TSPO-targeted PET and Optical Probes for the Detection and Localization of Premalignant and Malignant Pancreatic Lesions.

PURPOSE: Pancreatic cancer is among the most aggressive malignancies and is rarely discovered early. However, pancreatic "incidentalomas," particularly cysts, are frequently identified in asymptomatic patients through anatomic imaging for unrelated causes. Accurate determination of the malignant potential of cystic lesions could lead to life-saving surgery or spare patients with indolent disease undue risk. Current risk assessment of pancreatic cysts requires invasive sampling, with attendant morbidity and sampling errors. Here, we sought to identify imaging biomarkers of high-risk pancreatic cancer precursor lesions. EXPERIMENTAL DESIGN: Translocator protein (TSPO) expression, which is associated with cholesterol metabolism, was evaluated in premalignant and pancreatic cancer lesions from human and genetically engineered mouse (GEM) tissues. In vivo imaging was performed with [18F]V-1008, a TSPO-targeted PET agent, in two GEM models. For image-guided surgery (IGS), V-1520, a TSPO ligand for near-IR optical imaging based upon the V-1008 pharmacophore, was developed and evaluated. RESULTS: TSPO was highly expressed in human and murine pancreatic cancer. Notably, TSPO expression was associated with high-grade, premalignant intraductal papillary mucinous neoplasms (IPMNs) and pancreatic intraepithelial neoplasia (PanIN) lesions. In GEM models, [18F]V-1008 exhibited robust uptake in early pancreatic cancer, detectable by PET. Furthermore, V-1520 localized to premalignant pancreatic lesions and advanced tumors enabling real-time IGS. CONCLUSIONS: We anticipate that combined TSPO PET/IGS represents a translational approach for precision pancreatic cancer care through discrimination of high-risk indeterminate lesions and actionable surgery.

Combined blockade of EGFR and glutamine metabolism in preclinical models of colorectal cancer.

Improving response to epidermal growth factor receptor (EGFR)-targeted therapies in patients with advanced wild-type (WT) RAS colorectal cancer (CRC) remains an unmet need. In this preclinical work, we evaluated a new therapeutic combination aimed at enhancing efficacy by targeting cancer cell metabolism in concert with EGFR. We hypothesized that combined blockade of glutamine metabolism and EGFR represents a promising treatment approach by targeting both the "fuel" and "signaling" components that these tumors need to survive. To explore this hypothesis, we combined CB-839, an inhibitor of glutaminase 1 (GLS1), the mitochondrial enzyme responsible for catalyzing conversion of glutamine to glutamate, with cetuximab, an EGFR-targeted monoclonal antibody in preclinical models of CRC. 2D and 3D in vitro assays were executed following treatment with either single agent or combination therapy. The combination of cetuximab with CB-839 resulted in reduced cell viability and demonstrated synergism in several cell lines. In vivo efficacy experiments were performed in cell-line xenograft models propagated in athymic nude mice. Tumor volumes were measured followed by immunohistochemical (IHC) analysis of proliferation (Ki67), mechanistic target of rapamycin (mTOR) signaling (pS6), and multiple mechanisms of cell death to annotate molecular determinants of response. In vivo, a significant reduction in tumor growth and reduced Ki67 and pS6 IHC staining were observed with combination therapy, which was accompanied by increased apoptosis and/or necrosis. The combination showed efficacy in cetuximab-sensitive as well as resistant models. In conclusion, this therapeutic combination represents a promising new precision medicine approach for patients with refractory metastatic WT RAS CRC.

Pharmacological blockade of ASCT2-dependent glutamine transport leads to antitumor efficacy in preclinical models.

The unique metabolic demands of cancer cells underscore potentially fruitful opportunities for drug discovery in the era of precision medicine. However, therapeutic targeting of cancer metabolism has led to surprisingly few new drugs to date. The neutral amino acid glutamine serves as a key intermediate in numerous metabolic processes leveraged by cancer cells, including biosynthesis, cell signaling, and oxidative protection. Herein we report the preclinical development of V-9302, a competitive small molecule antagonist of transmembrane glutamine flux that selectively and potently targets the amino acid transporter ASCT2. Pharmacological blockade of ASCT2 with V-9302 resulted in attenuated cancer cell growth and proliferation, increased cell death, and increased oxidative stress, which collectively contributed to antitumor responses in vitro and in vivo. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in oncology, representing a new class of targeted therapy and laying a framework for paradigm-shifting therapies targeting cancer cell metabolism.

Optimized Translocator Protein Ligand for Optical Molecular Imaging and Screening.

Translocator protein (TSPO) is a validated target for molecular imaging of a variety of human diseases and disorders. Given its involvement in cholesterol metabolism, TSPO expression is commonly elevated in solid tumors, including glioma, colorectal cancer, and breast cancer. TSPO ligands capable of detection by optical imaging are useful molecular tracers for a variety of purposes that range from quantitative biology to drug discovery. Leveraging our prior optimization of the pyrazolopyrimidine TSPO ligand scaffold for cancer imaging, we report herein a new generation of TSPO tracers with superior binding affinity and suitability for optical imaging and screening. In total, seven candidate TSPO tracers were synthesized and vetted in this study; the most promising tracer identified (29, Kd = 0.19 nM) was the result of conjugating a high-affinity TSPO ligand to a fluorophore used routinely in biological sciences (FITC) via a functional carbon linker of optimal length. Computational modeling suggested that an n-alkyl linker of eight carbons in length allows for positioning of the bulky fluorophore distal to the ligand binding domain and toward the solvent interface, minimizing potential ligand-protein interference. Probe 29 was found to be highly suitable for in vitro imaging of live TSPO-expressing cells and could be deployed as a ligand screening and discovery tool. Competitive inhibition of probe 29 quantified by fluorescence and 3H-PK11195 quantified by traditional radiometric detection resulted in equivalent affinity data for two previously reported TSPO ligands. This study introduces the utility of TSPO ligand 29 for in vitro imaging and screening and provides a structural basis for the development of future TSPO imaging ligands bearing bulky signaling moieties.

Preclinical TSPO Ligand PET to Visualize Human Glioma Xenotransplants: A Preliminary Study.

Current positron emission tomography (PET) imaging biomarkers for detection of infiltrating gliomas are limited. Translocator protein (TSPO) is a novel and promising biomarker for glioma PET imaging. To validate TSPO as a potential target for molecular imaging of glioma, TSPO expression was assayed in a tumor microarray containing 37 high-grade (III, IV) gliomas. TSPO staining was detected in all tumor specimens. Subsequently, PET imaging was performed with an aryloxyanilide-based TSPO ligand, [18F]PBR06, in primary orthotopic xenograft models of WHO grade III and IV gliomas. Selective uptake of [18F]PBR06 in engrafted tumor was measured. Furthermore, PET imaging with [18F]PBR06 demonstrated infiltrative glioma growth that was undetectable by traditional magnetic resonance imaging (MRI). Preliminary PET with [18F]PBR06 demonstrated a preferential tumor-to-normal background ratio in comparison to 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG). These results suggest that TSPO PET imaging with such high-affinity radiotracers may represent a novel strategy to characterize distinct molecular features of glioma growth, as well as better define the extent of glioma infiltration for therapeutic purposes.

Acetate dependence of tumors.

Acetyl-CoA represents a central node of carbon metabolism that plays a key role in bioenergetics, cell proliferation, and the regulation of gene expression. Highly glycolytic or hypoxic tumors must produce sufficient quantities of this metabolite to support cell growth and survival under nutrient-limiting conditions. Here, we show that the nucleocytosolic acetyl-CoA synthetase enzyme, ACSS2, supplies a key source of acetyl-CoA for tumors by capturing acetate as a carbon source. Despite exhibiting no gross deficits in growth or development, adult mice lacking ACSS2 exhibit a significant reduction in tumor burden in two different models of hepatocellular carcinoma. ACSS2 is expressed in a large proportion of human tumors, and its activity is responsible for the majority of cellular acetate uptake into both lipids and histones. These observations may qualify ACSS2 as a targetable metabolic vulnerability of a wide spectrum of tumors.

Synthesis and structure-activity relationships of 5,6,7-substituted pyrazolopyrimidines: discovery of a novel TSPO PET ligand for cancer imaging.

Focused library synthesis and structure-activity relationship development of 5,6,7-substituted pyrazolopyrimidines led to the discovery of 2-(5,7-diethyl-2-(4-(2-fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide (6b), a novel translocator protein (TSPO) ligand exhibiting a 36-fold enhancement in affinity compared to another pyrazolopyrimidine-based TSPO ligand, 6a (DPA-714). Radiolabeling with fluorine-18 ((18)F) facilitated production of 2-(5,7-diethyl-2-(4-(2-[(18)F]fluoroethoxy)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)-N,N-diethylacetamide ((18)F-6b) in high radiochemical yield and specific activity. In vivo studies of (18)F-6b were performed which illuminated this agent as an improved probe for molecular imaging of TSPO-expressing cancers.

3'-Deoxy-3'-18F-fluorothymidine PET predicts response to (V600E)BRAF-targeted therapy in preclinical models of colorectal cancer.

UNLABELLED: Selective inhibition of oncogenic targets and associated signaling pathways forms the basis of personalized cancer medicine. The clinical success of (V600E)BRAF inhibition in melanoma, coupled with the emergence of acquired resistance, underscores the importance of rigorously validating quantitative biomarkers of treatment response in this and similar settings. Because constitutive activation of BRAF leads to proliferation in tumors, we explored 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET to noninvasively quantify changes in tumor proliferation that are associated with pharmacologic inhibition of (V600E)BRAF downstream effectors and that precede changes in tumor volume. METHODS: Human colorectal cancer (CRC) cell lines expressing (V600E)BRAF were used to explore relationships between upregulation of p27 and phosphorylation of BRAF downstream effectors on small-molecule (V600E)BRAF inhibitor exposure. Athymic nude mice bearing (V600E)BRAF-expressing human CRC cell line xenografts were treated with a small-molecule (V600E)BRAF inhibitor (or vehicle) daily for 10 d. Predictive (18)F-FLT PET was conducted before changes in tumor volume occurred. Correlations were evaluated among PET, inhibition of phosphorylated MEK (p-MEK) and phosphorylated-ERK (p-ERK) by Western blot, tumor proliferation by histology, and small-molecule exposure by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). RESULTS: Treatment of CRC cell lines with PLX4720 reduced proliferation associated with target inhibition and upregulation of p27. In vivo, PLX4720 treatment reduced (18)F-FLT uptake, but not (18)F-FDG uptake, in Lim2405 xenografts before quantifiable differences in xenograft volume. Reduced (18)F-FLT PET reflected a modest, yet significant, reduction of Ki67 immunoreactivity, inhibition of p-MEK and p-ERK, and elevated tumor cell p27 protein levels. Both (18)F-FLT PET and (18)F-FDG PET accurately reflected a lack of response in HT-29 xenografts, which MALDI imaging mass spectrometry suggested may have stemmed from limited PLX4720 exposure. CONCLUSION: We used preclinical models of CRC to demonstrate (18)F-FLT PET as a sensitive predictor of response to (V600E)BRAF inhibitors. Because (18)F-FLT PET predicted reduced proliferation associated with attenuation of BRAF downstream effectors, yet (18)F-FDG PET did not, these data suggest that (18)F-FLT PET may represent an alternative to (18)F-FDG PET for quantifying clinical responses to BRAF inhibitors.

Quantitative preclinical imaging of TSPO expression in glioma using N,N-diethyl-2-(2-(4-(2-18F-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide.

UNLABELLED: There is a critical need to develop and rigorously validate molecular imaging biomarkers to aid diagnosis and characterization of primary brain tumors. Elevated expression of translocator protein (TSPO) has been shown to predict disease progression and aggressive, invasive behavior in a variety of solid tumors. Thus, noninvasive molecular imaging of TSPO expression could form the basis of a novel, predictive cancer imaging biomarker. In quantitative preclinical PET studies, we evaluated a high-affinity pyrazolopyrimidinyl-based TSPO imaging ligand, N,N-diethyl-2-(2-(4-(2-(18)F-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide ((18)F-DPA-714), as a translational probe for quantification of TSPO levels in glioma. METHODS: Glioma-bearing rats were imaged with (18)F-DPA-714 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of (18)F-DPA-714 (130-200 MBq/0.2 mL). Blood was collected to derive the arterial input function (AIF), with high-performance liquid chromatography radiometabolite analysis performed on selected samples for AIF correction. Compartmental modeling was performed using the corrected AIF. Specific tumor cell binding of DPA-714 was evaluated by radioligand displacement of (3)H-PK 11195 with DPA-714 in vitro and displacement of (18)F-DPA-714 with an excess of DPA-714 in vivo. Immediately after imaging, tumor and healthy brain tissues were harvested for validation by Western blotting and immunohistochemistry. RESULTS: (18)F-DPA-714 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain. Infusion with DPA-714 (10 mg/kg) displaced (18)F-DPA-714 binding by greater than 60% on average. Tumor uptake of (18)F-DPA-714 was similar to another high-affinity TSPO imaging ligand, (18)F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline, and agreed with ex vivo assay of TSPO levels in tumor and healthy brain. CONCLUSION: These studies illustrate the feasibility of using (18)F-DPA-714 for visualization of TSPO-expressing brain tumors. Importantly, (18)F-DPA-714 appears suitable for quantitative assay of tumor TSPO levels in vivo. Given the relationship between elevated TSPO levels and poor outcome in oncology, these studies suggest the potential of (18)F-DPA-714 PET to serve as a novel predictive cancer imaging modality.

Quantitative, preclinical PET of translocator protein expression in glioma using 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline.

UNLABELLED: Translocator protein (TSPO), also referred to as peripheral benzodiazepine receptor (PBR), is a crucial 18-kDa outer mitochondrial membrane protein involved in numerous cellular functions, including the regulation of cholesterol metabolism, steroidogenesis, and apoptosis. Elevated expression of TSPO in oncology correlates with disease progression and poor survival, suggesting that molecular probes capable of assaying TSPO levels may have potential as cancer imaging biomarkers. In preclinical PET studies, we characterized a high-affinity aryloxyanilide-based TSPO imaging ligand, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline (18F-PBR06), as a candidate probe for the quantitative assessment of TSPO expression in glioma. METHODS: Glioma-bearing rats were imaged with 18F-PBR06 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of 18F-PBR06 (70-100 MBq/0.2 mL). Over the course of scanning, arterial blood was collected to derive the input function, with high-performance liquid chromatography radiometabolite analysis performed on selected samples for arterial input function correction. Compartmental modeling of the PET data was performed using the corrected arterial input function. Specific tumor cell binding of PBR06 was evaluated by radioligand displacement of 3H-PK 11195 with PBR06 in vitro and by displacement of 18F-PBR06 with excess PBR06 in vivo. Immediately after imaging, tumor tissue and adjacent healthy brain were harvested for assay of TSPO protein levels by Western blotting and immunohistochemistry. RESULTS: 18F-PBR06 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain, facilitating excellent contrast between tumor and adjacent tissue. Infusion with PBR06 (10 mg/kg) displaced 18F-PBR06 binding by approximately 75%. The accumulation of 18F-PBR06 in tumor tissues and adjacent brain agreed with the ex vivo assay of TSPO protein levels by Western blotting and quantitative immunohistochemistry. CONCLUSION: These preclinical studies illustrate that 18F-PBR06 is a promising tracer for visualization of TSPO-expressing tumors. Importantly, the close correlation between 18F-PBR06 uptake and TSPO expression in tumors and normal tissues, coupled with the high degree of displaceable binding from both tumors and the normal brain, represents a significant improvement over other TSPO imaging ligands previously evaluated in glioma. These data suggest the potential of 18F-PBR06 to elucidate the role of TSPO in oncology, as well as its potential development as a cancer imaging biomarker.

TIP-1 translocation onto the cell plasma membrane is a molecular biomarker of tumor response to ionizing radiation.

BACKGROUND: Tumor response to treatment has been generally assessed with anatomic and functional imaging. Recent development of in vivo molecular and cellular imaging showed promise in time-efficient assessment of the therapeutic efficacy of a prescribed regimen. Currently, the in vivo molecular imaging is limited with shortage of biomarkers and probes with sound biological relevance. We have previously shown in tumor-bearing mice that a hexapeptide (HVGGSSV) demonstrated potentials as a molecular imaging probe to distinguish the tumors responding to ionizing radiation (IR) and/or tyrosine kinase inhibitor treatment from those of non-responding tumors. METHODOLOGY/PRINCIPAL FINDINGS: In this study we have studied biological basis of the HVGGSSV peptide binding within the irradiated tumors by use of tumor-bearing mice and cultured cancer cells. The results indicated that Tax interacting protein 1 (TIP-1, also known as Tax1BP3) is a molecular target that enables the selective binding of the HVGGSSV peptide within irradiated xenograft tumors. Optical imaging and immunohistochemical staining indicated that a TIP-1 specific antibody demonstrated similar biodistribution as the peptide in tumor-bearing mice. The TIP-1 antibody blocked the peptide from binding within irradiated tumors. Studies on both of human and mouse lung cancer cells showed that the intracellular TIP-1 relocated to the plasma membrane surface within the first few hours after exposure to IR and before the onset of treatment associated apoptosis and cell death. TIP-1 relocation onto the cell surface is associated with the reduced proliferation and the enhanced susceptibility to the subsequent IR treatment. CONCLUSIONS/SIGNIFICANCE: This study by use of tumor-bearing mice and cultured cancer cells suggested that imaging of the radiation-inducible TIP-1 translocation onto the cancer cell surface may predict the tumor responsiveness to radiation in a time-efficient manner and thus tailor radiotherapy of cancer.

Radiation induces an antitumour immune response to mouse melanoma.

PURPOSE: Irradiation of cancer cells can cause immunogenic death. We used mouse models to determine whether irradiation of melanoma can enhance the host antitumour immune response and function as an effective vaccination strategy, and investigated the molecular mechanisms involved in this radiation-induced response. MATERIALS AND METHODS: For in vivo studies, C57BL6/J mice and the B16F0 melanoma cell line were used in a lung metastasis model, intratumoural host immune activation assays, and tumour growth delay studies. In vitro studies included a dendritic cell (DC) phagocytosis assay, detection of cell surface exposure of the protein calreticulin (CRT), and small interfering RNA (siRNA)-mediated depletion of CRT cellular levels. RESULTS: Irradiation of cutaneous melanomas prior to their resection resulted in more than 20-fold reduction in lung metastases after systemic challenge with untreated melanoma cells. A syngeneic vaccine derived from irradiated melanoma cells also induced adaptive immune response markers in irradiated melanoma implants. Our data indicate a trend for radiation-induced increase in melanoma cell surface exposure of CRT, which is involved in the enhanced phagocytic activity of DC against irradiated melanoma cells (VIACUC). CONCLUSION: The present study suggests that neoadjuvant irradiation of cutaneous melanoma tumours prior to surgical resection can stimulate an endogenous anti-melanoma host immune response.

Lithium-mediated protection of hippocampal cells involves enhancement of DNA-PK-dependent repair in mice.

Long-term neurological deficiencies resulting from hippocampal cytotoxicity induced by cranial irradiation (IR) present a challenge in the treatment of primary and metastatic brain cancers, especially in children. Previously, we showed that lithium protected hippocampal neurons from IR-induced apoptosis and improved neurocognitive function in treated mice. Here, we demonstrate accelerated repair of IR-induced chromosomal double-strand breaks (DSBs) in lithium-treated neurons. Lithium treatment not only increased IR-induced DNA-dependent protein kinase (DNA-PK) threonine 2609 foci, a surrogate marker for activated nonhomologous end-joining (NHEJ) repair, but also enhanced double-strand DNA end-rejoining activity in hippocampal neurons. The increased NHEJ repair coincided with reduced numbers of IR-induced gamma-H2AX foci, well-characterized in situ markers of DSBs. These findings were confirmed in vivo in irradiated mice. Consistent with a role of NHEJ repair in lithium-mediated neuroprotection, attenuation of IR-induced apoptosis of hippocampal neurons by lithium was dramatically abrogated when DNA-PK function was abolished genetically in SCID mice or inhibited biochemically by the DNA-PK inhibitor IC86621. Importantly, none of these findings were evident in glioma cancer cells. These results support our hypothesis that lithium protects hippocampal neurons by promoting the NHEJ repair-mediated DNA repair pathway and warrant future investigation of lithium-mediated neuroprotection during cranial IR, especially in the pediatric population.

SU11248 (sunitinib) sensitizes pancreatic cancer to the cytotoxic effects of ionizing radiation.

PURPOSE: SU11248 (sunitinib) is a small-molecule tyrosine kinase inhibitor which targets VEGFR and PDGFR isoforms. In the present study, the effects of SU11248 and ionizing radiation on pancreatic cancer were studied. METHODS AND MATERIALS: For in vitro studies human pancreatic adenocarcinoma cells lines were treated with 1 microM SU11248 1 h before irradiation. Western blot analysis was used to determine the effect of SU11248 on radiation-induced signal transduction. To determine if SU11248 sensitized pancreatic cancer to the cytotoxic effects of ionizing radiation, a clonogenic survival assay was performed using 0-6 Gy. For in vivo assays, CAPAN-1 cells were injected into the hind limb of nude mice for tumor volume and proliferation studies. RESULTS: SU11248 attenuated radiation-induced phosphorylation of Akt and ERK at 0, 5, 15, and 30 min. Furthermore, SU11248 significantly reduced clonogenic survival after treatment with radiation (p < 0.05). In vivo studies revealed that SU11248 and radiation delayed tumor growth by 6 and 10 days, respectively, whereas combined treatment delayed tumor growth by 30 days. Combined treatment with SU11248 and radiation further attenuated Brdu incorporation by 75% (p = 0.001) compared to control. CONCLUSIONS: SU11248 (sunitinib) sensitized pancreatic cancer to the cytotoxic effects of radiation. This compound is promising for future clinical trials with chemoradiation in pancreatic cancer.

Noninvasive assessment of cancer response to therapy.

Rapid assessment of cancer response to a therapeutic regimen can determine efficacy early in the course of treatment. Although biopsies of cancer can be used to rapidly assess pharmacodynamic response, certain disease sites are less accessible to repeated biopsies. Here, we simultaneously assess response in all sites of disease within days of starting therapy by use of peptide ligands selected for their ability to discern responding from nonresponding cancers. When conjugated to near-infrared imaging agents, the HVGGSSV peptide differentiates between these two types of cancer. Rapid, noninvasive assessment of the pharmacodynamic response within cancer promises to accelerate drug development and minimize the duration of treatment with ineffective regimens in cancer patients.

Hedgehog signaling in the murine melanoma microenvironment.

The Hedgehog intercellular signaling pathway regulates cell proliferation and differentiation. This pathway has been implicated to play a role in the pathogenesis of cancer and in embryonic blood vessel development. In the current study, Hedgehog signaling in tumor related vasculature and microenvironment was examined using human umbilical vein endothelial cells and B16F0 (murine melanoma) tumors models. Use of exogenous Sonic hedgehog (Shh) peptide significantly increased BrdU incorporation in endothelial cells in vitro by a factor of 2 (P < 0.001). The Hedgehog pathway antagonist cyclopamine effectively reduced Shh-induced proliferation to control levels. To study Hedgehog signaling in vivo a hind limb tumor model with the B16F0 cell line was used. Treatment with 25 mg/kg cyclopamine significantly attenuated BrdU incorporation in tumor cells threefold (P < 0.001), in tumor related endothelial cells threefold (P = 0.004), and delayed tumor growth by 4 days. Immunohistochemistry revealed that the Hedgehog receptor Patched was localized to the tumor stroma and that B16F0 cells expressed Shh peptide. Furthermore, mouse embryonic fibroblasts required the presence of B16F0 cells to express Patched in a co-culture assay system. These studies indicate that Shh peptide produced by melanoma cells induces Patched expression in fibroblasts. To study tumor related angiogenesis a vascular window model was used to monitor tumor vascularity. Treatment with cyclopamine significantly attenuated vascular formation by a factor of 2.5 (P < 0.001) and altered vascular morphology. Furthermore, cyclopamine reduced tumor blood vessel permeability to FITC labeled dextran while having no effect on normal blood vessels. These studies suggest that Hedgehog signaling regulates melanoma related vascular formation and function.

Histone deacetylase inhibitor NVP-LAQ824 sensitizes human nonsmall cell lung cancer to the cytotoxic effects of ionizing radiation.

Stage III nonsmall cell lung cancer is primarily treated with combined chemotherapy and radiation therapy. Relapses for progression of disease within irradiated sites remains a primary pattern of failure. To evaluate the interaction between histone deacetylase inhibitors and irradiation in nonsmall cell lung cancer, we studied NVP-LAQ824 in mouse models of human lung cancer. Colony formation assays were performed to determine whether LAQ824 sensitized nonsmall cell lung cancer to the cytotoxic effects of ionizing radiation. LAQ824 reduced clonogenic survival of the H23 and H460 cell lines five-fold compared with controls and four-fold compared with either agent alone (P<0.001). Western blot analysis of caspase cleavage, microscopic analysis of nuclei and Annexin-fluorescein isothiocyanate/propidium iodide flow cytometry assays showed that LAQ824 enhanced radiation-induced apoptosis and attenuated mitosis (P<0.001). Immunostaining for gamma-H2AX nuclear foci was performed to determine the effect of LAQ824 on radiation-induced DNA double-strand breaks. Combined modality treatment delayed the resolution of gamma-H2AX foci with over 30% of cells staining positive 6 h after treatment versus approximately 5 and 3% in cells treated with LAQ824 or radiation alone (P<0.001). Additionally, an in-vivo xenograft model was utilized to study the effects of fractioned irradiation and LAQ824 on tumor growth. Fractioned irradiation and LAQ824 delayed tumor growth by 19 days versus 7 and 4 days for treatment with LAQ824 and radiation alone. This study shows the effectiveness of histone deacetylase inhibitors to enhance the cytotoxic effects of radiation by attenuating DNA repair and inducing apoptosis in human nonsmall cell lung cancer.

HIV protease inhibitors enhance the efficacy of irradiation.

Tumor vascular endothelium is rather resistant to the cytotoxic effects of radiation. The HIV protease inhibitors (HPI) amprenavir, nelfinavir, and saquinavir have previously been shown to sensitize tumor cells to the cytotoxic effects of radiation. Additionally, this class of drug has been shown to inhibit angiogenesis and tumor cell migration. Therefore, in the current study, we wanted to determine whether HPIs could enhance the effect of radiation on endothelial function. Our study shows that HPIs, particularly nelfinavir, significantly enhance radiations effect on human umbilical vein endothelial cells (HUVEC) and tumor vascular endothelium. We show that pretreatment of HUVEC with nelfinavir results in enhanced cytotoxicity, including increased apoptosis, when combined with radiation. Moreover, using several functional assays, we show that combination treatment effectively blocks endothelial cell migration and organization. These findings were accompanied by attenuation of Akt phosphorylation, a known pathway for radioresistance. Last, in vivo analysis of tumor microvasculature destruction showed a more than additive effect for nelfinavir and radiation. This study shows that HPIs can enhance the effect of ionizing radiation on vascular endothelium. Therefore, the Food and Drug Administration-approved drug, nelfinavir, may be an effective radiosensitizer in the clinic.

Effects of vascular endothelial growth factor receptor inhibitor SU5416 and prostacyclin on murine lung metastasis.

The majority of patients with a diagnosis of cancer die from metastatic disease. Targeting specific steps in the metastatic process has the potential to improve patient outcomes. In this study, a novel lung metastasis model was developed by injecting DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbo-cyanine perchlorate)-labeled Lewis lung carcinoma cells into the tail vein of mice. The temporal development of tumor metastases was studied in the lung, liver and spleen. Additionally, the effects of vascular endothelial growth factor receptor inhibitor SU5416 and platelet activation inhibitor prostacyclin were tested in this metastasis model. Systemically injected Lewis lung carcinoma cells present in the lung at 15 min slowly accumulated in the liver and spleen reaching a peak at 4 days. After 8 days, tumor development was only evident in the lung. Use of SU5416 or prostacyclin lowered the initial density of Lewis lung carcinoma-labeled cells in the lung by a factor 1.8 and 2.3, respectively (P<0.05). Furthermore, treatment with prostacyclin or SU5416 decreased lung weight by over 50% and the number of visible metastatic nodes by over 90% (P<0.05). Combined treatment resulted in grossly normal lung tissue. Additionally, systemic treatment with prostacyclin reduced harvested metastatic cell adherence to endothelial cells by a factor of 10 and treatment with SU5416 attenuated vascular formation (P<0.001). In conclusion, SU5416 and prostacyclin effectively attenuated metastasis formation in this model. DiI labeling is an effective technique to monitor the temporal and spatial distribution of metastatic cells.

Histone deacetylase (HDAC) inhibitor LBH589 increases duration of gamma-H2AX foci and confines HDAC4 to the cytoplasm in irradiated non-small cell lung cancer.

Histone deacetylases (HDAC) have been identified as therapeutic targets due to their regulatory function in DNA structure and organization. LBH589 is a novel inhibitor of class I and II HDACs. We studied the effect of LBH589 and ionizing radiation (IR) on DNA repair in two human non-small cell lung cancer (NSCLC) cell lines (H23 and H460). gamma-H2AX foci present at DNA double-strand breaks (DSBs) were detected in the nuclei following 3 Gy irradiation for up to 6 hours. LBH589 administered before irradiation increased the duration of gamma-H2AX foci beyond 24 hours. Furthermore, radiation alone induced translocation of HDAC4 to the nucleus. In contrast, treatment with LBH589 followed by irradiation resulted in HDAC4 confinement to the cytoplasm, indicating that HDAC inhibition affects the nuclear localization of HDAC4. The findings that LBH589 confines HDAC4 to the cytoplasm and increases the duration of gamma-H2AX foci in irradiated cell lines suggest that HDAC4 participates in DNA damage signaling following IR. Annexin-propidium iodide flow cytometry assays, cell morphology studies, and cleaved caspase-3 Western blot analysis revealed a synergistic effect of LBH589 with IR in inducing apoptosis. Clonogenic survival showed a greater than additive effect when LBH589 was administered before irradiation compared with irradiation alone. In vivo tumor volume studies showed a growth delay of 20 days with combined treatment compared with 4 and 2 days for radiation or LBH589 alone. This study identifies HDAC4 as a biomarker of LBH589 activity and recognizes the ability of LBH589 to sensitize human NSCLC to radiation-induced DNA DSBs.