Joseph Knowles

Microbubble Therapy Enhances Anti-tumor Properties of Cisplatin and Cetuximab In Vitro and In Vivo

Objective. To determine if microbubble-mediated ultrasound therapy (MB-UST) can improve cisplatin or cetuximab cytotoxicity of head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo by increasing tumor-specific drug delivery by disruption of tumor cell membranes and enhancing vascular permeability. Study Design. In vitro and in vivo study. Setting. University medical center. Subjects. Immunodeficient mice (6 weeks old) and 4 HNSCC cell lines. Methods. Changes to cell permeability were assessed in vitro after MB-UST. Cellular apoptosis resulting from adjuvant MB-UST with subtherapeutic doses of cisplatin or cetuximab was assessed by cell survival assays in vitro. The in vivo effect of adjuvant MB-UST in flank tumors was assessed in vivo with histological analysis and diffusion-weighted magnetic resonance imaging (DW-MRI). Results. In vitro results revealed that MB-UST can increase cell permeability and enhance drug uptake and apoptosis in 4 HNSCC cell lines. In vivo adjuvant MB-UST with cetuximab or cisplatin showed a statistically significant reduction in tumor size when compared with untreated controls. TUNEL analysis yielded a larger number of cells undergoing apoptosis in tumors treated with cetuximab and adjuvant MB-UST than did cetuximab alone but was not significantly greater in tumors treated with cisplatin and adjuvant MB-UST compared with cisplatin alone. DW-MRI analysis showed more free water, which corresponds to increased cell membrane disruption, in tumors treated with MB-UST. Conclusion. MB-UST promotes disruption of cell membranes in tumor cells in vitro, which may be leveraged to selectively improve the uptake of conventional and targeted therapeutics in vivo.

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

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.

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.

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.

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 mm3), which was significantly lower than that of the EMMPRIN knockdown group (80±15 mm3; P=0.001) or the control group (240±41 mm3; 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 &mgr;m2) was significantly larger than that of group 4 (113±26 &mgr;m2; 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. Mol Cancer Res; 9(8); 1008–17. ©2011 AACR.

Ultrasound therapy amplifies chemotherapeutic effectiveness in head and neck cancer

Microbubble (MB) mediated ultrasound (US) therapy is a novel technique to non-invasively enhance cancer treatment by increasing cell membrane permeability and vascular extravasation. The objective of this experiment is to determine if MB-mediated US therapy can improve cancer therapy, specifically cisplatin or cetuximab, in head and neck cancer. In vitro experiments included combination chemotherapy and MB-mediated US therapy on head and neck cancer cell line, SCC-5, and dose curve responses to therapy. In vivo experimentation included a four week study of MB-mediated US therapy compared to current standard therapy of drug alone. Monitoring of tumor response to therapy included caliper measurements, diffusion-weighted magnetic resonance (DW-MR) imaging, and immunohistologic analysis. Anti-tumor effects increased by over 50% when combining MB-mediated US therapy with cetuximab and cisplatin (P = 0.01, P = 0.02). MBmediated US therapy was seen to produce as additive effect by increasing localized delivery of drug in head and neck cancer. This technique shows potential for improved localized therapy in cancer.

Ultrasound Therapy Boosts Drug Toxicity In Vitro and In Vivo

Objective: Ultrasound-mediated therapy (UST) utilizes mechanical vibration of microbubble contrast agent to enhance cell permeability and anti-cancer drug delivery. Here we determine if UST can improve cisplatin or cetuximab treatment of HNSCC in vitro and in vivo. Method: Cell permeability was assessed after UST in four HNSCC cell lines (SCC1, SCC5, Cal27, and FaDu). Combination of UST with cisplatin or cetuximab was assessed by cell survival in vitro. In vivo effects on flank tumors were measured in immune-suppressed mice. Results: Cell permeability was 48.2 ± 4.8% greater in cells treated with UST compared to controls (P in vitro, UST alone had no effect on cell survival (P = .59), but combination treatment with cetuximab (P = .002) or cisplatin (P in vivo) (P = .81), but combination with cetuximab (P = .04) orcisplatin (P = .01) enhanced their antitumor effects. TUNEL analysis of flank tumors demonstrated increased apoptosis in UST+ cetuximab (55% vs 44%, P = .05), and UST+ cisplatin (84% vs 77%, P = .24) compared with monotherapy with either drug. Conclusion: The antitumor effects of cetuximab and cisplatin in HNSCC are enhanced by concurrent treatment with ultrasound-mediated therapy in vitro and in vivo.

Preclinical Strategies to Optimize Optical Imaging in Head and Neck Cancer

Objective: There is a need for cancer imaging in the clinic and operating room. We have shown that fluorescently labeled antibodies can detect microscopic islands of head and neck cancer cells using modified intraoperative microscopes in preclinical animal studies. Here we evaluated the optimal targeting molecule for clinical translation. Method: We assessed in vitro binding of fluorescently labeled antibodies targeting EGFR (cetuximab/Erbitux and panitumumab/Vectibix) and an antibody mimetic (anti-EGFR affibody) in multiple cell lines (SCC5, OSC19, FADU). To compare in vivo imaging characteristics, HNSCC xenografts (SCC5, SCC1, FADU) were imaged after systemic injection of fluorescently labeled antibodies over 5 days. Results: The binding affinity was lower for the control antibody IgG (KD = 9.55 nM) compared with either panitumumab (KD = 6.82 nM) or cetuximab (KD = 2.86 nM). In vitro, panitumumab had a two fold increase in relative fluorescence compared with cetuximab and a four fold increase in fluorescence compared with control antibody (nonspecific IgG). Xenograft imaging in vivo demonstrated a variation in fluorescence intensity that correlated with circulating half-lives: peaks in fluorescence intensity were seen at 1.5 h (affibody) and 48 to 72 h (cetuximab, panitunumab). The pantitumumab and affibody images demonstrated greater tumor fluorescence and lower background signal compared to cetuximab. Conclusion: Panitumumab/Vectibix demonstrated optimal imaging characteristics which appeared to be related to its binding affinity and increased circulation time. These characteristics may allow for increased localization to the tumor site. Panitumumab may be the optimal antibody for clinical translation since it will decrease false positive rates associated with non-specific uptake.

TKI258 Is Efficacious as a Multitargeted TKI in Head and Neck Squamous Cell Carcinoma Models

Objective: Dovitinib (TKI28) is a multitargeted tyrosine kinase inhibitor that has not been previously investigated in the pre-clinical setting for the treatment of head and neck squamous cell carcinoma (HNSCC). Dovitinib is administered orally and targets receptors (FGFR1/2/3 and PDGF-beta) known to promote cell survival and proliferation and decrease cell apoptosis. Method: 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: Elevated expression of FGFR2, FGFR3, and PDGF-beta were indentified in HNSCC cell lines and human tumor specimens. In vitro, dovitinib reduced HNSCC cell proliferation (40-80%) and viability (40-60%). In a dose-dependent fashion, dovitinib also induced cytotoxicity and apoptosis in HNSCC cell lines. When co-cultured with fibroblasts, HNSCC cells proliferation was also decreased by dovitinib. In vivo, oral administration of dovitinib resulted in significant tumor regression and growth inhibition (67%). Disease-free survival at 75 days following completion of treatment was 33% and stable disease occurred in another 33%. Conclusion: This is the first report to demonstrate dovitinib inhibits HNSCC cell growth in vitro and in vivo.