Tumors In SCID Mice Research Articles

Gelsolin promotes cell growth and invasion through the upregulation of p-AKT and p-P38 pathway in osteosarcoma

The gelsolin (GSN) has been involved in the regulation of tumor formation and development, but the biological role of GSN in the pathogenesis of osteosarcoma (OS) has not been well characterized. In this study, we show that high expression of GSN was observed in OS tissues and correlated with tumor size, advanced Enneking stage, and poor patient prognosis. Knockdown of GSN significantly inhibited cell proliferation and invasiveness in the OS cell lines. Furthermore, stable overexpression of GSN in OS cells resulted in a significant increase in cell growth and motility with the downregulation of p-AKT and p-P38 pathway. Finally, overexpression of GSN promoted growth of OS tumors in SCID mice. Taken together, these results provided the first evidence that GSN might contribute to OS cancer development and could be an attractive therapeutic target for patients with OS.

Combined sonodynamic and antimetabolite therapy for the improved treatment of pancreatic cancer using oxygen loaded microbubbles as a delivery vehicle

In this manuscript we describe the preparation of an oxygen-loaded microbubble (O2MB) platform for the targeted treatment of pancreatic cancer using both sonodynamic therapy (SDT) and antimetabolite therapy. O2MB were prepared with either the sensitiser Rose Bengal (O2MB-RB) or the antimetabolite 5-fluorouracil (O2MB-5FU) attached to the microbubble (MB) surface. The MB were characterised with respect to size, physical stability and oxygen retention. A statistically significant reduction in cell viability was observed when three different pancreatic cancer cell lines (BxPc-3, MIA PaCa-2 and PANC-1), cultured in an anaerobic cabinet, were treated with both SDT and antimetabolite therapy compared to either therapy alone. In addition, a statistically significant reduction in tumour growth was also observed when ectopic human xenograft BxPC-3 tumours in SCID mice were treated with the combined therapy compared to treatment with either therapy alone. These results illustrate not only the potential of combined SDT/antimetabolite therapy as a stand alone treatment option in pancreatic cancer, but also the capability of O2-loaded MBs to deliver O2 to the tumour microenvironment in order to enhance the efficacy of therapies that depend on O2 to mediate their therapeutic effect. Furthermore, the use of MBs to facilitate delivery of O2 as well as the sensitiser/antimetabolite, combined with the possibility to activate the sensitiser using externally applied ultrasound, provides a more targeted approach with improved efficacy and reduced side effects when compared with conventional systemic administration of antimetabolite drugs alone.

Isolation and characterization of renal cancer stem cells from patient-derived xenografts.

As rapidly developing patient-derived xenografts (PDX) could represent potential sources of cancer stem cells (CSC), we selected and characterized non-cultured PDX cell suspensions from four different renal carcinomas (RCC). Only the cell suspensions from the serial xenografts (PDX-1 and PDX-2) of an undifferentiated RCC (RCC-41) adapted to the selective CSC medium. The cell suspension derived from the original tumor specimen (RCC-41-P-0) did not adapt to the selective medium and strongly expressed CSC-like markers (CD133 and CD105) together with the non-CSC tumor marker E-cadherin. In comparison, PDX-1 and PDX-2 cells exhibited evolution in their phenotype since PDX-1 cells were CD133high/CD105-/Ecadlow and PDX-2 cells were CD133low/CD105-/Ecad-. Both PDX subsets expressed additional stem cell markers (CD146/CD29/OCT4/NANOG/Nestin) but still contained non-CSC tumor cells. Therefore, using different cell sorting strategies, we characterized 3 different putative CSC subsets (RCC-41-PDX-1/CD132+, RCC-41-PDX-2/CD133-/EpCAMlow and RCC-41-PDX-2/CD133+/EpCAMbright). In addition, transcriptomic analysis showed that RCC-41-PDX-2/CD133− over-expressed the pluripotency gene ERBB4, while RCC-41-PDX-2/CD133+ over-expressed several tumor suppressor genes. These three CSC subsets displayed ALDH activity, formed serial spheroids and developed serial tumors in SCID mice, although RCC-41-PDX-1/CD132+ and RCC-41-PDX-2/CD133+ displayed less efficiently the above CSC properties. RCC-41-PDX-1/CD132+ tumors showed vessels of human origin with CSC displaying peri-vascular distribution. By contrast, RCC-41-PDX-2 originated tumors exhibiting only vessels of mouse origin without CSC peri-vascular distribution.Altogether, our results indicate that PDX murine microenvironment promotes a continuous redesign of CSC phenotype, unmasking CSC subsets potentially present in a single RCC or generating ex novo different CSC-like subsets.

Novel high-grade serous epithelial ovarian cancer cell lines that reflect the molecular diversity of both the sporadic and hereditary disease

Few cell line models of epithelial ovarian cancer (EOC) have been developed for the high-grade serous (HGS) subtype, which is the most common and lethal form of gynaecological cancer. Here we describe the establishment of six new EOC cell lines spontaneously derived from HGS tumors (TOV2978G, TOV3041G and TOV3291G) or ascites (OV866(2), OV4453 and OV4485). Exome sequencing revealed somatic TP53 mutations in five of the cell lines. One cell line has a novel BRCA1 splice-site mutation, and another, a recurrent BRCA2 nonsense mutation, both of germline origin. The novel BRCA1 mutation induced abnormal splicing, mRNA instability, resulting in the absence of BRCA1 protein. None of the cell lines harbor mutations in KRAS or BRAF, which are characteristic of other EOC subtypes. SNP arrays showed that all of the cell lines exhibited structural chromosomal abnormalities, copy number alterations and regions of loss of heterozygosity, consistent with those described for HGS. Four cell lines were able to produce 3D-spheroids, two exhibited anchorage-independent growth, and three (including the BRCA1 and BRCA2 mutated cell lines) formed tumors in SCID mice. These novel HGS EOC cell lines and their detailed characterization provide new research tools for investigating the most common and lethal form of EOC.

Therapeutic potential of thalidomide for gemcitabine-resistant bladder cancer.

Controversial effects of thalidomide for solid malignancies have been reported. In the present study, we evaluate the effects of thalidomide for transitional cell carcinoma (TCC), the most common type of bladder cancer. Thalidomide precipitates were observed when its DMSO solution was added to the culture medium. No precipitation was found when thalidomide was dissolved in 45% γ-cyclodextrin, and this concentration of γ-cyclodextrin elicited slight cytotoxicity on TCC BFTC905 and primary human urothelial cells. Thalidomide-γ-cyclodextrin complex exerted a concentration-dependent cytotoxicity in TCC cells, but was relatively less cytotoxic (with IC50 of 200 µM) in BFTC905 cells than the other 3 TCC cell lines, possibly due to upregulation of Bcl-xL and HIF-1α mediated carbonic anhydrase IX, and promotion of quiescence. Gemcitabine-resistant BFTC905 cells were chosen for additional experiments. Thalidomide induced apoptosis through downregulation of survivin and securin. The secretion of VEGF and TNF-α was ameliorated by thalidomide, but they did not affect cell proliferation. Immune-modulating lenalidomide and pomalidomide did not elicit cytotoxicity. In addition, cereblon did not play a role in the thalidomide effect. Oxidative DNA damage was triggered by thalidomide, and anti-oxidants reversed the effect. Thalidomide also inhibited TNF-α induced invasion through inhibition of NF-κB, and downregulation of effectors, ICAM-1 and MMP-9. Thalidomide inhibited the growth of BFTC905 xenograft tumors in SCID mice via induction of DNA damage and suppression of angiogenesis. Higher average body weight, indicating less chachexia, was observed in thalidomide treated group. Sedative effect was observed within one-week of treatment. These pre-clinical results suggest therapeutic potential of thalidomide for gemcitabine-resistant bladder cancer.

Abstract 5346: Notch3-mediated squamous cell differentiation shows anti-tumor effect on esophageal squamous cell carcinoma as well as reduces its resistance to 5-Fluorouracil

Abstract BACKGROUND: We have shown that Notch3 plays an important role in the regulation of esophageal squamous cell differentiation. However, it remains unclear how Notch3-mediated squamous cell differentiation influences the tumorigenicity of esophageal squamous cell carcinoma (ESCC) or its sensitivity to anti-cancer agents, including 5-Fluorouracil (5-FU). METHODS: We used 5-FU-resistant human ESCC cells, TE-11R, established by the step-wise continuous exposure of parental TE-11 cells to 5-FU. The TE-11R cells were stably transduced with intracellular domain of Notch3 (ICN3), an active form of Notch3, in a regulatable manner (Tet-On system). The anti-tumor effect of Notch3-overexpression was evaluated by implanting those cells into NOD SCID mice. 5-FU resistance was determined by calculating the 50% inhibitory concentration (IC50) of 5-FU using the WST-1 assay. Gene expression of squamous differentiation markers such as involucrin and cytokeratin 13 (CK13) as well as proliferative activity were determined. RESULTS: TE-11R cells formed bulky tumors in NOD SCID mice, and the tumor formation rate of TE-11R cells (6/6: 100%) was higher than that of TE-11 cells (1/8: 12.5%). TE-11R-derived tumors showed a less differentiated and more proliferative phenotype represented by fewer ‘keratin pearl’ formations with lower involucrin and Notch3 expressions as well as higher Ki67 expression in comparison with TE-11-derived tumors. Consistent with this, TE-11R cells showed lower gene expressions of Notch3, involucrin, and CK13, and exhibited approximately 1.4-fold higher proliferative activity than parental TE-11 cells in vitro. Notch3 overexpression resulted in the promotion of cell differentiation accompanied by increased expressions of involucrin and CK13, and suppressed cell growth in vitro as well as tumorigenicity in vivo. Finally, it clearly reduced the 5-FU resistance of TE-11R cells in vitro. IC50 values of TE-11R-ICN3 (DOX-) and TE-11R-ICN3 (DOX+) were 96.0 ± 19.7 and 21.6 ± 10.4 μM (P < 0.01), respectively. CONCLUSION: Our study showed that Notch3-mediated squamous cell differentiation reduced both the tumorigenicity and 5-FU resistance of ESCC cells. We suggest that a strategy to promote squamous cell differentiation may ameliorate the outcome associated with 5-FU-resistant ESCC. Citation Format: Osamu Kikuchi, Shinya Ohashi, Yukie Nakai, Yusuke Amanuma, Masahiro Yoshioka, Shin'ichi Miyamoto, Mitsuteru Natsuizaka, Hiroshi Nakagawa, Tsutomu Chiba, Manabu Muto. Notch3-mediated squamous cell differentiation shows anti-tumor effect on esophageal squamous cell carcinoma as well as reduces its resistance to 5-Fluorouracil. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2015-5346

PTU-143 Authentication and characterisation of a new oesophageal adenocarcinoma cell line: mfd-1

<h3>Introduction</h3> The United Kingdom has the highest age-standardised incidence of oesophageal adenocarcinoma (EAC) in the world. EAC has a narrow window for medical intervention and 2/3 of patients present with incurable disease. Even after potentially curative surgery survival rates are at best 50% at 5 years. The oesophageal International Cancer Genome Consortium (ICGC) is elucidating the genetic landscape of oesophageal adenocarcinoma but the impact of this initiative will only be fully tangible when suitable preclinical models to validate its results become available. We describe the generation and authentication of a novel EAC cell line in a tumour from a 55-year-old male (pT4N3M0) who died from disease recurrence 7 months after neo-adjuvant chemotherapy and oesophagectomy. <h3>Method</h3> The MFD-1 cell line was expanded directly from a resected EAC specimen without any recombinant-DNA transformation. Somatic genetic variations (SGV) in DNA from MFD-1, tumour, normal oesophagus, and leucocytes were analysed genome wide with the SNP6 DNA microarray (Affymetrix). WGS was performed in tumour and leucocyte DNA (Illumina). Functional studies of MFD-1 were performed <i>in vitro</i>(3D culture + immunohistochemistry (IHC) + flow cytometry) and <i>in vivo</i>(SCID mouse xenograft). <h3>Results</h3> MFD-1 showed 98% concordance with matched tumour on genome wide SNP6 analysis. MFD-1 and the source tumour shared identical SGVs in established cancer promoting genes in EAC including deletions of SMAD4 and p16. A genetic rearrangement in the TP53 gene was observed, consistent with a copy neutral SGV with loss of heterozygosity and a duplication of a mutant TP53 allele, the Arginine 273 Histidine mutation, found in the tumour and the MFD-1 cell line. A second somatic acquired mutation was found in the new EAC candidate DOCK2 gene in the tumour, whilst amplification of a wild type allele was documented in the cell line. In 3D culture, MFD-1 was invasive and proliferative. MFD-1 is Ep-CAM positive (on flow cytometry), and in IHC stains strongly for pan-Cytokeratin and moderately for CK 7, confirming its glandular epithelial phenotype. MFD-1 formed tumours in SCID mice that developed more rapidly than the historical EAC cell lines OE33 and Flo-1. <h3>Conclusion</h3> A new tool for the <i>in vitro</i>modelling of EAC has been validated using massive parallel genotyping and sequencing of parent and cell line genomes. A genetic mechanism of malignant transformation operating in the tumour/cell line is consistent with a duplication of a mutant allele in the TP53 gene with a loss of the wild-type allele which translates as a copy neutral SGV with loss of heterozygosity. <h3>Disclosure of interest</h3> None Declared.

Function of phosphorylation of NF-kB p65 ser536 in prostate cancer oncogenesis.

Majority of prostate cancer (PCa) patients carry TMPRSS2/ERG (T/E) fusion genes and there has been tremendous interest in understanding how the T/E fusion may promote progression of PCa. We showed that T/E fusion can activate NF-kB pathway by increasing phosphorylation of NF-kB p65 Ser536 (p536), but the function of p536 has never been studied in PCa. We report here that active p536 can significantly increase cell motility and transform PNT1a cells (an immortalized normal cell line), suggesting p536 plays a critical role in promoting PCa tumorigenesis. We have discovered a set of p536 regulated genes, among which we validated the regulation of CCL2 by p536. Based on all evidence, we favor that T/E fusion, NF-kB p536 and CCL2 form a signaling chain. Finally, PNT1a cells (not tumorigenic) can form tumors in SCID mice when overexpressing of either wild type or active p65 in the presence of activated AKT, demonstrating synergistic activities of NF-kB and AKT signals in promoting PCa tumorigenesis. These findings indicate that combination therapies targeting T/E fusion, NF-kB, CCL2 and/or AKT pathways may have efficacy in T/E fusion gene expressing PCa. If successful, such targeted therapy will benefit more than half of PCa patients who carry T/E fusions.

A novel phthalimide derivative, TC11, has preclinical effects on high-risk myeloma cells and osteoclasts.

Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the Cmax was 2.1 μM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.

Spontaneous immortalization of mouse liver sinusoidal endothelial cells.

The spontaneous immortalization of cells in vitro is a rare event requiring genomic instability, such as alterations in chromosomes and mutations in genes. In the present study, we report a spontaneously immortalized liver sinusoidal endothelial cell (LSEC) line generated from mouse liver. These immortalized LSECs showed typical LSEC characteristics with the structure of transcellular fenestrations, the expression of von Willebrand factor (VWF) and the ability to uptake DiI-acetylated-low density lipoprotein (DiI-Ac-LDL). However, these immortalized LSECs lost the ability to form capillary-like structures, and showed clonal and multilayer growth without contact inhibition. Moreover, their proliferation rate increased with the increase in the number of passages. In addition, these cells obained the expression of CD31 and desmin, and showed an upregulation of p53 protein expression; however, their karyotype was normal, and they could not form colonies in soft agar or tumors in SCID mice. In conclusion, in the present study, we successfully established a spontaneously immortalized LSEC line.

CXCL12/CXCR4 blockade by oncolytic virotherapy inhibits ovarian cancer growth by decreasing immunosuppression and targeting cancer-initiating cells.

Signals mediated by the chemokine CXCL12 and its receptor CXCR4 are involved in the progression of ovarian cancer through enhancement of tumor angiogenesis and immunosuppressive networks that regulate dissemination of peritoneal metastasis and development of cancer-initiating cells (CICs). In this study, we investigated the antitumor efficacy of a CXCR4 antagonist expressed by oncolytic vaccinia virus (OVV) against an invasive variant of the murine epithelial ovarian cancer cell line ID8-T. This variant harbors a high frequency of CICs that form multilayered spheroid cells and express the hyaluronan receptor CD44, as well as stem cell factor receptor CD117 (c-kit). Using an orthotopic ID8-T tumor model, we observed that i.p. delivery of a CXCR4 antagonist-expressing OVV led to reduced metastatic spread of tumors and improved overall survival compared with oncolysis alone. Inhibition of tumor growth with the armed virus was associated with efficient killing of CICs, reduced expression of ascitic CXCL12 and vascular endothelial growth factor, and decreases in i.p. numbers of endothelial and myeloid cells, as well as plasmacytoid dendritic cells. These changes, together with reduced recruitment of T regulatory cells, were associated with higher ratios of IFN-γ(+)/IL-10(+) tumor-infiltrating T lymphocytes, as well as induction of spontaneous humoral and cellular antitumor responses. Similarly, the CXCR4 antagonist released from virally infected human CAOV2 ovarian carcinoma cells inhibited peritoneal dissemination of tumors in SCID mice, leading to improved tumor-free survival in a xenograft model. Our findings demonstrate that OVV armed with a CXCR4 antagonist represents a potent therapy for ovarian CICs with a broad antitumor repertoire.

Bortezomib sensitizes non-small cell lung cancer to mesenchymal stromal cell-delivered inducible caspase-9-mediated cytotoxicity.

Delivery of suicide genes to solid tumors represents a promising tumor therapy strategy. However, slow or limited killing by suicide genes and ineffective targeting of the tumor has reduced effectiveness. We have adapted a suicide system based on an inducible caspase-9 (iC9) protein that is activated using a specific chemical inducer of dimerization (CID) for adenoviral-based delivery to lung tumors via mesenchymal stromal cells (MSCs). Four independent human non-small cell lung cancer (NSCLC) cell lines were transduced with adenovirus encoding iC9, and all underwent apoptosis when iC9 was activated by adding CID. However, there was a large variation in the percentage of cell killing induced by CID across the different lines. The least responsive cell lines were sensitized to apoptosis by combined inhibition of the proteasome using bortezomib. These results were extended to an in vivo model using human NSCLC xenografts. E1A-expressing MSCs replicated Ad.iC9 and delivered the virus to lung tumors in SCID mice. Treatment with CID resulted in some reduction of tumor growth, but addition of bortezomib led to greater reduction of tumor size. The enhanced apoptosis and anti-tumor effect of combining MSC-delivered Ad.iC9, CID and bortezomib appears to be due to increased stabilization of active caspase-3, as proteasomal inhibition increased the levels of cleaved caspase-9 and caspase-3. Knockdown of X-linked inhibitor of apoptosis protein (XIAP), a caspase inhibitor that targets active caspase-3 to the proteasome, also sensitized iC9-transduced cells to CID, suggesting that blocking the proteasome counteracts XIAP to permit apoptosis. Thus, MSC-based delivery of the iC9 suicide gene to human NSCLC effectively targets lung cancer cells for elimination. Combining this therapy with bortezomib, a drug that is otherwise inactive in this disease, further enhances the anti-tumor activity of this strategy.

Abstract 4517: Evaluation of the TOR kinase inhibitor CC214-2 in a mouse model of hormone-refractory prostate cancer

Abstract Prostate cancer is one of the most common malignancy diagnosed in men. Although androgen deprivation therapy is effective at the beginning of treatment, the majority of patients eventually progress to hormone refractory prostate cancer (HRPC), which leads to a poor outcome. HRPC mouse models have been developed to study transition from hormone dependent (HD) to hormone refractory (HR) disease; however, the exact mechanism behind this phenotypic conversion remains unclear. By using serial in vivo transplantation of LNCap xenograft tumors in castrated male SCID mice, we developed, validated and characterized a mouse model of HRPC, HR-LNCap. In vivo passaged tumor fragments form solid tumors in castrated male SCID mice. By comparison, HR-LNCap tumors grow more aggressively than parental LNCap tumors in SCID mice. A tumor cell line established from HR-LNCap xenograft tumors was able to grow in charcoal stripped serum containing medium, indicating it is androgen independent. HR-LNCap tumors lost sensitivity to androgen receptor antagonist bicalutamide, but remained sensitive to MDV-3100, a second generation AR antagonist approved for treating HRPC. Characterization of this model revealed that HR-LNCap tumors had unique AR signaling pathway profiles, including elevated AR expression and decreased expression of the AR-regulated genes, prostate specific antigen (PSA) and FKBP5, when compared with parental LNCap tumors. RNA sequencing was performed on HR-LNCap tumors to determine expression signature of the model. Interestingly, additional pathway analysis showed that HR-LNCap tumors have significantly higher levels of pAKT in comparison with parental LNCap tumors, indicating activation of AKT/mTOR pathway. Treatment of HR-LNCap tumor bearing mice with a small molecule TOR kinase inhibitor CC214-2 showed significant tumor growth inhibition. Further analysis demonstrated that treatment with CC214-2 inhibited TORC1 and TORC2 pathway markers, pAKT, pS6 and p4EBP1, in HR-LNCap tumors. Taken together, this study describes a mouse model of HRPC with a unique molecular profile, which can be used to evaluate novel therapeutics against HRPC. Citation Format: Wenqing Yang, Jing Jiu, Rama Krishna Narla, Heather K. Raymon. Evaluation of the TOR kinase inhibitor CC214-2 in a mouse model of hormone-refractory prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4517. doi:10.1158/1538-7445.AM2014-4517

Abstract 648: Semi-synthetic peptibodies are a novel personalized therapeutic with activity against lymphoma in vitro and in vivo

Abstract The complementarity determining region, or idiotype, of the surface immunoglobulin receptor is a true tumor-specific marker on B-cell lymphomas unique to each patient. Antibodies against idiotype can induce complete regression of lymphoma in patients, but since this requires the generation of a custom monoclonal antibody for each patient, it has not been practical. We have developed a method for targeting idiotype using a novel construct that we refer to as a “semi-synthetic peptibody”. It consists of a synthetic peptide ligand for idiotype that is covalently linked to the amino terminus of a recombinant IgG Fc domain. Peptide ligands for idiotype can be identified by high throughput screens of random peptide libraries and produced inexpensively by solid-phase synthesis. Linkage of these idiotype ligands to the Fc domain enhances their pharmacokinetics and augments their anti-tumor effect by activating innate immune effectors. Since each patient-specific synthetic peptide can be chemically linked to a common IgG Fc domain, this modular design yields a custom therapeutic that may be more practical to produce than a unique biologic monoclonal antibody. We demonstrate that semi-synthetic peptibodies bind specifically to the idiotype of tumor cells and induce apoptosis by crosslinking surface immunoglobulin. Additionally, they trigger antibody dependent cellular cytotoxicity, antibody mediated phagocytosis, and complement-mediated lysis of opsonized lymphoma cells in vitro. They possess a favorable pharmacokinetic profile and are sufficient to clear tumor in SCID mice challenged intravenously with a luciferase-labeled human lymphoma cell line. Tumor clearance in vitro and in vivo mediated by peptibody was superior to that achievable with an anti-idiotype monoclonal antibody. Thus, semi-synthetic anti-idiotype peptibodies demonstrate multimodal activity against lymphoma cells in vitro and clear human lymphoma in a disseminated xenograft model. This modular, semi-synthetic design may enable a personalized and targeted therapy that is feasible to produce for patients with B-cell lymphoma. Citation Format: James Torchia, Patrick Ng, Homer Chen, Kipp Weiskopf, Ronald Levy. Semi-synthetic peptibodies are a novel personalized therapeutic with activity against lymphoma in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 648. doi:10.1158/1538-7445.AM2014-648

Abstract 4809: Enhanced activity of aflibercept compared to bevacizumab in a patient-derived xenograft colorectal cancer model is associated with reduced tumor vascularity in humanized VEGF mice

Abstract VEGF inhibitors aflibercept (ziv-aflibercept in USA) and bevacizumab target vascular endothelial growth factor A (VEGF-A), and are approved for the 2nd line treatment of metastatic colorectal cancer (CRC) in combination with distinct chemotherapy regimens and after different 1st line therapies. Although both aflibercept and bevacizumab bind to VEGF-A (herein referred to as VEGF), aflibercept blocks additional VEGF family ligands PlGF and VEGF-B, which bind VEGF receptor 1 (VEGFR1), whereas bevacizumab does not. We recently utilized patient-derived xenograft (PDX) models of CRC to compare the pharmacological activity of aflibercept and bevacizumab. In a screening study in nude mice, aflibercept showed more potent anti-tumor activity in 39/48 CRC PDX compared to bevacizumab (Chiron M. et al, AACR-NCI-EORTC 2013). In these PDX, tumor levels of tumor-derived human VEGF (hVEGF) were much higher than host murine VEGF (mVEGF), however, a potential limitation was that bevacizumab does not bind mVEGF. To further rule out any contribution from mVEGF and to determine whether tumor cell expression of VEGFR1 played a role in the differential responses, we compared the effects of aflibercept and bevacizumab on selected PDX CRC tumors in SCID mice genetically engineered to express hVEGF from the mVEGF locus. In humanized VEGF mice, aflibercept was more potent in 3/7 PDX compared with bevacizumab but equivalent in 4/7 PDX. Tumors in PDX IMM-Colo-0018 and IMM-Colo-0010, with high and low VEGFR1 expression, respectively, regressed in response to aflibercept while bevacizumab resulted in growth stasis. PDX model CXF280 with high VEGFR1 expression, consistently showed significantly greater response to aflibercept in comparison to bevacizumab. In contrast, PDX model CFX260, also with high VEGFR1 expression, responded equally well to both drugs. There was no CRC PDX in humanized VEGF mice in which the activity of bevacizumab was greater than aflibercept. Immunohistochemistry analysis revealed that improved efficacy of aflibercept treatment over bevacizumab was associated with a greater reduction of tumor vessel density and sprouting in CFX280 tumors. Cumulatively, these results suggest that aflibercept is more potent than bevacizumab in certain PDX CRC tumor models. In this limited subset of PDX CRC models, differences in pharmacological activity observed between aflibercept and bevacizumab in humanized VEGF mice do not appear to correlate with tumor cell expression of VEGFR1, but instead are associated with a greater reduction of tumor blood vessels. We are further investigating the role of VEGFR1 and other factors that may explain enhanced anti-tumor efficacy of aflibercept in PDX CRC models in humanized mice. The results suggest that CRC patient subsets may exist where aflibercept is more active than therapies targeting only VEGF-A. Citation Format: Cristina L. Abrahams, Laura D. Winters, Baosheng Li, Asma A. Parveen, Tatjana Kloss, Rebecca G. Bagley, Donald A. Bergstrom, Gavin Thurston, Marielle Chiron. Enhanced activity of aflibercept compared to bevacizumab in a patient-derived xenograft colorectal cancer model is associated with reduced tumor vascularity in humanized VEGF mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4809. doi:10.1158/1538-7445.AM2014-4809

Establishment and characterization of a singaporean chinese lung adenocarcinoma cell line with four copies of the epidermal growth factor receptor gene.

We have established a lung adenocarcinoma cell line, ETCC016, from lung pleural effusion of a male Singaporean Chinese with advanced lung adenocarcinoma. The subject smoked 20 cigarettes per day for more than 30 years. The cell line arose from spontaneous transformation of cells grown in a collagen-coated culture dish. Transformed characteristics of the cell line include the ability to reach high confluency in a culture dish, low cell doubling time, ability to form colonies in soft agar, and ability to form solid tumor in immune-compromised SCID mice. Immunostaining showed that the cells originated from lung epithelial cells. Genomic analysis revealed a large amount of chromosomal aberrations (gain and loss of genetic materials, and loss of heterozygosity [LOH]), indicative of a long history of smoking. The cells have four copies of epidermal growth factor receptor (EGFR) and three copies of MYC, but have lost one copy of the RB1 gene. LOH was detected in TP53 and BRAF genes. There is no anaplastic lymphoma kinase (ALK) gene rearrangement. The ETCC016 lung adenocarcinoma cell line has demonstrated susceptibility towards inhibitors specific for EGFR/HER2 and ALK targets, but resistance to MYC-specific inhibitor. This cell line will be a useful model for further understanding of lung adenocarcinoma.

The RelB alternative NF-kappaB subunit promotes autophagy in 22Rv1 prostate cancer cells in vitro and affects mouse xenograft tumor growth in vivo.

BackgroundThe involvement of NF-κB signaling in prostate cancer (PCa) has largely been established through the study of the classical p65 subunit. Nuclear localization of p65 in PCa patient tissues has been shown to correlate with biochemical recurrence, while in vitro studies have demonstrated that the classical NF-κB signaling pathway promotes PCa progression and metastatic potential. More recently, the nuclear location of RelB, a member of the alternative NF-κB signaling, has also been shown to correlate with the Gleason score. The current study aims to clarify the role of alternative NF-κB in PCa cells by exploring, in vitro and in vivo, the effects of RelB overexpression on PCa biology.MethodsUsing a lentivirus-expression system, we constitutively overexpressed RelB or control GFP into 22Rv1 cells and monitored alternative transcriptional NF-κB activity. In vivo, tumor growth was assessed after the injection of 22Rv1-derived cells into SCID mice. In vitro, the impact of RelB on 22Rv1 cell proliferation was evaluated in monolayer culture. The anchorage-independent cell growth of derived-22Rv1 cells was assessed by soft agar assay. Apoptosis and autophagy were evaluated by Western blot analysis in 22Rv1-derived cells cultured in suspension using poly-HEMA pre-coated dishes.ResultsThe overexpression of RelB in 22Rv1 cells induced the constitutive activation of the alternative NF-κB pathway. In vivo, RelB expression caused a lag in the initiation of 22Rv1-induced tumors in SCID mice. In vitro, RelB stimulated the proliferation of 22Rv1 cells and reduced their ability to grow in soft agar. These observations may be reconciled by our findings that, when cultured in suspension on poly-HEMA pre-coated dishes, 22Rv1 cells expressing RelB were more susceptible to cell death, and more specifically to autophagy controlled death.ConclusionsThis study highlights a role of the alternative NF-κB pathway in proliferation and the controlled autophagy. Thus, the interplay of these properties may contribute to tumor survival in stress conditions while promoting PCa cells growth contributing to the overall tumorigenicity of these cells.

Fibroblast growth factor receptor 4: a putative key driver for the aggressive phenotype of hepatocellular carcinoma.

Recently, we found upregulation of fibroblast growth factor receptor 4 (FGFR4) in a subset of hepatocellular carcinoma (HCC). Here, we provide mechanistic insight into the role of FGFR4-mediated signalling for the aggressive behaviour of HCC cells. To overexpress FGFR4, hepatoma/hepatocarcinoma cells were transfected with a construct coding for FGFR4. For downmodulation of endogenous FGFR4, we used small interfering RNA or adenoviral infection with dominant-negative FGFR4 constructs being either kinase dead (kdFGFR4) or coding for the autoinhibitory soluble domain (solFGFR4). FGFR4 overexpression in non-tumourigenic hepatocarcinoma cells significantly reduced cell-matrix adhesion, enabled cells to grow anchorage-independently in soft agar, to disintegrate the lymph-/blood-endothelial barrier for intra-/extravasation of tumour cells and to form tumours in SCID mice. Transcriptome analysis revealed altered expression of genes involved in cell-matrix interactions. Conversely, in highly tumourigenic cell lines, kdFGFR4 or solFGFR4 lowered the proportion of cells in S phase of the cell cycle, enhanced the G0/G1 and G2/M-phase proportions, reduced anchorage-independent growth in vitro and attenuated disintegration of the lymph-/blood-endothelium and tumour formation in vivo. These findings were confirmed by altered expression profiles of genes being important for late stages of cell division. Deregulated FGFR4 expression appears to be one of the key drivers of the malignant phenotype of HCC cells. Accordingly, blockade of FGFR4-mediated signalling by soluble dominant-negative constructs, like solFGFR4, may be a feasible and promising therapeutic approach to antagonize aggressive behaviour of hepatoma/hepatocarcinoma cells.

Characterization of common marmoset dysgerminoma-like tumor induced by the lentiviral expression of reprogramming factors.

Recent generation of induced pluripotent stem (iPSCs) has made a significant impact on the field of human regenerative medicine. Prior to the clinical application of iPSCs, testing of their safety and usefulness must be carried out using reliable animal models of various diseases. In order to generate iPSCs from common marmoset (CM; Callithrix jacchus), one of the most useful experimental animals, we have lentivirally transduced reprogramming factors, including POU5F1 (also known as OCT3/4), SOX2, KLF4, and c-MYC into CM fibroblasts. The cells formed round colonies expressing embryonic stem cell markers, however, they showed an abnormal karyotype denoted as 46, X, del(4q), +mar, and formed human dysgerminoma-like tumors in SCID mice, indicating that the transduction of reprogramming factors caused unexpected tumorigenesis of CM cells. Moreover, CM dysgerminoma-like tumors were highly sensitive to DNA-damaging agents, irradiation, and fibroblast growth factor receptor inhibitor, and their growth was dependent on c-MYC expression. These results indicate that DNA-damaging agents, irradiation, fibroblast growth factor receptor inhibitor, and c-MYC-targeted therapies might represent effective treatment strategies for unexpected tumors in patients receiving iPSC-based therapy.

The American Pancreatic Association/Hirshberg Foundation Symposium Celebrating the 10th Anniversary of the Seed Grant Program

The Hirshberg Foundation for Pancreatic Cancer Research was founded by Agi Hirshberg in 1997, following her husband Ronald Hirshberg’s 8-month, 7-day battle with the disease. (http://www.pancreatic.org/). At the time, the Hirshberg family viewed pancreatic cancer as the ‘‘unknown enemy’’ (http:// www.pancreatic.org/site/c.htJYJ8MPIwE/b.8276835/k.66CB/ Agi_Hirshbergs_Story.htm). In the years since Ronald Hirshberg’s death, Agi Hirshberg has made it her mission to unmask the unknown for others facing the same enemy. When patients present with a new diagnosis of pancreatic cancer, most are already in an advanced stage of the disease. Pancreatic cancer is resistant to most chemotherapeutic agents and radiotherapy. These factors combine to make pancreatic cancer a highly lethal disease, and according to the National Cancer Institute (NCI) 2013 estimates, pancreatic cancer was diagnosed in approximately 45,220 US citizens, and some 38,460 died of the disease. 1 The Annual Report to the Nation on the Status of Cancer, 1975Y2009, shows that overall cancer death rates continued to decline in the United States among both men and women, among all major racial and ethnic groups, and for all of the many common cancer sites, including lung, colon and rectum, female breast, and prostate. However, this trend does not hold true for pancreatic cancer. The report demonstrates that death rates continued to increase during the latest time period (2000 through 2009) for pancreatic cancer. 2 Only about 23% of patients with cancer of the pancreas will still be living 1 year after diagnosis, and only about 4% will survive 5 years. Even for those diagnosed with pancreatic cancer that has not spread to other organs or systems, the overall 5-year survival rate approaches only 15%. The Hirshberg Foundation’s vision is to advance pancreatic cancer research and provide information, resources, and support to pancreatic cancer patients and their families. The mission of the Foundation is as follows: & To find a cure for pancreatic cancer in honor of Ron Hirshberg and the thousands of people who are diagnosed with this disease each year. & To create a premier Pancreatic Cancer Center where all needs of pancreatic cancer patients can be met in one location with the most advanced treatment options. & To be recognized as a patient support reference source for