Primary Subcutaneous Tumors Research Articles

Differential Proteome Analysis of Human Neuroblastoma Xenograft Primary Tumors and Matched Spontaneous Distant Metastases

Metastasis formation is the major cause for cancer-related deaths and the underlying mechanisms remain poorly understood. In this study we describe spontaneous metastasis xenograft mouse models of human neuroblastoma used for unbiased identification of metastasis-related proteins by applying an infrared laser (IR) for sampling primary tumor and metastatic tissues, followed by mass spectrometric proteome analysis. IR aerosol samples were obtained from ovarian and liver metastases, which were indicated by bioluminescence imaging (BLI), and matched subcutaneous primary tumors. Corresponding histology proved the human origin of metastatic lesions. Ovarian metastases were commonly larger than liver metastases indicating differential outgrowth capacities. Among ~1,900 proteins identified at each of the three sites, 55 proteins were differentially regulated in ovarian metastases while 312 proteins were regulated in liver metastases. There was an overlap of 21 and 7 proteins up- and down-regulated at both metastatic sites, respectively, most of which were so far not related to metastasis such as LYPLA2, EIF4B, DPY30, LGALS7, PRPH, and NEFM. Moreover, we established in vitro sublines from primary tumor and metastases and demonstrate differences in cellular protrusions, migratory/invasive potential and glycosylation. Summarized, this work identified several novel putative drivers of metastasis formation that are tempting candidates for future functional studies.

Targeting cytokine signaling checkpoint CIS activates NK cells to protect from tumor initiation and metastasis

ABSTRACTThe cytokine-induced SH2-containing protein CIS belongs to the suppressor of cytokine signaling (SOCS) protein family. Here, we show the critical role of CIS in suppressing natural killer (NK) cell control of tumor initiation and metastasis. Cish-deficient mice were highly resistant to methylcholanthrene-induced sarcoma formation and protected from lung metastasis of B16F10 melanoma and RM-1 prostate carcinoma cells. In contrast, the growth of primary subcutaneous tumors, including those expressing the foreign antigen OVA, was unchanged in Cish-deficient mice. The combination of Cish deficiency and relevant targeted and immuno-therapies such as combined BRAF and MEK inhibitors, immune checkpoint blockade antibodies, IL-2 and type I interferon revealed further improved control of metastasis. The data clearly indicate that targeting CIS promotes NK cell antitumor functions and CIS holds great promise as a novel target in NK cell immunotherapy.

The innate immune receptor Dectin-2 mediates the phagocytosis of cancer cells by Kupffer cells for the suppression of liver metastasis

Tumor metastasis is the cause of most cancer deaths. Although metastases can form in multiple end organs, the liver is recognized as a highly permissive organ. Nevertheless, there is evidence for immune cell-mediated mechanisms that function to suppress liver metastasis by certain tumors, although the underlying mechanisms for the suppression of metastasis remain elusive. Here, we show that Dectin-2, a C-type lectin receptor (CLR) family of innate receptors, is critical for the suppression of liver metastasis of cancer cells. We provide evidence that Dectin-2 functions in resident macrophages in the liver, known as Kupffer cells, to mediate the uptake and clearance of cancer cells. Interestingly, Kupffer cells are selectively endowed with Dectin-2-dependent phagocytotic activity, with neither bone marrow-derived macrophages nor alveolar macrophages showing this potential. Concordantly, subcutaneous primary tumor growth and lung metastasis are not affected by the absence of Dectin-2. In addition, macrophage C-type lectin, a CLR known to be complex with Dectin-2, also contributes to the suppression of liver metastasis. Collectively, these results highlight the hitherto poorly understood mechanism of Kupffer cell-mediated control of metastasis that is mediated by the CLR innate receptor family, with implications for the development of anticancer therapy targeting CLRs.

18F]MEL050 as a melanin-targeted PET tracer: Fully automated radiosynthesis and comparison to 18F-FDG for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases

IntroductionMelanoma is a highly malignant cutaneous tumor of melanin-producing cells. MEL050 is a synthetic benzamide-derived molecule that specifically binds to melanin with high affinity. Our aim was to implement a fully automated radiosynthesis of [18F]MEL050, using for the first time, the AllInOne™ synthesis module (Trasis), and to evaluate the potential of [18F]MEL050 for the detection of pigmented melanoma in mice primary subcutaneous tumors and pulmonary metastases, and to compare it with that of [18F]FDG. MethodsAutomated radiosynthesis of [18F]MEL050, including HPLC purification and formulation, were performed on an AllInOne™ synthesis module. [18F]MEL050 was synthesized using a one-step bromine-for-fluorine nucleophilic heteroaromatic substitution.Melanoma models were induced by subcutaneous (primary tumor) or intravenous (pulmonary metastases) injection of B16-F10-luc2 cells in NMRI mice. The maximum percentage of [18F]MEL050 Injected Dose per g of lung tissue (%ID/g Max) was determined on PET images, compared to [18F]FDG and correlated to in vivo bioluminescence imaging. ResultsThe automated radiosynthesis of [18F]MEL050 required an overall radiosynthesis time of 48min, with a yield of 13–18% (not-decay corrected) and radiochemical purity higher than 99%. [18F]MEL050 PET/CT images were concordant with bioluminescence imaging, showing increased radiotracer uptake in all primary subcutaneous tumors and pulmonary metastases of mice. PET quantification of radiotracers uptake in tumors and muscles demonstrated similar tumor-to-background ratio (TBR) with [18F]MEL050 and [18F]FDG in subcutaneous tumors and higher TBR with [18F]MEL050 than with [18F]FDG in pulmonary metastases. ConclusionWe successfully implemented the radiosynthesis of [18F]MEL050 using the AllInOne™ module, including HPLC purification and formulation. In vivo PET/CT validation of [18F]MEL050 was obtained in mouse models of pigmented melanoma, where higher [18F]MEL050 uptake was observed in sub-millimetric pulmonary metastases, comparatively to [18F]FDG.

Abstract 5133: Targeting the eicosanoid 20-HETE suppresses pancreatic cancer growth and metastasis through regulation of inflammation

Abstract Pancreatic cancer incidence and death rates are on the rise. Most available therapies fail partly due to the uniquely inflammatory pancreatic tumor stromal compartment. Prolonged and unresolved inflammation is implicated in the pathogenesis of pancreatic cancer, yet the critical inflammatory mediators remain largely unknown. The role of lipid autacoids in pancreatic cancer, which are metabolites of arachidonic acid and other related fatty acids, has not been thoroughly studied. Among these, metabolites of the cytochrome P450 (CYP) epoxygenases and ω-hydroxylases, including EETs and 20-HETE, respectively, have been mostly neglected in cancer. We previously showed that epoxyeicosatrienoic acids (EETs) stimulate multi-organ metastasis and tumor dormancy escape. Here we show that elevated levels of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of ω-hydroxylases of the CYP4A/F subfamilies, stimulated pancreatic primary tumor growth and metastasis. We measured 20-HETE and other lipid autacoids via LC/MS/MS in multiple cell lines and found that 20-HETE was produced primarily by pancreatic cancer cells, which express the 20-HETE generating enzyme, CYP4F2. Overexpression of CYP4F2 and exogenous 20-HETE induced Ras activity in HEK293 cells or pancreatic cancer and macrophage cell lines, respectively. 20-HETE attracted human macrophages in vitro, while macrophage migration was reduced towards pancreatic cancer cells treated with an inhibitor of 20-HETE synthesis, HET0016. Inhibition of 20-HETE synthesis in human macrophages and pancreatic cancer cells decreased HB-EGF and M-CSF production. Systemic administration of 20-HETE promoted primary human and murine pancreatic tumor growth in subcutaneous and orthotopic murine models. Transgenic mice overexpressing 20-HETE (Tie2-CYP4F2 Tr) exhibited spontaneous liver and mediastinal lymph node metastasis following resection of subcutaneous primary pancreatic tumors, in contrast to wild type control mice. Targeting 20-HETE by HET0016 or siRNA towards CYP4F2 suppressed primary pancreatic tumor growth. Systemic treatment of orthotopic pancreatic tumors with HET0016, 14,15-EEZE, and gemcitabine regressed established pancreatic tumors and prolonged survival without overt toxicity. Thus, 20-HETE critically regulates Ras activation, inflammation, and pancreatic tumor growth and metastasis. Our studies offer a mechanistic rationale for pharmacologically targeting 20-HETE as a novel approach to treat pancreatic cancer. Citation Format: Yael Gus-Brautbar, Julia Piwowarski, Jessica A. Casper, Diane R. Bielenberg, Jennifer Cheng, Birgitta Schmidt, Bruce D. Hammock, Darryl C. Zeldin, Mark W. Kieran, Dipak Panigrahy. Targeting the eicosanoid 20-HETE suppresses pancreatic cancer growth and metastasis through regulation of inflammation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5133.

Abstract A23: Reversible metabolic changes in human melanoma cells enable distant metastasis

Abstract Metastasis is a complex multistep process: cancer cells have to detach from the bulk of the primary tumor, intravasate into the blood, survive anoikis and high shear forces in the blood stream while existing as single cells, extravasate into a distal site, and finally proliferate to form metastatic nodules. Currently our understanding of the mechanisms that govern the metastatic cascade is very limited. Our laboratory has developed a model of melanoma metastasis using patient-derived xenografts in immunocompromised NOD/SCID gamma (NSG) mice. This model is predictive of the clinical outcome in patients: stage III melanomas that metastasize efficiently in NSG mice also form distant metastases in patients, whereas melanomas that metastasize inefficiently in mice do not readily metastasize in patients. Using this model we have isolated melanoma cells from primary subcutaneous tumors, peripheral blood, and metastatic nodules from visceral organs, and tested their capacity to form tumors at different sites. The blood and distant sites were hostile environments that prevented efficient tumorigenesis, compared to the primary site. Cells from metastatic nodules formed tumors at higher frequency than cells from the blood or primary subcutaneous tumors, when transplanted directly into visceral organs. To examine the mechanism underlying the ability of metastatic cells to grow in visceral organs, we have performed metabolomic profiling of these cell populations and discovered that compared to cells from primary subcutaneous tumors, both circulating tumor cells (CTCs) as well as cells from metastatic nodules have a low Glutathione (GSH)/oxidized glutathione (GSSG) ratio as well as high levels of reactive oxygen species (ROS). These data strongly indicate that melanoma cells experience high levels of oxidative stress in the blood and in visceral organs, suggesting this might be the reason why solid cancers are highly inefficient at forming distant metastases. Furthermore, chronic treatment of mice with antioxidants such as N-acetylcysteine had no effect on the growth of the primary subcutaneous tumors but caused a significant increase in circulating tumor cell frequency and the overall metastatic burden in distant organs. These data suggest that the ability of the cells to detoxify ROS may be directly linked to their ability to metastasize. We found that reversible metabolic changes in efficiently metastasizing melanomas increased their capacity to withstand oxidative stress. Depletion of NADPH-producing enzymes in the folate pathway had a detrimental effect on both circulating tumor cell frequency as well as overall metastatic burden. Thus, oxidative stress limits distant metastasis by melanoma cells in vivo, and successful metastasizers rely on upregulation of antioxidant mechanisms particularly through the folate pathway. This work provides insight into the metabolism of metastasis and suggests that specific folate pathway enzymes may represent potential novel therapeutic targets. Citation Format: Elena Piskounova, Michalis Agathocleous, Sara Mann, Ralph DeBerardinis, Sean J. Morrison. Reversible metabolic changes in human melanoma cells enable distant metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A23.

Photodynamic diagnosis of gastric cancer using HPPH-CD

Gastric cancer is associated with a poor prognosis and is the fourth most common carcinoma and the second leading cause of cancer-related mortality worldwide due to its late-stage diagnosis and recurrence. New strategies are urgently needed to improve this condition. 2-[1′-Hexyloxyethyl]-2-devinyl pyropheophorbide-(HPPH)-cyanine dye (CD) mediated photodynamic diagnosis (PDD) has attracted great attention as a relatively new modality for tumor diagnosis. However, the efficacy of HPPH-CD-PDD on gastric cancer is poorly understood. The present study evaluates the effectiveness of PDD on gastric cancer diagnosis using HPPH-CD. We first evaluated the imaging potential of HPPH-CD on ICR mice bearing S180 subcutaneous tumors to make a preliminary definition of the ideal conditions for diagnosis. Subsequently, animal models with gastric cancer were established by giving 3,4-benzopyrene (B(a)P) to KM mice and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) to Wistar rats. The fluorescence signals of the stomach were then detected after HPPH-CD delivery, followed by imaging under a standard bright field. Furthermore, the tumors were collected, fixed and processed for histologic review. We found that subcutaneous S180 tumors and orthotopic primary gastric tumors could be easily visualized with fluorescence imaging after the delivery of HPPH-CD, and all malignant lesions with strong fluorescence were histologically confirmed. These results revealed the potential of HPPH-CD as a novel photosensitizer for the enhanced visualization of tumors in gastric cancer.

Anthropomorphic Phantoms for Confirmation of Linear Accelerator-Based Small Animal Irradiation.

Three dimensional (3D) scanning and printing technology is utilized to create phantom models of mice in order to assess the accuracy of ionizing radiation dosing from a clinical, human-based linear accelerator. Phantoms are designed to simulate a range of research questions, including irradiation of lung tumors and primary subcutaneous or orthotopic tumors for immunotherapy experimentation. The phantoms are used to measure the accuracy of dose delivery and then refine it to within 1% of the prescribed dose.

Primary subcutaneous myxoid liposarcoma: a clinicopathologic review of three cases with molecular confirmation and discussion of the differential diagnosis.

Myxoid liposarcoma typically presents as a deep-seated mass in the lower extremity of adults. Presentation as a primary subcutaneous tumor is rare. Here we discuss clinicopathologic characteristics of three such cases and their differential diagnosis to alert dermatopathologists to this unusual clinical presentation of a potentially aggressive entity. Cases of myxoid liposarcoma were retrieved from archives and consultation files. Inclusion required location above the subcutaneous fascia with no evidence of a metastatic origin. Clinicopathologic features were retrospectively reviewed. Fluorescence in situ hybridization for DDIT3 (CHOP) gene rearrangement was performed on all cases. The tumors affected young adults (two males and one female, mean 36 years, range 32-40 years). No prior history of myxoid liposarcoma or deep soft tissue mass was identified. The tumors occurred in the foot, thigh and hand. All demonstrated multilobular architecture with abundant myxoid stroma, prominent branching capillary vascular network and lipoblastic differentiation. No dermal involvement was seen. Round cell features were identified in one case and represented <5% of the tumor. All patients remain disease-free following local excision only at 6, 8 and 13 months. Myxoid liposarcoma can rarely present as a primary subcutaneous mass and should be considered in the differential diagnosis of cutaneous myxoid tumors in adults.

Abstract 4833: The eicosanoid 20-HETE triggers Ras activation, pancreatic cancer growth and metastasis through inflammatory macrophage signaling

Abstract Prolonged, unresolving inflammation is increasingly implicated in the pathogenesis of pancreatic cancer, but the critical inflammatory mediators remain largely unknown. Most efforts to elucidate these mediators have focused on protein cytokines rather than lipid autacoids. Lipid autacoids are metabolites of arachidonic acid and other related fatty acids that regulate inflammation and tumor growth. Among these, the metabolites of the cytochrome P450 (CYP) epoxygenases and ω-hydroxylases, including EETs and 20-HETE, respectively, have been largely neglected in cancer. Recently, we showed that epoxyeicosatrienoic acids (EETs) stimulate multi-organ metastasis and tumor dormancy escape. We report for the first time, that elevated levels of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of the ω-hydroxylases CYP4A/F, stimulate primary pancreatic tumor growth and metastasis. Using genetic and pharmacological manipulation of endogenous 20-HETE, we demonstrate that systemic administration of 20-HETE promotes primary human and murine pancreatic cancer in subcutaneous and orthotopic models. Furthermore, a transgenic murine model of endogenously high 20-HETE level (Tie2-CYP4F2 Tr), exhibited spontaneous liver and mediastinal lymph node metastasis after resection of subcutaneous primary pancreatic tumors. In contrast, wild-type mice did not develop liver and mediastinal lymph node metastasis after primary tumor resection. We show that 20-HETE activated Ras in human pancreatic tumor cell lines and the 20-HETE antagonist, HET0016, decreased Ras activity. The p65 subunit of NF-κB also increased in a 20-HETE-dependent manner in these tumor cell lines. 20-HETE induced migration of human macrophages in vitro, and tumor-associated macrophages expressed the 20-HETE generating enzyme CYP4F2. Depletion of macrophages with clodronate liposomes impaired CYP-metabolite-stimulated tumor growth. In addition, we show that 14,15-EET promotes pancreatic tumor growth in vivo. Moreover, inhibitors of soluble epoxide hydrolase, the enzyme that metabolizes EETs, elevated EET levels and promoted pancreatic tumor growth. Either the 20-HETE antagonist HET0016, or the EET antagonist 14,15-EEZE alone significantly inhibited primary tumor growth. Remarkably, systemic administration of both antagonists together resulted in sustained regression of established pancreatic tumors. This combination of 20-HETE and EET antagonists also suppressed ascites formation and prolonged survival in an orthotopic pancreatic cancer model without toxic effects. Thus, 20-HETE and EET production by macrophages in the tumor microenvironment critically regulates Ras activation, pancreatic tumor growth and metastasis. Our studies offer a mechanistic rationale for using 20-HETE and EET antagonists as a novel approach and anti-cancer therapeutics for pancreatic cancer. Citation Format: Yael Gus-Brautbar, Jessica Casper, Diane R. Bielenberg, Jennifer Cheng, Birgitta Schmidt, Bruce D. Hammock, Darryl C. Zeldin, Mark W. Kieran, Dipak Panigrahy. The eicosanoid 20-HETE triggers Ras activation, pancreatic cancer growth and metastasis through inflammatory macrophage signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4833. doi:10.1158/1538-7445.AM2014-4833

P17.67 * SUBCUTANEOUS METASTASES FROM A GLIOBLASTOMA MULTIFORME: CASE REPORT AND REVIEW OF LITERATURE

OBJECTIVES: We expose the case of a long-survival glioblastoma multiforme (‘GBM’) located at posterior fossa which presented a subcutaneous metastases four years after the surgery without evidence of recurrence throughout Central Nervous System (‘CNS’) previous at the secondary diagnosis. METHODS: A 57-year-old came to the Emergency Department with vomits, instability and pulsating headache. The neurological exploration showed posterior fossa signs and the Magnetic Resonance Image (MRI) confirmed a mass located on the right cerebellum hemisphere, with radiological characteristic of high-grade glioma, without associated hydrocephalus. A total gross resection was performed, being forced to return to the operating room due to a post-operative hematoma. After that, the clinical evolution was satisfactory and the patient could receive adjuvant treatments, finishing them with a Karnofsky Performance Status of 80. After four years of progression-free survival, we observed a stiff and painful area over the scar that was completely removed. Confirmed the ‘GBM’ diagnosis, the patient received chemotherapy without good response and, after five years of overall survival and one year after the second diagnosis, the patient died due to a neural and systemic tumoral progression. RESULTS: The cerebellar lesion met the anatomopathological criteria to be diagnosed as a ‘GBM’: cellular atypia, mitosis, endovascular proliferation and necrosis, with an extent oligodendroglial component. For this reason, we made the test for 1p/19q codeletion, which was negative. The subcutaneous tumor could be diagnosed as a ‘GBM’ metastases by cytomorphology and the similar immunohistochemical results between both samples. There was a positive GFAP cellular proliferation with a MIB-1 index about 20%. Since 2002, nine cases of confirmed subcutaneous metastases of 'GBM' have been reported, but their overall survival were shorter than this case, maybe because of the important oligodendroglial component and the infratentorial location of the primary lesion in this patient. CONCLUSIONS: Due to the extreme aggressiveness and the natural history of ‘GBM’, the incidence of metastases is very low and its appearance in the subcutaneous tissue is exceptional. Because of their low incidence and the short survival of these patients, the subcutaneous metastases are often misdiagnosed as primary subcutaneous tumors. There is a strong relationship between metastases and invasive procedures, like craneotomies and the placement of Ommaya reservories or ventricular drainages. The clinical history and, specially, the anatomopathological and immunohistochemical results of both lesions are essential for the correct diagnosis.

Use of electrochemotherapy in a case of neck skin metastasis of oral squamous cell carcinoma: Case report and considerations

BackgroundSquamous cell carcinoma (SCC) is the most common oral cavity malignant tumor. Surgery, radiotherapy, and chemotherapy have been the major options for its treatment. Electrochemotherapy (ECT) is a novel local treatment successfully used in secondary or primary skin or subcutaneous tumors. This new cancer treatment is a modality in which a locally applied electrical field enhances cell membrane permeability, thereby allowing greater intracellular accumulation of a chemotherapeutic agent.MethodsWe report a case of a man affected by an ulcerated SCC. The man was treated with ECT with good results.ResultsIn our case, ECT was successful in the management of extensive metastasis of SCC in clinical conditions, whereas other approaches were rejected.ConclusionThis case shows good clinical results; however, other studies are necessary to show that ECT should be considered as a promising treatment option. © 2014 The Authors. Head & Neck Published by Wiley Periodicals, Inc. Head Neck 36: E86–E90, 2014

Use of nanocurcumin to inhibit proliferation and metastases of hepatocellular carcinoma via NF-κB mediated matrix metalloproteinase-9 downregulation.

e22103 Background: Curcumin regulates the expression and secretion of MMPs, and has potent anti-cancer properties in several human cancer cell lines and animal carcinogenesis models. In this study we try to investigate the effects of polymeric nanoparticle encapsulated formulation of curcumin– nanocurcumin (NC) on Hepatocellular carcinoma (HCC) in vitro and in vivo. Methods: The effects of NC alone and in combination with sorafenib (SO) were investigated on HCC cell lines, Huh7 and MHCCLM3 in vitro by using proliferation and invasion assay, western blot, qRT-PCR, enzyme-linked immunosorbent assay and immunohistochemistry staining, and the subcutaneous and orthotopic HCC xenograft nude mice models (MHCCLM3) were used to evaluate primary tumor growth and metastasis after treatments. Results: NC alone (or combined with SO)inhibited HCC cell proliferation (p&lt;0.05) and invasion in vitro (p&lt;0.01), and remarkably decreased both the subcutaneous and orthotopic primary tumor growth and lung metastases in vivo. NC and/or SO could down-regulating the expressions of Matrix Metalloproteinase-9 (MMP9), p-ERK1 and NF-kB/p65 (p&lt;0.05). Conclusions: NC showed potent anti-invasion and metastasis properties in HCC via NF-κB mediated MMP9 down-regulation, which may provide a new strategy to the treatment of HCC patients and prevention of tumor recurrence after operation.

Abstract 960: Blocking the Notch pathway induces mesenchymal-to-epithelial transition in lung adenocarcinoma: A novel approach to overcoming chemoresistance.

Abstract Backgroung: The Epithelial-Mesenchymal Transition (EMT) is a key developmental program that is often activated during cancer invasion and metastasis. EMT cells possess the characteristics of cell motility, invasiveness, and have stem cells properties. EMT expression profile correlates with resistance to chemotherapy and poor outcome in multiple tumors and there is evidence suggesting an integral role of Notch pathway in mediating an EMT status. In this study, we investigated Notch activity and EMT status and the correlation with chemoressitance in lung adenocarcinoma. Results: Transduction of multiple lung adenocarcinoma cell lines, NCI-H1299, NCI-H460, NCI-H358, NCI-H441 with a Notch GFP-reporter construct identified a subset of cells with high Notch activity (GFP-positive). We compared cell migration capacity of GFP-positive and -negative cells utilizing a transwell invasion assay. Cells with high Notch activity, as evidenced by GFP expression, had a significantly higher migration capability as compared to GFP-negative cells. To evaluate resistance to chemotherapy, we treated GFP-pos and GFP-neg cells with docetaxel or cisplatin and measured apoptosis by Annexin V expression with flow cytometry. GFP-pos cells showed major resistance to both agents as compared to GFP-neg cells. When GFP-pos cells were exposed to a Notch activity blocker, gamma secretase inhibitor (GSI), these cells were rendered sensitive to cisplatin or docetaxel exposure as evident by an increase in Annexin V expression. Using RNA microarray analysis and RT-PCR assays, there was an increased expression of EMT related genes (Vimentin, N-Cadherin, Snail-1,….) in GFP-pos cells as compared to GFP-neg cells. Furthermore, NOD/SCID mice with subcutaneous primary lung tumor xenografts treated with GSI showed decreased expression of downstream effectors of Notch as as well as vimentin. Mice treated with combination of GSI and docetaxel had better tumor control as compared to those treated with GSI and docetaxel as single agents. Conclusion: These studies suggest that the Notch pathway is an important regulator of EMT status and that its inhibition induces mesenchymal to epithelial transition overcoming chemoresistnace. Notch inhibitors in combination with standard chemotherapy present a potential effective approach to achieve better disease control in lung cancer. Citation Format: Khaled Hassan, Luo Wang, Hasan Korkaya, Paige O'Dowd, Sarah Dubaisi, Rayyan Kadi, Rawad Nasr, Binghui Zhang, April Davis, Gregory Kalemkerian, Max Wicha. Blocking the Notch pathway induces mesenchymal-to-epithelial transition in lung adenocarcinoma: A novel approach to overcoming chemoresistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 960. doi:10.1158/1538-7445.AM2013-960

Reduced Latency in the Metastatic Niche Contributes to the More Aggressive Phenotype of LM8 Compared to Dunn Osteosarcoma Cells

Metastasis is the major cause of death of osteosarcoma patients and its diagnosis remains difficult. In preclinical studies, however, forced expression of reporter genes in osteosarcoma cells has remarkably improved the detection of micrometastases and, consequently, the quality of the studies. We recently showed that Dunn cells equipped with a lacZ reporter gene disseminated from subcutaneous primary tumors as frequently as their highly metastatic subline LM8, but only LM8 cells grew to macrometastases. In the present time-course study, tail-vein-injected Dunn and LM8 cells settled within 24 h at the same frequency in the lung, liver, and kidney of mice. Furthermore, Dunn cells also grew to macrometastases, but, compared to LM8, with a delay of two weeks in lung and one week in liver and kidney tissue, consistent with prolonged survival of the mice. Dunn- and LM8-cell-derived ovary and spine metastases occurred less frequently. In vitro, Dunn cells showed less invasiveness and stronger contact inhibition and intercellular adhesion than LM8 cells and several cancer- and dormancy-related genes were differentially expressed. In conclusion, Dunn cells, compared to LM8, have a similar capability but a longer latency to form macrometastases and provide an interesting new experimental system to study tumor cell dormancy.

Inhibition of Cdc42-Interacting Protein 4 (CIP4) Impairs Osteosarcoma Tumor Progression

In patients with Osteosarcoma (OS), larger primary tumor size correlates with higher metastasis incidence and lower overall survival. Identifying the mechanisms that control primary tumor growth can lead to the new therapeutic approaches and improve prognosis. Cytoskeleton regulatory molecules play an important role in tumor cells proliferation and motility. In the current study, we investigated the role of scaffolding protein Cdc42-interacting protein 4 (CIP4) in OS.Murine OS cells, DLM8 were stably transfected with shCIP4 plasmid and their tumorigenic activity was studied in vitro and in vivo. The effect of CIP4 downregulation on cytoskeleton was studied by immunohistochemical analysis.In vivo studies revealed that downregulation of CIP4 in OS tumor cells can inhibit primary subcutaneous tumor growth and delay spontaneous metastases growth in the animal lungs. In vitro studies confirmed that inhibiting CIP4 expression significantly reduced tumor cells invasiveness and migration. Changes in CIP4 expression altered its cellular localization pattern and tumor cell morphology, which could explain changes in OS cell behavior in vivo and in vitro. Cells with downregulated CIP4 showed lower levels of actin in cells along with fewer perinuclear actin stress filaments and actin bridges to the lipid bilayer of the cell membrane than control cells.This study presents compelling evidence that inhibition of CIP4 expression in OS cells impairs metastatic behavior of cells in vitro and prolongs survival of animals by inhibiting primary tumor growth. Although the lack of CIP4 in tumor cells did not prevent formation of metastasis, it did substantially delay their formation in the animals' lungs.

Melanoma Cells Use Thy-1 (CD90) on Endothelial Cells for Metastasis Formation

The cell adhesion molecule Thy-1 (CD90) mediates the adhesion of melanoma cells to activated human endothelial cells (EC) via the interaction with the αvβ3-integrin on the tumor cells invitro. Here, we report a strong expression of Thy-1 on both blood vessel and lymphatic EC in melanoma and melanoma metastases. Vascular endothelial growth factor and tumor necrosis factor-α were identified as inducers of Thy-1 expression on EC invitro. The physiological role of Thy-1 for lymphogenic and hematogenic metastasis of melanoma cells was substantiated in an experimental metastasis model using B16/F10 melanoma cells. Mice lacking Thy-1 showed markedly diminished experimental lung metastasis after injection of B16/F10 melanoma cells compared to wild-type littermate controls. In addition, on generation of a primary subcutaneous tumor, metastasis to regional lymph nodes was clearly reduced in Thy-1(-/-) mice. However, Thy-1 deletion did not affect subcutaneous primary tumor growth, tumor-induced recruitment of inflammatory cells or T cells, angiogenesis, or T-cell activation. In conclusion, Thy-1 contributes to metastasis of melanoma cells by mechanisms likely involving a Thy-1-mediated adhesion of melanoma cells to EC.

Abstract A39: Dietary supplementation with curcumin enhances metastatic growth of Lewis lung carcinoma in mice

Abstract The present study investigated the effects of dietary supplementation with curcumin (the principal curcuminoid of the popular Indian spice turmeric) on spontaneous metastasis of Lewis lung carcinoma (LLC) in female C57/BL6 mice. Mice were fed the AIN93G control diet or that diet supplemented with 2% or 4% curcumin for 5 weeks at which time they were injected subcutaneously with 2.5 x 105 viable LLC cells. The subcutaneous primary tumor was surgically removed when it reached approximately 8 mm in diameter, and the experiment was terminated 10 days after the surgery. There was no difference in pulmonary metastatic yield among the groups. Curcumin supplementation at either dietary level did not significantly affect tumor growth. However, the combine data from both curcumin groups showed that curcumin treatment increased tumor cross-sectional area by 46% (p &amp;lt; 0.05) and tumor volume by 70% (p &amp;lt; 0.05) compared with the controls. Curcumin supplementation increased plasma concentrations of the angiogenic factors fibroblast growth factor-basic (p &amp;lt; 0.05) and vascular endothelial growth factor (p &amp;lt; 0.05), as well as the inflammatory cytokines interleukin-1β (p &amp;lt; 0.05) and monocyte chemotactic protein-1 (p &amp;lt; 0.05) compared with the controls. These results demonstrate that curcumin does not prevent metastasis, and indicate that it can enhance metastatic growth of LLC in mice, perhaps through up-regulation of angiogenesis and inflammation. Citation Format: Lin Yan. Dietary supplementation with curcumin enhances metastatic growth of Lewis lung carcinoma in mice. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A39.

Inhibition of Cdc42-Interacting Protein 4 (CIP4) Impairs Osteosarcoma Tumor Progression

In patients with Osteosarcoma (OS), larger primary tumor size correlates with higher metastasis incidence and lower overall survival. Identifying the mechanisms that control primary tumor growth can lead to the new therapeutic approaches and improve prognosis. Cytoskeleton regulatory molecules play an important role in tumor cells proliferation and motility. In the current study, we investigated the role of scaffolding protein Cdc42-interacting protein 4 (CIP4) in OS. Murine OS cells, DLM8 were stably transfected with shCIP4 plasmid and their tumorigenic activity was studied in vitro and in vivo. The effect of CIP4 downregulation on cytoskeleton was studied by immunohistochemical analysis. In vivo studies revealed that downregulation of CIP4 in OS tumor cells can inhibit primary subcutaneous tumor growth and delay spontaneous metastases growth in the animal lungs. In vitro studies confirmed that inhibiting CIP4 expression significantly reduced tumor cells invasiveness and migration. Changes in CIP4 expression altered its cellular localization pattern and tumor cell morphology, which could explain changes in OS cell behavior in vivo and in vitro. Cells with downregulated CIP4 showed lower levels of actin in cells along with fewer perinuclear actin stress filaments and actin bridges to the lipid bilayer of the cell membrane than control cells. This study presents compelling evidence that inhibition of CIP4 expression in OS cells impairs metastatic behavior of cells in vitro and prolongs survival of animals by inhibiting primary tumor growth. Although the lack of CIP4 in tumor cells did not prevent formation of metastasis, it did substantially delay their formation in the animals' lungs.

Therapeutic efficacy of an hTERT promoter-driven oncolytic adenovirus that expresses apoptin in gastric carcinoma

The efficacy and specificity of treatment are the major challenges for cancer gene therapy. Oncolytic virotherapy is an attractive drug delivery platform of cancer gene therapy. Previous studies have determined that apoptin is a p53-independent, Bcl-2-insensitive apoptotic protein that has the ability to induce apoptosis specifically in tumor cells. In this study, we show that the administration of a dual cancer-specific oncolytic adenovirus construct, Ad-hTERT-E1a-apoptin [in which the adenovirus early region 1a (E1a) gene is driven by the cancer-specific promoter of human telomerase reverse transcriptase (hTERT) and that expresses apoptin simultaneously], suppresses tumor growth in gastric carcinoma cells in vitro and reduces the tumor burden in vivo in xenografted nude mice. The observation that infection with the Ad-hTERT-E1a-apoptin construct significantly inhibited the growth of gastric cancer cells and protected normal human gastric epithelium from growth inhibition confirmed the induction of cancer cell-selective adenovirus replication, growth inhibition and apoptosis by this therapeutic approach. In vivo assays were performed using BALB/c nude mice that had established primary tumors. Subcutaneous primary tumor volume was reduced not only in the intratumoral injection group but also in the systemic delivery mice following treatment with Ad-hTERT-E1a-apoptin. Furthermore, treatment of primary models with Ad-hTERT-E1a-apoptin increased the mouse survival time. These data reinforce previous research and highlight the potential therapeutic application of Ad-hTERT-E1a-apoptin for the treatment of neoplastic diseases in clinical trials.