Molecular targeting of ultrasonographic contrast agent for detection of head and neck squamous cell carcinoma.

OBJECTIVE: To investigate the feasibility of ultrasonographic (US) imaging of head and neck cancer with targeted contrast agents both in vitro and in vivo. We hypothesize that conjugation of microbubble contrast agent to tumor-specific antibodies may improve US detection of head and neck squamous cell carcinoma (HNSCC). DESIGN: Preclinical blinded assessment of anti-EGFR and anti-CD147 microbubble contrast agents for US imaging of HNSCC. SETTING: Animal study. SUBJECTS: Immunodeficient mice. INTERVENTION: Injection of targeted microbubbles. MAIN OUTCOME MEASURE: Microbubble uptake in tumors as detected by US. RESULTS: In vitro assessment of anti-epidermal growth factor receptor (EGFR) and anti-CD147-targeted microbubbles in 6 head and neck cancer cell lines yielded a 6-fold improvement over normal dermal fibroblasts (P < .001). Binding of targeted agents had a positive correlation to both epidermal growth factor receptor (EGFR) (R(2) = 0.81) and CD147 (R(2) = 0.72) expression among all cell lines. In vivo imaging of flank tumors in nude mice (N = 8) yielded enhanced resolution of anti-EGFR-and anti-CD147-targeted microbubble agents over IgG control (P < .001), while dual-targeted contrast agents offered enhanced imaging over single-targeted contrast agents (P = .02 and P = .05, respectively). In a blinded in vivo assessment, targeted contrast agents increased intratumoral enhancement of flank tumors over controls. Targeted US contrast agents to both EGFR and CD147 were 100% sensitive and 87% specific in the detection of flank tumors. CONCLUSION: This preclinical study demonstrates feasibility of using molecular US to target HNSCC for contrast-enhanced imaging of HNSCC tumor in vivo.

Fetal mouse skin heals scarlessly in a chick chorioallantoic membrane model system.

In mammals, the early-gestation fetus has the regenerative ability to heal skin wounds without scar formation. This observation was first reported more than 3 decades ago, and has been confirmed in a number of in vivo animal models. Although an intensive research effort has focused on unraveling the mechanisms underlying scarless fetal wound repair, no suitable model of in vitro fetal skin healing has been developed. In this article, we report a novel model for the study of fetal wound healing. Fetal skin from gestational day 16.5 Balb/c mice (total gestation, 20 days) was grafted onto the chorioallantoic membrane of 12-day-old chicken embryos and cultured for up to 7 days. At 48 hours postengraftment, circular wounds (diameter = 1 mm) were made in the fetal skin using a rotating titanium sapphire laser (N = 45). The tissue was examined daily by visual inspection to look for signs of infection and ischemia. The grafts and the surrounding host tissue were examined histologically. In all fetal skin grafts, the wounds completely reepithelialized by postinjury day 7, with regeneration of the dermis. Fetal mouse skin xenografts transplanted onto the chorioallantoic membrane of fertilized chicken eggs provides a useful model for the study of fetal wound healing. This model can be used as an adjunct to traditional in vivo mammalian models of fetal repair.

(18)F-FDG PET/CT imaging detects therapy efficacy of anti-EMMPRIN antibody and gemcitabine in orthotopic pancreatic tumor xenografts.

PURPOSE: The objective of this study is to evaluate the therapeutic response to a novel monoclonal antibody targeting human extracellular matrix metalloproteinase inducer (EMMPRIN) in combination with gemcitabine in a pancreatic-tumor xenograft murine model by sequential 2-deoxy-2-[18F]fluoro-D-glucose ((18)F-FDG) positron emission tomography/computed tomgraphy (PET/CT) imaging. PROCEDURES: Four groups of SCID mice bearing orthotopic pancreatic tumor xenografts were injected with phosphate-buffered saline, gemcitabine (120 mg/kg BW), anti-EMMPRIN antibody (0.2 mg), or combination, respectively, twice weekly for 2 weeks, while (18)F-FDG PET/CT imaging was performed weekly for 3 weeks. Changes in mean standardized uptake value (SUV(mean)) of (18)F-FDG and volume of tumors were determined. RESULTS: The tumor SUV(mean) change in the group receiving combination therapy was significantly lower than those of the other groups. Tumor-volume changes of groups treated with anti-EMMPRIN monotherapy or combined therapy were significantly lower than that of the control group. CONCLUSIONS: These data provide support for clinical studies of anti-EMMPRIN therapy with gemcitabine for pancreatic cancer treatment.

Disruption of the AKT pathway inhibits metastasis in an orthotopic model of head and neck squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: MK-2206 is an orally active, allosteric inhibitor of AKT, a component of the phosphatidylinositol-3 kinase (PI3K) pathway. The PI3K-AKT pathway is a downstream signaling pathway that has recently been found to play an important role in head and neck squamous cell carcinoma (HNSCC). The objective of this study is to examine the role AKT inhibition may play in treatment of HNSCC. STUDY DESIGN: In vivo and in vitro study. METHODS: Cell migration after 24-hour treatment with subtherapeutic doses of MK-2206 was assessed using an enzyme-linked immunosorbent assay in four HNSCC cell lines: CAL27, FaDu, SCC-1, and SCC-5. In vitro effect of MK-2206 on cell migration was assessed by making linear scratches in culture plates after cell lines were grown to confluency. Images were taken at 8, 16, and 24 hours. In vivo analysis was performed on nude mice with human SCC1-orthotopic tongue tumors. After tumors were allowed to grow for 7 days, mice were treated with oral dosing of 120 mg/kg of MK-2206 every other day for 2 weeks. Tumor size was assessed after each treatment using a pair of digital calipers. At the end of the treatment period, mice were sacrificed and cervical lymph nodes were assessed for metastasis using fluorescent imaging of tumor cell markers. RESULTS: Subtherapeutic doses of MK-2206 were sufficient to significantly reduce cell migration in FaDu, SCC-1, and SCC-5 cell lines (P < .001) but not in Cal27 (P = .09). In vitro scratch test results in SCC-1 cells yielded significant reduction in cell movement at 8, 16, and 14 hours (P < .001). In vivo orthotopic model yielded significant reduction in primary tumor size (P = .04) and reduction in positive cervical lymph nodes (P = .01) between treatment and control mice. In addition we found 100% survival of MK-2206 treated mice after 2 weeks of treatment compared with 70% survival in our control group (P = .03). CONCLUSIONS: Treatment with MK-2206 is sufficient to inhibit HNSCC chemotaxis and migration in vitro. In an orthotopic model, treatment with MK-2206 reduces primary tumor size and cervical metastasis while improving survival. MK-2206 currently is being used in phase II clinical trials for combination treatment of metastatic solid tumors and may be useful for treating HNSCC as well.

Extracelluar matrix metalloproteinase as a novel target for pancreatic cancer therapy.

The objective of this study was to evaluate extracellular matrix metalloproteinase (EMMPRIN) as a novel target in orthotopic pancreatic cancer murine models. MIA PaCa-2 human pancreatic tumor cells were implanted in groups 1 and 3-7, whereas MIA PaCa-2 EMMPRIN knockdown cells were implanted in group 2. Dosing with anti-EMMPRIN antibody started immediately after implantation for groups 1-3 (residual tumor model) and at 21 days after cell implantation for groups 4-7 (established tumor model). Groups 3, 5, and 7 were treated with anti-EMMRPIN antibody (0.2-1.0 mg) twice weekly for 2-3 weeks, whereas the other groups served as the control. In the residual tumor model, tumor growth of anti-EMMPRIN-treated group was successfully arrested for 21 days (15 ± 4 mm(3)), which was significantly lower than that of the EMMPRIN knockdown group (80 ± 15 mm(3); P=0.001) or the control group (240 ± 41 mm(3); P<0.001). In the established tumor model, anti-EMMPRIN therapy lowered tumor volume increase by approximately 40% compared with the control, regardless of the dose amount. Ki67-expressed cell density of group 5 was 939 ± 150 mm(-2), which was significantly lower than that of group 4 (1709 ± 145 mm(-2); P=0.006). Microvessel density of group 5 (30 ± 6 mm(-2)) was also significantly lower than that of group 4 (53 ± 5 mm(-2); P=0.014), whereas the microvessel size of group 5 (191 ± 22 µm(2)) was significantly larger than that of group 4 (113 ± 26 µm(2); P=0.049). These data show the high potential of anti-EMMPRIN therapy for pancreatic cancer and support its clinical translation.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells.

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.

Anti-EMMPRIN antibody treatment of head and neck squamous cell carcinoma in an ex-vivo model.

Targeting the molecular pathways associated with carcinogenesis remains the greatest opportunity to reduce treatment-related morbidity and mortality. Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as CD147, is a cell surface molecule known to promote tumor growth and angiogenesis in preclinical studies of head and neck carcinoma making it an excellent therapeutic target. To evaluate the feasibility of anti-EMMPRIN therapy, an ex-vivo human head and neck cancer model was established using specimens obtained at the time of surgery (n=22). Tumor slices were exposed to varying concentrations of anti-EMMPRIN monoclonal antibody and cetuximab for comparison purposes. Cetuximab is the only monoclonal antibody currently approved for the treatment of head and neck carcinoma. After treatment, tumor slices were assessed by immunohistochemistry and western blot analysis for apoptosis (TUNEL) and EMMPRIN expression. Of the tumor specimens 33% showed a significant reduction in mean ATP levels after treatment with cetuximab compared with untreated controls, whereas 58% of the patients responded to anti-EMMPRIN therapy (P<0.05). Samples, which showed reactivity to anti-EMMPRIN, also had greater EMMPRIN expression based on immunohistochemistry staining (49%) when compared with nonresponders (25%, P=0.06). In addition, TUNEL analysis showed a larger number of cells undergoing apoptosis in antibody-treated tumor slices (77%) compared with controls (30%, P<0.001) with activation of apoptotic proteins, caspase 3 and caspase 8. This study shows the potential of anti-EMMPRIN to inhibit proliferation and promote apoptosis and suggests its future role in the targeted treatment of head and neck carcinoma.

Adult and fetal wound healing.

Mammalian wound healing is an intricate process involving the coordinated interaction of a variety of tissue types and cellular lineages. This is governed by a complex array of physical, biological and chemical signals. In adult tissue, the successful completion of wound healing inevitably results in the formation of scar. However, animal studies of embryonic tissue have shown that early gestation fetal wounds heal with complete restoration of tissue structure and function. The exact mechanisms underlying scarless healing in the fetus remain to be elucidated.