Fibrosarcoma Cell Line Research Articles

Abstract 5112: Traditional Chinese medicine formula LQ inhibits spontaneous and cyclophosphamide-enhanced metastasis.

Abstract TCM (Traditional Chinese Medicine) formula LQ, an herbal cocktail, has shown anti-metastasis and anti-tumor efficacy without overt side-effects. In the present study, the human HT1080 fibrosarcoma cell line was labeled with both red fluorescent protein (RFP) in the cytoplasm and green fluorescent protein (GFP) in the nucleus. Dual-color HT1080 cells were injected into the footpad of mice following LQ treatment. Dual-color HT1080 cells were also injected into epigastric cranialis vein of the mice pre-treated with cyclophosphamide (CYC) or a combination of CYC and LQ. The efficacy was monitored through dynamic subcellular imaging of trafficking of cancer cells in lymph nodes and blood vessels in live mice. LQ significantly inhibited HT1080 spontaneous metastases to their local lymph nodes in the foot pad injection model. In the control group, 100% of mice had lymph node metastasis, compared to 12.5% of the mice in the LQ group. LQ also inhibited cyclophosphamide-enhanced experimental metastasis in the epigastric cranialis vein injection model. LQ prolonged the overall survival of mice in the spontaneous lymph node metastatic model. LQ therefore shows promise as an antimetastatic agent. Citation Format: Lei Zhang, Chengyu Wu, Yong Zhang, Robert M. Hoffman. Traditional Chinese medicine formula LQ inhibits spontaneous and cyclophosphamide-enhanced metastasis. [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 5112. doi:10.1158/1538-7445.AM2013-5112

Abstract 875: Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitors of Nuclear Export (SINE).

Abstract Abstract SINE are novel small molecules in human phase I clinical trials for advanced cancers. SINEs inhibit nuclear export through covalent binding to Exportin 1 (XPO1/CRM1) leading to forced nuclear retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, PTEN, pRB and IKB, leading to selective death of cancer cells. Resistant cells were created by treating the sensitive fibrosarcoma cell line HT1080 with increasing concentrations of SINE over 12 months. Gene chip analysis of parental-sensitive and drug-resistant cells that were treated with SINE resulted in activation of distinct pathways that mediate mechanisms of response and drug resistance. Methods Resistant cells were generated by selection in increasing concentrations of SINE. Cell viability was assayed by MTT. Immunofluorescence was used to compare nuclear export of TSPs. FACS, qPCR and immunoblots were used to measure effects on cell cycle, gene expression and cell death. RNA from naïve and drug treated sensitive/resistant cells was analyzed by Affymetrix microarrays. Results Treatment of SINE-sensitive fibrosarcoma cell line HT1080 (IC50 = 13.6nM) with gradually increasing concentrations of SINE for ∼1 year resulted with > 100 fold decrease in sensitivity to SINE cytotoxicity (IC50 = 1.7μM). Resistant cells displayed prolonged cell cycle (∼72 vs 24hrs) compared to parental cells. Resistant cells did not show increased PgP activity, suggesting that resistance to SINE was not the result of the induction of the multidrug resistance mechanism. Sequencing of CRM1 from the SINE resistant cells revealed no mutations in the SINE / cargo binding pocket including the reactive Cys528. Upon exposure to SINE, resistant cells had reduced nuclear accumulation of p53, p21, FOXO1A, IKB and p27 compared with SINE-sensitive cells. Interestingly, in SINE-sensitive cells, but not in resistant cells, SINE treatment resulted in potent pRB dephosphorylation (growth suppressive form of pRB), nuclear localization and the activation of p53 as measured by the induction of p53-downstream targets p21 and MIC1 (GDF15). In addition, SINE reduced levels of the anti-apoptotic protein Mcl-1, and induced the apoptotic markers Caspase 3 and PARP cleavage in sensitive but not in SINE resistant cells. Genes involved in cell adhesion, cytoskeleton formation, tight junctions, vesicle transport, cell cycle, and apoptosis were identified as key pathways correlating with drug response and resistance. Conclusions This study identified key pathways and genes that are likely mediating response and resistance to SINE antagonists. The extensive selection time (∼1 year) needed to achieve drug resistance suggests that generation of resistance may be difficult and that drug response may be prolonged. Citation Format: Yosef Landesman, Sharon Shechter, Jean-Richard Saint-Martin, Trinayan Kashyap, Marsha Crochiere, William Senapedis, Paul Ippolitti, Boris Klebanov, Sharon Tamir, Diego del Alamo, Mwanasha Hamuza, Gali Golan, Ori Kalid, Erkan Baloglu, Dilara McCauley, Michael Kauffman, Sharon Shacham. Deciphering mechanisms of drug sensitivity and resistance to Selective Inhibitors of Nuclear Export (SINE). [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 875. doi:10.1158/1538-7445.AM2013-875

Dentine matrix protein 1 (DMP-1) is a marker of bone-forming tumours

Dentin matrix protein 1 (DMP-1) is highly expressed by osteocytes and is a non-collagenous matrix protein found in dentin and bone. In this study, we determined the expression of DMP-1 in mature and immature human bone and examined whether DMP-1 is useful in distinguishing osteoid/bone-forming tumours from other primary and secondary bone tumours. DMP-1 expression was immunohistochemically determined in paraffin sections of a wide range of benign and malignant primary bone tumours and tumour-like lesions (n = 353). DMP-1 mRNA expression was also examined in osteosarcoma and fibrosarcoma cell lines as well as bone tumour specimens (n = 5) using real-time PCR. In lamellar and woven bone, DMP-1 was expressed in the matrix around osteocyte lacunae and canaliculi; osteoblasts and other cell types in the bone were negative. Matrix staining of the osteoid and bone was seen in bone-forming tumours including osteoma, osteoid osteoma, osteoblastoma and osteosarcoma. DMP-1 staining was also seen in fibrous dysplasia, osteofibrous dysplasia and chondroblastoma and in reactive bone in solitary bone cysts and aneurysmal bone cysts. DMP-1 was not expressed in the tumour component of other bone neoplasms including Ewing sarcoma, chondrosarcoma, leiomyosarcoma, fibrosarcoma, giant cell tumour of bone and metastatic carcinoma. DMP-1 mRNA was expressed in osteosarcoma cell lines and tumour samples. DMP-1 is a matrix marker expressed around osteocytes in human woven and lamellar bone and is useful in identifying osteosarcoma and other bone-forming tumours.

Low molecular weight heparin suppresses receptor for advanced glycation end products‐mediated expression of malignant phenotype in human fibrosarcoma cells

The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor and its engagement by ligands such as high mobility group box 1 (HMGB1) is implicated in tumor growth and metastasis. Low molecular weight heparin (LMWH) has an antagonistic effect on the RAGE axis and is also reported to exert an antitumor effect beyond the known activity of anticoagulation. However, the link between the anti-RAGE and antitumor activities of LMWH has not yet to be fully elucidated. In this study, we investigated whether LMWH could inhibit tumor cell proliferation, invasion, and metastasis by blocking the RAGE axis using in vitro and in vivo assay systems. Stably transformed HT1080 human fibrosarcoma cell lines were obtained, including human full-length RAGE-overexpressing (HT1080(RAGE)), RAGE dominant-negative, intracellular tail-deleted RAGE-overexpressing (HT1080(dnRAGE)), and mock-transfected control (HT1080(mock)) cells. Confocal microscopy showed the expression of HMGB1 and RAGE in HT1080 cells. The LMWH significantly inhibited HMGB1-induced NFκB activation through RAGE using an NFκB-dependent luciferase reporter assay and the HT1080 cell lines. Overexpression of RAGE significantly accelerated, but dnRAGE expression attenuated HT1080 cell proliferation and invasion in vitro, along with similar effects on local tumor mass growth and lung metastasis in vivo. Treatment with LMWH significantly inhibited the migration, invasion, tumor formation, and lung metastasis of HT1080(RAGE) cells, but not of HT1080(mock) or HT1080(dnRAGE) cells. In conclusion, this study revealed that RAGE exacerbated the malignant phenotype of human fibrosarcoma cells, and that this exacerbation could be ameliorated by LMWH. It is suggested that LMWH has therapeutic potential in patients with certain types of malignant tumors.

Inhibition of Growth and Induction of Apoptosis in Fibrosarcoma Cell Lines by Echinophora platyloba DC: In Vitro Analysis.

Echinophora platyloba DC plant (Khousharizeh) is one of the indigenous medicinal plants which is used as a food seasoning and medicine in Iran. The objective of this study was to examine the in vitro cytotoxic activity and the mechanism of cell death of crude methanolic extracts prepared from Echinophora platyloba DC, on mouse fibrosarcoma cell line (WEHI-164). Cytotoxicity and viability of methanolic extract was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dye exclusion assay. Cell death ELISA was employed to quantify the nucleosome production result from nuclear DNA fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling (TUNEL) assay. Our results demonstrated that the extract decreased cell viability, suppressed cell proliferation, and induced cell death in a time- and dose-dependent manner in WEHI-164 cells (IC50 = 196.673 ± 12.4 μg/mL) when compared with a chemotherapeutic anticancer drug, Toxol. Observation proved that apoptosis was the major mechanism of cell death. So the Echinophora platyloba DC extract was found to time- and dose-dependently inhibit the proliferation of fibrosarcoma cell possibly via an apoptosis-dependent pathway.

P53-dependent radiobiological responses to internalised indium-111 in human cells

The p53 tumour suppressor protein plays a pivotal role in the response of mammalian cells to DNA damage. It regulates cell cycle progression, apoptosis and DNA repair mechanisms and is therefore likely to influence response to targeted radionuclide therapy. This study investigated the role of p53 in the cellular response to the Auger-emitting radionuclide indium-111. Two stable clones of a HT1080 fibrosarcoma cell line, differing only in p53 status due to RNAi-mediated knockdown of p53 expression, were incubated for 1 h with [¹¹¹In]-oxinate (0-10 MBq/ml). Radiopharmaceutical uptake into HT1080 cells was measured in situ using a non-contact phosphorimager method. Cellular sensitivity and DNA damage were measured by, respectively, clonogenic survival analysis and the single cell gel electrophoresis (Comet) assay. Mean uptake of [¹¹¹In]-oxinate in HT1080 cells was unaffected by p53 status, reaching a maximum of 9Bq/cell. [¹¹¹In]-oxinate-induced cytotoxicity was also identical in both clones, as measured by IC50 (0.68 MBq/ml). However the formation of DNA damage, measured immediately after treatment with [¹¹¹In]-oxinate, was found to be up to 2.5-fold higher in the p53-deficient HT1080 clone. The increased DNA damage induced in p53-deficient HT1080 cells suggests an early deficiency in the repair of DNA damage during the treatment period. However, the similarity in cellular sensitivity, irrespective of p53 status, suggests that reduced p53 leads to a concomitant reduction in p53-dependent cytotoxicity despite the persistence of DNA damage. The results may provide insight into how tumours that differ in p53 status respond to therapeutic radionuclides.

Synthesis and in vitro Antitumor Activity of 6-Chloro-4-piperazinquinazolinethiosemicarbazone Derivatives

Three new quinazoline derivatives 5a~5c bearing piperazine have been designed and synthesized by four-step reactions of cyclization,chlorination,substitution and condensation using 2-amino-5-chlorobenzoic acid and formamide as starting materials.Their structures were confirmed by 1H NMR,13C NMR,ESI-MS and IR.The in vitro cytotoxicities against SGC-7901(human gastric cancer),KB(human oral epidermoid cancer) and HT-1080(human fibrosarcoma) cell lines of compounds 5a~5c were tested with colorimetric MTT assay.The results indicated that compounds 5a~5c had poor anticancer activities against SGC-7901 and HT-1080 cell lines,while had no notable inhibitory activity against KB cell lines.

Derivation of feline vaccine-associated fibrosarcoma cell line and its growth on chick embryo chorioallantoic membrane – a new in vivo model for veterinary oncological studies

Feline vaccine associated fibrosarcomas are the second most common skin tumor in cats. Methods of treatment are: surgery, chemotherapy and radiotherapy. Nevertheless, the usage of cytostatics in feline vaccine associated sarcoma therapy is limited due to their adverse side effects, high toxicity and low biodistribution after i.v. injection. Therefore, much research on new therapeutic drugs is being conducted. In human medicine, the chick embryo chorioallantoic membrane (CAM) model is used as a cheap and easy to perform assay to assess new drug effectiveness in cancer treatment. Various human cell lines have different tumors growth on CAM. In veterinary medicine such model has not been described yet. In the present article derivation of feline vaccine associated fibrosarcoma cell line and its growth on CAM is described. The cell line and the tumor grown were confirmed by histopathological and immunohistochemical examination. As far as we believe, this is the first attempt to create such model, which may be used for further in vivo studies in veterinary oncology.

Mutant IDH1 is required for IDH1 mutated tumor cell growth

Frequent somatic hotspot mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in gliomas, acute myeloid leukemias, chondrosarcomas, and other cancers, providing a likely avenue for targeted cancer therapy. However, whether mutant IDH1 protein is required for maintaining IDH1 mutated tumor cell growth remains unknown. Here, using a genetically engineered inducible system, we report that selective suppression of endogenous mutant IDH1 expression in HT1080, a fibrosarcoma cell line with a native IDH1(R132C) heterozygous mutation, significantly inhibits cell proliferation and decreases clonogenic potential. Our findings offer insights into changes that may contribute to the inhibition of cell proliferation and offer a strong preclinical rationale for utilizing mutant IDH1 as a valid therapeutic target.

Extracellular Matrix Remodeling in Takayasu's Arteritis: Role of Matrix Metalloproteinases and Adventitial Inflammation

Takayasu's arteritis (TA) is an inflammatory fibrosing arteritis affecting predominately the aorta and its main branches. Pathogenesis of this disease remains enigmatic. Despite the numerous studies, the role of adventitia in vascular lesion formation in the setting of TA has been ignored. Virtually nothing is known about the mechanism regulating inflammation in the adventitia in the setting of TA. The present study included subjects with Takayasu's arteritis and normal healthy control subjects. Isolated T cells from peripheral blood mononuclear cells (PBMCs) using nylon wool and HUT-78 (human cutaneous T lymphoma cell line) were stimulated with PHA for 24 h. Stimulated cell were fixed with paraformaldehyde and fractionated into membrane, cytosolic and nuclear fractions. These cellular fractions were co-cultured with human fibrosarcoma cell line (HT-1080) and transcriptional expression of matrix metalloproteinases (MMP-1, 3, 9 and TIMP-1) was determined using semiquantitative RT-PCR. Stimulation of MMPs-TIMP synthesis by HT-1080 cells was mimicked by a membranous fraction derived from activated T-cell isolated from TA subjects and activated HUT-78 cells, whereas cytosolic and nuclear fractions were ineffective. In conclusion, for the first time we provide evidence for the presence of a cell surface-specific antigenic moiety on T-cells of TA subjects, which is responsible for activation of fibroblasts (cells predominantly present in adventitia) to enhance MMP production and, therefore, may lead to extracellular matrix degradation.

A novel derivative of decursin, CSL‐32, blocks migration and production of inflammatory mediators and modulates PI3K and NF‐κB activities in HT1080 cells

Decursin and related coumarin compounds in herbal extracts have a number of biological activities against inflammation, angiogenesis and cancer. We have analysed a derivative of decursin (CSL-32) for activity against inflammatory activation of cancer cells, such as migration, invasion and expression of pro-inflammatory mediators. The human fibrosarcoma cell line, HT1080, was treated with TNFα (tumour necrosis factor α) in the presence or absence of CSL-32. The cellular responses and modification of signalling adapters were analysed with respect to the production of pro-inflammatory mediators, as also migration, adhesion and invasion. Treatment of HT1080 cells with CSL-32 inhibited their proliferation, without affecting cell viability, and TNFα-induced expression of pro-inflammatory mediators, such as MMP-9 (matrix metalloproteinase-9) and IL-8 (interleukin-8). CSL-32 also suppressed phosphorylation and degradation of IκB (inhibitory κB), phosphorylation of p65 subunit of NF-κB (nuclear factor-κB) and nuclear translocation of NF-κB, which are required for the expression of pro-inflammatory mediators. In addition, CSL-32 inhibited invasion and migration of HT1080 cells, as also cellular adhesion to fibronectin, an ECM (extracellular matrix) protein. CSL-32 treatment resulted in a dose-dependent inhibition of PI3K (phosphoinositide 3-kinase) activity, required for the cellular migration. The analyses show that CSL-32 inhibits processes associated with inflammation, such as the production of pro-inflammatory mediators, as well as adhesion, migration and invasion in HT1080 cells.

Tankyrase 1 regulates centrosome function by controlling CPAP stability

CPAP--a gene mutated in primary microcephaly--is required for procentriole formation. Here we show that CPAP degradation and function is controlled by the poly(ADP-ribose) polymerase tankyrase 1. CPAP is PARsylated by tankyrase 1 in vitro and in vivo. Overexpression of tankyrase 1 leads to CPAP proteasomal degradation, preventing centriole duplication, whereas depletion of tankyrase 1 stabilizes CPAP in G1, generating elongated procentrioles and multipolarity. Tankyrase 1 localizes to centrosomes exclusively in G1, coinciding with CPAP degradation. Hence, tankyrase 1-mediated PARsylation regulates CPAP levels during the cell cycle to limit centriole elongation and ensure normal centrosome function.

Targeting a Newly Established Spontaneous Feline Fibrosarcoma Cell Line by Gene Transfer

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas.

Do Anti-Angiogenic VEGF (VEGFxxxb) Isoforms Exist? A Cautionary Tale

Splicing of the human vascular endothelial growth factor-A (VEGF-A) gene has been reported to generate angiogenic (VEGFxxx) and anti-angiogenic (VEGFxxxb) isoforms. Corresponding VEGFxxxb isoforms have also been reported in rat and mouse. We examined VEGFxxxb expression in mouse fibrosarcoma cell lines expressing all or individual VEGF isoforms (VEGF120, 164 or 188), grown in vitro and in vivo, and compared results with those from normal mouse and human tissues. Importantly, genetic construction of VEGF164 and VEGF188 expressing fibrosarcomas, in which exon 7 is fused to the conventional exon 8, precludes VEGFxxxb splicing from occurring. Thus, these two fibrosarcoma cell lines provided endogenous negative controls. Using RT-PCR we show that primers designed to simultaneously amplify VEGFxxx and VEGFxxxb isoforms amplified only VEGFxxx variants in both species. Moreover, only VEGFxxx species were generated when mouse podocytes were treated with TGFβ-1, a reported activator of VEGFxxxb splice selection in human podocytes. A VEGF164/120 heteroduplex species was identified as a PCR artefact, specifically in mouse. VEGFxxxb isoform-specific PCR did amplify putative VEGFxxxb species in mouse and human tissues, but unexpectedly also in VEGF188 and VEGF164 fibrosarcoma cells and tumours, where splicing to produce true VEGFxxxb isoforms cannot occur. Moreover, these products were only consistently generated using reverse primers spanning more than 5 bases across the 8b/7 or 8b/5 splice junctions. Primer annealing to VEGFxxx transcripts and amplification of exon 8b primer ‘tails’ explained the artefactual generation of VEGFxxxb products, since the same products were generated when the PCR reactions were performed with cDNA from VEGF164/VEGF188 ‘knock-in’ vectors used in the generation of single VEGF isoform-expressing transgenic mice from which the fibrosarcoma lines were developed. Collectively, our results highlight important pitfalls in data interpretation associated with detecting VEGFxxxb isoforms using current methods, and demonstrate that anti-angiogenic isoforms are not commonly expressed in mouse or human tissues.

Immunization with E1A expressing tumor cell line enhances antigen specific tumor immunity (74.5)

Abstract The stable expression of Adenovirus (Ad) E1A in tumor cells reduces tumorigenicity by 1,000 fold, an effect dependent on the elicitation of an NK cell and T cell mediated antitumor response. We generated B6 fibrosarcoma cell lines (MCA tumor cells) that stably expressed ovalbumin (MCA-OVA) or full-length E1A fused to ovalbumin (MCA-E1A-OVA) to test the hypotheses that E1A could function as a molecular adjuvant to induce a robust anti-tumor immune response to other weakly or non-immunogenic tumor antigens. MCA E1A-OVA was more susceptible to killing by NK in vitro and 1000 fold less tumorigenic than MCA or MCA-OVA in vivo. Immunization with 1X10^5 live MCA-OVA or MCA- E1A-OVA tumor cells elicited comparable numbers of IFNγ- and TNFα -producing OVA specific CD8 T cells that had similar cytolytic activity against OVA-expressing target cells in in vivo CTL killing assays. Immunization of MCA-OVA or MCA-E1A-OVA cells also increased the number of MCA-OVA cells required to form tumors by 1,000 fold in an anatomically distant site. However, primary tumors developed at the site of immunization with MCA-OVA but not MCA-E1A-OVA cells. These data demonstrate that immunization of live MCA-E1A or MCA-OVA tumor cells can elicit a robust, systemic anti-tumor immune response. However, primary tumor progression occurs at the site of inoculation following immunization with MCA-OVA cells despite the induction of an anti-tumor immune response.

Abstract 890: Interleukin 11: A novel therapeutic target in IL-11Rα expressing sarcomas

Abstract Interleukin-11 (IL-11) is a member of the IL-6 cytokine family, which mediate signaling via a common signal-transducing gp130 component and a cytokine-specific subunit (IL-11Rβ). Upon binding to the IL-11Rα, a signaling cascade activates several pathways, including JAK kinases, STAT transcription factors (STAT3 and STAT1), MAPK, src-family kinases and PI3K. IL-11 has been implicated in experimental models of chronic inflammation and associated tumorigenesis. Oncomine analysis of publicly available microarray data from patient biopsies showed that IL-11Rα mRNA is expressed in several sarcomas and not in the matched normal tissue counterparts. Surgical resection and cytotoxic chemotherapy are the standard of care for most locally advanced and metastatic sarcomas, the identification of new markers and the development of targeted therapies are needed to increase long-term survival of these patients. Previous studies demonstrated IL-11Rα is expressed in human primary and metastatic osteosarcomas but not in normal bone, suggesting that IL-11Rα is a potential target for sarcoma therapy. We analyzed a panel of 45 human sarcoma cell lines for the expression of IL-11Rα and IL-11. Interestingly, IL-11Rα protein is expressed at high levels in the majority of cell lines examined. In addition, we found that nearly all osteosarcoma, rhabdomyosarcoma, fibrosarcoma and Ewing sarcoma cell lines express high constitutive levels of IL-11 mRNA, indicating that IL-11 autocrine signaling may play a role in these tumor types. Two lentiviral vectors, used for the delivery of shRNA targeting IL-11 in HT-1080, produced an 80-90% decrease in IL-11 mRNA expression when compared to the luciferase control shRNA; however, IL-11 mRNA levels remained significantly high in HT1080 knock-down cells when compared to the constitutive levels of non sarcoma cancer types. Decreasing levels of IL-11 in HT-1080 resulted in a 50% inhibition of cell growth, demonstrating that IL-11 has a role in cell proliferation. Further experiments aimed to assess the expression of IL-11 and IL-11Rβ in xenografts and to explore the role of IL-11 in sarcoma pathogenesis, propagation and response to therapy are ongoing. These data suggest that neutralization of IL-11 or IL-11Rα may be a potential target for the development of cancer therapeutics for sarcomas. Funded by NCI Contract No. HHSN261200800001E. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 890. doi:1538-7445.AM2012-890

Abstract 2678: Traditional Chinese Medicine (TCM) in combination with S. typhimurium A1-R for cancer treatment

Abstract Traditional Chinese Medicine (TCM) has long shown promise for the treatment of cancer. We have previously developed a bacterial cancer therapy strategy using the genetically-engineered strain, Salmonella typhimurium A1-R, which is auxotrophic for arg and leu, which attenuates growth in normal tissue but allows high tumor virulence. S. typhimurium A1-R is effective against metastatic human prostate, breast, and pancreatic cancer cell lines as well as osteosarcoma, fibrosarcoma, and glioma cell lines in clinically-relevant nude-mouse and syngeneic-mouse models. The current study evaluated TCM as combination treatment during bacterial therapy in metastatic mouse models of human cancer. TCM prevented hemorrhagic spots on the skin, liver, and spleen after bacterial infection. Fur discoloration and reduced activity was also observed in mice treated with S. typhimurium A1-R. These symptoms were ameliorated when bacterial treatment was combined with TCM. In a syngeneic lung cancer metastatic model in immunocompetent mice, the combination of TCM and S. typhimurium A1-R enabled S. typhimurium to be safely administered at a dose of 2×105 CFU twice a week i.v. in immunocompotent mice with increased treatment efficacy compared to monotherapy with S. typhimurium A1-R. The results of the present study suggest that combination of S. typhimurium A1-R and TCM has important potential for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2678. doi:1538-7445.AM2012-2678

Abstract 2685: Imaging the various mechanisms of cancer-cell killing by Salmonella typhimurium A1-R

Abstract We have developed a genetically-modified bacterial strain of Salmonella typhimurium, auxotrophic for leucine and arginine, which also expresses green fluorescent protein (GFP), termed S. typhimurium A1-R. S. typhimurium A1-R was found to be effective against metastatic human prostate, breast, and pancreatic cancer cell lines as well as osteosarcoma, fibrosarcoma and glioma cell lines in clinically-relevant nude mouse models. To understand the tumor-killing mechanism of S. typhimurium A1-R-GFP, we have studied their interaction with fluorescence-labeled cancer cells in vitro and in vivo. S. typhimurium-GFP invasion, proliferation, and virulence in the cancer cells were visualized by confocal fluorescence microscopy with the Olympus FV1000. We found that A1-R-induced cancer cell death had different mechanisms in different cancer cell lines. Some S. typhimurium A1-R-treated cancer cell lines became apoptotic and necrotic, and other cell lines burst after extensive intracellular bacterial growth. The time for S. typhimurium A1-R-GFP to kill the majority of the cancer cells varied from line to line, ranging from 2 hours to 72 hours. The understanding of the various mechanisms of cell killing by S. typhimurium A1-R will be important for its use as a general therapeutic for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2685. doi:1538-7445.AM2012-2685

Abstract 4396: Combination of intratumoral regulated IL-12 gene delivery and systemic chemotherapy with Palifosfamide (IPM-Tris) enhances anti-tumor effects in breast cancer (4T1) model

Abstract A major obstacle for the development of effective immunotherapy is the ability of tumors to escape detection and/or destruction by the immune system. This study explored pairing a potent immunomodulatory cytokine (IL-12) with a bifunctional alkylating agent currently in Phase III clinical trials for soft tissue sarcoma (Palifosfamide, IPM-Tris). In this study intratumorally administered adenoviral vector, Ad-RTS-mIL-12, expressing IL-12 under the control of Rheoswitch Therapeutic System®, (RTS®), a novel inducible promoter system was used. Expression of IL-12 is regulated by oral administration of a small molecule activator ligand, INXN-1001 (AL). The in vitro transduction of the human fibrosarcoma cell line, HT1080, with Ad-RTS-hIL12 + AL at a MOI of 400 pfu, produced >6000-fold induction of human IL-12 above background, while IPM-Tris (1-3 uM) was cytotoxic in a variety of cancer cell lines. The effect of Ad-RTS-mIL12 + AL in combination with IPM-Tris was evaluated in a subcutaneous 4T1 syngeneic BALB/c mouse mammary tumor model at low doses, which were chosen to have minimal effect on tumor growth rate. Indeed, IPM-Tris alone (40 - 120 mg/m2) had little effect on tumor growth rate (< 30%) compared to vehicle. In contrast, a single intratumoral injection of Ad-RTS-mIL12 (1x1010 vp) and oral administration of AL (15, 30, 75 or 150 mg/m2) once daily for 5 days led to significant AL dose-related tumor growth inhibition (28, 29, 62, 59%, respectively; p<0.05) with the Ad-RTS-mIL12 having no effect relative to control by Day 35. Combined treatment with Ad-RTS-mIL12 + AL (30 mg/m2) and IPM-Tris (40 or 120 mg/m*2 administered IP QD for 3 days) significantly inhibited tumor growth inhibition (∼71-90%, p<0.001) and increased median survival rate by 8-14 days when compared to the single agents alone, without overt toxicity as assessed by change in body weight. These results indicate that local intratumoral immunotherapy combined with low dose chemotherapy with palifosfamide offers a distinct advantage over either therapeutic modality alone. These results are supportive of our hypothesis that cytotoxic agents at low doses may prime the immune system to enhance immunotherapy, which could potentially be translated into a safe clinical regimen for the treatment of metastatic breast cancer. Additional studies are ongoing to explore in depth the immunologic mechanism(s) that account for the more potent immunotherapeutic effects of IL-12 when combined with IPM-Tris. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4396. doi:1538-7445.AM2012-4396

Abstract 5296: The role of VEGF receptors 1 and 2 on tumor vascularization and progression in single VEGF isoform expressing tumors

Abstract Background Murine fibrosarcoma cell lines that express single VEGF isoforms (VEGF120 VEGF165 and VEGF188) and a wild-type control (VEGFWT) have been developed. The most extreme differences in vascular properties are observed between VEGF120 and VEGF188 tumors, with VEGF120 tumors developing more immature vessels. These differences impact on therapeutic outcome. We hypothesised that differential signaling through VEGF receptors 1 and 2 underlie the isoform-specific differences. Methods VEGFWT, VEGF120 and VEGF164 tumors were implanted sub-cutaneously (s/c), and VEGF188 tumors s/c or in dorsal skin-flap window-chambers (WC) in SCID mice. Mice were treated with ImClone neutralizing antibodies against VEGFR-1 (MF-1) or VEGFR-2 (DC101). Excised s/c tumors were stained for blood vessels (CD31), pericytes (αSMA), necrosis (H&E) or apoptosis (Caspase III). Tumor vascular development in WCs was imaged using intra-vital microscopy. Results VEGFWT and VEGF188 tumors were most sensitive to growth inhibition following treatment with DC101, whereas MF-1 had no effect on growth of any tumor type. In vitro, DC101 had no effect on proliferation of any tumor cell types. Immunohistochemical studies showed no changes in necrosis or apoptosis in VEGF120 tumors, but a significant decrease in vascular area with DC101. VEGF188 tumors showed no change in apoptosis, but both treatments increased necrosis. Vascular area was significantly reduced post DC101 treatment but significantly increased post MF-1 treatment. Both antibodies significantly increased αSMA staining, primarily due to myofibroblast infiltration. WC studies using VEGF188 tumors supported the effects observed with DC101, showing a significantly reduced blood vessel number and length. Furthermore, MF-1 treatment (either immediately post tumor implantation or to established tumors) resulted in more hemorrhagic tumors with large peripheral feeder vessels. Early DC101 treatment impaired tumor development, and once tumors developed on cessation of treatment, the vessels were highly chaotic. Conclusions DC101-induced tumor growth retardation, and corresponding vascular effects in VEGF188 tumors, confirms a role for VEGFR2 signaling in tumor progression and vascular development. However, the lack of growth effect in VEGF120 and VEGF164 tumors following treatment with either antibody suggests a level of receptor redundancy. MF-1 increased angiogenesis in VEGF188 tumors, despite no change in growth rate, and these neo-vessels appeared hemorrhagic, implying increased permeability. These data suggest a role for VEGFR-1 in tumor angiogenesis, involving vascular maturation, at least where VEGF188 is highly expressed. This may occur via specific VEGFR-1 signaling and/or sequestration of VEGF to control signaling via VEGFR-2. Acknowledgements Supported by CR-UK We thank ImClone Systems for providing MF-1 and DC101. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5296. doi:1538-7445.AM2012-5296