopic oral cavity SCID mouse model demonstrated a reduction of tumor volume using MMP-9 and MMP-2 null fibroblasts (48% and 49%, respectively) compared to WT fibroblasts. Consistent with in vitro studies, MT1-MMP null fibroblasts when co-injected with FaDu cells resulted in a 90% reduction in tumor volume compared to FaDu cells injected with WT fibroblasts.ConclusionThese data suggest a role for fibroblast-derived MMP-2 and MT1-MMP in HNSCC tumor invasion in vitro and tumor growth in vivo.

In vivo detection of head and neck cancer orthotopic xenografts by immunofluorescence.

Purpose: To determine whether Cy5.5‐labeled antiepidermal growth factor (EGFR) antibody could be used to detect head and neck squamous cell carcinoma (HNSCC) xenografts in vivo.

Anti-EMMPRIN Monoclonal Antibody as a Novel Agent for Therapy of Head and Neck Cancer

Purpose: Extracellular matrix metalloprotease inducer (EMMPRIN) is a tumor surface protein that promotes growth and is overexpressed in head and neck cancer. These features make it a potential therapeutic target for monoclonal antibody (mAb)–based therapy. Because molecular therapy is considered more effective when delivered with conventional cytotoxic agents, anti-EMMPRIN therapy was assessed alone and in combination with external beam radiation. Experimental Design: Using a murine flank model, loss of EMMPRIN function was achieved by transfection with a small interfering RNA against EMMPRIN or treatment with a chimeric anti-EMMPRIN blocking mAb. Cytokine expression was assessed for xenografts, tumor cells, fibroblasts, and endothelial cells. Results: Animals treated with anti-EMMPRIN mAb had delayed tumor growth compared with untreated controls, whereas treatment with combination radiation and anti-EMMPRIN mAb showed the greatest reduction in tumor growth (P = 0.001). Radiation-treated EMMPRIN knockdown xenografts showed a reduction in tumor growth compared with untreated knockdown controls (P = 0.01), whereas radiation-treated EMMPRIN–expressing xenografts did not show a delay in tumor growth. Immunohistochemical evaluation for Ki67 and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) resulted in a reduction in proliferation (P = 0.007) and increased apoptosis in anti-EMMPRIN mAb–treated xenografts compared with untreated controls (P = 0.087). In addition, we provide evidence that EMMPRIN suppression results in decreased interleukin 1β (IL-1β), IL-6, and IL-8 cytokine production, in vitro and in vivo. Conclusions: These data suggest that anti-EMMPRIN antibody inhibits tumor cell proliferation in vivo and may represent a novel targeted treatment option in head and neck squamous cell carcinoma.

Extracellular Matrix Metalloprotease Inducer-Expressing Head and Neck Squamous Cell Carcinoma Cells Promote Fibroblast-Mediated Type I Collagen Degradation In vitro

Until recently, tumor progression has been considered a multistep process defined by tumor cell mutations and the importance of the surrounding stroma poorly understood. It is now recognized that matrix-degrading enzymes that promote tumor cell invasion are elaborated by both tumor cells and fibroblasts in vivo. To determine the relative role of tumor cell–derived proteases compared with fibroblast-derived proteases, coculture experiments were done with each cell type using an in vitro model of type I collagen degradation. Head and neck squamous cell carcinoma cells in coculture with normal dermal fibroblasts showed matrix degradation, but neither cell type alone produced this effect. Manipulating the in vitro coculture environment showed that collagenolysis in this model was a result of fibroblast-derived matrix metalloproteases (MMP). To explore the possible role of extracellular matrix metalloprotease inducer (EMMPRIN) in this interaction, transfection of EMMPRIN into a cell line with low endogenous EMMPRIN expression was done and showed a significant increase in collagenolysis. Inhibition of collagenolysis with a tissue inhibitor of metalloprotease-2 (TIMP-2) and a synthetic furin inhibitor was observed but not with TIMP-1, which suggested a possible role for membrane type-1 MMP. These results suggest that fibroblast-derived MMPs but not those from tumor cells are important for in vitro collagenolysis and that this process is promoted by tumor cell–expressed EMMPRIN.

Optical imaging predicts tumor response to anti-EGFR therapy.

To evaluate cetuximab treatment in head and neck squamous cell carcinoma xenografts and cell lines, we investigated a preclinical model of head and neck squamous cell carcinoma. Head and neck squamous cell carcinoma cell lines SCC-1, FaDu, CAL27, UM-SCC-5 and UM-SCC-22A were used to generate subcutaneous flank xenografts in SCID mice. Mice were divided into control and cetuximab treatment groups, mice in the latter group received 250 μg cetuximab once weekly for four weeks. After completion of therapy, SCC-1 (P < 0.001), UM-SCC-5 (P < 0.001), UM-SCC-22A (P = 0.016) and FaDu (P = 0.007) tumors were significantly smaller than control, while CAL27 tumors were not different from controls (P = 0.90). Mice were systemically injected with 50 μg of the Cy5.5-cetuximab bioconjugate and imaged by stereomicroscopy to determine if tumor fluorescence predicted tumor response. Intact tumor fluorescence did not predict response. Tissue was harvested from untreated xenografts to evaluate ex vivo imaging. Cell lines were then evaluated in vitro for fluorescence imaging after Cy5.5-cetuximab bioconjugate labeling. The location of fluorescence observed in labeled cells was significantly different for cell lines that responded to treatment, relative to unresponsive cells. Tumors from cell lines that showed low internalized signal in vitro responded best to treatment with cetuximab. This preclinical model may aid in determining which cancer patients are best suited for cetuximab therapy.

Stereomicroscopic fluorescence imaging of head and neck cancer xenografts targeting CD147

Purpose: To demonstrate that systemically administered fluorescently labeled anti-CD147 antibody can detect head and neck squamous cell carcinoma xenografts in vivo. Experimental design: In vivo immunodeficient murine model.Methods: To determine if fluorescently labeled anti-CD147 antibody was specific for tumors in vivo, anti-CD147 and non-specific IgG1k antibody were labeled with a near infrared fluorophore (Cy5.5) and administered systemically to immunodeficient mice bearing SCC-1 xenografts. Imaging was performed over a 72 hour period using brightfield and fluorescent (685-735 nm) stereomicroscopy. To determine if fluorescence varied with receptor expression, SCID mice were xenografted with cell lines expressing variable amounts of CD147: FaDu (control vector transfected), FaDu/siE (siRNA CD147 knockdown) or FaDu/E (CD147 overexpressing) cells. Results: Peak tumor fluorescence was visualized by near infrared stereomicroscopy in SCC-1 tumors at 24 hours after systemic injection of anti-CD147:Cy5.5 bioconjugate. SCC-1 xenografts demonstrated significantly higher fluorescent intensity after administration of CD147:Cy5.5 (48 au, P < 0.0001) compared to IgG1k:Cy5.5 isotype control antibody (9 au). FaDu tumors overexpressing CD147 (FaDu/E) demonstrated higher fluorescence (53 au) compared to control vector transfected cells (FaDu, 33 au, P < 0.0001) which was higher than CD147 knockdown cells (FaDu/siE, 5 au, P < 0.0001). Conclusions: This data suggests fluorescently labeled anti-CD147 may have clinical utility in detection of HNSCC.

Therapy of head and neck squamous cell carcinoma with replicative adenovirus expressing tissue inhibitor of metalloproteinase-2 and chemoradiation.

Recent studies have demonstrated the efficacy of targeted therapy combined with radiotherapy in head and neck squamous cell carcinoma (HNSCC). We hypothesized that a combination treatment including a replicating adenovirus armed with tissue inhibitor of metalloproteinase-2 (TIMP-2), radiation and Cisplatin will augment treatment response and reduce tumor growth in vivo of HNSCC xenografts. Both single-agent (TIMP-2 virus, radiation and Cisplatin) and the combination therapies were evaluated in vitro and in vivo. The efficacy of both single-agent and combination therapies in vivo was determined by monitoring tumor growth and immunohistochemistry. Treatment with replicative Ad-TIMP-2 virus and radiation decreased cell viability in vitro and resulted in an additional antiangiogenic response in vivo. Tumor response rates to treatment with replicative Ad-TIMP-2, radiation, Cisplatin or combination therapies ranged from limited inhibition of tumor growth of the single-agent therapy to a statistically significant additive antitumor response with the combination therapies. Replicative Ad-TIMP-2+radiation+Cisplatin in the SCC1 nude mice demonstrated the greatest response rates in tumor growth and angiogenesis. Combination of Ad-TIMP-2 gene therapy with radiation and the triple treatment group resulted in an augmented therapeutic response. This is the first report of the potential benefits of combining radiation and MMP inhibitor treatment.

Modulation of Tumor Cell Growth In Vivo by Extracellular Matrix Metalloprotease Inducer

OBJECTIVE To investigate if loss of extracellular matrix metalloprotease inducer (EMMPRIN) will inhibit the growth of head and neck squamous cell carcinoma (HNSCC) tumor cell lines in vivo. Tumor cell-derived EMMPRIN is highly overexpressed in HNSCC and is thought to be induced by surrounding fibroblasts to stimulate matrix metalloproteases, which modulate tumor cell invasion, growth, and angiogenesis. DESIGN In vivo study using FaDu tumor xenografts. SETTING Academic research facility. SUBJECTS Severe combined immunodeficiency (SCID) mice. INTERVENTIONS The HNSCC cell line FaDu was transfected with EMMPRIN (FaDu/E), control vector (FaDu), or plasmid-expressing small-interfering RNA against EMMPRIN (FaDu/siE). Tumor cells combined with fibroblast cells were xenografted onto the flank of SCID mice. Tumors were measured biweekly over 4 weeks, at which time the mice were killed, and tumor samples were analyzed for proliferation (Ki-67 immunohistochemical analysis), vascularization (factor VIII staining), and apoptosis (TUNEL [terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling] assay). MAIN OUTCOME MEASURE Growth of head and neck cancer cell lines genetically engineered to express variable levels of EMMPRIN. RESULTS Tumor growth positively correlated and animal survival negatively correlated with increasing EMMPRIN expression. FaDu/E tumor growth was significantly larger at 4 weeks compared with FaDu tumors (P = .006). Similarly, the control vector-transfected FaDu tumors were significantly larger than FaDu/siE (P < .001). Immunohistochemical analysis demonstrated increased Ki-67 in EMMPRIN-transfected cells, without a significant change in the rate of apoptosis between groups. Vascular density and tumor formation rate also increased significantly with EMMPRIN expression. CONCLUSION This study suggests that anti-EMMPRIN-targeted therapy may prove to be a novel treatment option in HNSCC.

Extracellular Matrix Metalloprotease Inducer Stimulates Fibroblast‐Mediated Tumor Growth In Vivo

Hypothesis: Extracellular matrix metalloprotease inducer (EMMPRIN) is a molecule expressed on the cell surface of tumor cells that has been shown to induce both tumor cells and fibroblasts to express matrix metalloproteases in vitro. We hypothesize that fibroblasts are stimulated by EMMPRIN to create a microenvironment favorable to tumor growth.

EMMPRIN expression is required for response to bevacizumab therapy in HNSCC xenografts

The HNSCC cell line, FaDu was stably transfected with control vector (FaDu) or with plasmid expressing small interfering RNA against EMMPRIN (FaDu/siE). Tumor cells were treated with bevacizumab (0, 25, 50, and 75 ng/ml) in vitro, and then cell counts were performed at 72 h. For in vivo analysis, tumor cells were xenografted onto the flank of SCID mice, and were treated with 100 microg bevacizumab twice weekly for three weeks. Xenograft samples from the control and treatment groups were analyzed for microvessel density. Escalating doses of bevacizumab had no effect on the growth of tumor cells in vitro (P.or=0.086). However, tumor xenografts expressing EMMPRIN responded to bevacizumab treatment (P=0.0013), whereas the EMMPRIN knockdown cell line did not (P=0.7942). Immunohistochemical analysis demonstrated that microvascular density was reduced in the treated FaDu tumors (P=0.005), but not in the FaDu/siE tumors (P=0.48). Currently there is limited information on biomarkers to predict response to bevacizumab. By demonstrating effectiveness of bevacizumab therapy in tumors that express EMMPRIN, but not in tumors with silenced EMMPRIN expression, this study suggests that EMMPRIN may serve as a biomarker for response to bevacizumab treatment.