Orthotopic Pancreatic Xenografts Research Articles

Impact of the PI3K-alpha inhibitor alpelisib on everolimus resistance and somatostatin receptor expression in an orthotopic pancreatic NEC xenograft mouse model.

The mechanistic target of rapamycin complex 1 (mTORC1) inhibitor everolimus is one of the few approved therapies for locally advanced and metastatic neuroendocrine tumours (NETs). However, after initial disease stabilisation, most patients develop resistance within 1 year. Our aim was to overcome resistance to everolimus by additional treatment with the PI3K-alpha inhibitor alpelisib in an everolimus-resistant orthotopic pancreatic neuroendocrine carcinoma xenograft mouse model. Female SCID mice underwent laparoscopic pancreatic transplantation of everolimus-sensitive (BON1KDMSO) or everolimus-resistant (BON1RR2) NET cells. Both groups were further divided into four treatment groups: placebo, everolimus, alpelisib, and everolimus + alpelisib (combination). Oral treatment was started at a tumour volume of approximately 140 mm3 and continued until 1900-2000 mm3, validated by weekly MRI. Somatostatin receptor expression and tumour viability were analysed by 68Ga-DOTATOC and 18F-FDG PET/CT. Everolimus resistance of the BON1RR2 tumours was confirmed. In the everolimus-sensitive group, everolimus alone, alpelisib alone, and combination treatment significantly prolonged survival, compared to placebo, while in the BON1RR2 group, only combination treatment significantly prolonged survival compared to placebo, but neither everolimus nor alpelisib alone. Placebo-treated everolimus-sensitive tumours grew more rapidly (median survival 45 days), compared to placebo-treated everolimus-resistant tumours (60 days). Within the everolimus-sensitive group, the combination-treated mice showed the longest median survival (52 days). Of all groups, the everolimus-resistant combination-treated group survived longest (69 days). Combination treatment with everolimus and alpelisib seems promising to overcome everolimus resistance in neuroendocrine neoplasms, and should be further examined in a clinical trial.

ImmunoPET Imaging of Pancreatic Tumors with 89Zr-Labeled Gold Nanoparticle-Antibody Conjugates.

Targeted delivery in vivo remains an immense roadblock for the translation of nanomaterials into the clinic. The greatest obstacle is the mononuclear phagocyte system (MPS), which sequesters foreign substances from general circulation and causes accumulation in organs such as the liver and spleen. The purpose of this study was to determine whether attaching an active targeting antibody, 5B1, to the surface of gold nanoparticles and using clodronate liposomes to deplete liver and splenic macrophages could help to minimize uptake by MPS organs, increase targeted delivery to CA19.9-positive pancreatic tumors, and enhance pancreatic tumor delineation. To produce the antibody-gold nanoparticle conjugate (Ab-AuNP), the Ab was conjugated to p-isothiocyanatobenzyl-desferrioxamine (p-SCN-DFO) and subsequently conjugated to NHS-activated gold nanoparticles. The Ab-AuNP was characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Modified Lindmo assay was performed to assess binding affinity and internalization potential in vitro. The Ab-AuNP was radiolabeled with 89Zr and injected into CA19.9-positive BxPc-3 pancreatic orthotopic tumor-bearing mice pretreated with or without clodronate liposomes for PET imaging and biodistribution studies. Inductively coupled plasma-optical emission spectrometry (ICP-OES) analysis was used to confirm delivery of gold nanoparticles to BxPc-3 pancreatic subcutaneous xenografts. Mice pretreated with clodronate liposomes in an orthotopic setting demonstrated decreased liver uptake at early time points (12.2 ± 2.3% ID/g vs. 22.8 ± 3.8% ID/g at 24h) and increased tumor uptake at 120h (13.8 ± 8.0% ID/g vs. 6.0 ± 1.2% ID/g). This allowed for delineation of orthotopic pancreatic xenografts in significantly more mice treated with clodronate (6/6) than in mice not treated with clodronate (2/6) or mice injected with gold nanoparticles labeled with a nonspecific antibody (0/5). The combination of clodronate liposomes and an active targeting antibody on the surface of gold nanoparticles allowed for PET/CT imaging of subcutaneous and orthotopic pancreatic xenografts in mice.

Abstract 4060: Discovery and preclinical development of KRAS-targeting siRNA therapeutic modality for the treatment of pancreatic cancer

Abstract Pancreatic cancer is the third most common cause of death from cancer in the US. The disease has a poor prognosis with a five-year survival rate under 10%. Mutations in KRAS are driving tumor growth in about 95% of patients with pancreatic cancer. Currently there are no approved drugs that target KRAS. Recently there has been progress targeting a specific mutation, KRAS G12C with small molecules in lung cancer, but this mutation accounts for only 1% of patients with Pancreatic Ductal Adenocarcinoma (PDAC). RNA interference has the potential to directly target KRAS, but delivery to pancreas has been challenging. We have developed a lipid nanoparticle formulation that is able to distribute to pancreatic tumor tissue. The proprietary lipid nanoparticle (LNP) is comprised of an ionizable, non-immunogenic, biodegradable lipid and the composition has been tuned to favor enrichment in pancreatic tumor by screening in orthotopic pancreatic tumor mouse models. A potent siRNA with IC50 in the range of 10pM targeting KRAS directly has been identified and the activity on downstream pathways and kinetics have been demonstrated. Transfection of ND-05-1192 led to more impeded cell growth in pancreatic cancer cells (PANC-1, AsPC-1, Capan-1, SW1990 and BxPC-3) than normal cells (human hepatocyte, pancreatic cells and HUVEC cells). Western blot results demonstrated effective downregulations of key proteins involved in MAPK and PI3K signaling pathways and upregulated activities of proteins involved in apoptotic signaling pathways, such as BIM, caspase 3/7 and cleaved-PARP following inhibition of KRAS protein expression. In vivo biodistribution and tumor cell uptake study results demonstrated that LNP transported ND-05-1192 siRNA molecules to the pancreas site and pancreatic orthotopic tumor tissues with the majority of tumor cells showing LNP product uptake. The application of ND-05-1192/LNP product significantly suppressed tumor growth in PANC-1 and AsPC-1 bioluminescent orthotopic pancreatic xenograft models. Inhibition efficiency revealed by tumor weight and bioluminescent response (BLR) as the treatment end points reached up to 50% in the PANC-1 orthotopic xenografts following once weekly intravenous administration. In the orthotopic AsPC-1 model, the average BLR was significantly reduced in a dose-dependent manner. Additionally, in AsPC-1 orthotopic xenografts, the survival rate of the ND-05-1192/LNP treatment group was significantly prolonged at a 4 mg/kg dose compared to the vehicle control group. In summary, we conclude that ND-05-1192/LNP is delivered to the orthotopic pancreatic tumor and its considerable uptake by tumor cells results in KRAS target downregulation, tumor growth inhibition and overall survival benefit as demonstrated in our orthotopic xenograft models. Citation Format: Zhihong (Julie) OBrien, Li Wang, Cima Cina, Bharat Majeti, James Chu, Xiaoen Wang, Robiel Baclig, James Lin, Jose Ibarra, Dhrubamitra Chatterjee, Jung-Kang Jin, Jean-Pierre Clamme, Jennifer Daniels, Hai Wang, Jens Harborth, Wenbin Ying. Discovery and preclinical development of KRAS-targeting siRNA therapeutic modality for the treatment of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4060.

Multimodal Targeted Nanoparticle-Based Delivery System for Pancreatic Tumor Imaging in Cellular and Animal Models.

Pancreatic ductal adenocarcinoma (PDAC), which ranks forth on the cancer-related death statistics still is both a diagnostic and a therapeutic challenge. Adenocarcinoma of the exocrine human pancreas originates in most instances from malignant transformation of ductal epithelial cells, alternatively by Acinar-Ductal Metaplasia (ADM). RA-96 antibody targets to a mucin M1, according to the more recent nomenclature MUC5AC, an extracellular matrix component excreted by PDAC cells. In this study, we tested the usability of multimodal nanoparticle carrying covalently coupled RA-96 Fab fragments for pancreatic tumor imaging. In order to make and evaluate a novel, better targeting, theranostic nanoparticle, iron nanoparticles and the optical dye indocyanin green (ICG) were encapsulated into the cationic sphingomyelin (SM) consisting liposomes. RA-96 Fab fragment was conjugated to the liposomal surface of the nanoparticle to increase tumor homing ability. ICG and iron nanoparticle-encapsulated liposomes were studied in vitro with cells and (i) their visibility in magnetic resonance imaging (MRI), (ii) optical, (iii) Magnetic particle spectroscopy (MPS) and (iv) photoacoustic settings was tested in vitro and also in in vivo models. The targeting ability and MRI and photoacoustic visibility of the RA-96-nanoparticles were first tested in vitro cell models where cell binding and internalization were studied. In in vivo experiments liposomal nanoparticles were injected into the tail vain using an orthotopic pancreatic tumor xenograft model and subcutaneous pancreatic cancer cell xenografts bearing mice to determine in vivo targeting abilities of RA-96-conjugated liposomes Results: Multimodal liposomes could be detected by MRI, MPS and by photoacoustic imaging in addition to optical imaging showing a wide range of imaging utility. The fluorescent imaging of ICG in pancreatic tumor cells Panc89 and Capan-2 revealed an increased association of ICG-encapsulated liposomes carrying RA-96 Fab fragments in vitro compared to the control liposomes without covalently linked RA-96. Fluorescent molecular tomography (FMT) studies showed increased accumulation of the RA96-targeted nanoparticles in the tumor area compared to non-targeted controls in vivo. Similar accumulation in the tumor sites could be seen with liposomal ferric particles in MRI. Fluorescent tumor signal was confirmed by using an intraoperative fluorescent imaging system, which showed fluorescent labeling of pancreatic tumors. These results suggest that RA-96-targeted liposomes encapsulating ICG and iron nanoparticles can be used to image pancreatic tumors with a variety of optical and magnetic imaging techniques. Additionally, they might be a suitable drug delivery tool to improve treatment of PDAC patients.

Preclinical study of oncolytic adenovirus-mediated cytotoxic gene therapy with gemcitabine for chemoresistance of desmoplastic tumor.

e15507 Background: Pancreatic cancer is a highly lethal disease. Excessive accumulation of tumor extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT) phenotype are two main contributors to drug resistance in desmoplastic pancreatic tumors. Methods: To overcome the limitations of various conventional therapeutics against desmoplastic pancreatic cancer, we utilized an oncolytic adenovirus (oAd) for combination therapy with gemcitabine, a standard chemotherapeutic for pancreatic cancer treatment. Here, we report the results of preclinical pharmacology and efficology studies conducted in pancreatic cancer model. Results: Efficacy studies in orthotopic pancreatic xenograft tumor model demonstrated that oAd effectively attenuated tumor cell proliferation. oAd further prevented metastasis of pancreatic cancer and sensitized pancreatic tumor to gemcitabine treatment. Furthermore, oAd augmented drug penetration and dispersion within pancreatic tumor xenografts and patient-derived pancreatic tumor spheroids. These results suggest that oAd enhance the therapeutic effect of chemotherapeutic agent in chemo-resistant and desmoplastic pancreatic tumor, thus overcoming the preexisting limitations of standard treatments. Conclusions: Overall, these results demonstrate that local administration of an oncolytic adenovirus with gemcitabine is well tolerated and support moving this investigational approach into human trials.

Abstract 4855: AMC303 inhibits tumor growth and metastasis in animal models by targeting CD44v6, a co-receptor of multiple oncogenic receptor tyrosine kinases

Abstract Introduction: CD44v6, a member of the CD44 family, is an essential co-receptor for c-MET, RON and VEGFR-2. The receptor tyrosine kinases (RTKs) c-MET and RON, are key players in tumorigenic pathways, are activated by the ligands HGF and MSP, respectively and homo- or heterodimerize with each other or can be autoactivated due to amplification. The RTK VEGFR-2 is critical for angiogenesis in solid epithelial tumors. The peptidergic inhibitor AMC303, which allosterically and selectively binds to CD44v6, was investigated for its inhibitory effects on ligand-induced and autophosphorylated c-MET activation, and its impact on tumor growth and metastases in vivo. Methods: In vitro, tumor cells were incubated with AMC303 for 30 min prior to induction with an RTK-ligand. Analysis of RTK activation was carried out by Western blotting. For in vivo xenotransplantation human pancreatic tumor cells (L3.6pl) were orthotopically injected. Animals were treated with AMC303 i.p. at 0.1, 1, 10 mg/kg QOD or QWK for 3 weeks to address dose-dependency, the optimal treatment schedule was investigated with one, three or five times weekly administrations. Regression of metastases was investigated at 1 mg/kg QOD. Survival was evaluated with a dose of 1 mg/kg QOD. Immunohistochemical (IHC) analysis was performed for cleaved caspase 6, smooth muscle actin and CD31. Reduction of vessel permeability was assessed in Balb/C mice with the Miles assay and quantification of intra-tumoral FITC dextran after i.v. injection. Results: In vitro, AMC303 inhibited c-MET, VEGFR2 and RON phosphorylation induced by their respective ligands. Notably, AMC303 inhibited c-MET activation in cells with enhanced c-MET signaling caused by the exon 14 skipping mutation, but not c-MET auto-phosphorylation due to gene amplification. In vivo, AMC303 treatment attenuated primary xenograft tumor growth and metastasis and significantly prolonged survival. IHC on treated xenograft-tumors revealed an increase in apoptosis and necrosis along with a reduction in myofibroblast infiltration, angiogenesis and vessel permeability. Pharmacokinetic properties after i.p. administration of AMC303 revealed an terminal half-life of 6h. Administration schedules of AMC303 (1mg/kg) given one, three or five times per week had no significant impact on tumor growth attenuation. AMC303 treatment could be paused for 10 days without incurring significant tumor regrowth. Conclusions: AMC303 inhibits c-MET in vitro in a ligand-dependent fashion and in vivo significantly improved overall survival in an orthotopic pancreatic xenograft mouse tumor model. AMC303 effectively attenuated both primary and metastatic tumor growth by increasing apoptosis, reduction of angiogenesis and vascular normalization. These findings demonstrate the benefit of targeting multiple oncogenic RTK signaling pathways by AMC303. Citation Format: Tina Heumann, Vanessa Al-Rawi, Thorsten Läufer, Martin Augsten, Alexandra Matzke-Ogi, Oliver Coutelle. AMC303 inhibits tumor growth and metastasis in animal models by targeting CD44v6, a co-receptor of multiple oncogenic receptor tyrosine kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4855.

Oncolytic Ad co-expressing decorin and Wnt decoy receptor overcomes chemoresistance of desmoplastic tumor through degradation of ECM and inhibition of EMT.

Pancreatic cancer is a highly lethal disease. Excessive accumulation of tumor extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT) phenotype are two main contributors to drug resistance in desmoplastic pancreatic tumors. To overcome desmoplasia and chemoresistance of pancreatic cancer, we utilized an oncolytic adenovirus (Ad) co-expressing decorin and soluble Wnt decoy receptor (HEmT-DCN/sLRP6). An orthotopic pancreatic xenograft tumor model was established in athymic nude mice using Mia PaCa-2 cells, and the antimetastatic and antitumor efficacy of systemically administered HEmT-DCN/sLRP6 was evaluated. Immunohistochemical analysis of tumor tissues was performed to assess ECM degradation, induction of apoptosis, viral dispersion, and inhibition of the Wnt/β-catenin signaling pathway. HEmT-DCN/sLRP6 effectively degraded tumor ECM and inhibited EMT, leading to enhanced viral distribution, induction of apoptosis, and attenuation of tumor cell proliferation in tumor tissue. HEmT-DCN/sLRP6 prevented metastasis of pancreatic cancer. Importantly, HEmT-DCN/sLRP6 sensitized pancreatic tumor to gemcitabine treatment. Furthermore, HEmT-DCN/sLRP6 augmented drug penetration and dispersion within pancreatic tumor xenografts and patient-derived tumor spheroids. Collectively, these results illustrate that HEmT-DCN/sLRP6 can enhance the dispersion of both oncolytic Ad and a chemotherapeutic agent in chemoresistant and desmoplastic pancreatic tumor, effectively overcoming the preexisting limitations of standard treatments.

Tumor selective uptake of drug-nanodiamond complexes improves therapeutic outcome in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths and novel treatment approaches are urgently needed. Here we show that poly(ethylene glycol)-functionalized nanodiamonds loaded with doxorubicin (ND-PEG-DOX) afforded a considerable improvement over free drug in an orthotopic pancreatic xenograft model. ND-PEG-DOX complexes were also superior to free DOX in 3-dimensional (3D) tumor spheroids of PDAC. ND-PEG showed no cytotoxicity towards macrophages, and histopathological analysis showed no abnormalities of major organs upon in vivo administration of ND-PEG-DOX. These results provide evidence that ND-mediated drug delivery may serve as a means of improving the therapeutic outcome in PDAC.

Development of apratoxin S10 (Apra S10) as an anti-pancreatic cancer agent and its preliminary evaluation in an orthotopic patient-derived xenograft (PDX) model.

Despite the significant progress in the field of cancer therapeutics, the incidence of pancreatic cancer (PC) has continuously increased. One possible mechanism for this increasing burden is impaired drug delivery and drug resistance resulting from a unique tumor microenvironment and genetic mutations. Apratoxins are potent anticancer agents and cotranslational translocation inhibitors with potential therapeutic applications to treat cancers with active secretory pathways. Here, we developed apratoxin S10 (Apra S10) as an anti-pancreatic cancer agent which potently inhibited the growth of both established and patient-derived primary pancreatic cancer cells. We validated its mechanism of action on pancreatic cancer cells by demonstrating the downregulation of multiple receptor tyrosine kinases and inhibition of growth factor and cytokine secretion. Apra S10 also inhibited a number of cytokines secreted by stromal cells, suggesting that Apra S10 not only inhibited pancreatic cancer cell secretion, but also reduced the level of factors secreted by other cell types active within the tumor microenvironment. As Apra S10 tissue distribution indicated its high enrichment in pancreas tissue, an orthotopic pancreatic patient-derived xenograft mouse model that closely mimics the human pancreatic tumor microenvironment was for the first time used in apratoxin studies. Apra S10 showed promising antitumor effect in this pancreatic cancer model and this effect was mediated through anti-proliferation properties.

Tumor-Specific Labeling of Pancreatic Cancer Using a Humanized Anti-CEA Antibody Conjugated to a Near-Infrared Fluorophore.

Development of a humanized fluorophore-conjugated antibody that can improve contrast for fluorescence-guided oncologic surgeries. BxPC-3-GFP pancreatic cancer cells were injected into flanks of nude mice. Fragments of subcutaneous tumors were grafted onto the pancreatic tail of recipient mice to create orthotopic xenograft models of pancreatic cancer. After tumors developed for 4weeks, a humanized anti-carcinoembryonic antigen antibody conjugated to an 800nm near-infrared fluorescent dye (hM5A-IR800) was injected intravenously. Mice were imaged at 6, 12, 24, 48, and 72h after injection. Fluorescence imaging showed that hM5A-IR800 specifically localized to BxPC-3 human pancreatic cancer cells. The fluorescent probe localized to cell surfaces in vitro and specifically co-localized with green fluorescent protein-labeled tumors in an orthotopic pancreatic xenograft model in vivo. Serial imaging at specific time points showed peak signal intensity of the orthotopic pancreatic tumor at 48h; this time point corresponded with a maximal tumor-to-background ratio (TBR) of 16.6 at 48h. hM5A-IR800 was successfully able to specifically label orthotopic pancreatic tumors in situ. The longer wavelength allowed deeper tissue penetration, particularly in tumor areas covered by normal pancreatic parenchyma. The probe had expected kinetics for an antibody-fluorophore conjugate, with the peak signal intensity reached at 48h. A clear tumor signal was observed with a TBR>5 at all time points, with high contrast (TBR of 16.6) at 48h. hM5A-IR800 demonstrated excellent tumor localization and a very bright signal. It is a promising agent for future clinical fluorescence-guided surgery applications.

EGFR-Targeted Metal Chelating Polymers (MCPs) Harboring Multiple Pendant PEG2K Chains for MicroPET/CT Imaging of Patient-Derived Pancreatic Cancer Xenografts.

In this study, we developed a new generation of metal chelating polymer (MCP) reagents that carry multiple polyethylene glycol (PEG) pendant groups to provide stealth to MCP-based radioimmunoconjugates (RICs). We describe the MCP synthesis for covalent attachment to panitumumab F(ab')2 fragments (pmabF(ab')2) in which different numbers of pendant methoxy-PEG chains [M = 2000, ∼45 ethylene glycol (EG) repeat units, referred to as PEG2K] are incorporated into the polymer backbone. The pendant PEG2K chains were designed to provide a protein-repellant corona so that metal chelators attached closer to the polymer backbone will be less apparent to the physiological environment. DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) groups to chelate 64Cu were installed on these conjugates to be employed for PET imaging. The conjugation of MCPs to pmabF(ab')2 was based on a UV quantifiable bis-aromatic hydrazone formation under mild conditions (pH 5-6) between an aromatic aldehyde introduced on ε-NH2 groups of lysines in the F(ab')2 fragments and a hydrazinonicotinamide (HyNic) group installed on the initiating end of the MCP. Three MCPs with 17 polyglutamide (PGlu) repeat units, DOTA chelators and with an average of 2, 4, and 8 pendant PEG2K chains were studied to examine their in vitro and in vivo characteristics, as well as their potential for PET/CT imaging. A pmabF(ab')2-MCP conjugate carrying 2 PEG2K and one carrying 8 PEG2K pendant chains in the polymer were selected for microPET/CT imaging and biodistribution studies in tumor-bearing mice. Orthotopic pancreatic patient-derived xenografts tumors were visualized by PET/CT imaging. These RICs showed low levels of liver and spleen uptake along with even lower levels of kidney uptake. These encouraging results confirm the stealth properties of the MCPs with pendant PEG2K chains.

Tissue Transglutaminase Activates Cancer-Associated Fibroblasts and Contributes to Gemcitabine Resistance in Pancreatic Cancer

Resistance to chemotherapy is a hallmark of pancreatic ductal adenocarcinoma (PDA) and has been partly attributed to the dense desmoplastic stroma, which forms a protective niche for cancer cells. Tissue transglutaminase (TG2), a Ca2+-dependent enzyme, is secreted by PDA cells and cross-links proteins in the tumor microenvironment (TME) through acyl-transfer between glutamine and lysine residues, promoting PDA growth. The objective of the current study was to determine whether secreted TG2 by PDA cells alters the response of pancreatic tumors to gemcitabine. Orthotopic pancreatic xenografts and co-culture of PDA and stromal cells were employed to determine the mechanisms by which TG2 alters tumor-stroma interactions and response to gemcitabine. Analysis of the pancreatic The Cancer Genome Atlas (TCGA) database demonstrated that increased TG2 expression levels correlate with worse overall survival (hazard ratio=1.37). Stable TG2 knockdown in PDA cells led to decreased size of pancreatic xenografts and increased sensitivity to gemcitabine in vivo. However, TG2 downregulation did not increase cytotoxicity of gemcitabine in vitro. Additionally, multivessel density and gemcitabine uptake in pancreatic tumor tissue, as measured by mass spectrometry (MS-HPLC), were not significantly different in tumors expressing TG2 versus tumors in which TG2 was knocked down. Fibroblasts, stimulated by TG2 secreted by PDA cells, secrete laminin A1, which protects cancer cells from gemcitabine-induced cytotoxicity. In all, our results demonstrate that TG2 secreted in the pancreatic TME orchestrates the cross talk between cancer cells and stroma, impacting tumor growth and response to chemotherapy. Our study supports TG2 inhibition to increase the antitumor effects of gemcitabine in PDA.

Abstract 1318: Pancreatic cancer therapy using polyion micelles co-loaded with cyclopamine and paclitaxel

Abstract Sonic hedgehog (SHH) signaling pathway is an important regulator during the initiation and progression of pancreatic cancer, and sustained activation of this pathway contributes to excessive deposition of tumor stroma and maintenance of tumor-initiating cells. Cyclopamine, a potent inhibitor of the SHH pathway, has shown promising antitumor efficacy. However, cyclopamine is insoluble in water and is highly toxic, and cyclopamine monotherapy did not effectively inhibit tumor growth in a range of preclinical pancreatic cancer models. We hypothesized that polyion complex polymeric micelles that simultaneously deliver cyclopamine and paclitaxel (M-CPA/PTX) would display greater efficacy against pancreatic tumor than micelles loaded with cyclopamine alone (M-CPA) or paclitaxel alone (M-PTX). We tested this hypothesis in cell cultures and in an orthotopic human pancreatic cancer Miapaca-2 xenograft model and a genetically engineered mouse model of pancreatic ductal carcinoma (PDAC), the KPC model. Our results showed that M-CPA interrupted the SHH pathway by antagonizing the smoothened receptor, reduced expression of Gli-1 transcription factor, and inhibited proliferation of Miapaca-2 cells and stroma-producing human pancreatic stellate cells. In Miapaca-2 cells, M-CPA decreased the fraction of CD133+ cells (cancer stem cells) and decreased tumorsphere formation, whereas M-TXL increased the fraction of CD133+ cells and tumorsphere formation. M-CPA negated the stimulation of human pancreatic stellate cells by exogenous SHH ligands. In vivo, M-CPA/PTX reduced tumor cell proliferation, depleted tumor stroma, and increased tumor vasculature density, leading to effective inhibition of tumor growth in Miapaca-2 xenografts. In the KPC model, M-CPA/PTX significantly prolonged media survival of mice with PDAC compared to non-treatment control mice. In conclusion, M-CPA/PTX was an effective strategy to treat pancreatic cancer. (supported in part by the Viragh Family Foundation and the John S. Dunn Foundation) Citation Format: Jun Zhao, Chunhui Wu, Xiaoxia Wen, Junjie Li, Qian Huang, Ying Ma, Stephen Ullrich, Huamin Wang, Jason Fleming, Chun Li. Pancreatic cancer therapy using polyion micelles co-loaded with cyclopamine and paclitaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1318.

Tissue Transglutaminase Mediated Tumor-Stroma Interaction Promotes Pancreatic Cancer Progression.

Aggressive pancreatic cancer is commonly associated with a dense desmoplastic stroma, which forms a protective niche for cancer cells. The objective of the study was to determine the functions of tissue transglutaminase (TG2), a Ca(2+)-dependent enzyme that cross-links proteins through transamidation and is abundantly expressed by pancreatic cancer cells in the pancreatic stroma. Orthotopic pancreatic xenografts and coculture systems tested the mechanisms by which the enzyme modulates tumor-stroma interactions. We show that TG2 secreted by cancer cells effectively molds the stroma by cross-linking collagen, which, in turn, activates fibroblasts and stimulates their proliferation. The stiff fibrotic stromal reaction conveys mechanical cues to cancer cells, leading to activation of the YAP/TAZ transcription factors, promoting cell proliferation and tumor growth. Stable knockdown of TG2 in pancreatic cancer cells leads to decreased size of pancreatic xenografts. Taken together, our results demonstrate that TG2 secreted in the tumor microenvironment orchestrates the cross-talk between cancer cells and stroma fundamentally affecting tumor growth. Our study supports TG2 inhibition in the pancreatic stroma as a novel strategy to block pancreatic cancer progression.

Abstract 1484: Pancreatic cancer and normal pancreas water content and its impact in metabolite quantification

Abstract The absence of early symptoms in pancreatic cancer creates a critical need for identifying new noninvasive biomarkers. Magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) are being evaluated in the diagnosis of several solid malignancies. A hallmark of most solid tumors is the detection of elevated phosphocholine (PC) and total choline (tCho). We previously observed elevated levels of tCho in several pancreatic cancer cell lines and tumor xenografts (1). Initial single voxel studies performed in humans suggest that the tCho signal normalized to water may be relatively high in normal pancreas compared to pancreatic tumor (2). Since metabolites are normalized to the water signal it is important to determine differences in its content for accurate quantification. Here, we quantified tCho in orthotopic and subcutaneous Panc1 pancreatic xenografts, and in normal pancreas. 1H MRSI acquired on a 9.4T spectrometer showed heterogeneous tCho signal in orthotopic tumors. To further determine differences in tCho, tumor tissues and pancreas were embedded in agarose and imaged ex vivo with 1H MRSI. Concentration of tCho was 3.38 ± 0.95 mM in orthotopic tumors, 1.32 ± 0.59 mM in subcutaneous tumors, and 1.27 ± 0.52 mM in normal pancreas (n = 2), when using the uncorrected water signal for normalization. Despite a much higher tCho signal in the subcutaneous tumors, tCho concentrations were comparable to the pancreas. We next estimated the water content of the tumors and the pancreas as a ratio of wet weight to dry weight (measured after 72h of lyophilization) and confirmed a significantly higher water content in tumors compared to the pancreas (wet to dry weight ratio of ∼ 6 vs ∼ 4). A separate set of tumors were freeze-clamped and used for high-resolution 1H MRS. Tumor and pancreas extracts were obtained using a dual-phase extraction method and 1H MR spectra were acquired as previously described (3). To determine the tCho concentration, peak integrations from spectra for choline, PC and glycerophosphocholine were compared to an internal standard. Integrals of the metabolites of interest were determined and normalized first to the tissue wet weight. Once the tCho concentration in tumors and pancreas was corrected for differences in water content, a two-fold higher tCho concentration was observed in tumor tissue compared to normal pancreas. These data support the use of 1H MRSI that provides a tCho map rather than the placement of single voxels to address heterogeneities in the pancreas and in pancreatic cancers. The results highlight the importance of quantifying water content in the calculation of metabolite concentration when comparing different tissues. Work supported by NIH P50CA103175. (1) Penet et al., Clin Cancer Res (2014). (2) Ma et al., Journal of computer assisted tomography (2011). (3) Shah et al., NMR Biomed (2012). Citation Format: Marie-France Penet, Balaji Krishnamachary, Tariq Shah, Yelena Mironchik, Anirban Maitra, Zaver M. Bhujwalla. Pancreatic cancer and normal pancreas water content and its impact in metabolite quantification. [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 1484. doi:10.1158/1538-7445.AM2015-1484

Abstract A46: The Ets-transcription factor Etv1 induces epithelial-mesenchymal transition (EMT) and invasion as well as expands the stromal compartment in vivo

Abstract Introduction: The Ets-transcription factor Etv1 is involved in epithelial-mesenchymal interactions during pancreatic development and shows enhanced expression in PanIN as well as in pancreatic ductal adenocarcinoma (PDAC). Therefore, we aimed to identify the mechanistic roles of Etv1 in EMT and invasion in PanIN and PDAC. Methods: Cells were isolated from Pdx1Cre;KrasG12D/+-mice (PanIN) and Pdx1Cre;KrasG12D/+;p53R175H/+-mice (PDAC) and lentiviral tranduction was used to induce overexpression or knockdown (shRNA) of Etv1, respectively. Cells were grown in 3D organotypic culture to analyze morphology and growth behavior. Invasion and sphere formation (self-renewal) assays were performed. FACS-sorting was used to isolate CD44+/CD24+ cells as a putative cancer stem cell population in PDAC and analyzed for Etv1 expression. To assess the role of Etv1 in PDAC in vivo, orthotopic pancreatic xenograft transplantation of Etv1 overexpressing PDAC cells was performed. Results: Etv1 overexpression in PanIN cells grown in 3D-organotypic culture induces a spindle shaped morphology and highly perturbed cyst architecture in contrast to control PanIN cells that form spheroid cysts that resemble normal pancreatic ductal structures. Concurrently and consistently, EMT-related genes (snail, twist, zeb1 and zeb2, as well as vimentin and N-cadherin) were found upregulated by >2-fold in PanIN-mEtv1 cells. Moreover, the expression of Mmp3 and Mmp9 was significantly increased. Functionally, the invasive capacity of PanIN-mEtv1 cells was more than twice that of control cells; knockdown of Etv1 in PDAC-cells significantly abrogated their invasive capacity. In sphere forming assays PanIN-mEtv1 cells revealed an increased self-renewal capacity, whereas sphere formation is reduced by knockdown of Etv1 in PanIN as well as in PDAC cells. FACS-sorting and subsequent expression analysis revealed increased Etv1 levels in the putative cancer stem cell population of CD44+/CD24+ PDAC-cells. Data from orthotopic xenografts showed significantly larger tumors, significantly increased stromal expansion measured by trichrome staining, and increased local tumor invasion in Etv1-overexpressing cells compared to controls. The increased stromal expansion significantly correlated to the increased tumor volume observed in Etv1 over-expressing xenografts. Conclusion: Our novel data indicate that Etv1 induces EMT as well as invasion both in vitro and in vivo and mediates self-renewal capacity of premalignant cells derived from PanIN- as well as PDAC-cells. Our in vivo data suggest a role for Etv1 in expanding the stromal compartment. These striking data suggest that Etv1 is biologically important in EMT. In vivo experiments with conditional knockout of Etv1 in Pdx1Cre;KrasG12D/+;p53R175H/+-mice are ongoing to further elucidate the role of Etv1 in PanIN and PDAC initiation and progression. Citation Format: Koushik K. Das, Steffen Heeg, Maximilian Reichert, Shigetsugu Takano, Basil S. Bakir, Gregory P. Botta, Christopher Hahn, Andrew D. Rhim, Anil K. Rustgi. The Ets-transcription factor Etv1 induces epithelial-mesenchymal transition (EMT) and invasion as well as expands the stromal compartment in vivo. [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 A46.

Aspirin counteracts cancer stem cell features, desmoplasia and gemcitabine resistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is characterized by an extremely poor prognosis. An inflammatory microenvironment triggers the pronounced desmoplasia, the selection of cancer stem-like cells (CSCs) and therapy resistance. The anti-inflammatory drug aspirin is suggested to lower the risk for PDA and to improve the treatment, although available results are conflicting and the effect of aspirin to CSC characteristics and desmoplasia in PDA has not yet been investigated. We characterized the influence of aspirin on CSC features, stromal reactions and gemcitabine resistance. Four established and 3 primary PDA cell lines, non-malignant cells, 3 patient tumor-derived CSC-enriched spheroidal cultures and tissues from patients who did or did not receive aspirin before surgery were analyzed using MTT assays, flow cytometry, colony and spheroid formation assays, Western blot analysis, antibody protein arrays, electrophoretic mobility shift assays (EMSAs), immunohistochemistry and in vivo xenotransplantation. Aspirin significantly induced apoptosis and reduced the viability, self-renewal potential, and expression of proteins involved in inflammation and stem cell signaling. Aspirin also reduced the growth and invasion of tumors in vivo, and it significantly prolonged the survival of mice with orthotopic pancreatic xenografts in combination with gemcitabine. This was associated with a decreased expression of markers for progression, inflammation and desmoplasia. These findings were confirmed in tissue samples obtained from patients who had or had not taken aspirin before surgery. Importantly, aspirin sensitized cells that were resistant to gemcitabine and thereby enhanced the therapeutic efficacy. Aspirin showed no obvious toxic effects on normal cells, chick embryos or mice. These results highlight aspirin as an effective, inexpensive and well-tolerated co-treatment to target inflammation, desmoplasia and CSC features PDA.

Targeted Noninvasive Imaging of EGFR-Expressing Orthotopic Pancreatic Cancer Using Multispectral Optoacoustic Tomography

Detection of orthotopic xenograft tumors is difficult due to poor spatial resolution and reduced image fidelity with traditional optical imaging modalities. In particular, light scattering and attenuation in tissue at depths beyond subcutaneous implantation hinder adequate visualization. We evaluate the use of multispectral optoacoustic tomography (MSOT) to detect upregulated epidermal growth factor (EGF) receptor in orthotopic pancreatic xenografts using a near-infrared EGF-conjugated CF-750 fluorescent probe. MSOT is based on the photoacoustic effect and thus not limited by photon scattering, resulting in high-resolution tomographic images. Pancreatic tumor-bearing mice with luciferase-transduced S2VP10L tumors were intravenously injected with EGF-750 probe before MSOT imaging. We characterized probe specificity and bioactivity via immunoblotting, immunocytochemistry, and flow cytometric analysis. In vitro data along with optical bioluminescence/fluorescence imaging were used to validate acquired MSOT in vivo images of probe biodistribution. Indocyanine green dye was used as a nonspecific control to define specificity of EGF-probe accumulation. Maximum accumulation occurred at 6 hours postinjection, demonstrating specific intratumoral probe uptake and minimal liver and kidney off-target accumulation. Optical bioluminescence and fluorescence imaging confirmed tumor-specific probe accumulation consistent with MSOT images. These studies demonstrate the utility of MSOT to obtain volumetric images of ligand probe biodistribution in vivo to detect orthotopic pancreatic tumor lesions through active targeting of the EGF receptor.

Preliminary evaluation of 1′-[18F]fluoroethyl-β-D-lactose ([18F]FEL) for detection of pancreatic cancer in nude mouse orthotopic xenografts

IntroductionEarly detection of pancreatic cancer could save many thousands of lives. Non-invasive diagnostic imaging, including PET with [18F]FDG, has inadequate resolution for detection of small (2–3mm) pancreatic tumours. We demonstrated the efficacy of PET imaging with an 18F-labelled lactose derivative, [18F]FEDL, that targets HIP/PAP, a biomarker that is overexpressed in the peritumoural pancreas. We developed another analogue, 1-[18F]fluoroethyl lactose ([18F]FEL), which is simpler to synthesise, for the same application. We conducted a preliminary evaluation of the new probe and its efficacy in detecting orthotopic pancreatic carcinoma xenografts in mice. MethodsXenografts were developed in nude mice by injecting L3.6pl/GL+ pancreatic carcinoma cells into the pancreas of each mouse. Tumour growth was monitored by bioluminescence imaging (BLI); accuracy of BLI tumour size estimates was verified by MRI in two representative mice. When the tumour size reached approximately 2–3mm, the animals were injected with [18F]FEL (3.7MBq) and underwent static PET/CT scans. Blood samples were collected at 2, 5, 10, 20 and 60min after [18F]FEL injection to track blood clearance. Following imaging, animals were sacrificed and their organs and tumours/pancreatic tissue were collected and counted on a gamma counter. Pancreas, including tumour, was frozen, sliced and used for autoradiography and immunohistochemical analysis of HIP/PAP expression. ResultsTumour growth was rapid, as observed by BLI and MRI. Blood clearance of [18F]FEL was bi-exponential, with half-lives of approximately 3.5min and 40min. Mean accumulation of [18F]FEL in the peritumoural pancreatic tissue was 1.29±0.295 %ID/g, and that in the normal pancreas of control animals was 0.090±0.101 %ID/g. [18F]FEL was cleared predominantly by the kidneys. Comparative analysis of autoradiographic images and immunostaining results demonstrated a correlation between [18F]FEL binding and HIP/PAP expression. Conclusion[18F]FEL may be useful for non-invasive imaging of early-stage pancreatic tumours by PET. The results warrant further studies.

Estimating tumour volume in a primary orthotopic mouse model of human pancreatic cancer using rapid acquisition magnetic resonance imaging

Pancreatic cancer has a mortality rate that closely approximates its incidence. This statistic highlights a dismal prognosis that has not improved despite decades of clinical trials. There is optimism that more sophisticated in vivo studies will better predict for clinical efficacy. This includes utilizing more relevant, orthotopic, primary xenograft models and incorporating imaging for tumour burden assessment. There has been hesitation in the routine incorporation of imaging in preclinical studies related primarily to the length and cost of imaging protocols. We hypothesized that rapid acquisition MR scans that balance efficiency with tumour visualization, could be effectively used to provide accurate and timely tumour size estimates. Mice bearing orthotopic primary pancreatic xenografts were study subjects. Animals were imaged on a 7 Tesla preclinical MR scanner, using the most rapid acquisition that provided adequate assessment of tumour size. Two different tumour volume estimates were made -one calculated from 2 dimensions obtained on imaging, and the other a software-based (MIPAV) volumetric analysis. Both were compared with tumour mass (range: 237 to 2575 mg). Although both calculated tumour volume (V=½ (LxW 2 )) and software-based volumetric analysis underestimated tumour size, they correlated well with tumour mass (correlation co-efficient 0.88 (p<0.0001) and 0.94 (p<0.0001) respectively). Our data demonstrate that rapid acquisition MR protocols can provide accurate tumour volume estimates to allow for longitudinal assessment of growth kinetics. This data has implications for the routine adoption of small animal imaging for preclinical studies using orthotopic pancreatic xenograft models.