Xenograft Model Of Pancreatic Ductal Adenocarcinoma Research Articles

Abstract B25: Development of 5B1, an anti-CA19.9 monoclonal antibody, as a near-infrared fluorescent probe for intraoperative imaging of pancreatic cancer

Abstract Background: For patients diagnosed with pancreatic ductal adenocarcinoma (PDAC), resection offers the best chance of survival. However, for those 20% of patients who qualify for resection, the 5-year survival rate is only 25% due to the high incidence of undiscovered metastases. Along with identifying metastases, properly defining the margin between tumor and healthy tissue is also a crucial determinant of the success of resection. The intraoperative use of near-infrared fluorescence (NIRF) imaging agents to facilitate the identification of previously undetected metastases and to define proper resection margins has the potential to improve the outcomes in patients. Monoclonal antibodies (mABs) are one promising platform for the development of NIRF imaging agents due to their high-specificity and low background in healthy tissues. CA19.9 is a well-established biomarker for PDAC, and 5B1, a fully human, anti-CA19.9 antibody has already shown promise both as a potential therapeutic and as a platform for development of a positron emission tomography agent. Based on previous studies we investigated 5B1’s potential as a NIRF imaging agent for use in an intraoperative setting. Methods: IRDye800CW-NHS and IRDye650-NHS were conjugated to 5B1 and a mouse IgG via lysine residues to generate 5B1-FL800, 5B1-FL650, and IgG-FL800. Each construct and the free acid of the IR800Dye were characterized using in vitro binding assays with two CA19.9-positive cell pancreatic cancer derived lines (BxPC3 and L3.6pl) and a negative cell line (PanC-1). The in vivo dose needed achieve an optimal signal to background ratio was determined in female, athymic nude mice with varying doses of 5B1-FL800. The potential of the constructs for use as NIRF agents for image-guided tumor identification and surgery was examined in a subcutaneous xenograft model using the previously mentioned cell lines as well as an L3.6pl orthotopic pancreas xenograft model. Images were obtained at 24, 48, 96, and 144 hours (750nm excitation/800nm emission). Post mortem, NIRF image-guided surgery was performed to identify and remove the tumor tissue and the relative uptake was compared to healthy tissue. Immunohistochemistry (IHC) as well as hematoxylin and eosin staining (H&E) were performed on excised tumors to confirm specific uptake in tumor tissue. Results: The NIRF dyes were conjugated to 5B1 and a non-specific mouse IgG with a dye:mAB ratio of 1.1−3.2 (>98% purity). In vitro binding assays showed negligible binding to any cell line by the NIRF-labeled IgG and unconjugated dye. The two NIRF 5B1 constructs bound to both CA19.9 positive cell lines with low nanomolar affinity while no specific binding to PanC1 was observed. Specific binding could be inhibited by addition of excess 5B1. The binding was visualized in BxPC3 and L3.6pl cells by fluorescence microscopy. Pilot imaging studies in a small cohort of mice showed that 5B1-FL800 was superior to 5B1-FL650 in achieving a high signal-to-background ratio in vivo and that 0.5 nmol per mouse dose was an optimal dose for in vivo imaging. Imaging studies showed excellent tumor uptake in CA19.9-positive tumor bearing mice with an optimal tumor:background ratio being achieved at 96 hours post injection. The ratio of NIRF signal in the excised tumors was 8-30x higher than any healthy organ. The 5B1-800FL signal was used to successfully identify and guide the removal of tumor tissue from mice, including small portions of the tumor that were invisible to the naked eye. IHC and HE staining confirmed specific binding of the NIRF-conjugated 5B1 constructs to CA19.9 positive tumor tissue. Conclusions: IRDye800CW and IRDye650 were conjugated to the anti-CA19.9, fully-human mAB, 5B1, which was successfully used for in vivo NIRF imaging in both subcutaneous and orthotopic xenograft models of PDAC. 5B1 demonstrated exceptional potential for use in delineation of pancreatic tumor margins, NIRF guided-resection, and identification of metastases overlooked by other imaging techniques. Citation Format: Jacob L. Houghton, Dalya A. Abdel-Atti, Ritsuko Sawada, Wolfgang W. Scholz, Jason S. Lewis. Development of 5B1, an anti-CA19.9 monoclonal antibody, as a near-infrared fluorescent probe for intraoperative imaging of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B25.

Efficacy and safety of the hypoxia-activated prodrug TH-302 in combination with gemcitabine and nab-paclitaxel in human tumor xenograft models of pancreatic cancer

Tumors often contain hypoxic regions resistant to chemo- and radiotherapy. TH-302 (T) is an investigational hypoxia-activated prodrug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. This study evaluated the efficacy and safety profile of combining T with gemcitabine (G) and nab-paclitaxel (nP) in human pancreatic ductal adenocarcinoma (PDAC) xenograft models in mice. Antitumor activity of the G + nP + T triplet was assessed and compared with T-alone or the G + nP doublet in the Hs766t, MIA PaCa-2, PANC-1, and BxPC-3 PDAC xenograft models. Efficacy was assessed by tumor growth kinetic analysis. Body weight, blood cell counts, blood chemistry, and the von Frey neuropathy assay were analyzed to evaluate safety profiles. Pharmacodynamic changes after the treatment were determined by immunohistochemistry of cell proliferation, DNA damage, apoptosis, hypoxia, and tumor stroma density. The G + nP + T triplet exhibited enhanced efficacy compared with T-alone or the G + nP doublet. Compared with vehicle (V), G + nP induced body weight loss, reduced neutrophil and lymphocyte counts, increased the levels of liver function parameters, and induced neurotoxicity. However, when T was added to G + nP, there was no statistically increased impairment compared to G + nP. The triplet significantly increased DNA damage, apoptosis, and tumor necrosis. Furthermore, the triplet further inhibited cell proliferation and reduced stroma density and intratumoral hypoxia. The triplet combination of G + nP + T exhibited superior efficacy but additive toxicity was not evident compared to the G + nP doublet in this study. This study provides a translational rationale for combining G, nP, and T in the clinical setting to assess efficacy and safety. A Phase I clinical trial of the triplet combination is currently underway (NCT02047500).

Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302.

BackgroundHypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models.ResultsThree human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized 13C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors.ConclusionsUsing metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s40170-014-0026-z) contains supplementary material, which is available to authorized users.

Abstract 2691: Smo-targeted sonic hedgehog signaling inhibitor enhances therapeutic antibody deposition and efficacy in a patient-derived pancreatic ductal adenocarcinoma model

Abstract Tumor stroma supports growth, invasion, and treatment resistance of pancreatic ductal adenocarcinoma (PDAC), and in conjunction with hypovascularity, constitutes a barrier that minimizes tumor cell drug exposure. Targeting tumor stroma is an attractive strategy in PDAC, and Smo-binding inhibitors are reported to mediate stromal thinning and increased microvessel density in PDAC models. No information exists as to the effect of sHHI upon deposition of macromolecular therapeutic agents such as monoclonal antibodies (mAb). Therefore we investigated effects of sHHI LDE225 upon tumor vascularity, mAb deposition, and mAb therapeutic efficacy in a patient-derived xenograft (PDX) model of PDAC in SCID mice. PDAC tumor #18269 was established in SCID mice and has retained abundant stroma and low microvessel density reminiscent of the original patient tumor. It expresses EGF receptor. Tumor-bearing mice were dosed orally with LDE225 (40 mg/kg) for 10 days, and pharmacodynamic biomarkers of sHHI effect were quantified. Mouse-specific TaqMan probes demonstrated high Gli1 expression in stromal cells, which was deeply suppressed within 3 days of sHHI dosing. Human-specific probes showed little Gli1 expression in tumor cells and no effect of sHHI treatment. Over 10 days of sHHI treatment, tumor microvessel density did not change significantly (based on CD31 staining), but the fraction of functional vessels increased significantly by day 7 (based on staining with fluorescent Lycopersicon esculentum lectin injected IV prior to tumor harvest). Fluorescently labeled mAbs that were tumor-targeted (cetuximab; anti-EGFR) or non-targeted (anti-topotecan) were co-injected on d7 of sHHI treatment. Compared to untreated controls, tumor exposure (AUC) of cetuximab more than doubled, and was observed to distribute to regions of the tumor enriched in tumor cells. Deposition of non-targeted mAb also was increased relative to controls, but distributed more to stromal areas than tumor cell areas. To investigate whether the enhanced deposition of cetuximab mediated by sHHI pretreatment would enhance efficacy, cetuximab was administered on d 5, 8, 11 of a 10-day sHHI or vehicle (control) regimen. This sequence was repeated 6 times, with an inter-cycle gap of 5 days. Rituximab (anti-human CD20) was the non-targeted control. Cetuximab alone showed no therapeutic benefit. However, cetuximab combined with sHHI suppressed tumor progression for more than 80 days, and median time to study endpoint was more than 250% longer than for all other treatment groups except sHHI-alone, which mediated a 30% increase. Conclusions: increased perfusion and vascular permeability mediated by sHHI pretreatment permits access of therapeutic mAb to otherwise inaccessible regions of the tumor interior, and reveals anti-tumor activity of a mAb that previously failed clinically for PDAC treatment. Citation Format: Jun Wang, Yang Qu, Rose Pitoniak, Sheryl A. Trueman-Fatallah, Bonnie L. Hylander, Elizabeth A. Repasky, Wen Wee Ma, Robert M. Straubinger. Smo-targeted sonic hedgehog signaling inhibitor enhances therapeutic antibody deposition and efficacy in a patient-derived pancreatic ductal adenocarcinoma model. [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 2691. doi:10.1158/1538-7445.AM2014-2691

Abstract 5463: ICG-001 induces cell cycle arrest to inhibit in vitro and in vivo pancreatic cancer growth

Abstract Wnt/β-catenin signaling is an important mediator of pancreatic ductal adenocarcinoma (PDAC) initiation and progression, warranting its exploration as a target of therapeutic intervention. Wnt modulators with acceptable in vivo bioavailability and efficacy as Wnt/β-catenin transcriptional inhibitors have only recently become available and include the small molecule compound ICG-001. Discovered in a library screen for inhibitors of Wnt/β-catenin reporter activity, ICG-001 interacts with cyclic AMP-response element-binding protein (CBP) to block its function as a co-transcriptional activator of β-catenin/TCF-mediated transcription. ICG-001 inhibits both in vitro and in vivo colon cancer growth and has demonstrated efficacy in a variety of other in vivo Wnt-driven disease models. Having recently shown subsets of PDAC tumors are dependent on increased levels of Wnt ligand-dependent autocrine signaling, we addressed the efficacy and function of ICG-001 on in vitro and in vivo PDAC growth. As a single agent, ICG-001 significantly inhibited anchorage-dependent and -independent growth of PDAC cell lines in vitro and increased survival in an orthotopic xenograft model of PDAC (HR=0.207, 95%CI: 0.079-0.543, P=0.001). ICG-001 also synergized with gemcitabine to inhibit anchorage-dependent growth in vitro and improve survival in the orthotopic xenograft model (HR= 0.074, 95%CI: 0.026, 0.212, P<0.001). Mechanistically, ICG-001 treatment did not significantly increase apoptosis in PDAC cell lines, but instead dramatically inhibited cell cycle progression via G1 arrest. Although ICG-001 inhibited β-catenin/TCF-mediated transcription as measured by luciferase-based reporter assays and endogenous target gene expression, these changes occurred at concentrations above those seen to induce G1 cell cycle arrest, suggesting ICG-001 growth inhibitory effects may be unrelated to its specific effects as a Wnt pathway inhibitor. Gene expression microarray analysis revealed ICG-001 treatment alters the expression of known Wnt target genes, as well as a much larger number of genes involved in DNA replication and cell cycle control. In conclusion, this study indicates ICG-001 is a promising small-molecule therapeutic for pancreatic adenocarcinoma and identifies numerous target genes whose altered expression provide insights into mechanism of action and candidate biomarkers for following therapeutic response. . Citation Format: Michael D. Arensman, Anna R. Lay, David W. Dawson. ICG-001 induces cell cycle arrest to inhibit in vitro and in vivo pancreatic cancer growth. [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 5463. doi:10.1158/1538-7445.AM2014-5463

Abstract C287: Efficacy and safety of the TH-302, gemcitabine, and nab-paclitaxel combination in xenograft models of pancreatic cancer.

Abstract Background: Tumors often consist of hypoxic regions which are resistant to chemo- and radiotherapy. TH-302 (T) is an investigational hypoxia-targeted drug that selectively releases the DNA cross-linker bromo-isophosphoramide mustard under hypoxic conditions. A randomized Phase 2 clinical trial in pancreatic ductal adenocarcinoma (PDAC) demonstrated a significant increase in progression-free survival from the addition of T to gemcitabine (G) vs. G only (NCT01144455). A Phase 3 trial of G + T compared to G + placebo is currently underway (NCT01746979). G + nab-Paclitaxel (nP) significantly prolonged overall survival in a recent Phase 3 trial (NCT00844649). We investigated the efficacy and safety profile of combining T with G and nP in PDAC xenograft models. Methods: Four PDAC xenograft models were established by s.c. implantation of Hs766t, MIA PaCa-2, PANC-1 and BxPC-3 cells into the flank of nude mice. A ‘same day’ administration of T, nP and G with a Q3Dx5 regimen was employed: T, 50mg/kg, ip; nP: 30mg/kg, iv; and G: 60mg/kg ip. Saline treated animals served as vehicle control (V). Antitumor activity of the G + nP + T triplet was assessed, and compared with the G + nP doublet. Hematological, blood chemistry, and immunohistochemical-based analyses were also performed in PANC-1 bearing nude and/or CD-1 mice. Samples were collected 24 hours after the last treatment. Neuropathy assays were performed in CD-1 mice. Results: Using tumor growth inhibition, and Kaplan-Meier analysis of median time to tumor size of 1000mm3 to evaluate antitumor activity, G + nP + T triplet exhibited enhanced efficacy compared with T-alone or the G + nP in all four models. Of note, the complete response rate (CR) of G + nP + T in the PANC-1 model reached 100%, compared to 10% and 50% in the T-alone and G + nP, respectively. G + nP or T-alone treatment reduced neutrophils or lymphocytes compared with V, but there was no additional decrease in neutrophil or lymphocyte count when T was added to G + nP compared to G + nP. Plasma ALT and AST levels were elevated after G + nP treatment, and G + nP + T did not further increase the levels of these liver function parameters. By immunostaining, Ki67-positive cells (a marker of cell proliferation) were significantly reduced after the drug treatment in the PANC-1 xenograft, and to a greater magnitude in G + nP + T. The necrotic region in G + nP + T was increased compared with all other groups. Employing the von Frey neuropathy assay, G + nP treated mice exhibited mechanical allodynia in the hind paw 9 days after initiation of treatment whereas mice treated with T did not, and there was no additive neuropathy in G + nP + T. Conclusions: The triplet combination of G + nP + T exhibited superior efficacy but additive toxicity was not evident compared to G + nP. These studies provide a translational rationale for combining G, nP, and T in the clinical setting to assess efficacy and safety. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C287. Citation Format: Jessica D. Sun, Qian Liu, Dharmendra Ahluwalia, Wenwu Li, Yan Wang, Ayesha S. Ruprell, Charles P. Hart. Efficacy and safety of the TH-302, gemcitabine, and nab-paclitaxel combination in xenograft models of pancreatic cancer. [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 C287.

Autophagy and enhanced chemosensitivity in experimental pancreatic cancers induced by noninvasive radiofrequency field treatment

Patients with pancreatic ductal adenocarcinoma (PDAC) have limited therapeutic options and poor response to the standard gemcitabine (GCB)-based chemotherapy. In the current study, the authors investigated the feasibility of noninvasive short-wave radiofrequency (RF) electric fields to improve the cytotoxic effect of GCB on PDAC cells and determined its mechanism of action. The cytotoxicity of RF alone and in combination with GCB was studied in vitro on normal pancreatic human pancreatic ductal epithelial cells and different PDAC cell lines by flow cytometry, and in vivo on ectopic and orthotopic human PDAC xenograft models in mice. The mechanism of RF activity was studied by Western blot analysis and immunohistochemistry. Toxicity was determined by histopathology. Exposure of different PDAC cells to 13.56-megahertz radio waves resulted in a substantial cytotoxic effect, which was accompanied by the induction of autophagy but not apoptosis. These effects of RF were found to be absent in normal cells. Excessive numbers of autophagosomes in cancer cells persisted 24 to 48 hours after RF exposure and then declined. The addition of a subtoxic dose of GCB to RF treatment inhibited the recovery of cancer cells from the RF-induced autophagy and enhanced the cytotoxic effect of the latter on cancer cells. The treatment of PDAC in situ in mice with the combination of noninvasive RF and GCB was found to have a superior antitumor effect compared with the use of RF or GCB alone, yet there was no evidence of systemic toxicity. Noninvasive RF treatment induced autophagy but not apoptosis in cancer cells and demonstrated potential as an enhancer of chemotherapy for treating patients with pancreatic cancer without toxicity to normal cells.

Abstract 935: Inhibition of pancreatic cancer growth via MEK-targeted therapy is significantly enhanced by concomitant inhibition of EGFR and Her2.

Abstract Introduction: MAPK and EGFR family signaling promote tumor proliferation in pancreatic ductal adenocarcinoma (PDAC). Previously we have shown that lapatinib (EGFR/Her2 inhibitor) significantly improved the ability of trametinib (MEK1/2 inhibitor) to inhibit PDAC tumor growth. Here, we investigated whether combined panitumumab (EGFR inhibitor), trastuzumab (Her2 inhibitor) and trametinib treatment would more effectively inhibit tumor growth than trametinib monotherapy in a patient-derived (PD), orthotopic, xenograft model of PDAC. Methods: In vitro proliferation assays were performed with PD-PDAC cell line MAD09-366 exposed to trametinib, panitumumab, trastuzumab, and combination therapies. Western blot analysis was performed on treated cell lysates. Athymic, nude mice were orthotopically implanted with 3 different PD-PDAC xenografts (MAD09-366, 08-608, and 08-738). Established murine tumors were treated with control, trametinib (0.3mg/kg,PO, qDay), panitumumab (500ug, IP, BIW), trastuzumab (200ug, IP, BIW) or combination for 2-4 weeks. Interval volumetric MRI was used to measure tumor growth. Phospho-RTK and MAPK arrays were used to assess tumor molecular response. Results: In vitro studies demonstrated improved growth inhibition of MAD09-366 (Kras mut.) cells exposed to combination therapy with all 3 inhibitors relative to control or each inhibitor alone. Western blot analysis revealed that EGF stimulation increased Ras pathway signaling in this Kras mutant cell line. Under conditions of EGF stimulation, the greatest inhibition of Ras pathway signaling was seen in cells exposed to all 3 inhibitors. In vivo studies in all PD-PDAC xenografts revealed that triple inhibitor therapy significantly decreased the rate of tumor growth relative to control, trametinib alone, panitumumab alone, or panitumumab plus trastuzumab. In two of three PD-PDACs assessed, triple therapy was superior to trametinib plus panitumumab. The greatest response was seen in MAD08-738 (Kras wt) triple-therapy-treated mice whose tumor size decreased by 9.3%. Phospho-array analysis demonstrated increased activity of Akt and its downstream effectors in trametinib treated tumors. Relative Akt phosphorylation returned to levels seen in control treated tumors with the addition of panitumumab and trastuzumab under triple therapy. Conclusions: Combination therapy with trametinib, panitumumab, and trastuzumab produced the most complete in vitro Ras signaling blockade and most effective in vivo growth inhibition. Phospho-array data suggests that a compensatory increase in pro-survival PI3K/Akt signaling in response to Ras pathway blockade alone is mitigated by the concomitant inhibition of EGFR/Her2 signaling. This combination treatment strategy should be considered for a future clinical trial in pancreatic cancer patients. Citation Format: James M. Lindberg, Timothy E. Newhook, Sara J. Adair, Clifford A. Cutchins, Alison Kim, J. Thomas Parsons, Todd W. Bauer. Inhibition of pancreatic cancer growth via MEK-targeted therapy is significantly enhanced by concomitant inhibition of EGFR and Her2. [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 935. doi:10.1158/1538-7445.AM2013-935

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Antitumoral activity of parvovirus-mediated IL-2 and MCP-3/CCL7 delivery into human pancreatic cancer: implication of leucocyte recruitment

Pancreatic ductal adenocarcinoma (PDAC) represents the fourth leading cause of cancer-related death in western countries. The patients are often diagnosed in advanced metastatic stages, and the prognosis remains extremely poor with an overall 5-year survival rate less than 5 %. Currently, novel therapeutic strategies are being pursued to combat PDAC, including oncolytic viruses, either in their natural forms or armed with immunostimulatory molecules. Natural killer cells are critical players against tumours and infected cells. Recently, we showed that IL-2-activated human NK cells displayed killing activity against PDAC cells, which could further be enhanced through the infection of PDAC cells with the rodent parvovirus H-1PV. In this study, the therapeutic efficacy of parvovirus-mediated delivery of three distinct cyto/chemokines (Il-2, MCP-3/CCL7 and IP-10/CXCL10) was evaluated in xenograft models of human PDAC. We show here that activated NK and monocytic cells were found to be recruited by PDAC tumours upon infection with parvoviruses armed with IL-2 or the chemokine MCP-3/CCL7, resulting in a strong anti-tumour response.

Abstract 1326: Magnetization transfer MRI in pancreatic cancer xenograft models

Abstract Tumor desmoplasia is a common hallmark of pancreatic ductal adenocarcinoma (PDAC), the 4th leading cause of cancer-related mortality. Recent studies clearly indicate that desmoplasia actively facilitates disease progression, metastasis, and drug resistance. Precise quantification of tumor fibrosis levels in PDAC may be critically important for patient staging and for the prediction or monitoring of responses to therapy. The current gold-standard for characterizing desmoplasia_histologic analysis of biopsy samples_is invasive and cannot avoid misrepresentation by reason of sampling error. Therefore, new methods to permit noninvasive quantification of fibrosis levels in PDAC would be particularly useful. Magnetization transfer (MT) MRI can be used to non-invasively probe dynamic physical processes involving the exchange of magnetization between sub-populations of free water protons and those water protons bound to tissue macromolecules. MT-MRI studies have demonstrated that MT contrast (MTC) can be highly sensitive to tissue collagen concentration, the primary component of desmoplasia. Thus, we hypothesized that desmoplasia-associated fibrosis levels in PDAC may be directly reflected in MT-MRI magnetization transfer ratio (MTR) measurements. The purpose of this study was to compare non-invasive MTR measurements to invasive histologic assessments of tumor fibrosis-levels in three mouse xenograft models of PDAC. In this study, MT MRI measurements were performed in 3 PDAC mouse xenograft models. For each of 28 PDAC xenografts, MTR were calculated and compared to histologic fibrosis levels from reference standard trichrome staining. MTR was found to be significantly higher in tumors grown using BxPC-3 cell line (39.4 ± 5.1, mean ± SD) compared to the MTR for the tumors grown from Panc-1 (32.4 ± 2.8) and Capan-1 (27.3 ± 2.9) cell lines (P < 0.05 for each comparison). Histologic measurements showed a similar trend with BxPC-3 tumors demonstrating significantly higher fibrosis levels (percentage of fibrotic tissue area, 6.48 ± 2.59) when compared to Panc-1 (3.54 ± 2.18) and Capan-1 (2.07 ± 1.60) tumors. MTR measurements were well correlated to quantitative fibrosis levels (r = 0.69, P = 0.01). Our results indicated that MTR measurements offer the potential to serve as a valuable in vivo biomarker of desmoplasia in PDAC. 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 1326. doi:1538-7445.AM2012-1326

An Epigenetic Approach to Pancreatic Cancer Treatment: The Prospective Role of Histone Deacetylase Inhibitors

Pancreatic ductal adenocarcinoma (PDAC) is quite resistant to conventional treatments, and gemcitabine, the standard chemotherapeutic agent, offers only a small benefit. Development and progression of PDAC are complex processes involving dysregulation of multiple signal transduction pathways arising from not only genetic but also epigenetic alterations. This makes the epigenetic approach to the treatment of PDAC of great interest. Histone deacetylases, a family of enzymes that, by removal of acetyl groups from a variety of histone and nonhistone proteins, play an important role in the epigenetic regulation of gene expression, are frequently dysregulated in PDAC. In particular, overexpression of class I histone deacetylases has been related to higher tumor grade, poor prognosis and development of chemoresistance. Histone deacetylase inhibitors (HDACIs), a novel class of agents endowed with pleiotropic antitumor effects, appear promising either for their preferential toxicity towards transformed as compared to normal cells and their ability to synergistically enhance the anticancer activity of radiotherapy and many chemotherapeutic agents. Many HDACIs have been shown to increase the antiproliferative and proapoptotic effects of gemcitabine, 5-fluorouracil and bortezomib, a new proteasome inhibitor, in vitro and in vivo PDAC xenograft models. MGCD0103, romidepsin, panobinostat, vorinostat and valproic acid, are currently being tested in association with radiotherapy or chemotherapy (gemcitabine, fluoropyrimidines, proteasome inhibitors) in phase I-II clinical trials in patients with locally advanced or metastatic PDAC.

Abstract C173: APE1/Ref-1-STAT3 dual-targeting synergize to effectively inhibit pancreatic cancer cell survival.

Abstract Pancreatic cancer remains a largely incurable disease, with patients facing the worst 5-year survival rate of any cancer. The challenge is to identify the molecular effectors that critically regulate the survival of pancreatic ductal adenocarcinoma (PDAC) cells, to devise effective molecular-targeted strategies that can prevent or minimize the selection of resistant tumor variants, and overcome the protective role of the tumor-associated fibrosis and stroma. Increasing evidence supports the need for strategies targeting multiple molecular effectors in PDAC. Thus, a strategy is to identify critical molecules that regulate multiple signaling mediators (as transcription factors), and on intracellular mechanisms with direct effects on multiple pathways (as antioxidant and redox mediators) critical for PDAC functions. APE1/Ref-1 is a dual function protein, which in addition to DNA repair activity also exerts redox control of transcription factors, including NF- B, p53, AP-1, CREB, HIF-1 and others. Treatment with E3330, a unique small molecule redox signaling inhibitor that recognizes an alternate, redox active conformation of APE1/Ref-1, markedly inhibits the DNA binding and transcriptional activity of NF-κB, AP-1, and HIF-1. Our previous work established APE1/Ref-1 as a potential molecular target in pancreatic cancer. We demonstrated that human adenocarcinoma and peri-pancreatic metastases exhibit increased APE1/Ref-1 expression, and blocking APE1/Ref-1 redox activity delays tumor progression in xenograft models of PDAC, including patient-derived tumor cells. STAT3 is a transcription factor that regulates critical cell functions and has been shown to play important roles in several cancers. STAT3 signaling has been implicated in pancreatic cancer biology, namely by mediating or regulating cell survival, proliferation, tumor angiogenesis and metastasis. Although STAT3 signaling can be engaged and modulated by different processes, the mechanisms regulating STAT3 transcriptional activity in PDAC cells are largely unknown, namely the impact of oxidative stress and its redox status. A recent report demonstrated that STAT3 activity is under redox control and identified the critical oxidation-sensitive cysteines in the STAT3 DNA binding domain. However, the modifier of STAT3 which converts it from an oxidized to a reduced form was not identified. It has been shown that APE1/Ref-1 physically interacts with STAT3 on the VEGF promoter and enhances IL-6-induced DNA binding activity of STAT3 in HepG2 cells. However, it is unknown whether APE-1/Ref-1 is involved in the redox control of STAT3 activity, and whether the cellular redox status affects STAT3 signaling in PDAC cells. Here, we demonstrate that APE1/Ref-1 redox activity regulates the STAT3 DNA binding and transcriptional activity using gene silencing, overexpression of WT or redox-defective APE1/Ref-1, and redox-selective pharmacological inhibition. Blockade of APE1/Ref-1 redox synergizes with STAT3 selective antagonists to markedly inhibit the proliferation and survival of human PDAC cells, inducing cell apoptosis. These studies identify the mechanism by which APE1/Ref-1 regulates STAT3 signaling, and establishes the rationale for the development of APE1/STAT3 dual-targeting strategies for the treatment of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C173.

EMAP II-Based Antiangiogenic-Antiendothelial In Vivo Combination Therapy of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) frequently resists conventional cytotoxic therapy. The antitumor effects of endothelial monocyte-activating polypeptide II (EMAP) have been attributed to its antiendothelial and antiangiogenic activities. We tested the hypothesis that a combination of EMAP with bevacizumab (Bev) and gemcitabine (Gem) targets different pathways of PDAC progression and represents more effective treatment. Proliferation of PDAC and endothelial cell lines was evaluated in vitro. In vivo tumor growth and survival PDAC xenograft experiments were performed with EMAP, Bev, and Gem, either alone or in combination. Intratumoral microvessel density and proliferative activity were analyzed by immunostaining with PECAM-1 and proliferating cell nuclear antigen antibodies, and apoptotic activity was measured by the TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) procedure. Compared with controls, net reduction in tumor growth in EMAP, Bev, Gem, EMAP + Bev, EMAP + Gem, Bev + Gem, and EMAP + Bev + Gem groups was 58, 40, 40, 67, 68, 69, and 96%, respectively. Addition of EMAP to the Bev + Gem group statistically significantly improved survival at a median of >8 days while inducing long-term survival in some animals after maintenance therapy. Combination treatment of EMAP with Bev and Gem reduced proliferation of endothelial but not of PDAC cells. Addition of EMAP to Bev and Gem statistically significantly decreased proliferative activity while maintaining a comparable rate of microvessel density and apoptosis. Addition of antiendothelial EMAP to a Bev and Gem regimen improves antitumor effects in a xenograft model of PDAC. This multitargeting strategy to prevent PDAC progression shows therapeutic promise and may overcome limitations by combinations of Gem with anti-vascular endothelial growth factor agents alone.