Tumors In SCID Mice Research Articles

Abstract C238: Polyacetal polymer-based anti-HER2 antibody-drug conjugate employing cysteine bioconjugation through thioether linkage allows a high drug loading of dolastatin-derived payload with excellent pharmacokinetics and potent anti-tumor activity.

Abstract The application of biodegradable, polyacetal polymers to antibody-drug conjugate (ADC) design can provide numerous advantages, including significantly higher capacity for drug payload (∼20 drugs per antibody), the use of payloads with low potency that are not suitable for direct conjugation, the improvement of physicochemical properties for ADC, especially with highly hydrophobic payloads and the use of protein recognition scaffolds beyond the commonly used IgGs. The basis of this novel polyacetal polymer-based conjugation system is a hydrophilic, fully biodegradable polyacetal carrier (PHF or poly(1-hydroxymethylethylene hydroxymethylformal, or Fleximer®) modified with chemically orthogonal linkers. One linker is used to covalently attach a targeting moiety (mAb or alternative), while a second, chemically distinct linker is used to attach a drug payload and to control the mechanism and rate of drug release. Previously, we have reported potent anti-tumor activity with trastuzumab-s-Dolaflexin™, an anti-HER2 ADC composed of trastuzumab and a proprietary dolastatin derivative coupled to a Fleximer scaffold (Dolaflexin™). In that example, Dolaflexin was conjugated to the antibody through interchain cysteine residues via a hindered disulfide linkage (AACR Annual Meeting 2013 Abstract #4331). Unlike direct drug-cysteine linked ADCs that can result in destabilization of antibody by disruption of interchain disulfide bridges, we have shown that Dolaflexin conjugation via cysteines in the antibody hinge region stabilizes the resulting ADCs through the formation of inter-chain bridge structures. Trastuzumab-s-Dolaflexin ADC exhibited a prolonged plasma half-life, tumor-specific accumulation, and potent anti-tumor activity in vivo. Here, we report a novel trastuzumab-m-Dolaflexin ADC using a maleimide linker that further enhances the pharmacokinetics of the ADC and demonstrates complete regressions of established HER2+ BT-474 xenograft tumors in SCID mice even at a single dose of 2.5 mg/kg. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C238. Citation Format: Joshua Thomas, Alex Yurkovetskiy, Natalya Bodyak, Mao Yin, Patrick Conlon, Cheri Stevenson, Alex Uttard, Liu Qin, Dmitry Gumerov, Elena Ter-Ovaneysan, Michael DeVit, Laura L. Poling, Peter U. Park, Timothy B. Lowinger. Polyacetal polymer-based anti-HER2 antibody-drug conjugate employing cysteine bioconjugation through thioether linkage allows a high drug loading of dolastatin-derived payload with excellent pharmacokinetics and potent anti-tumor activity. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C238.

Abstract CN03-02: Conditionally reprogrammed cells for personalized medicine.

Abstract CN03-02: Conditionally reprogrammed cells for personalized medicine.

Expression of the Chemokine Receptor CXCR7 in CXCR4-Expressing Human 143B Osteosarcoma Cells Enhances Lung Metastasis of Intratibial Xenografts in SCID Mice

More effective treatment of metastasizing osteosarcoma with a current mean 5-year survival rate of less than 20% requires more detailed knowledge on mechanisms and key regulatory molecules of the complex metastatic process. CXCR4, the receptor of the chemokine CXCL12, has been reported to promote tumor progression and metastasis in osteosarcoma. CXCR7 is a recently deorphanized CXCL12-scavenging receptor with so far not well-defined functions in tumor biology. The present study focused on a potential malignancy enhancing function of CXCR7 in interaction with CXCR4 in osteosarcoma, which was investigated in an intratibial osteosarcoma model in SCID mice, making use of the human 143B osteosarcoma cell line that spontaneously metastasizes to the lung and expresses endogenous CXCR4. 143B osteosarcoma cells stably expressing LacZ (143B-LacZ cells) were retrovirally transduced with a gene encoding HA-tagged CXCR7 (143B-LacZ-X7-HA cells). 143B-LacZ-X7-HA cells co-expressing CXCR7 and CXCR4 exhibited CXCL12 scavenging and enhanced adhesion to IL-1β-activated HUVEC cells compared to 143B-LacZ cells expressing CXCR4 alone. SCID mice intratibially injected with 143B-LacZ-X7-HA cells had significantly (p<0.05) smaller primary tumors, but significantly (p<0.05) higher numbers of lung metastases than mice injected with 143B-LacZ cells. Unexpectedly, 143B-LacZ-X7-HA cells, unlike 143B-LacZ cells, also metastasized with high incidence to the auriculum cordis. In conclusion, expression of the CXCL12 scavenging receptor CXCR7 in the CXCR4-expressing human 143B osteosarcoma cell line enhances its metastatic activity in intratibial primary tumors in SCID mice that predominantly metastasize to the lung and thereby closely mimic the human disease. These findings point to CXCR7 as a target, complementary to previously proposed CXCR4, for more effective metastasis-suppressive treatment in osteosarcoma.

Multi-targeted DATS prevents tumor progression and promotes apoptosis in ectopic glioblastoma xenografts in SCID mice via HDAC inhibition

Glioblastoma, the most lethal brain tumor, remains incurable despite aggressive chemotherapy and surgical interventions. New chemotherapeutics for glioblastoma have been explored in preclinical models and some agents have reached the clinical setting. However, success rates are not significant. Previous investigations involving diallyl trisulfide (DATS), a garlic compound, indicated significant anti-cancer effects in glioblastoma in vitro. DATS has also been shown to inhibit histone deacetylase activity and impede glioblastoma tumor progression. We hypothesized that DATS would block ectopic U87MG tumor by multiple pro-apoptotic pathways via inhibiting histone deacetylase (HDAC). To prove this, we developed ectopic U87MG tumors in SCID mice and treated them daily with intraperitoneal injections of DATS for 7 days. Results indicated that DATS (10 μg/kg-10 mg/kg) dose-dependently reduced tumor mass and number of mitotic cells within tumors. Histological and biochemical assays demonstrated that DATS reduced mitosis in tumors, decreased HDAC activity, increased acetylation of H3 and H4, inhibited cell cycle progression, decreased pro-tumor markers (e.g., survivin, Bcl-2, c-Myc, mTOR, EGFR, VEGF), promoted apoptotic factors (e.g., bax, mcalpian, active caspase-3), and induced DNA fragmentation. Our data also demonstrated an increase in p21Waf1 expression, which correlated with increased p53 expression and MDM2 degradation following DATS treatment. Finally, histological assessment and enzyme assays showed that even the highest dose of DATS did not negatively impact hepatic function. Collectively, our results clearly demonstrated that DATS could be an effective therapeutic agent in preventing tumor progression and inducing apoptosis in human glioblastoma in vivo, without impairing hepatic function.

Amplification of tumor inducing putative cancer stem cells (CSCs) by vitamin A/retinol from mammary tumors

Solid tumors contain a rare population of cancer stem cells (CSCs) that are responsible for relapse and metastasis. The existence of CSC however, remains highly controversial issue. Here we present the evidence for putative CSCs from mammary tumors amplified by vitamin A/retinol signaling. The cells exhibit mammary stem cell specific CD29hi/CD49fhi/CD24hi markers, resistance to radiation and chemo therapeutic agents and form highly metastatic tumors in NOD/SCID mice. The cells exhibit indefinite self renewal as cell lines. Furthermore, the cells exhibit impaired retinol metabolism and do not express enzymes that metabolize retinol into retinoic acid. Vitamin A/retinol also amplified putative CSCs from breast cancer cell lines that form highly aggressive tumors in NOD SCID mice. The studies suggest that high purity putative CSCs can be isolated from solid tumors to establish patient specific cell lines for personalized therapeutics for pre-clinical translational applications. Characterization of CSCs will allow understanding of basic cellular and molecular pathways that are deregulated, mechanisms of tumor metastasis and evasion of therapies that has direct clinical relevance.

Myxoma Virus Infection Promotes NK Lysis of Malignant Gliomas In Vitro and In Vivo

Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas.

Origin of the vasculature supporting growth of primary patient tumor xenografts

BackgroundStudies of primary patient tumor xenografts grown in immunodeficient mice have shown that these tumors histologically and genetically closely resemble the original tumors. These patient xenograft models are becoming widely used for therapeutic efficacy studies. Because many therapies are directed at tumor stromal components and because the tumor microenvironment also is known to influence the response of a tumor to therapy, it is important to understand the nature of the stroma and, in particular, the vascular supply of patient xenografts.MethodsPatient tumor xenografts were established by implanting undisrupted pieces of patient tumors in SCID mice. For this study, formalin fixed, paraffin embedded specimens from several types of solid tumors were selected and, using species-specific antibodies which react with formalin fixed antigens, we analyzed the species origin of the stroma and blood vessels that supported tumor growth in these models. Additionally, we investigated the kinetics of the vascularization process in a colon tumor and a mesothelioma xenograft. In mice bearing a head and neck xenograft, a perfusion study was performed to compare the functionality of the human and mouse tumor vessels.ResultsIn patient tumors which successfully engrafted, the human stroma and vessels which were engrafted as part of the original tumor did not survive and were no longer detectable at the time of first passage (15–25 weeks). Uniformly, the stroma and vessels supporting the growth of these tumors were of murine origin. The results of the kinetic studies showed that the loss of the human vessels and vascularization by host vessels occurred more rapidly in a colon tumor (by 3 weeks) than in a mesothelioma (by 9 weeks). Finally, the perfusion studies revealed that while mouse vessels in the periphery of the tumor were perfused, those in the central regions were rarely perfused. No vessels of human origin were detected in this model.ConclusionsIn the tumors we investigated, we found no evidence that the human stromal cells and vessels contained in the original implant either survived or contributed in any substantive way to the growth of these xenografts.

Abstract 2700: BRCA1-IRIS overexpression promotes formation of aggressive breast cancer.

Abstract Introduction: Women with Her2+ or triple negative/basal-like (TN/BL) breast cancers succumb to their cancer rapidly and early in life, due in part to Herceptin resistance that develops in Her2+ patients and because of the lack of appropriate therapies for TN/BL patients. Great effort is being made now to uncover appropriate drug targets to overcome these shortcomings. In this report we examine whether BRCA1-IRIS can be a valuable treatment target for Her2+ and/or TN/BL tumors. Methods: Immunohistochemistry (IHC) staining of breast tumor samples using a newly generated mouse monoclonal anti-BRCA1-IRIS antibody was performed. BRCA1-IRIS expression was correlated to that of AKT1, AKT2, p-AKT, survivin, ERα, PR, and BRCA1/p220 expression detected using commercial antibodies. Where available, patients’ tumor grade, tumor stage, age at diagnosis, and node positivity were also compared. We also generated subcutaneous tumors in SCID mice using human mammary epithelial (HME) cells overexpressing BRCA1-IRIS and then compared AKT and survivin expression, and tumor phenotype and aggressiveness to RasV12-induced tumors. Results: The majority of human breast tumors, especially Her2+ and TN/BL tumors, were BRCA1-IRIS-positive. These tumors also expressed high levels of AKT1, AKT2, p-AKT, and survivin, but were ERα-, PR-, and BRCA1/p220-negative. BRCA1-IRIS overexpressing Her2+ and TN/BL tumors were high-grade tumors, and showed lymph-node positivity and/or distant metastasis compared to those not overexpressing BRCA1-IRIS. BRCA1-IRIS overexpression also significantly reduced the age at which patients with Her2+ or TN/BL tumors were diagnosed compared to patients with the same tumors but negative for BRCA1/IRIS (51.4±16.2 vs. 64.5±13.2, p≤0.05 for Her2+ and 48.9±10.1 vs. 70.8±6.8, p≤0.01 for TN/BL). Surprisingly, TN/BL/BRCA1-IRIS-positive tumors were larger than Her2+/BRCA1-IRIS-positive tumors (30.6±19.3 vs. 16.1±9.7, p≤0.05). HME cells overexpressing BRCA1-IRIS developed tumors when injected subcutaneously in SCID mice. These tumors were invasive and showed high expression levels of AKT1, AKT2, and p-AKT when compared to RasV12-induced tumors. These tumors also overexpressed the mesenchymal protein vimentin, but did not express the epithelial proteins CK19 and p63, and were BRCA1/p220-negative. In contrast, the less aggressive RasV12-induced tumors expressed CK19, p63, and BRCA1/p220, but not vimentin. Conclusion: These data demonstrate that BRCA1-IRIS overexpression triggers aggressive breast tumor formation, especially in patients with tumors of the Her2+ or TN/BL subtypes. We propose that BRCA1-IRIS inhibition may be used as a novel therapeutic option for the treatment of aggressive breast tumors of the Her2+ and/or TN/BL subtype. Citation Format: Wael M. El Shamy. BRCA1-IRIS overexpression promotes formation of aggressive breast cancer. [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 2700. doi:10.1158/1538-7445.AM2013-2700

Abstract 2779: Establishment and molecular characterization of a panel of Asian patient-derived tumor xenograft models .

Abstract Rodent tumor models with histological and molecular resemblance of human tumors and improved predictive value for clinical drug response are highly desired for oncology drug discovery and development. Patient-derived xenograft (PDX) tumor models are believed to better preserve features of human malignancy than cancer cell line-derived xenograft models. We have established more than 170 PDX models from Asian-prevalent human tumors in SCID or nude mice. Here we report molecular and pharmacological profiling of a panel of pancreatic adenocarcinoma (PAC) and hepatocellular carcinoma (HCC) models. Among 13 PAC models, Sequenom analysis showed KRAS mutation in all models, p53 mutation in 3, p16/CDKN2A deletion in 9 and SMAD4 deletion in 4 models. Comparison of transcriptomes by Affymetrix U133Plus2 between the original tumor and derived xenografts at different passages (P1-P6) in one PAC model revealed a high degree of similarity (R2 =0.92-0.97). Also, the gene mutational status and histological characters remained unchanged across the parental tumor and different passages of the PDX model. Evaluation of response to Gemzar treatment (60 mpk, Q4D x 3) showed significant tumor regression in 6 PAC models and partial tumor growth inhibition in 4 PAC models. Notably, the regression cohort and the partial response cohort displayed differential gene expression patterns. In addition, primary tumor cells derived from PAC models via tissue digestion were tested in vitro with Gemzar. The in vitro sensitivity to Gemzar of derived cells correlated with in vivo response of the parental PAC models. Gene expression analysis in 11 HCC models found aberrant gene expression involving several signaling pathways such as WNT, EGF/IGF and TGF-β, which recapitulate the alteration of these pathways in human HCC. These HCC PDX models exhibited major features of three HCC subclasses defined by the study in human HCC: S1 with activation of WNT and TGF-β pathways, S2 with enriched AKT and MYC activation and S3 with β-catenin activation. The response of these models to Sorafenib was also examined and varying degrees of sensitivity to Sorafenib were observed. Together, we have successfully generated PDX tumor models that preserve the histological and biological properties of the original human tumor and represent a heterogeneous patient population with varying degree of response to current stand of care. These models are a relevant and powerful tool for evaluation of anticancer therapeutics. Whole genome sequencing and gene expression profiling by RNAseq on PDX models are being preformed. Citation Format: Xiaoran Qin, Zhonghua Tang, Gang Hu, Kedong Ouyang, Ke Wang, Fu Li, Fubo Xie, Qiuming Pan, Min Shi, Gang Zhao, Yixin Zhang, Chunchao Zhu, Danyi Wen, Weikang Tao. Establishment and molecular characterization of a panel of Asian patient-derived tumor xenograft models . [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 2779. doi:10.1158/1538-7445.AM2013-2779

Abstract 245: Phenotypically diverse cancer cell subpopulations in a luminal-like breast cancer xenograft model are associated with different signaling pathways.

Abstract High degree of intratumor cellular diversity represent a major challenge when attempting to cure cancer. The presence of tumor cell subpopulations with enhanced in vivo tumorigenic capacity (tumor initiating cells, TICs), and high resistance to conventional cancer therapy, compared with the “bulk” tumor cell populations, has for the last decade been the focus of many breast cancer research groups. The cellular heterogeneity of an orthotopic luminal-like breast cancer xenograft model was investigated using IHC, flow cytometry, whole genome expression profiling and mass spectrometry-based proteomics combined with in vivo tumorigenicity and targeted therapy assays. Epithelial cell adhesion molecule, EpCAM, highly specific for the human tumor epithelial cells, was used to separate human tumor cells from the mouse stromal compartment. Further flow analysis of the EpCAM positive tumor cell population revealed diverse expression of several cell surface markers, including CD24 and SSEA-4 (stage specific embryonic antigen 4). SSEA-4-/CD24-, SSEA-4+/CD24- and SSEA-4-/CD24+ populations were capable of initiating tumors in NOD SCID mice while SSEA-4+/CD24+ cells were non-tumorigenic. Tumors resulting from the SSEA-4+/CD24- subpopulation did not express CD24, while tumors arising from the SSEA-4-/CD24- and SSEA-4-/CD24+ populations, contained all four subpopulations. As measured by whole genome expression analysis and mass spectrometry-based proteomics, the molecular differences were most pronounced between tumorigenic subpopulations and the non-tumorigenic subpopulation. While the mRNA expression data revealed a high degree of similarity among the four subpopulations, the proteomics data suggested that several signaling pathways might have distinct and different activity across the populations. The effects of targeted therapy against ER and WNT signaling on heterogeneity and tumorigenicity have been evaluated by in vivo experiments. Citation Format: Nirma Skrbo, Kristin Andersen, Alexandr Kristian, Linn Antberg, Geir Olav Hjortland, Olav Egenbråten, Peter James, Gunhild Mari Mælandsmo, Therese Sørlie. Phenotypically diverse cancer cell subpopulations in a luminal-like breast cancer xenograft model are associated with different signaling pathways. [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 245. doi:10.1158/1538-7445.AM2013-245

281 INTRAHEPATIC ULTRASOUND-MEDIATED GENE DELIVERY

Quiescence is a strategy that cancer stem cells (CSCs) adopt in stressful environments such are those triggered by antineoplastic treatments. CSCs form micrometastases that stay dormant for variable time periods and regain proliferation after unknown stimuli. cMYC induces pluripotency in differentiated cells and may have a role in dormant cells activation. Aim: To analyze cMYC role in the switch from dormant (generated by HCC line BCLC5) to growing tumors formed by BCLC5 cells transfected with cMYC (BCLC5-cMYC), compared to BCLC9 already expressing cMYC. Methods: Creation of a stable BCLC5 cell line expressing cMYC by transfection of pCMV6-cMYC, selection of cells expressing cMYC was done by G418 treatment. cMYC levels were confirmed by PCR/Western Blot (WB). Subcutaneous (s.c.) injection of cells into SCID mice: 10 BCLC9, BCLC5 and BCLC5-cMYC cells were s.c. injected. Stable cell lines derived from xenograft tumors: eight lines (t1 to t8) were derived from a BCLC5-cMYC tumor and 3 cell lines (c1, c2, c4) from tumors produced by injection of BCLC5-cMYC t7 cells. Gene expression of proangiogenic genes (VEGFA, CD13), epithelial– mesenchymal markers (CDH1, VIM), invasion markers (NM23H1, cMET, ITGB1, MMP2, MMP9) and genes associated with cell pluripotency (OCT4, SOX2, NANOG, CD133, EPCAM) were assessed by real-time PCR, immunohistochemistry and WB. Results: BCLC9 cells produced tumors in SCID mice while BCLC5 cells developed viable tumors <1–2mm in size that remained stable after 6 months of injection. BCLC5-cMYC cells generated tumors with high proliferation and cell lines showed increased expression of VEGFA, CD13 and CDH1 and decreased VIM, cMET and MMP2 compared to BCLC5. Tumor derived cell lines keep expressing pluripotency related genes and acquire EPCAM’s, which was originally expressed only by BCLC9. Second generation of tumors BCLC5-cMYC and their cell lines showed increased expression of proangiogenic markers, genes associated to epithelial phenotype and those related to CSC while markers associated with invasive behavior decreased. Conclusion: cMYC activation induces angiogenesis and epithelial markers while preserving pluripotent phenotype and reducing invasive potential. These changes consolidate along tumor evolution explaining the aggressive behavior of residual disease after antineoplastic treatment.

A recombinant scFv-FasLext as a targeting cytotoxic agent against human Jurkat-Ras cancer

BackgroundTargeted therapy of human cancers is an attractive approach and has been investigated with limited success. We have developed novel cytotoxic agents for targeted therapy of human cancers based on the extracellular cytotoxicity domain of CD178 (FasL) and the specificity offered by single chain antibodies (scFv) against dominant human tumor Ag TAG-72 (cc49scFv) and TAL6 (L6scFv).ResultsThe cc49scFv-FasLext is highly effective in in vitro killing of human TAG-72+ Jurkat-Ras tumor cells with a 30,000 fold greater cytotoxicity as compared to soluble FasL (sFasL). On the other hand, L6scFv-FasLext only increased cytotoxicity 500-fold as compared with sFasL against TAL6+ HeLa cells in in vitro assays. The high specificity and strong cytotoxicity of cc49scFv-FasLext made it feasible to cure IP-implanted Jurkat-Ras tumors in SCID mice.ConclusionOur study demonstrated that scFv-FasLext with a strong cytotoxicity against sensitive human tumor targets may be useful as effective chemotherapeutic agents.

Non-invasive Monitoring of Ultrasound-Stimulated Microbubble Radiation Enhancement Using Photoacoustic Imaging

Modulation of the tumour microvasculature has been demonstrated to affect the effectiveness of radiation, stimulating the search for anti-angiogenic and vascular-disrupting treatment modalities. Microbubbles stimulated by ultrasound have recently been demonstrated as a radiation enhancer when used with different cancer models including PC3. Here, photoacoustics imaging technique was used to assess this treatment’s effects on haemoglobin levels and oxygen saturation. Correlations between this modality and power doppler assessments of blood flow, and histology measurements of vascular integrity and cell death were also investigated. Xenograft prostate tumours in SCID mice were treated with 0, 2, or 8 Gy radiation combined with microbubbles exposed to 500 kHz ultrasound at a peak negative pressure of 0, 570, and 750 kPa. Tumours were assessed and levels of total haemoglobin, oxygen saturation were measured using photoacoustics before and 24 hours after treatment along with power doppler measured blood flow. Mice were then sacrificed and tumours were assessed for cell death and vascular composition using immunohistochemistry. Treatments using 8 Gy and microbubbles resulted in oxygen saturation decreasing by 28 ± 10% at 570 kPa and 25 ± 29% at 750 kPa, which corresponded to 44 ± 9% and 40 ± 14% respective decreases in blood flow as measured with power doppler. Corresponding histology indicated 31 ± 5% at 570 kPa and 37 ± 5% at 750 kPa in terms of cell death. There were drops in intact vasculature of 15 ± 2% and 20 ± 2%, for treatments at 570 kPa and 750 kPa. In summary, photoacoustic measures of total haemoglobin and oxygen saturation paralleled changes in power doppler indicators of blood flow. Destruction of tumour microvasculature with microbubble-enhanced radiation also led to decreases in blood flow and was associated with increases in cell death and decreases in intact vasculature as detected with CD31 labeling.

Microenvironment-Dependent Phenotypic Changes in a SCID Mouse Model for Malignant Mesothelioma

Background and Aims: Malignant mesothelioma is an aggressive, therapy-resistant tumor. Mesothelioma cells may assume an epithelioid or a sarcomatoid phenotype, and presence of sarcomatoid cells predicts poor prognosis. In this study, we investigated differentiation of mesothelioma cells in a xenograft model, where mesothelioma cells of both phenotypes were induced to form tumors in severe combined immunodeficiency mice.Methods: Xenografts were established and thoroughly characterized using a comprehensive immunohistochemical panel, array comparative genomic hybridization (aCGH) of chromosome 3, fluorescent in situ hybridization, and electron microscopy.Results: Epithelioid and sarcomatoid cells gave rise to xenografts of similar epithelioid morphology. While sarcomatoid-derived xenografts had higher growth rates, the morphology and expression of differentiation-related markers was similar between xenografts derived from both phenotypes. aCGH showed a convergent genotype for both xenografts, resembling the original aggressive sarcomatoid cell sub-line.Conclusion: Human mesothelioma xenografts from sarcomatoid and epithelioid phenotypes converged to a similar differentiation state, and genetic analyses suggested that clonal selection in the mouse microenvironment was a major contributing factor. This thoroughly characterized animal model can be used for further studies of molecular events underlying tumor cell differentiation.

Intratumoral acetic acid injection eradicates human prostate cancer tumors in a murine model

To treat localized prostate cancer without substantial morbidity, an ideal treatment would be an effective local therapy with minimal morbidity. Direct injections have been used to treat benign prostatic hyperplasia without major complications, but in limited cases. We evaluated the local oncotoxic effects of acetic acid in a prostate cancer xenograft murine model. PC3 and LNCaP human prostate cancer cell lines were used to grow subcutaneous tumors in SCID mice. For each cell line, 14 mice underwent intratumor injection with 25% acetic acid (0.05 ml/100 cm3 of tumor) after the tumor was >300 mm3. Post-treatment one mouse/group was euthanized after 2 h, 24 h, 1 and 2 weeks; remaining mice (n = 10) were killed at 120 days. Control mice (8/group) were euthanized after they met the humane criteria for tumor burden and overall health. Tumor necrosis was noted immediately post-injection; by 24 h, ulceration and crusting of overlying skin were noted, which healed into scars by 23 ± 5 days. Histological examination showed tumor degeneration and necrosis with blood vessel obstruction. Ten treated mice in both groups survived for 120 days, which was much longer than the mean survival of PC3 (40 ± 9 days) and LNCaP (56 ± 10) control mice. Direct injection of acetic acid successfully eradicated both tumors. This treatment option could potentially be used in humans for treatment of early localized prostate cancer and nonoperative management of locally advanced cases. This is the first report of successful local chemical therapy for prostate cancer.

Targeting B-Cell Lymphoma with Idiotype-Specific Peptibodies: Toward a Personalized and Tumor-Specific Therapy

Abstract 3713 Background:The complementarity determining region, or idiotype, of the surface immunoglobulin receptor is a tumor-specific marker on B-cell lymphomas that is unique to each patient. Antibodies against idiotype can induce complete regression of lymphoma in patients, but since this therapeutic approach requires the generation of a custom monoclonal antibody for each patient, it has not been practical. Objective:Here we describe a method for targeting the idiotype on the surface of a B-cell lymphoma by using synthetic idiotype-ligands covalently linked to a recombinant IgG Fc domain (Figure 1A). These peptide idiotype-ligands can be identified through oligopeptide library screens and produced inexpensively by automated solid-phase synthesis. Linkage of idiotype-ligands to the Fc domain serves two purposes: to enhance their pharmacokinetics and to augment their anti-tumor effect by activating immune effector functions. Since each patient-specific peptide can be chemically linked to a common IgG Fc domain, this modular construct design yields a patient-specific therapeutic that does not require the production of a custom biologic macromolecule for each patient. Results:Idiotype peptide-ligands were produced by solid-phase synthesis and covalently linked to the amino-terminus of a recombinant mouse IgG2a Fc domain by native chemical ligation, a method for site-selective polypeptide ligation. The resulting peptibody demonstrated targeted killing of a human lymphoma cell line in vitro by crosslinking surface immunoglobulin and triggering activation-induced death. Additionally, the peptibody triggered complement-mediated cytotoxicity of opsonized lymphoma cells in vitro and activated natural killer cells co-cultured with opsonized lymphoma cells. The peptibody exhibited a favorable pharmacokinetic profile and peptibody treatment was sufficient to clear tumor in SCID mice challenged intravenously with a luciferase-labeled human lymphoma cell line (p = 0.0018; Figure 1B-D). Conclusions:Idiotype-specific peptibodies demonstrate multimodal activity against lymphoma cells in vitro and clear human lymphoma in a disseminated xenograft model. The modular design of this therapeutic may enable a personalized and targeted therapy that is feasible to produce for patients with B-cell lymphoma. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

IL-6 Stimulates STAT3 and Pim-1 Kinase in Pancreatic Cancer Cell Lines

We investigated the signaling pathways activated in response to interleukin 6 (IL-6) in pancreatic cell lines, with a focus on signal transducer and activator of transcription 3 (STAT3) and proto-oncogene serine/threonine-protein (Pim-1) kinase. Interleukin 6 receptor (IL-6R) expression and IL-6-induced cell signaling was measured by Western blotting in human pancreatic cell lines. Cucurbitacin I was used as a pharmacological tool to investigate the role of STAT3 in Pim-1 activation. Stably overexpressing Pim-1 kinase cell lines were characterized for their response to IL-6 in vitro and for their growth rate as flank tumors in scid mice. Interleukin 6 receptor was expressed across multiple cancer cell lines. In Panc-1 cells, IL-6 treatment increased expression of phosphorylation of signal transducer and activator of transcription 3 and Pim-1 kinase. Cucurbitacin I treatment alone increased pErk1/2 expression in wild-type and Pim-1-overexpressing cell lines and resulted in exaggerated Pim-1 kinase protein levels in control and IL-6-stimulated cells, suggesting that up-regulation of Pim-1 may be partially STAT3 independent. Pim-1 overexpression did not significantly affect growth rate in vitro or in vivo in Panc-1 or MiaPaCa2 cell lines. Interleukin 6 activates STAT3 and stimulates Pim-1 kinase in pancreatic cell line models. The regulation and consequence of Pim-1 expression seems to be highly context dependent.

Prox1 suppresses the proliferation of neuroblastoma cells via a dual action in p27-Kip1 and Cdc25A

Neuroblastoma is a pediatric tumor that originates from precursor cells of the sympathetic nervous system with less than 40% long-term survival in children diagnosed with high-risk disease. These clinical observations underscore the need for novel insights in the mechanisms of malignant transformation and progression. Accordingly, it was recently reported that Prox1, a homeobox transcription regulator, is expressed in higher levels in human neuroblastoma with favorable prognosis. Consistently, we have recently shown that Prox1 exerts a strong antiproliferative effect on neural precursor cells during embryonic development. Thus, Prox1 is a candidate gene with a critical role in suppressing malignant neuroblastoma transformation. Here, we provide evidence that Prox1 strongly suppresses the proliferation of mouse and human neuroblastoma cell lines and blocks the growth of neuroblastoma tumors in SCID mice. Conversely, short hairpin RNA (shRNA) -mediated knockdown of basal Prox1 expression significantly induces proliferation, genomic instability and the ability of neuroblastoma cells to form tumors. Mechanistically, analysis of an inducible Prox1-overexpressing Neuro2A cell line indicates that Prox1 is sufficient to suppress CyclinD1, CyclinA and CyclinB1, consistent with a role in cell cycle arrest. Surprisingly, Prox1 strongly induces CyclinE1 expression in the same system despite its action on blocking cell cycle progression, which could account for the context dependent oncogenic function of Prox1. Most importantly, Prox1 was sufficient to decrease Cdc25A and induce p27-Kip1, but not p21-Cip1 or p53. By alleviating the Prox1 action in Cdc25A and p27-Kip1 expression, we were able to rescue its effect on cell cycle arrest. Together these data suggest that Prox1 negatively regulates neuroblastoma carcinogenesis through suppression of Cdc25A and induction of p27-Kip1 to counteract CyclinE1 overexpression and block cell cycle progression. Furthermore, these observations render Prox1 a candidate target for the treatment of neuroblastoma tumors.

Abstract 2311: microRNA-196b regulates HOXB7 in cervical cancer

Abstract Introduction: microRNAs (miRNAs) have been shown to play an important biological role in many human malignancies. The down-regulation of miR-196b has been previously reported in cervical cancer (CaCx), but its contribution to tumor progression remains unelucidated. Materials and Methods: miRNA expression was measured in frozen tissues from CaCx biopsies (n=79) and normal cervix (n=11), plus three CaCx cell lines (ME-180, SiHa and HT-3) using a quantitative real-time PCR (qPCR) approach simultaneously measuring 377 miRNAs. SiHa and ME-180 cells were transfected with 30 nmol/L of pre-miR-196b, pre-miR Negative Control (NC), siHOXB7, siVEGF, or Negative Control siRNA. Cell viability, clonogenicity and migration/invasion were analyzed using the Trypan blue exclusion assay, clonogenic assay and trans-well migration assays, respectively. Protein levels of HOXB7 and VEGF were measured by immunoblotting and ELISA, respectively. To determine candidate mRNA targets of miR-196b, a tri-modal approach was utilized by combining: a) all predicted targets from five target prediction databases; b) genes upregulated in CaCx patients; and c) genes down-regulated after in vitro miR-196b over-expression. A luciferase reporter assay was used to confirm the binding of miR-196b to the HOXB7 3′ UTR. Tumor formation was monitored in xenograft tumors in SCID mice; CD31 and Ki67 immunostaining were performed on tumors 25 days after implantation. Results: Significant down-regulation of miR-196b was observed in the CaCx cells lines and tissues. Patients with low miR-196b expression (n=39) experienced worse disease-free survival compared to those with high (n=39) miR-196b expression (p=0.02, HR=0.39). Pre-miR-196b transfection reduced cell viability (25% after 48h; 41% after 72h), clonogenicity (57%) and invasion (32% vs. 63% for cells treated with NC). In vivo, pre-miR-196b slightly decreased tumor growth, but associated with significantly reduced CD31 (69%) and Ki-67 expression (46% vs. 53% for NC cells). The luciferase reporter assay verified that HOXB7 is a direct and specific target of miR-196b. Decreased VEGF mRNA (43%) and protein (78%) levels after HOXB7 knockdown demonstrated that VEGF was a downstream mediator of HOXB7 activation. siRNA knockdown of HOXB7 or VEGF recapitulated the biological effects of miR-196b over-expression, including reduced cell viability (66% for siHOXB7 at 72h; 60% for VEGF at 72h), clonogenicity (69% for siHOXB7; 78% for siVEGF) and invasion (52% for siHOXB7 and 42% for siVEGF vs. 74% for Negative Control cells); thus corroborating the miR-196b/HOXB7/VEGF axis in CaCx progression. Conclusion: We have newly identified the miR-196b/HOXB7/VEGF pathway as an important dysregulated axis contributing to human CaCx progression; hence therapeutic targeting of this pathway could potentially improve patient outcome. 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 2311. doi:1538-7445.AM2012-2311

Abstract 1406: A new ovarian cancer metastasis model using multicellular spheroids generated from human ovarian cancers tissues

Abstract Objective Most research aimed at dissecting the mechanism by which epithelial ovarian cancer (EOC) metastasizes has focused on the ability of individual cells to attach and create tumor implants. However, multicellular aggregates known as spheroids are frequently found in ovarian cancer ascites and these aggregates are increasingly thought to play an important role in metastasis not only for ovarian cancer but also other solid tumors where similar phenomena have been observed. We investigated characteristics of spheroids compared to cells grown in monolayer to further characterize these spheroids. Methods IRB approval was obtained for collection of human tissue. Fresh specimens of ovarian cancer were minced, enzymatically digested, rinsed and incubated either in DMEM to generate monolayer cultures or Mammosphere media to generate spheroids. Tumorigenicity was assessed by inoculating individual spheroids or an equal number of dissociated cells into the peritoneal cavity of SCID mice. Gene expression was examined using Western blot and quantitative real-time PCR. Results We found that cells from most ovarian cancers do generate spheroids and most of the spheroids generated are able to reliably induce peritoneal implants in SCID mice. At a molecular level, the spheroids were enriched for expression of specific markers including CD133, CD44, NANOG and OCT4, associated with populations of cancer initiating cells (CICs) as well as other stem cell markers. MiR-26b, characteristic of CICs, was significantly up regulated in spheroids compared to monolayer cells. Spheroids from CD133 positive cells induced ovarian and abdominal tumors in SCID mice, however, CD44 spheroids only generated localized tumor, suggesting that CD133 is important in metastasis of ovarian cancer. Conclusion Our results indicate that populations of cells from human EOC are capable of spontaneously generating multicellular aggregates that resemble the spheroids found in ovarian cancer ascites de novo. These spheroids are enriched for expression of markers including CD133, CD44, NANOG and OCT4, suggesting that spheroid formation enhances stem cell-like markers. CD133 might also serve as a necessary marker for metastasis of ovarian cancer. The increased expression of miR-26b in spheroids compared to monolayer culture is a novel finding that needs further investigation. 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 1406. doi:1538-7445.AM2012-1406