Soft Tissue Sarcoma Cell Lines Research Articles

Abstract 2073: The parp inhibitor olaparib (AZD2281) greatly enances the effect of temozolomide in soft tissue sarcoma cell lines.

Abstract Sarcomas are heterogeneous mesenchymal neoplasms which have very limited treatment options. Chemotherapies for advanced sarcoma include DNA damaging agents such as doxorubicin, dacarbazine and gemcitabine with very low response rates. We elected to study the effects of olaparib (AZD2281), a novel, potent orally active PARP inhibitor in combination with standard chemotherapy Temozolomide (TMZ) in various sarcoma subtypes. We examined the effects of olaparib and TMZ in a panel of 7 sarcoma cell lines including leiomyosarcoma (SK-LMS, SK-UT1, SK-UT1b), liposarcoma (LS141, DDLS) and malignant peripheral nerve sheath tumors (MPNST, ST8814). In vitro results have shown inhibition of cell viability with combination treatment in all cell lines at concentrations of 300uM TMZ and 1 uM olaparib. After 48 hrs of drug treatment, we also examined biochemical effects showing PAR inhibition, p53 induction and γH2AX increase, indicative of DNA damage. Flow cytometric analysis of each cell line indicated G2/M cell cycle arrest with combination treatment. These observations correlated with enhanced apoptosis as measured by biochemical PARP cleavage and DNA content (sub-G1 population). Taking these observations and results, there is strong evidence to support combining standard chemotherapy TMZ with the PARP inhibitor olaparib in various sarcoma subtypes. Citation Format: Elgilda Musi, Grazia Ambrosini, Gary K. Schwartz. The parp inhibitor olaparib (AZD2281) greatly enances the effect of temozolomide in soft tissue sarcoma cell lines. [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 2073. doi:10.1158/1538-7445.AM2013-2073

Abstract 3453: Selective sensitivity of Ewing's Sarcoma to the PARP inhibitor BMN 673.

Abstract Background: A principal function of Poly-ADP-Ribose Polymerase (PARP) proteins is to help orchestrate the DNA damage response by recruiting a multitude of proteins to sites of DNA damage. PARP inhibitors have shown particular promise in settings where a preexisting DNA damage repair defect exists, such as in tumors that lack a functional homologous recombination (HR) pathway. PARPs also have roles in epigenetic modulation and PARP inhibition has been shown to affect the cell cycle and cellular stress response. As such, PARP inhibitors may be efficacious in tumors besides those known to have HR defects. We hypothesized that subsets of bone and soft tissue sarcomas could potentially be selectively sensitive to BMN 673, an oral, selective PARP-1/2 inhibitor currently in phase 1 trials (AACR-NCI-EORTC Abstract #B-64, #B-67, 2011). Methods: We determined the IC50G of BMN 673 across 50 human soft tissue and bone sarcoma cell lines representing 5 main histologies by exposing the lines to six BMN 673 concentrations over a 6-day incubation and using direct cell counts. Synergy was determined by calculating cell growth inhibition in cell lines exposed to BMN 673 along with gemcitabine and SN-38. Translocations in Ewings sarcoma lines were available from public sources. In vivo response was evaluated by monitoring the changes in tumor volume of established subcutaneous xenografts in 6-8wk old CD-1 athymic, nude mice treated daily with BMN 673 by gavage. Baseline gene expression profiles of the cell lines were generated using the Agilent microarray platform. Results: BMN 673 had a potent anti-proliferative activity in low nanomolar range with 20/24 Ewing's sarcoma lines demonstrating IC50G<24 nM. The IC50G of Ewing's lines to BMN 673 (Mean IC50G: 10.4 nM; n=24 lines) was significantly lower compared to osteosarcoma (Mean IC50G: 108.6 nM, n=11; p=0.0001) and liposarcoma (Mean IC50G: 197.4 nM, n=4; p=0.0001) but not statistically significant compared to rhabdomyosarcoma (Mean IC50G: 19.8 nM, n=9; p=0.18). Ewing's sarcoma cells harboring EWS-FLI1 and EWS-ERG translocations both appear to be highly sensitive to BMN 673 (EWS-FLI1: Mean IC50G: 10.8 nM, n=18; EWS-ERG: Mean IC50G: 9.0 nM, n=5, p=0.61). PARP1 mRNA levels were not different among the very sensitive and the moderately sensitive Ewings lines. Combination of BMN 673 with gemcitabine or SN-38 generated tumor growth inhibition more potent than either agent alone. An in vivo dose-dependent sensitivity to BMN 673 was noted in xenograft studies using 3 Ewing's and 1 osteosarcoma cell line. Microarray analyses for predictors of response/resistance are currently ongoing. Conclusions: A large panel of Ewings sarcoma cell lines demonstrate selective in vitro and in vivo sensitivity to BMN 673, this response may be augmented by the addition of cytotoxic chemotherapy. Citation Format: Arun Singh, Bartosz Chmielowski, Weiping Jia, Kareem Clarke, Neil O'Brien, Judy Dering, Dylan Conklin, Yuqiao Shen, Leonard Post, Richard S. Finn, Dennis J. Slamon. Selective sensitivity of Ewing's Sarcoma to the PARP inhibitor BMN 673. [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 3453. doi:10.1158/1538-7445.AM2013-3453

Effects of TRAIL and taurolidine on apoptosis and proliferation in human rhabdomyosarcoma, leiomyosarcoma and epithelioid cell sarcoma

Soft tissue sarcomas (STS) are a heterogeneous group of malignant tumours representing 1% of all malignancies in adults. Therapy for STS should be individualised and multimodal, but complete surgical resection with clear margins remains the mainstay of therapy. Disseminated soft tissue sarcoma still represents a therapeutic dilemma. Commonly used chemotherapeutic agents such as doxorubicin and ifosfamide have proven to be effective in fewer than 30% in these cases. Therefore, we tested the apoptotic and anti-proliferative in vitro effects of TNF-related apoptosis-inducing ligand (TRAIL) and taurolidine (TRD) on rhabdomyosarcoma (A-204), leiomyosarcoma (SK-LMS-1) and epithelioid cell sarcoma (VA-ES-BJ) cell lines. Viability, apoptosis and necrosis were quantified by FACS analysis (propidium iodide/Annexin V staining). Gene expression was analysed by DNA microarrays and the results validated for selected genes by rtPCR. Protein level changes were documented by western blot analysis. Cell proliferation was analysed by BrdU ELISA assay. The single substances TRAIL and TRD significantly induced apoptotic cell death and decreased proliferation in rhabdomyosarcoma and epithelioid cell sarcoma cells. The combined use of TRAIL and TRD resulted in a synergistic apoptotic effect in all three cell lines, especially in rhabdomyosarcoma cells leaving 18% viable cells after 48 h of incubation (p<0.05). Analysis of the differentially regulated genes revealed that TRD and TRAIL influence apoptotic pathways, including the TNF-receptor associated and the mitochondrial pathway. Microarray analysis revealed remarkable expression changes in a variety of genes, which are involved in different apoptotic pathways and cross talk to other pathways at multiple levels. This in vitro study demonstrates that TRAIL and TRD synergise in inducing apoptosis and inhibiting proliferation in different human STS cell lines. Effects on gene expression differ relevantly in the sarcoma entities. These results provide experimental support for in vivo trials assessing the effect of TRAIL and TRD in STS and sustain the approach of individualized therapy.

Effect of Costunolide and Dehydrocostus Lactone on Cell Cycle, Apoptosis, and ABC Transporter Expression in Human Soft Tissue Sarcoma Cells

Human soft tissue sarcomas represent a rare group of malignant tumours that frequently exhibit chemotherapeutic resistance and increased metastatic potential following unsuccessful treatment. In this study, we investigated the effects of costunolide and dehydrocostus lactone, which have been isolated from Saussurea lappa using activity-guided isolation, on three soft tissue sarcoma cell lines of various origins. The effects on cell proliferation, cell cycle distribution, apoptosis induction, and ABC transporter expression were analysed. Both compounds inhibited cell viability dose- and time-dependently. IC50 values ranged from 6.2 µg/mL to 9.8 µg/mL. Cells treated with costunolide showed no changes in cell cycle, little in caspase 3/7 activity, and low levels of cleaved caspase-3 after 24 and 48 h. Dehydrocostus lactone caused a significant reduction of cells in the G1 phase and an increase of cells in the S and G2/M phase. Moreover, it led to enhanced caspase 3/7 activity, cleaved caspase-3, and cleaved PARP indicating apoptosis induction. In addition, the influence of costunolide and dehydrocostus lactone on the expression of ATP binding cassette transporters related to multidrug resistance (ABCB1/MDR1, ABCC1/MRP1, and ABCG2/BCRP1) was examined using real-time RT-PCR. The expressions of ABCB1/MDR1 and ABCG2/BCRP1 in liposarcoma and synovial sarcoma cells were significantly downregulated by dehydrocostus lactone. Our data demonstrate for the first time that dehydrocostus lactone affects cell viability, cell cycle distribution and ABC transporter expression in soft tissue sarcoma cell lines. Furthermore, it led to caspase 3/7 activity as well as caspase-3 and PARP cleavage, which are indicators of apoptosis. Therefore, this compound may be a promising lead candidate for the development of therapeutic agents against drug-resistant tumours.

Oncolytic virotherapy for human bone and soft tissue sarcomas using live attenuated poliovirus

The poliovirus receptor CD155, is essential for poliovirus to infect and induce death in neural cells. Recently, CD155 has been shown to be selectively expressed on certain types of tumor cells originating from the neural crest, including malignant glioma and neuroblastoma. However, the expression pattern of CD155 in soft tissue sarcoma has not been examined. Therefore, we first examined CD155 expression in sarcoma cell lines, and found the expression of both CD155 mRNA and protein in 12 soft and bone tissue sarcoma cell lines. Furthermore, we examined the effect of live attenuated poliovirus (LAPV) on 6 bone and soft tissue sarcoma cell lines invitro, and found that LAPV induced apoptosis by activating caspases7 and3 in all of these cell lines. Furthermore, in BALB/c nu/nu mice xenotransplanted with HT1080 fibrosarcoma cells, administration of live attenuated poliovirus caused growth suppression of the tumors. These results suggest that oncolytic therapy using a LAPV may represent a new option for the treatment of bone and soft tissue sarcomas.

Preclinical study of trabectedin (TR) and poly(ADP-ribose)polymerase 1 (PARP-1) inhibitor combination in soft tissue sarcoma (STS).

10066 Background: TR is an alkylating agent approved in Europe for the treatment of advanced STS as second/third line therapy. TR binds to the minor groove of DNA and interferes with gene transcription and nucleotide excision repair mechanism, inducing DNA double strand breaks (DSBs), and S/G2 cell cycle arrest. There is a strong clinical interest to increase TR activity combining it with other anti-cancer drugs. PARP-1 inhibitors disable DNA base-excision repair mechanism causing the accumulation of DSBs and look like a reasonable TR partner to be explored. We focused our in vitro studies on the effects of the combination of TR with the PARP-1 inhibitor Olaparib (OL). Methods: We explored the activity of TR-OL combination against a panel of different histotypes of STS cell lines, evaluating cell viability after 72h treatment with escalating doses of TR (0-2 nM), OL (0-20 µM), and their constant combination. Following colony formation, cell cycle, apoptosis (annexin V+/PI+) and DNA damage (phospho-histone H2AX - Ser139) were checked. Results: The TR-OL combination strongly affects STS cell viability, showing synergism (Combination Index &lt; 1, based on Chou–Talalay method) on 8 out of 13 cell lines tested. We observed a strong synergism, as a massive reduction of colony growth (402.91, MES-SA, and DMR-SN-8.4.98 lines) induced by the combination if compared with each single agent (78% vs. ~27% : p&lt;0.05). OL potentiates the S/G2 cell-cycle arrest caused by TR at 48h (Control= 29%, TR= 33.4%, OL= 31.5%, TR-OL= 80.9%), and induces a strong increase of the apoptotic cells at 72h (Control= 17.4%, TR= 28.3%, OL= 33.5%, TR-OL= 56.8%). Furthermore, the TR-OL synergism on DNA damage is confirmed by a significant increase of the DSBs marker (Control= 8.7%, TR= 59.5%, OL= 23.8%, TR-OL= 76.5%). Conclusions: These results validate the biological rationale to combine TR and PARP-1 inhibitors in STS and suggest assessing this drug combination in the clinical setting.

Abstract 3763: Treatment of soft tissue sarcoma cells by EGFR and/or STAT3 inhibitors

Abstract Background: We previously found Epidermal Growth Factor Receptor (EGFR) inhibitor can block liposarcoma cell EGFR (HER1) survival signalling. However, it did not interfere with HER2 regulated Janus Kinases (JAK), and Signal Transducer and Activator of Transcription (STAT) signal transduction. Our research suggested JAK/STAT may be one mechanism for EGFR inhibitor resistance. Targeting both EGFR and STAT3, which has never been tested in sarcoma may overcome this problem. The principal aim of this preliminary study was to investigate the effect and mechanism of both STAT3 inhibitor alone and in combination with EGFR inhibitor in the treatment of soft tissue sarcoma (STS) cell lines. Methods: STAT3 inhibitor (S3I-201) mono-therapy or in combination with EGFR inhibitor Gefitinib was investigated in 7 STS cell lines (449b, 778, SW872, SW684, SW982, GCT and HT1080). Crystal-violet colorimetric and clonogenic assays were used to measure drug effects. For assessing a potential role of treatment, we investigated expression and activity of STAT and EGFR by Western blot and immunohistochemistry staining before and after treatment. Data were analysed using Chou &amp; Talalay method and CalcuSyn software. Results: All 7 STS cell lines expressed phosphorylated/total STAT3 and EGFR. Anti-proliferative and anti-clonogenic effects of S3I-201 monotherapy on STS were dose- and time-dependent. Sensitive cells to S3I-201 (IC50 Δ50μM) were HT1080, SW684, GCT and SW982 (90% HT1080 were inhibited at day 3 post-administration of 25μM drug), partially sensitive cells were 449B and 778, and non-sensitive cells (IC50 ≤ 200μM) were SW872 (only 29% SW872 were inhibited at day 5 with 200μM S3I-201). Western blot analysis of whole-cell lysates from HT1080 showed phosphorylated STAT3 (pSTAT3) levels were significantly diminished after S3I-201 treatment, whereas pSTAT3 were not down-regulated in resistant cells SW872. IC50 of Gefitinib monotherapy on STS were 20 - 40μM, and 778 had minor effect (33% cells were inhibited after 5 days treatment). Combination therapy with Gefitinib and S3I-201 achieved synergistic antiproliferative effect (mean CIs &amp;lt; 0.90) in 6/7 STS cell lines (CI: 449b (0.3-1), 778 (0.1-0.2), SW872 (0.6-0.7), SW684 (0.2-0.5), GCT (0.3-0.4) and HT1080 (0.5-0.6)), except SW982 (1-1.2). For the most synergistic 778 cells, the drug reduction index for Gefitinib and S3I-201 was 5.9 and 12.7. Although treatment with Gefitinib alone inhibited pSTAT3 expression on serum-starved 778, EGF stimulation reversed the inhibition function. However in combination therapy, STAT3 phosphorylation was not induced by EGF stimulation. Conclusion: Combination therapy targeting both EGFR and STAT3 is a worthwhile treatment to pursue in anti-sarcoma therapy. Further studies will focus on potential mechanisms as well as the anti-sarcoma effect in the animal models. These results will have a clinical implication in treatment of STS in the future. 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 3763. doi:1538-7445.AM2012-3763

Effective Oncolytic Vaccinia Therapy for Human Sarcomas

Approximately one fourth of bone and soft-tissue sarcomas recur after prior treatment. GLV-1h68 is a recombinant, replication-competent vaccinia virus that has been shown to have oncolytic effects against many human cancer types. We sought to determine whether GLV-1h68 could selectively target and lyse a panel of human bone and soft-tissue sarcoma cell lines invitro and invivo. GLV-1h68 was tested in a panel of four cell lines including: fibrosarcoma HT-1080, osteosarcoma U-2OS, fibrohistiocytoma M-805, and rhabdomyosarcoma HTB-82. Gene expression, infectivity, viral proliferation, and cytotoxicity were characterized invitro. HT-1080 xenograft flank tumors grown invivo were injected intratumorally with a single dose of GLV-1h68. All four cell lines supported robust viral transgene expression invitro. At a multiplicity of infection (MOI) of five, GLV-1h68 was cytotoxic to three cell lines, resulting in >80% cytotoxicity over 7 d. Invivo, a single injection of GLV-1h68 into HT-1080 xenografts exhibited localized intratumoral luciferase activity peaking at d 2-4, with gradual resolution over 8 d and no evidence of spread to normal tissues. Treated animals exhibited near-complete tumor regression over a 28-d period without observed toxicity. GLV-1h68 has potent direct oncolytic effects against human sarcoma invitro and invivo. Recombinant vaccinia oncolytic virotherapy could provide a new platform for the treatment of patients with bone and soft tissue sarcomas. Future clinical trials investigating oncolytic vaccinia as a therapy for sarcomas are warranted.

Immunoglobulin G Locus Events in Soft Tissue Sarcoma Cell Lines

Recently immunoglobulins (Igs) have been found to be expressed by cells other than B lymphocytes, including various human carcinoma cells. Sarcomas are derived from mesenchyme, and the knowledge about the occurrence of Ig production in sarcoma cells is very limited. Here we investigated the phenomenon of immunoglobulin G (IgG) expression and its molecular basis in 3 sarcoma cell lines. The mRNA transcripts of IgG heavy chain and kappa light chain were detected by RT-PCR. In addition, the expression of IgG proteins was confirmed by Western blot and immunofluorescence. Immuno-electron microscopy localized IgG to the cell membrane and rough endoplasmic reticulum. The essential enzymes required for gene rearrangement and class switch recombination, and IgG germ-line transcripts were also identified in these sarcoma cells. Chromatin immunoprecipitation results demonstrated histone H3 acetylation of both the recombination activating gene and Ig heavy chain regulatory elements. Collectively, these results confirmed IgG expression in sarcoma cells, the mechanism of which is very similar to that regulating IgG expression in B lymphocytes.

Up-regulation of the DR5 Expression by Proteasome Inhibitor MG132 Augments TRAIL-Induced Apoptosis in Soft Tissue Sarcoma Cell Lines

PurposeCurrent chemotherapeutics for treating locally advanced or metastatic soft tissue sarcomas (STS) are limited. Accordingly, the present in vitro study was conducted to evaluate the effects of treatment of STS cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) applied as a single agent or in combination with a proteasome inhibitor, MG132.Materials and MethodsSensitivity to TRAIL and activity of TRAIL-induced apoptotic pathways were analyzed in four STS cell lines: HTB-82 (rhabdomyosarcoma), HT-1080 (fibrosarcoma), HTB-93 (synovial sarcoma), and HTB-94 (chondrosarcoma). Reduction of the dye dimethylthiazolyl 2,5 diphenyltetrazolium bromide (MTT) was used to evaluate cytotoxic activity; western blots were used to evaluate TRAIL-induced apoptosis.ResultsTRAIL induced apoptosis in HTB-93 cells, but had little effect in HTB-82, HT-1080, or HTB-94 cells. Expression of TRAIL receptor-1 and -2 did not correlate with sensitivity to TRAIL. Co-incubation of cells with TRAIL and a proteasome inhibitor, MG132, augmented the apoptotic effect of TRAIL in both TRAIL-sensitive and TRAIL-resistant cells. This effect was due to up-regulation of TRAIL receptors and members of the pro-apoptotic BCL-2 family by MG132.ConclusionThese data show that combining TRAIL with MG132 enhances apoptosis and overcomes TRAIL resistance. This restoration of TRAIL sensitivity occurs through an increase in the expression of death receptor 5 and of pro-apoptotic BCL-2 family members such as BAX.

Abstract 5047: Survival signal pathways as potential therapeutic targets in soft tissue sarcoma cell lines

Abstract Introduction: Soft tissue sarcomas (STS) are rare malignancies but have poor survival rate. Surgery with standard chemotherapy has been the mainstay of treatment but newer strategies are necessary to improve survival. The investigations for targeted therapy are underway but little is known about potential targets until now. We previously presented potential molecular targets in osteosarcoma (OS) cell lines (101st AACR annual meeting 2010, abstract No.2451). We tried to compare the protein and mRNA profilings between the OS and STS cell lines to suggest the feasibility for developing molecular targeted agents in STS. Methods: We examined the protein expression of c-Met, Akt, mTOR, and their phosphorylated status, and PTEN in seven OS (MG-63, HOS, KHOS/NP, SK-ES-1, U-2OS, Saos-2, and G-292) and five STS cell lines (SK-UT-1, A-673, SW-871, SW-982, HT-1080) using western blotting. The gene expression profilings of the cell lines were performed by 44K microarray (Agilent Inc.). Results: Microarray data showed that whole genome expression pattern of STS was different from OS cell lines. But, the expressions of 94 mRNAs within the signal transduction pathways, which represent the currently utilized drug targets, revealed similar pattern between two groups of cell lines. To verify the possibility of developing molecular targeted agents in STS, we examined the activation patterns of signal transduction pathway proteins in each STS cell line. All STS cell lines except A-673 with low c-Met, but high p-Met, expressed moderate level of c-Met and p-Met. PI3K was expressed in all cell lines, with higher p-PI3K in SK-UT-1 and A-673 than in other STS cell lines. All but SW-872 cell line expressed Akt, and SK-UT-1, SW-872 and HT-1080 had higher levels of p-Akt. SW872 showed negligible PTEN and high level of p-Akt. SW982 generally repressed level of p-Met, p-PI3K, p-Akt, p-mTOR comparing with other STS cell lines. Meanwhile, mTOR and p-mTOR were expressed relatively even in all STS cell lines. Conclusion: We presented that STS cell lines had variously activated signal pathway status as in OS cell lines. Common potential targets such as mTOR was revealed, and the specific targets including PI3K or Akt were also presented. It suggests the possibility of developing specific molecular targeted agents in each type of STSs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5047. doi:10.1158/1538-7445.AM2011-5047

Abstract 4350: Translating diagnostic gene expression profiles for pediatric solid tumors

Abstract BACKGROUND: Features of cancer genomics including gene expression levels can be employed to create biomarker profiles that can predict prior to therapy the diagnosis and prognosis of an individual patient. We aim to translate robust and reproducible diagnostic and prognostic profiles for pediatric solid tumors into clinical tools applicable to all tumor specimens, fresh frozen (FF) or formalin fixed (FFPE). RESULTS: We used Affymetrix GeneChip Human Exon 1.0 ST (HuEx) arrays on 24 cell lines or tumors (4 fusion-positive rhabdomyosarcoma [RMSpos], 5 fusion-negative RMS [RMSneg], 6 Ewing sarcoma family of tumors [ESFT], 5 neuroblastoma [NB], and 4 osteosarcoma [OS]) to identify a 41-feature diagnostic metagene that clearly distinguished both the original test samples and a set of validation samples. We then incorporated the 41-feature metagene into a 48-gene panel. To translate this diagnostic signature, we compared the performance of HuEx arrays with several mid-plex assay platforms including Fluidigm's quantitative reverse-transcriptase PCR (q-RT-PCR) and NanoString's nCounter™ Digital Analyzer by measuring gene expression on an 18-cell line panel (4 RMSpos, 4 RMSneg, 5 ESFT, 1 NB, and 4 OS). With q-RT-PCR, we observed r2 values of 0.703 ± 0.085 (RMSpos), 0.558 ± 0.138 (RMSneg), 0.689 ± 0.070 (ESFT), and 0.607 ± 0.121 (OS). nCounter reported r2 values of 0.672 ± 0.171 (RMSpos), 0.620 ± 0.143 (RMSneg), 0.675 ± 0.094 (ESFT), and 0.583 ± 0.129 (OS). Hierarchical clustering with all platforms was able to distinguish the RMSpos, ESFT, and NB cell lines; however, the RMSneg and OS cell lines were less clearly distinguished because some of the OS cell lines lacked high expression of genes characteristic of OS tumors. Cross-platform comparisons yielded good correlation between nCounter and Q-RT-PCR (average r2 = 0.710 ± 0.183). An 18-sample dilution series revealed that EWS-FLI1 type I or PAX3-FKHR transcripts were consistently detected by q-RT-PCR or nCounter even at 1:1000 RNA dilution. Among biological duplicates, q-RT-PCR reported an average r2 = 0.916 ± 0.039 while nCounter obtained average r2 = 0.983 ± 0.011 including raw cell lysates. In addition, we also compared Fluidigm's q-RT-PCR with Applied Biosystems’ Taqman Low-Density Arrays (TLDA) on five NB patient bone marrow samples to detect residual tumor cells. We observed an average r2 = 0.929 ± 0.119 across five NB genes and one housekeeping gene. Finally, we have also successfully profiled FF vs. FFPE tumors on HuEx and are currently applying these mid-plex technologies to paired FF and FFPE samples in order to create diagnostic profiles applicable to routine FFPE material. CONCLUSIONS: Mid-plex platform can reliably distinguish bone and soft tissue sarcoma cell lines and can be used to translate the application of diagnostic signatures. These data warrant further studies to analyze FFPE samples, to compare additional mid-plex platforms, and to test potential prognostic signatures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4350. doi:10.1158/1538-7445.AM2011-4350

Abstract 5415: Preclinical evaluation of telomerase-specific oncolytic virotherapy for human bone and soft tissue sarcomas

Abstract Purpose: Bone and soft tissue sarcomas frequently occur in young children and show aggressive progression, resistance to conventional chemotherapy and poor prognosis, indicating a requirement for novel antitumor therapy to improve the clinical outcome. Tumor-specific replication-selective oncolytic virotherapy is a promising antitumor therapy for induction of cell death in tumor, but not of normal, cells. We previously developed an oncolytic adenovirus, OBP-301, that kills human epithelial malignant cells in a telomerase-dependent manner. Recent evidence suggests that non-epithelial malignant cells, which have low telomerase activity, maintain telomere length through alternative lengthening of telomeres (ALT). However, it remains unclear if OBP-301 is cytopathic for non-epithelial malignant cells. Here, we evaluated the antitumor effect of OBP-301 on human bone and soft tissue sarcoma cells. Experimental Design: The cytopathic activity of OBP-301, coxsackie and adenovirus receptor (CAR) expression, and telomerase activity were examined in 10 bones (OST, U2OS, HOS, HuO9, MNNG/HOS, SaOS-2, NOS-2, NOS-10, NDCS-1 and OUMS-27) and in 4 soft tissue (CCS, NMS-2, SYO-1 and NMFH-1) sarcoma cell lines. OBP-301 antitumor effects were assessed using orthotopic tumor xenograft models. The fiber-modified-OBP-301, (OBP-405), was used to confirm an antitumor effect on OBP-301-resistant sarcomas. Results: OBP-301 was cytopathic for 12 sarcoma cell lines but not for the non-CAR-expressing OUMS-27 and NMFH-1 cells. Sensitivity to OBP-301 was dependent on CAR expression, not on telomerase activity. ALT-type sarcomas were also sensitive to OBP-301 because of upregulation of human telomerase reverse transcriptase (hTERT) mRNA following virus infection. Intratumoral injection of OBP-301 significantly suppressed the growth of OST and SYO-1 tumors. Furthermore, fiber-modified OBP-405 showed antitumor effects on OBP-301-resistant OUMS-27 and NMFH-1 cells. Conclusions: We showed that OBP-301 has antitumor effects on both non-ALT-type and ALT-type sarcoma cells through upregulation of human telomerase reverse transcriptase mRNA. Furthermore, coxsackie and adenovirus receptor-negative sarcoma cells were efficiently killed by fiber-modified OBP-301-derived-OBP-405 through virus-integrin binding. A telomerase-specific oncolytic adenovirus is a promising antitumor reagent for the treatment of bone and soft tissue sarcomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5415. doi:10.1158/1538-7445.AM2011-5415

Abstract 2665: NBTXR3 hafnium oxide nanoparticle activated by ionizing radiation demonstrates marked radio-enhancement and antitumor effect via high energy deposit in human soft tissue sarcoma

Abstract Local and systemic control of Soft Tissue Sarcoma (STS) remains a clinical challenge. Radiation therapy is part of the standard of care of STS. The narrowness of its therapeutic window represents the main concern for different clinical settings. Thus, local delivery of radiation doses is critical to ensure optimal benefit-risk ratio. NBTXR3, biocompatible hafnium oxide nanoparticles were designed as therapeutics to be activated by ionizing radiation to achieve tumor control by enhancement of local energy deposition. A global non clinical program was implemented in mesenchymal tumor models. In vitro clonogenic survival assays were performed in two human fibrosarcoma cell lines (HT1080 and Hs913t) and a human liposarcoma cell line (SW872) to evaluate the cellular response to radiation-activated NBTXR3 at increasing concentrations. A marked radio-enhancement was observed in human STS cell lines with dose enhancement factors estimated at 4 Gy ranging from 1.48 up to 5.36 according to NBTXR3 concentrations. Further, in human immortalized MRC5V1 fibroblasts, no significant decrease in clonogenic surviving fraction was observed upon activation of NBTXR3 under similar conditions suggesting a differential radiation response between fibroblasts and fibrosarcoma, liposarcoma cell lines. This differential radiation response was further confirmed between HT1080 fibrosarcoma cell line and MRC5V1 fibroblasts upon activation of NBTXR3 at increasing concentration using high energy gamma-rays. Mechanistic studies have demonstrated in HT1080 cells that NBTXR3 activation induces an increase of 53BP1 foci associated with more “complex” DNA damages than ionizing radiation alone. The level of unrepaired DNA damages was also estimated by flow cytometry analysis showing a higher level of gamma-H2AX at 48h following NBTXR3 activation than after radiotherapy alone. DNA fragmentation and Annexin V staining have demonstrated that these unrepaired DNA damages upon NBTXR3 activation induces apoptosis in HT1080 fibrosarcoma cells. In vivo, tolerance and tumor growth delay were investigated in two human xenografted tumor models, HT1080 fibrosarcoma and a patient derived liposarcoma LPS80T3 (poorly differentiated grade 3). No toxicity related to NBTXR3 was reported. In both models, a significant advantage was demonstrated in terms of tumor growth inhibition and survival when compared to radiation therapy alone. These findings establish a novel approach of treatment, which may be applied to a broad range of tumors. Therefore, incrementing the amount of energy deposited within the tumor through the activation of NBTXR3 crystalline nanoparticles constitutes a revolutionary approach for future clinical investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2665. doi:10.1158/1538-7445.AM2011-2665

Suppression of Soft Tissue Sarcoma Growth by a Host Defense-Like Lytic Peptide

BackgroundSoft tissue sarcoma (STS) is an anatomically and histologically heterogeneous neoplasia that shares a putative mesenchymal cell origin. The treatment with common chemotherapeutics is still unsatisfying because of association with poor response rates. Although evidence is accumulating for potent oncolytic activity of host defense peptides (HDPs), their potential therapeutic use is often limited by poor bioavailability and inactivation in serum. Therefore, we tested the designer host defense-like lytic D,L-amino acid peptide [D]-K3H3L9 on two STS cell lines in vitro and also in an athymic and syngeneic mouse model. In recent studies the peptide could show selectivity against prostate carcinoma cells and also an active state in serum.Methods In vitro the human synovial sarcoma cell line SW982, the murine fibrosarcoma cell line BFS-1 and primary human fibroblasts as a control were exposed to [D]-K3H3L9, a 15mer D,L-amino acid designer HDP. Cell vitality in physiological and acidic conditions (MTT-assay), cell growth (BrdU) and DNA-fragmentation (TUNEL) were investigated. Membrane damage at different time points could be analyzed with LDH assay. An antibody against the tested peptide and recordings using scanning electron microscopy could give an inside in the mode of action. In vivo [D]-K3H3L9 was administered intratumorally in an athymic and syngeneic (immunocompetent) mouse model with SW982 and BFS-1 cells, respectively. After three weeks tumor sections were histologically analyzed.ResultsThe peptide exerts rapid and high significant cytotoxicity and antiproliferating activity against the malignant cell lines, apparently via a membrane disrupting mode of action. The local intratumoral administration of [D]-K3H3L9 in the athymic and syngeneic mice models significantly inhibited tumor progression. The histological analyses of the tumor sections revealed a significant antiproliferative, antiangiogenic activity of the treatment group.ConclusionThese findings demonstrate the in vitro and in vivo oncolytic activity of [D]-K3H3L9 in athymic and syngeneic mouse models.

Preclinical Evaluation of Telomerase-Specific Oncolytic Virotherapy for Human Bone and Soft Tissue Sarcomas

Tumor-specific replication-selective oncolytic virotherapy is a promising antitumor therapy for induction of cell death in tumor cells but not of normal cells. We previously developed an oncolytic adenovirus, OBP-301, that kills human epithelial malignant cells in a telomerase-dependent manner. Recent evidence suggests that nonepithelial malignant cells, which have low telomerase activity, maintain telomere length through alternative lengthening of telomeres (ALT). However, it remains unclear whether OBP-301 is cytopathic for nonepithelial malignant cells. Here, we evaluated the antitumor effect of OBP-301 on human bone and soft tissue sarcoma cells. The cytopathic activity of OBP-301, coxsackie and adenovirus receptor (CAR) expression, and telomerase activity were examined in 10 bone (OST, U2OS, HOS, HuO9, MNNG/HOS, SaOS-2, NOS-2, NOS-10, NDCS-1, and OUMS-27) and in 4 soft tissue (CCS, NMS-2, SYO-1, and NMFH-1) sarcoma cell lines. OBP-301 antitumor effects were assessed using orthotopic tumor xenograft models. The fiber-modified OBP-301 (termed OBP-405) was used to confirm an antitumor effect on OBP-301-resistant sarcomas. OBP-301 was cytopathic for 12 sarcoma cell lines but not for the non-CAR-expressing OUMS-27 and NMFH-1 cells. Sensitivity to OBP-301 was dependent on CAR expression and not on telomerase activity. ALT-type sarcomas were also sensitive to OBP-301 because of upregulation of human telomerase reverse transcriptase (hTERT) mRNA following virus infection. Intratumoral injection of OBP-301 significantly suppressed the growth of OST and SYO-1 tumors. Furthermore, fiber-modified OBP-405 showed antitumor effects on OBP-301-resistant OUMS-27 and NMFH-1 cells. A telomerase-specific oncolytic adenovirus is a promising antitumor reagent for the treatment of bone and soft tissue sarcomas.

Establishment and Characterization of a Canine Soft Tissue Sarcoma Cell Line

Stringently controlled in vitro experiments are a necessary part of translational research. Cell lines are useful for exploring the underlying biology of cancer. Very few canine soft tissue sarcoma cell lines exist. This report describes the establishment of a new canine soft tissue sarcoma cell line (MBSa1) derived from a high-grade, metastatic neurofibrosarcoma. The primary tumor tissue was obtained from a 12-year-old neutered male German Shepherd Dog and was maintained in tissue culture for a minimum of 20 passages over 7 months. MBSa1 was injected into athymic mice to determine tumorigenicity. Five million cells were injected into the subcutis of the right flank of athymic nude mice. Nine of the 10 mice grew tumors 1 cm or larger within 8 weeks of cell injection. The large number of in vitro passages coupled with solid tumor formation in athymic nude mice demonstrates that MBSa1 has been immortalized and is tumorigenic.

Antitumor Activity of Polyuridylic Acid in Human Soft Tissue and Bone Sarcomas

The immunomodulatory properties of polyuridylic acid (PolyU) make it a promising agent in cancer immunotherapy. However, there is limited information on its direct effects on tumor cells. TLR8 mRNA and protein expression in soft tissue sarcoma (STS) and bone sarcoma (BS) cell lines were determined by PCR and flow cytometry, respectively. Apoptosis and proliferation assays were performed using annexin V staining and BrdU incorporation assays, respectively. A relative cell enumeration was evaluated with WST-1 reagent. Expression levels of apoptotic proteins were evaluated by Western blotting. We demonstrate that PolyU treatment resulted in a significant decrease in STS and BS cell count by inducing apoptosis and inhibition of cell proliferation. All cell lines examined expressed TLR8 and the effect of PolyU was partially mediated through TLR8. Several apoptotic proteins including caspases were activated or increased in STS cells after treatment with PolyU. Administration PolyU resulted in significant growth inhibition of STS without any observable adverse effects in mouse xenograft tumor models. These results elucidate the effect of PolyU in STS and BS cells and demonstrate that PolyU may be a potential therapeutic agent for STS and BS.

Targeting CHK1 pathway to induce cytotoxic response to histone deactylase inhibitors and evaluating the synergistic effects of CHK1 and HDAC inhibitors in human soft tissue sarcoma cell lines and primary tumor xenografts.

10049 Background: Histone deacetylase inhibitors (HDACis) are promising anticancer drugs; however, the molecular mechanisms leading to cell death by HDACis have not been well understood and no clear mechanism of resistance has been elucidated to explain limited efficacy of HDACis in clinical trials. Methods: In vitro treatment of cell lines with HDACi and Chk1 inhibitors, time course experiments, protein analysis by western blot, and ex vivo evaluation of sarcoma xenografts were performed. Results: In HDACi- treated sarcoma cell lines, Chk1 downregulation was functional as indicated by decreased inhibitory phosphorylation of cdc25c and its downstream target cdc2. Chk1 downregulation occurred upon HDACi treatment and preceded apoptosis, as assessed by poly(ADP-ribose) polymerase cleavage. Pharmacological inhibition of Chk1 showed strong synergistic effect with LBH589 in sarcoma cells in vitro. Serial sampling of mouse xenografts of these sarcoma cell lines using FNAB indicates that downregulation of Chk1 upon HDACi treatment occurs in vivo and can be used to assess and predict efficacy of treatment. Conclusions: Taken together these results suggest that Chk1 could be an early pharmacodynamic marker to assess HDACi efficacy in clinical samples. A combination of drugs targeting HDAC and Chk1 enzymes may be clinically useful for sarcoma treatment. We show that expression of checkpoint kinase 1 (Chk1), which has a major role in G2 cell cycle checkpoint regulation, was markedly reduced in multiple sarcoma cell lines both in vitro and in vivo as well as in human sarcoma samples tested ex vivo when cells were treated with the HDACi LBH589, scriptaid, valproic acid, or apicidin. No significant financial relationships to disclose.

Abstract 1041: Restoration of p53 function in soft tissue sarcomas correlates with increased sensitivity to TNFα

Abstract Background: Sarcomas are rare, poorly understood cancers, highly aggressive and refractory to standard therapies. In these tumors mutations of p53 or Mdm2 amplification are frequently found. Isolated limb perfusion (ILP) with TNFα and melphalan is an innovative approach to treat unresectable soft tissue sarcomas (STS) of extremities. TNF is known to increase endothelium permeability for melphalan, and to disrupt tumoral vessels. However, the direct apoptotic effect of TNF on sarcoma cells remains controversial. Several reports indicate an association of sensitivity to TNF with p53 activity in in vitro models. In the present study we attempted to test the ability of TNF to induce apoptosis of sarcoma cells and investigated the role of p53 in the regulation of this effect. Methods: Ex vivo study was performed on sarcoma tumor pieces from patients. Tumor slices were treated with TNF and cytotoxic effect was assessed with TUNEL method. In vitro studies were carried on human soft tissue sarcoma cell lines with different p53 status and Mdm2 expression, representing 5 histological types. Cytotoxic effect of TNF was assessed with DiOC6/PI test. Activation of p53 was verified by the detection of expression of proteins encoded by p53 target genes. Expression of TNF receptor was examined by flow cytometry. Results: In tumor slices treated ex vivo with TNF we observed apoptosis of not only endothelial, but also sarcoma cells. In vitro studies revealed that cell lines with active p53 were sensitive to TNF (80% and 40% of apoptosis), while 6 cell lines were resistant: 2 with wt p53 and Mdm2 amplification (2-4% of apoptosis), 2 with p53 mutation and 2 with p53 deletion (5-15% of apoptosis). TNFR I was expressed by all the cell lines. We next tested sensitivity to TNF after restoration of p53 activity. For this purpose we used either of two compounds: Nutlin-3a, an inhibitor of Mdm2, or CP-31398, a molecule restoring wild-type conformation to mutant TP53. In cell lines with Mdm2 amplification treatment with Nutlin alone resulted in decreased cell viability, while in combined treatment induced strong apoptosis synergistically with TNF. Similarly, treatment of cell lines expressing mutated p53 with CP-31398 led to a significant enhancement of sensitivity to TNF. Conclusions: The results of our ex vivo and in vitro studies suggest, that during ILP, TNF may directly kill sarcoma cells and its cytotoxic activity is correlated with the functionality of p53. Restoration of p53 activity may be advantageous not only in therapies based on DNA disruption, but also based on the use of TNFα. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1041.