Experimental Metastasis Model Research Articles

Endothelial cell-anchored tissue factor pathway inhibitor regulates tumor metastasis to the lung in mice.

Tissue factor pathway inhibitor (TFPI) is a physiological inhibitor of the tissue factor (TF)-initiated coagulation pathway. Both circulating and tumor cell-associated TFPI significantly reduce tumor cell-induced coagulation activation and lung metastasis. However, the significance of endothelial cell-anchored TFPI in cancer biology remains largely unexplored. We generated mice with full-length disruption of TFPI (including TFPIα and TFPIβ isoforms) in endothelial cells, using a Cre-LoxP system and gene inactivation (GI) strategy. Experimental pulmonary tumor metastasis models were used with TFPI-deficient mice to evaluate the role of endothelial cell-anchored TFPI in cancer progression. Finally, lung microvascular permeability and microenvironment were investigated. TFPI-deficient mice were viable and fertile, and showed decreased plasma TFPI levels and lung TFPI levels as compared with their control littermates. TFPI deficiency in endothelial cells promoted pulmonary tumor metastasis with an increased vascular permeability and altered lung microenvironment. Our observations suggest that endothelial cell-anchored TFPI controls lung tumor metastasis, and does so largely through the inhibition of local TF-induced thrombin generation and the regulation of the lung microenvironment in mice.

Direct delivery of a cytotoxic anticancer agent into the metastatic lymph node using nano/microbubbles and ultrasound.

Direct injection of an anticancer agent into a metastatic lymph node (LN) has not been used as a standard treatment because evidence concerning the efficacy of local administration of a drug into a metastatic LN has not been established. Here we show that the combination of intralymphatic drug delivery with nano/microbubbles (NMBs) and ultrasound has the potential to improve the chemotherapeutic effect. We delivered cis-diamminedichloroplatinum (II) (CDDP) into breast carcinoma cells in vitro and found that apoptotic processes were involved in the antitumor action. Next, we investigated the antitumor effect of intralymphatic chemotherapy with NMBs and ultrasound in an experimental model of LN metastasis using MXH10/Mo-lpr/lpr mice exhibiting lymphadenopathy. The combination of intralymphatic chemotherapy with NMBs and ultrasound has the potential to improve the delivery of CDDP into target LNs without damage to the surrounding normal tissues. The present study indicates that intralymphatic drug delivery with NMBs and ultrasound will potentially be of great benefit in the clinical setting.

A preclinical evaluation of SKLB261, a multikinase inhibitor of EGFR/Src/VEGFR2, as a therapeutic agent against pancreatic cancer.

The clinical prognosis of pancreatic cancer remains rather disappointing despite tremendous efforts in exploring medical treatments in the past two decades. Development of more effective treatment strategies is still desperately needed to improve outcomes in patients with pancreatic cancer. SKLB261 is a multikinase inhibitor obtained recently through a lead optimization. In this investigation, we shall evaluate its anti-pancreatic cancer effects both in vitro and in vivo. SKLB261 is a multikinase inhibitor potently inhibiting EGFR, Src, and VEGFR2 kinases. It could significantly inhibit cell proliferation, migration, and invasion, and induce apoptosis in cellular assays of human pancreatic cancer cells that are sensitive or resistant to dasatinib and/or gemcitabine. Western blot analysis showed that SKLB261 inhibited the activation of EGFR and Src kinases as well as their downstream signaling proteins, including FAK, ERK, and STAT3. SKLB261 also showed potent antiangiogenic effects in transgenic zebrafish models. In vivo, SKLB261 displayed more potent antitumor activities than dasatinib, gemcitabine, or erlotinib in pancreatic cancer xenografts, including BxPC-3, PANC-1, AsPC-1, and HPAC. Furthermore, mice receiving SKLB261 therapy showed significant survival advantage compared with vehicle-treated and gemcitabine-treated groups in an experimental metastasis model of pancreatic cancer. These data, together with the good pharmacokinetic properties and low toxicity of this compound, provide a rationale for the ongoing clinical evaluation of SKLB261 in the treatment of pancreatic cancer.

LAMC2 enhances the metastatic potential of lung adenocarcinoma.

Lung cancer is the number one cancer killer, and metastasis is the main cause of high mortality in lung cancer patients. However, mechanisms underlying the development of lung cancer metastasis remain unknown. Using genome-wide transcriptional analysis in an experimental metastasis model, we identified laminin γ2 (LAMC2), an epithelial basement membrane protein, to be significantly upregulated in lung adenocarcinoma metastatic cells. Elevated LAMC2 increased traction force, migration, and invasion of lung adenocarcinoma cells accompanied by the induction of epithelial-mesenchymal transition (EMT). LAMC2 knockdown decreased traction force, migration, and invasion accompanied by EMT reduction in vitro, and attenuated metastasis in mice. LAMC2 promoted migration and invasion via EMT that was integrin β1- and ZEB1-dependent. High LAMC2 was significantly correlated with the mesenchymal marker vimentin expression in lung adenocarcinomas, and with higher risk of recurrence or death in patients with lung adenocarcinoma. We suggest that LAMC2 promotes metastasis in lung adenocarcinoma via EMT and may be a potential therapeutic target.

Abstract IA18: A brain metastasis-promoting role for cathepsin S identified from analysis of tumor- and stroma-supplied proteolytic networks

Abstract Cancer cells in an aggressive primary tumor are adept at exploiting their local tissue microenvironment. In contrast, when metastatic cells leave these favorable surroundings, they must possess or acquire traits that will allow them to survive and colonize foreign, potentially hostile tissue environments. The obstacles that metastasizing tumor cells encounter vary from organ to organ, and are highly influenced by non-cancerous stromal cells of the tumor microenvironment. For example, the blood-brain barrier, composed of endothelial cells, astrocytes and pericytes, presents a far more formidable structure for tumor cells to penetrate, compared to the fenestrated capillaries in the bone marrow. While the primary tumor microenvironment has emerged as an important regulator of cancer progression, it is less well understood how different tissue environments influence metastatic processes. We used a dual species-specific microarray platform to uncover tumor-stroma interactions that modulate organ tropism of brain, bone and lung metastasis in animal models of cancer. Among the differentially regulated tumor- and stroma-specific genes, we identified cathepsin S as a novel regulator of breast-to-brain metastasis. In breast cancer patients, high cathepsin S expression at the primary site correlated with decreased brain metastasis-free survival. Both macrophages and tumor cells produce cathepsin S, and only the combined depletion significantly reduced brain metastasis in experimental models in vivo. We show that cathepsin S specifically mediates blood-brain barrier transmigration via proteolytic processing of the junctional adhesion molecule (JAM)-B. Pharmacological inhibition of cathepsin S significantly reduced experimental brain metastasis, supporting its consideration as a therapeutic target for this disease. Citation Format: Lisa Sevenich, Robert Bowman, Steve Mason, Daniela Quail, Franck Rapaport, Benelita Elie, Edi Brogi, Priscilla Brastianos, William Hahn, Leslie Holsinger, Joan Massagué, Christina Leslie, Johanna A. Joyce. A brain metastasis-promoting role for cathepsin S identified from analysis of tumor- and stroma-supplied proteolytic networks. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr IA18. doi:10.1158/1538-7445.CHTME14-IA18

Abstract B19: Brain metastasis depends on tumor cell initiated coagulation

Abstract Background: The incidence of brain metastasis exceeds that of primary brain tumors tenfold and is most frequently associated with lung cancer, breast cancer and melanoma. Despite a prognosis of only 6-9 months for patients with brain metastases, mechanisms of tumor cell brain colonization from the blood stream are unknown. Understanding this step could enable effective therapeutic strategies to prevent the development of incurable metastatic brain disease. We found that blood borne metastatic cancer cells reside within the cerebral microvasculature for several days and associate with platelets while crossing the blood brain barrier. Intravascular tumor cell survival and extravasation during this time may critically limit the success of brain metastasis. Activation of coagulation, platelets and fibrin formation contribute to tumor progression, cancer-associated thrombosis and metastatic spread to peripheral organs. However the function of coagulation in the seeding of brain metastases is unknown. Aims: We evaluated whether tumor cell-expressed tissue factor, a key activator of coagulation, promotes tumor cell seeding of brain metastases by initiating critical tumor cell-vascular interactions. Methods: We developed models of experimental brain metastasis to study how intravascular tumor cells cooperate with the coagulation system during early stages of brain colonization to survive and cross the blood brain barrier. Human breast cancer cells, injected into the left cardiac ventricle of immune deficient mice, are tracked during the initial phase of brain metastasis and progressive metastatic brain disease by detailed and quantitative histological analyses. We address how coagulation contributes to brain metastasis by targeting human tissue factor with specific inhibitory antibodies. We use ex-vivo bioluminescence imaging and immunohistochemical analyses at later stages of brain metastasis to determine the extent to which an early, transient treatment will result in long-term inhibition of brain metastatic disease. Results: We demonstrate that inhibition of tissue factor expressed by tumor cells reduces brain colonization. Targeting of tissue factor inhibits development of breast cancer brain metastasis and extends animal survival. We find that tissue factor properties which initiate coagulation or promote cytoprotective signaling pathways differentially contribute to brain metastasis development. Our findings indicate that the inhibition of coagulation can prevent the seeding of breast cancer cells into the brain. Conclusions: Our studies provide mechanistic insights into the process of tumor cell brain colonization and identify targets for development of therapeutics to prevent cancer metastasis to the brain and enhance patient survival. Citation Format: Laurie J. Gay, John Day, Sarah LeBoeuf, Melissa Ritland, Zaverio M. Ruggeri, Wolfram Ruf, Brunhilde H. Felding. Brain metastasis depends on tumor cell initiated coagulation. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B19. doi:10.1158/1538-7445.CHTME14-B19

Deregulation of SLIT2-Mediated Cdc42 Activity Is Associated with Esophageal Cancer Metastasis and Poor Prognosis

SLIT2, a secreted protein, has been found to inactivate Cdc42 GTPase to modulate neural cell migration. However, alteration of SLIT2-mediated Cdc42 in terms of migration regulation remains undefined in esophageal squamous cell carcinoma (ESCC). We report here in ESCC cell, animal, and clinical models that SLIT2 acts as a migration suppressor and serves as a prognostic biomarker. The immunohistochemistry data indicated that 31.8% (49 of 154) of tumors from ESCC patients showed low expression of SLIT2 protein which correlated with poor overall survival and disease-free survival. DNA methylation analysis suggested that promoter hypermethylation is responsible for low expression of SLIT2 in ESCC. Knockdown of SLIT2 increased ESCC cell migration, while SLIT2 stable overexpression reduced cell migration. ESCC cells treated with conditioned media from cells overexpressing SLIT2 also suppressed cell migration. Importantly, silencing of SLIT2 decreased the complex formation, and thus induced Cdc42 activity and promoted membrane localization of focal adhesion kinase and Paxillin. Anti-metastatic effect of SLIT2 was confirmed in an experimental metastasis model of SLIT2 knockdown ESCC cells. Our results provide novel evidence that low expression of SLIT2 correlates with poor prognosis and promotes metastasis in ESCC, which may be regulated by the Cdc42-mediated pathways.

Inhibition of spontaneous and experimental lung metastasis of soft-tissue sarcoma by tumor-targeting Salmonella typhimurium A1-R.

Prognosis of patients with lung metastases of soft-tissue sarcoma is still poor. Therefore, novel systemic therapy is needed to improve the survival of soft-tissue sarcoma. In the present study, tumor-targeting therapy with a genetically-modified auxotrophic strain of Salmonella typhimurium, termed A1-R, was evaluated. Mouse models of primary soft tissue sarcoma and spontaneous lung metastasis were obtained by orthotopic intra-muscular injection of HT1080-RFP human fibrosarcoma cells. S. typhimurium A1-R was administered from day 14, once a week for two weeks. On day 28, lung samples were excised and observed with a fluorescence imaging system. The number of lung metastasis was 8.8 ± 3.4 in the untreated group and 0.8 ± 0.8 in the treated group (P = 0.024). A mouse model of experimental lung metastasis was obtained by tail vein injection of HT1080-RFP cells. The mice were treated with S. typhimurium A1-R (i.v.) on day 7, once a week for three weeks. S. typhimurium A1-R significantly reduced lung metastases and improved overall survival (P = 0.004). S. typhimurium A1-R bacterial therapy has future potential for treating advanced soft tissue sarcoma and improving prognosis of patients with lung metastasis.

Efficacy of a cathepsin K inhibitor in a preclinical model for prevention and treatment of breast cancer bone metastasis.

Cathepsin K (CatK) is essential for osteoclast-mediated bone resorption. CatK expression is also detected in breast cancer cells that metastasize to bone. Here, the CatK inhibitor L-235 dosed in prevention (10, 30, and 100 mg/kg, p.o., b.i.d.) or treatment regimen (30 mg/kg) was compared with the bisphosphonate zoledronic acid (ZOL, 7.5 μg/kg/wk, s.c.) in the intratibial injection model of MDA-MB-231 breast carcinoma in nude rats. Progression of osteolysis, skeletal tumor burden, and local metastasis was evaluated by radiography through 42 days and ex vivo μCT and histology. IHC and RT-PCR confirmed the increases in CatK protein and mRNA levels in human breast cancer primary and metastatic tumors. In the experimental model of breast cancer bone metastasis, L-235 dosed in preventive mode resulted in a dose-related reduction of osteolysis of 72%, 75%, and 87% respectively, compared with ZOL by 86% versus intact. Similarly, L-235 significantly reduced intratibial tumor volume by 29%, 40%, and 63%, respectively, compared with 56% by ZOL versus vehicle. Efficacy of L-235 and ZOL on reduction of osteolytic lesions and tumor burden was comparable in treatment versus preventive regimens. All L-235 doses inhibited cortical disruption and extraskeletal tumor growth to a level comparable with ZOL. Assessment of local metastasis demonstrated that treatment with the CatK inhibitor was more effective than ZOL in reducing breast cancer invasion. These data support the role of CatK in breast cancer skeletal growth and metastasis and CatK inhibitors may represent a novel oral therapy for treatment of metastatic breast cancer.

447 Novel, quantitative in vivo shRNA screening approach identifies new molecular targets to block cancer metastasis

Background: The identification of novel therapeutic targets to block cancer metastasis is a key step towards the reduction of cancer-related patient deaths. For prostate cancer, the most commonly-diagnosed cancer in men, the 5 year survival rate for those with metastatic disease is less than 10%. We have recently established that intravasation, a critical step of metastasis, is dependent upon production cell migration. In this study, we have utilized a quantitative in vivo screening approach that relies on the assessment of in vivo cell migration as a primary readout. Methods: Our screen approach utilizes an ex ovo avian embryo platform combined with real-time, high-resolution 3D visualization of human cancer metastasis. When injected intravenously, fluorescent human cancer cells form hundreds of individual metastatic colonies that can be simultaneously visualized in vivo. A customized Matlab-based analysis software was developed to interpret 3D images from this model to generate a metastatic colony invasiveness index (C.I.). As a proof of principle, we showed that previously described inhibitors of cell migration and metastasis significantly reduces the C.I., which closely correlates with their antimetastatic potential. In the full screen, human head and neck cancer cells (Hep3) and prostate cancer cells (PC3) were transduced with a whole genome shRNA and injected intravenously into the CAM. Metastatic colonies with C.I. below that of control cells were excised and expanded. shRNA identities were determined by high-throughput sequencing. Results: More than fifty novel genetic regulators of metastasis were identified that have not been previously described using other approaches. Of these, there were eleven druggable targets that included cancer cell invasion and cell cycle regulating protein kinases, cell surface and nuclear receptors and cytoskeleton modulating enzymes. shRNA-mediated knockdown of these genes blocked human cancer cell metastasis in both experimental and spontaneous mouse models of metastasis. Lead hit serine-threonine kinase SRPK1 was significantly associated with prostate cancer invasion and metastasis using publicly-available gene expression data. We demonstrate that SRPK1 directly modulates the splicing isoform profile of several key cell adhesion molecules, impacting cell migration in vitro and in vivo. Inhibition of SRPK1 decreases the ability of prostate cancer cells to metastasize in vivo. Conclusion: We developed a novel and robust in vivo screening platform that provides a rapid and quantitative assessment of therapeutic targets of metastasis. Using this platform, we have identified numerous promising therapeutic targets that that are functionally involved in cancer invasion and metastasis.

448 Exposure to EGFR inhibitors influences release of extracellular vesicles by tumor cells

Background: The identification of novel therapeutic targets to block cancer metastasis is a key step towards the reduction of cancer-related patient deaths. For prostate cancer, the most commonly-diagnosed cancer in men, the 5 year survival rate for those with metastatic disease is less than 10%. We have recently established that intravasation, a critical step of metastasis, is dependent upon production cell migration. In this study, we have utilized a quantitative in vivo screening approach that relies on the assessment of in vivo cell migration as a primary readout. Methods: Our screen approach utilizes an ex ovo avian embryo platform combined with real-time, high-resolution 3D visualization of human cancer metastasis. When injected intravenously, fluorescent human cancer cells form hundreds of individual metastatic colonies that can be simultaneously visualized in vivo. A customized Matlab-based analysis software was developed to interpret 3D images from this model to generate a metastatic colony invasiveness index (C.I.). As a proof of principle, we showed that previously described inhibitors of cell migration and metastasis significantly reduces the C.I., which closely correlates with their antimetastatic potential. In the full screen, human head and neck cancer cells (Hep3) and prostate cancer cells (PC3) were transduced with a whole genome shRNA and injected intravenously into the CAM. Metastatic colonies with C.I. below that of control cells were excised and expanded. shRNA identities were determined by high-throughput sequencing. Results: More than fifty novel genetic regulators of metastasis were identified that have not been previously described using other approaches. Of these, there were eleven druggable targets that included cancer cell invasion and cell cycle regulating protein kinases, cell surface and nuclear receptors and cytoskeleton modulating enzymes. shRNA-mediated knockdown of these genes blocked human cancer cell metastasis in both experimental and spontaneous mouse models of metastasis. Lead hit serine-threonine kinase SRPK1 was significantly associated with prostate cancer invasion and metastasis using publicly-available gene expression data. We demonstrate that SRPK1 directly modulates the splicing isoform profile of several key cell adhesion molecules, impacting cell migration in vitro and in vivo. Inhibition of SRPK1 decreases the ability of prostate cancer cells to metastasize in vivo. Conclusion: We developed a novel and robust in vivo screening platform that provides a rapid and quantitative assessment of therapeutic targets of metastasis. Using this platform, we have identified numerous promising therapeutic targets that that are functionally involved in cancer invasion and metastasis.

Haematopoietic focal adhesion kinase deficiency alters haematopoietic homeostasis to drive tumour metastasis.

Metastasis is the main cause of cancer-related death and thus understanding the molecular and cellular mechanisms underlying this process is critical. Here, our data demonstrate, contrary to established dogma, that loss of haematopoietic-derived focal adhesion kinase (FAK) is sufficient to enhance tumour metastasis. Using both experimental and spontaneous metastasis models, we show that genetic ablation of haematopoietic FAK does not affect primary tumour growth but enhances the incidence of metastasis significantly. At a molecular level, haematopoietic FAK deletion results in an increase in PU-1 levels and decrease in GATA-1 levels causing a shift of hematopoietic homeostasis towards a myeloid commitment. The subsequent increase in circulating granulocyte number, with an increase in serum CXCL12 and granulocyte CXCR4 levels, was required for augmented metastasis in mice lacking haematopoietic FAK. Overall our findings provide a mechanism by which haematopoietic FAK controls cancer metastasis.

The Rac Inhibitor EHop-016 Inhibits Mammary Tumor Growth and Metastasis in a Nude Mouse Model

Metastatic disease still lacks effective treatments, and remains the primary cause of cancer mortality. Therefore, there is a critical need to develop better strategies to inhibit metastatic cancer. The Rho family GTPase Rac is an ideal target for anti-metastatic cancer therapy, because Rac is a key molecular switch that is activated by a myriad of cell surface receptors to promote cancer cell migration/invasion and survival. Previously, we reported the design and development of EHop-016, a small molecule compound, which inhibits Rac activity of metastatic cancer cells with an IC50 of 1μM. EHop-016 also inhibits the activity of the Rac downstream effector p21-activated kinase (PAK), lamellipodia extension, and cell migration in metastatic cancer cells. Herein, we tested the efficacy of EHop-016 in a nude mouse model of experimental metastasis, where EHop-016 administration at 25mg/kg body weight (BW) significantly reduced mammary fat pad tumor growth, metastasis, and angiogenesis. As quantified by UPLC MS/MS, EHop-016 was detectable in the plasma of nude mice at 17 to 23ng/ml levels at 12h following intraperitoneal (i.p.) administration of 10 to 25mg/kg BW EHop-016. The EHop-016 mediated inhibition of angiogenesis In Vivo was confirmed by immunohistochemistry of excised tumors and by In Vitro tube formation assays of endothelial cells. Moreover, EHop-016 affected cell viability by down-regulating Akt and Jun kinase activities and c-Myc and Cyclin D expression, as well as increasing caspase 3/7 activities in metastatic cancer cells. In conclusion, EHop-016 has potential as an anticancer compound to block cancer progression via multiple Rac-directed mechanisms.

Abstract 4876: Characterization of malignant melanoma growth triggered by the brain microenvironment in experimental metastasis models

Abstract The brain offers a unique microenvironment to which malignant melanoma frequently spreads leading to dismal prognosis for the patients. The molecular determinants that promote melanoma brain metastases however, remain largely unknown. Here, we show an experimental strategy utilizing two species of immune-compromised animals in a metastasis model to generate in vivo-cultivated metastatic tissues along with their corresponding host tissues in order to identify molecular events associated with brain metastases. The study was based on the melanoma cell lines, Melmet 1 and Melmet 5 with opposing phenotypes (invasive/proliferative, respectively) in the in vitro setting and distinct metastatic patterns in vivo, where Melmet 1 showed a preference to the brain. Transcriptional changes in the melanoma cells, as induced by the brain-microenvironment in both host species, revealed the opportunistic nature of melanoma in this context. We detected transitioning from the invasive phenotype to the proliferative phenotype, as the brain metastases passed from early to late growth phase. Of significance, we identified a brain-adaptive phenotype with synaptic characteristics and where glutamate signaling and Ca2+-dependent effectors played a central role. The brain-adaptive phenotype was more prominent in the earlier metastatic growth phases compared to a later phase. Analysis of host tissue uncovered a cooperative inflammatory microenvironment formed by activated host cells. Altogether, this suggests that acquisition of the brain-adaptive phenotype in a permissive environment might be critical for the establishment of metastatic growth in the brain microenvironment and further, targeting glutamate signaling may represent a therapeutic approach in brain metastasis. Global network modeling and functional annotation analysis revealed that several of the central molecular factors and pathways mediating melanoma brain metastasis in this study, are also central to other pathological diseases, in particular neurodegenerative diseases. The identification of essential molecular networks that operate to promote the brain-adaptive phenotype is of clinical relevance, as they represent leads to urgently needed therapeutic targets. Citation Format: Vigdis Nygaard, Lina Prasmickaite, Kotryna Vasiliauskaite, Trevor Clancy, Eivind Hovig. Characterization of malignant melanoma growth triggered by the brain microenvironment in experimental metastasis models. [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 4876. doi:10.1158/1538-7445.AM2014-4876

Abstract 4953: Establishment and validation of models for metastasis developed from patient xenogragrafts (PDX)

Abstract While surgery, chemotherapy and radiation mostly enable a successful treatment of primary tumors, an efficient treatment of metastasis still remains challenging. A prerequisite for the development of new drugs is the availability of relevant tumor models for preclinical testing. In the last years, patient derived xenograft (PDX) models have proven to be more relevant for drug screening and translational research than cell line derived xenograft models. While several experimental models of metastasis are already available (J. Hoffmann, et al., Neuro-Oncology, 2009), it was the aim of our study to extent the panel of validated metastasis models derived from patients. We screened PDX for their ability to metastasize in mice. Metastatic outcome was characterized in dependence of injection route, cell number and mouse strain. Time to develop metastases, number of visible metastases in the lung and liver, as well the rate of human DNA detected in these organs were used as evaluation parameter of metastasis. Method:Tumors from stably growing PDX (I. Fichtner et al., Clin Cancer Res., 2008) were resected and used to produce a single cell suspension. These cells were injected into different mouse strains by routes indicated below to induce metastasis. At defined time points, or when mice became moribund, mice were sacrificed and inspected for macroscopic metastases. Quantification of early disseminated tumor cells was done by TaqMan PCR (Becker et al., 2002). Normalised CT values (CTnorm = ΔCTnegative control - (ΔCTsample) were used as degree of metastasis. Results: Eight out of 14 PDX models including lung (2x), colon (2x), breast, melanoma, sarcoma, and pancreatic carcinoma have been shown to develop metastases depending on the mouse strain and injection (i.v., i.p., s.c., or orthotopic) route. Best results were obtained for melanoma (MEL1956) and lung carcinoma (LU9313). Both models developed strong metastasis in lung and liver with CTnorm values > 14 (lung) and 7.9 (liver), if cells were injected i.v. Lu 9313M cells could also be injected orthotopically causing a high degree of metastasis (CTnorm ∼ 10 after 25 days). Metastasis of colon carcinoma was less effective when initiated via the intravenous route, but was much better if cells were injected i.p. with CTnorm values above 8. Other model with high metastasis rate were the sarcoma SA10133 (i.v. route, CTnorm values > 7.5) and the pancreatic carcinoma Panc 9617 (s.c. route, CTnorm 4 - 9). Three breast cancer PDX were also investigated, but only MA 4296 developed metastases, derived from orthotopically implanted tumor cells with a CTnorm of ∼ 9.8 for the lung. Outlook: EPO GmbH established a panel of eight different tumor models from PDX to induce metastasis, each with specific conditions. The results of this study may contribute to improve the treatment of cancer by providing models with high clinic relevance for the testing of novel antimetastatic treatments. Citation Format: Jens Hoffmann, Andrea Orthmann, Annika Hoffmann, Reiner Zeisig, Iduna Fichtner. Establishment and validation of models for metastasis developed from patient xenogragrafts (PDX). [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 4953. doi:10.1158/1538-7445.AM2014-4953

Abstract LB-116: Cep55, a master regulator of cytokinesis in breast cancer pathogenesis

Abstract Defects in the number, structure, and function of centrosomes can generate mono- or multipolar mitotic spindles and cytokinesis failure resulting in aneuploidy and chromosome instability, which are common characteristics of tumour cells. Centrosomal proteins thus play central roles in the execution of proper cell division. Cep55 is a centrosomal protein required for proper segregation of two daughter cells during cytokinesis. It often elevated in wide range of cancer types including breast cancer. To ascertain the functional role of Cep55 in breast cancer pathogenesis, we performed publically available data mining as well as IHC staining in a cohort of breast cancer patients and found that elevated levels of Cep55 is significantly associated with p53 mutation, basal like subtype, relapse free and distant-metastasis free survival. Immunoblot analysis of non-malignant and breast cancer cell lines revealed higher expression of Cep55 protein in basal like subtype. RNAi mediated Cep55 depletion in cultured human breast cancer cells resulted in decreased cell proliferation, induction of apoptosis in-vitro and failure to establish xenografts in-vivo. Moreover, these cells failed to establish lung colonisation due to less proliferative, migrative and invasive capacity in experimental metastasis models. In an initial effort to identify causal effective of Cep55 mediated breast cancer pathogenesis, we found that Cep55-depleted cells that survive following long term culture have restored their ploidy compared with aneuploid cells that have constitutively high levels of Cep55. Moreover, these non-tumorigenic cells also have defect in major signalling networks assessed by Kinexus proteomic array. In line with this finding, we found that constitutive overexpression of Cep55 in non-transformed mammary epithelial cells, MCF-10a induced ERK1/2 and STAT3 signalling possibly due to increase in chromosome instability. Taken together, these results indicate that Cep55 promotes breast cancer initiation, progression and lung colonisation through pleiotropic effect and genomic instability that likely contribute to aneuploidy state of breast cancer tumours Citation Format: Murugan Kalimutho, Jacinta Simmons, Winnie Fernando, Jodi Saunus, Kevin Spring, Sunil Lakhani, Fares Al-Ejeh, Kum Kum Khanna. Cep55, a master regulator of cytokinesis in breast cancer pathogenesis. [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 LB-116. doi:10.1158/1538-7445.AM2014-LB-116

Abstract 4592: Etirinotecan pegol accumulates in breast cancer brain metastases and prolongs survival in an experimental model of brain metastases of human triple negative breast cancer

Abstract Background: Breast cancer brain metastases (BCBM) remain a challenging consequence of advanced breast cancer. The blood-brain barrier prevents many chemotherapeutic agents used to control systemic disease from reaching effective concentration in brain metastases. Etirinotecan pegol (EP) is the first long acting topoisomerase 1 inhibitor that provides continuous exposure to SN38 with reduced peak concentrations, which results in a better efficacy and safety profile than irinotecan (IRN). EP resulted in a RECIST response rate of 39% (7/18) in patients with metastatic TNBC. Here we present encouraging brain metastases distribution and survival results for EP in an experimental mouse model of TNBC brain metastasis using IRN as a control. Methods: For efficacy, mice with established MDA-MB-231Br BCBM (treatment started 21 days after intracardiac injection of MDA-MB-231Br Luc cells) were treated IV with vehicle, 50 mg/kg IRN, or 50 mg/kg EP q7d. Efficacy was assessed using bioluminescent imaging and survival. After reaching the study endpoint, brain samples were harvested, sectioned, and analyzed for size and number of metastases. For brain metastases distribution, mice with established MDA-MB-231 brain metastases received 50 mg/kg 14C-EP or 14C-IRN. At planned times, animals were sacrificed and brain was harvested, sectioned, and mounted on slides for quantitation of radioactivity in brain metastases and brain tumor distant tissue (BDT). Results: In the efficacy study, tumor bioluminescence in vehicle and IRN treated animals steadily increased. Vehicle animals died on days 16-23 post start of treatment and IRN animals died on day 21, leading to a median survival of 21 days in both groups. In contrast, bioluminescence decreased in EP treated animals over time, and 50% of animals survived to the end of study on day 70. Based on microscopic assessment of brain sections, EP treated animals had significantly reduced number of tumors per brain slice (0.5 for EP vs. 14.5 for IRN), and significantly smaller lesion size (0.04 mm2 for EP vs. 0.25 mm2 for IRN). Radiographic quantitation of brain sections confirmed that EP preferentially distributes into brain metastases compared to BDT. Brain metastases concentrations after EP were >4-fold higher compared to IRN treatment. Conclusions: EP shows promising efficacy in an experimental model of breast cancer brain metastases. EP selectively penetrates the compromised blood-brain barrier at the metastatic lesion, reduces the size and number of metastatic lesions, and prolongs survival. Plasma SN38 trough concentrations observed in this study are achieved clinically with the 145 mg/m2 q21d EP regimen used in the Phase 3 BEACON study in patients with metastatic BC. Assessment of the clinical relevance of these data is planned in the subset of BEACON patients with brain metastases. Citation Format: Mohamed Nounou, Ute Hoch, Chris E. Adkins, Tori B. Terrell, Heidi Villalba, Michael E. Eldon, Paul R. Lockman. Etirinotecan pegol accumulates in breast cancer brain metastases and prolongs survival in an experimental model of brain metastases of human triple negative breast cancer. [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 4592. doi:10.1158/1538-7445.AM2014-4592

Abstract 991: Brain metastasis depends on tumor cell initiated coagulation

Abstract Background: The incidence of brain metastasis exceeds that of primary brain tumors tenfold and is most frequently associated with lung cancer, breast cancer and melanoma. Despite a prognosis of only 6-9 months for patients with brain metastases, mechanisms of tumor cell brain colonization from the blood stream are unknown. Understanding this step could enable effective therapeutic strategies to prevent the development of incurable metastatic brain disease. We found that blood borne metastatic cancer cells reside within the cerebral microvasculature for several days and associate with platelets while crossing the blood brain barrier. Intravascular tumor cell survival and extravasation during this time may critically limit the success of brain metastasis. Activation of coagulation, platelets and fibrin formation contribute to tumor progression, cancer-associated thrombosis and metastatic spread to peripheral organs. However the function of coagulation in the seeding of brain metastases is unknown. Aims: We evaluated whether tumor cell-expressed tissue factor, a key activator of coagulation, promotes tumor cell seeding of brain metastases by initiating critical tumor cell-vascular interactions. Methods: We developed models of experimental brain metastasis to study how intravascular tumor cells cooperate with the coagulation system during early stages of brain colonization to survive and cross the blood brain barrier. Human breast cancer cells, injected into the left cardiac ventricle of immune deficient mice, are tracked during the initial phase of brain metastasis and progressive metastatic brain disease by detailed and quantitative histological analyses. We address how coagulation contributes to brain metastasis by targeting human tissue factor with specific inhibitory antibodies. We use ex-vivo bioluminescence imaging and immunohistochemical analyses at later stages of brain metastasis to determine the extent to which an early, transient treatment will result in long-term inhibition of brain metastatic disease. Results: We demonstrate that inhibition of tissue factor expressed by tumor cells reduces brain colonization. Targeting of tissue factor inhibits development of breast cancer brain metastasis and extends animal survival. We find that tissue factor properties which initiate coagulation or promote cytoprotective signaling pathways differentially contribute to brain metastasis development. Blockade of tissue factor-initiated coagulation inhibits intravascular tumor cell survival and prevents brain metastases in vivo. Our findings indicate that the inhibition of coagulation can prevent the seeding of breast cancer cells into the brain. Conclusions: Our studies provide mechanistic insights into the process of tumor cell brain colonization and identify targets for development of therapeutics to prevent cancer metastasis to the brain and enhance patient survival. Citation Format: Laurie J. Gay, John Day, Sarah LeBoeuf, Melissa Ritland, Zaverio Ruggeri, Wolfram Ruf, Brunhilde H. Felding. Brain metastasis depends on tumor cell initiated coagulation. [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 991. doi:10.1158/1538-7445.AM2014-991

Abstract 146: Combining molecular targeted drugs to inhibit both cancer cells and activated stromal cells in colon cancer

Abstract Recent studies have shown that tumor growth and metastasis are not determined by cancer cells alone but also by a variety of stromal cells. We have shown previously that platelet-derived growth factor receptors (PDGF-Rs) are overexpressed by various stromal cell populations, but not by cancer cells themselves in human colon cancer tissues. Blockade of PDGF-R signaling pathways in tumor-associated stromal cells has been shown to inhibit the stromal reaction. Activation of phosphoinositide 3-kinase (PI3K)/Akt/ mammalian target of rapamycin (mTOR) signaling is frequently observed in many cancer types and has been implicated in the pathogenesis of various diseases. mTOR is a central regulatory kinase that increases the production of proteins that stimulate key cellular processes such as cell proliferation, metabolism, survival, and angiogenesis. However, the effects of mTOR inhibitors on the stromal component of tumor tissues have not been studied. In the present study, the effects of nilotinib (PDGF-R tyrosine kinase inhibitor) and everolimus (mTOR inhibitor) on tumor development and metastasis, were examined using orthotopic and experimental liver metastatic models of human colon cancer, whereby KM12SM cells were implanted into the cecum and spleen of nude mice, respectively. Groups of mice received saline (control), nilotinib (100mg/kg/day) or everolimus (1mg/kg/day), or a combination of nilotinib (100mg/kg/day) and everolimus (1mg/kg/day) by daily oral gavage. After 4 weeks of treatment, primary tumors and metastatic lesions were resected, and specimens were analyzed histologically and immunohistochemically for comparison between the treatment group and control group. After treatment with nilotinib, the stromal reaction was significantly decreased. After treatment with everolimus, the stromal reaction was not decreased, but tumor cell proliferation and microvessel density were decreased. With the combination therapy, both tumor cell proliferation and the stromal reaction were decreased and apoptosis of tumor cells was increased, resulting in a remarkable decrease in tumor size and the number of metastatic liver lesions. Therefore, concurrent inhibition of tumor cells and activated stromal cells by combination therapy may represent a novel molecular-targeted approach for patients with colon cancer. Citation Format: Ryo Yuge, Yasuhiko Kitadai, Kei Shinagawa, Mieko Onoyama, Yuichiro Tanaka, Shinji Tanaka, Wataru Yasui, Kazuaki Chayama. Combining molecular targeted drugs to inhibit both cancer cells and activated stromal cells in colon cancer. [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 146. doi:10.1158/1538-7445.AM2014-146

Chemopreventive and Anticancer Activities of Allium victorialis var. platyphyllum Extracts.

Background:Allium victorialis var. platyphyllum is an edible perennial herb and has been used as a vegetable or as a Korean traditional medicine. Allium species have received much attention owing to their diverse pharmacological properties, including antioxidative, anti-inflammatory, and anticancer activities. However, A. victorialis var. platyphyllum needs more study.Methods:The chemopreventive potential of A. victorialis var. platyphyllum methanol extracts was examined by measuring 12-O-tetra-decanoylphorbol 13-acetate (TPA)-induced superoxide anion production in the differentiated HL-60 cells, TPA-induced mouse ear edema, and Ames/Salmonella mutagenicity. The apoptosis-inducing capabilities of the extracts were evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, 4’,6-diamidino-2-phenylindole staining, and the DNA fragmentation assay in human colon cancer HT-29 cells. Antimetastatic activities of the extracts were also investigated in an experimental mouse lung metastasis model.Results:The methanol extracts of A. victorialis var. platyphyllum rhizome (AVP-R) and A. victorialis var. platyphyllum stem (AVP-S) dose-dependently inhibited the TPA-induced generation of superoxide anion in HL-60 cells and TPA-induced ear edema in mice, as well as 7,12-dimethylbenz[a]anthracene (DMBA) and tert-butyl hydroperoxide (t-BOOH) -induced bacterial mutagenesis. AVP-R and AVP-S reduced cell viability in a dose-related manner and induced apoptotic morphological changes and internucleosomal DNA fragmentation in HT-29 cells. In the experimental mouse lung metastasis model, the formation of tumor nodules in lung tissue was significantly inhibited by the treatment of the extracts.Conclusions:AVP-R and AVP-S possess antioxidative, anti-inflammatory, antimutagenic, proapoptotic, and antimetastatic activities. Therefore, these extracts can serve as a beneficial supplement for the prevention and treatment of cancer.