Pancreatic Cancer Patient-derived Xenograft Research Articles

Abstract 4071: FGFR1 inhibitor reverses Smoothened inhibition-induced epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma

Abstract The majority of Pancreatic cancer (PaCA) patients present with metastatic disease and receive little benefit from chemotherapy. Although nanoparticulate carriers can potentially deposit sustained and high doses of chemotherapeutics in tumors, PaCA tumors are poorly perfused, which limits drug deposition. Ten days of oral dosing with NVP-LDE225 (40 mg/kg), an inhibitor of the sonic hedgehog (SHH) pathway (sHHI), increased tumor deposition of blood-borne, fluorescently labeled, sterically-stabilized lipidic (SSL) nanoparticles by enhancing the perfusion and permeability of the otherwise-impermeable microvasculature of PaCA patient-derived xenograft (PDX) tumors. However, literature indicates sHH ablation is associated with increased Epithelial-to-Mesenchymal (EMT) transition and invasiveness in PaCA. Here we investigated the compensatory role of FGFR1 in sHHI-induced tumor progression. Four to 8 daily treatments with the sHHI resulted in no significant reduction of tumor volume, but markedly increased the ratio of tumor cells (human mitochondria+) to Collagen1 in the stroma, in two s.c. low-passage PDX models of varying stromal content. Immunostaining of sHHI-treated tumors revealed no alteration in total FGFR1 expression, but showed a significant elevation in the (1) percent of FGFR1+ pixels correlating with DAPI+ nuclei in tumor (human mitochondria+) cells and stromal cells, (2) number of vimentin+ (EMT) cells, and (3) proliferative index marked by Ki67+ nuclei, compared to controls, suggesting accelerated tumor progression. Nuclear FGFR1 was associated with vimentin+ cells, but was excluded from the nuclei of Ki67+ cells in all groups, implicating nuclear translocation of FGFR1 in EMT. NVP-BGJ398, a small-molecule pan-FGFR inhibitor (FGFRI), administered p.o. for 3 days (15 mg/kg/day) after 7d of sHHI-pretreatment (1) significantly reduced nuclear localization of FGFR1 compared to sHHI-treated animals, (2) reversed the ratio of human mitochondria+ tumor cells to Collagen 1+ stroma, (3) depleted vimentin+ structures by >2 fold, and (4) reduced Ki67+ cells to control levels. FGFRI without sHHI-pretreatment resulted in decreased nuclear FGFR1 relative to controls, but did not impact proliferation, EMT, or tumor cell-to-stroma ratio. Finally, the sHHI/FGFRI sequence increased functional (FITC-L. esculentum lectin+) tumor vessel density and area the perfused by vascular permeability probes (fluorescent SSL) compared to both controls and sHHI-treated animals. FGFRI administration in vehicle-treated controls had no impact on vascular permeability or perfusion. We conclude that the combination of sHHI in sequence with FGFRI may reverse potentially deleterious effects of sHHI and mediate a transient enhancement of tumor permeability, and increase deposition and penetration of nanoparticulate SSL drug formulations into pancreatic cancers. Citation Format: Tista Roy Chaudhuri, Qingxiang Lin, Ninfa L. Straubinger, Wen Wee Ma, Robert M. Straubinger. FGFR1 inhibitor reverses Smoothened inhibition-induced epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4071. doi:10.1158/1538-7445.AM2017-4071

Abstract 1135: DRD2 is critical for pancreatic cancer and promises pharmacological therapy by already established antagonists

Abstract Introduction and aims: Although the overall five-year survival of all patients with cancer stands at 63%, for pancreatic cancer patients, it is a disheartening 8% - a number that remains largely unchanged for three decades. Of the patients diagnosed with pancreatic cancer, about 85% exhibit pancreatic ductal adenocarcinoma (PDAC). Most of these patients die within 4 to 6 months after diagnosis. The poor prognosis is caused by the detection at only late stages, and lack of effective options for chemotherapy. The widely used chemotherapeutic agent gemcitabine, confers a median survival advantage of only 6 months, and resistance to therapy develops in the vast majority of patients. Given this poor prognosis of patients with PDAC, there is an urgent need to find more effective therapies. Experimental procedures: Microarrays were used to perform global gene expression profiling in 195 PDAC and 41 normal pancreatic tissue samples. Using these profiling data, we undertook an extensive analysis of PDAC transcriptome by superimposing the pathway context and interaction networks of aberrantly expressed genes to identify factors with central roles in PDAC pathways. Next, tissue microarray analysis (TMA) were used to verify the expression of the candidate target in independent set of 152 samples comprising 40 normal pancreatic tissues, 49 chronic pancreatitis sections (CP) and 63 PDAC samples. We further validated the functional relevance of the candidate molecule through RNA interference (RNAi) and pharmacological inhibition in vitro and in vivo. Results: We identified dopamine receptor D2 (DRD2) as a key modulator of cancer pathways in PDAC. DRD2 up-regulation at the protein level was validated in a large independent sample cohort. Most importantly, we found that blockade of DRD2, through RNAi or pharmacological inhibition using FDA-approved antagonists hampers the proliferative and invasive capacities of pancreatic cancer cells while modulating cAMP and endoplasmic reticulum stress pathways. Also, we observed a potent effect of DRD2 antagonists on inhibition of cancer cell proliferation using different model of primary and metastatic tumor cells derived from spontaneous pancreatic cancer mouse models and patient-derived pancreatic adenocarcinoma mouse xenograft (PDX) models. Conclusions: Our findings demonstrate that inhibiting DRD2 represents a novel therapeutic approach for PDAC. Since DRD2 inhibitors are already in the clinic for the management of schizophrenia, our results from this study could support a drug repurposing strategy to expedite clinical evaluation of these agents as novel therapy against pancreatic cancer. Citation Format: Pouria Jandaghi, Hamed S. Najafabadi, Andrea Bauer, Andreas I. Papadakis, Matteo Fassan, Anita Hall, Anie Monast, Maryam Safisamghabadi, Magnus von Knebel Doeberitz, John P. Neoptolemos, Eithne Costello, William Greenhalf, Aldo Scarpa, Bence Sipos, Daniel Auld, Mark Lathrop, Morag Park, Markus W. Büchler, Oliver Strobel, Thilo Hackert, Nathalia Giese, George Zogopoulos, Veena Sangwan, Sidong Huang, Jörg D. Hoheisel, Yaser Riazalhosseini. DRD2 is critical for pancreatic cancer and promises pharmacological therapy by already established antagonists [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1135. doi:10.1158/1538-7445.AM2017-1135

Orthotopic Patient-Derived Pancreatic Cancer Xenografts Engraft Into the Pancreatic Parenchyma, Metastasize, and Induce Muscle Wasting to Recapitulate the Human Disease

Limitations associated with current animal models serve as a major obstacle to reliable preclinical evaluation of therapies in pancreatic cancer (PC). In an effort to develop more reliable preclinical models, we have recently established a subcutaneous patient-derived xenograft (PDX) model. However, critical aspects of PC responsible for its highly lethal nature, such as the development of distant metastasis and cancer cachexia, remain underrepresented in the flank PDX model. The purpose of this study was to evaluate the degree to which an orthotopic PDX model of PC recapitulates these aspects of the human disease. Human PDX-derived PC tumors were implanted directly into the pancreas of NOD.Cg-Prkdc Il2rg/SzJ mice. Tumor growth, metastasis, and muscle wasting were then evaluated. Orthotopically implanted PDX-derived tumors consistently incorporated into the murine pancreatic parenchyma, metastasized to both the liver and lungs and induced muscle wasting directly proportional to the size of the tumor, consistent of the cancer cachexia syndrome. Through the orthotopic implantation technique described, we demonstrate a highly reproducible model that recapitulates both local and systemic aspects of human PC.

Abstract 4937: Comparison of orthotopic and subcutaneous implantation of pancreatic cancer patient-derived xenograft models for drug development

Abstract Patient derived xenograft (PDX) models of pancreatic cancer provide an excellent platform for understanding human disease, characterizing tumor-initiating cells, and testing of novel therapeutics. Herein, we compared the natural pancreatic microenvironment via orthotopic implantation and the traditional approach of subcutaneous implantation for effects on PDX tumor growth, tumor morphology, cell population diversity, tumorigenic potential, metastatic potential, and response to therapeutics. Two approaches were used: (1) primary human cancer tissue fragments were implanted in orthotopic and subcutaneous environments; and, (2) PDX previously established by subcutaneous engraftment were reimplanted into either an orthotopic or a subcutaneous environment. We report that orthotopically-engrafted primary PDX more closely reflect the gross tumor morphology of the original patient than subcutaneously-derived tumors. In models derived from subcutaneous engraftment, variation of tumor morphology, tumor growth, cell population diversity, and tumorigenic potential between tumors grown in orthotopic and subcutaneous environments were dependent on PDX line; and, stromal infiltration and mucin production were more prominent in the orthotopic background for some PDX models. In addition, we observe increased metastatic potential in orthotopic tumor-bearing mice compared with their subcutaneous counterparts. Tumor metastasis occurred in distal filtering organs, including the lung, liver, lymph nodes, and peritoneal cavity, among others. This result is consistent with the observation of increased circulating tumor cells in the blood of orthotopic tumor bearing mice. Comparison of therapeutic efficacy with gemcitabine chemotherapy and antibody-drug conjugate (ADC) therapy in orthotopic and subcutaneous tumors showed therapeutic response was dependent on PDX line. Interestingly, for PDX lines that exhibited morphological differences between orthotopic and subcutaneous backgrounds, treatment was less effective in the orthotopic microenvironment suggesting the microenvironment alters the ability of therapeutics to impede tumor growth. Reduced therapeutic response was also found to be consistent with reduced ADC uptake and vascularity in orthotopic tumors. Taken together, the orthotopic PDX model serves as a more accurate representation of human pancreatic cancer by displaying stromal-rich morphology, exhibiting metastatic cell behavior, and recapitulating therapeutic response challenges observed in patients. Citation Format: Sarah L. Fong, Marybeth A. Pysz, Kristen D. McKnight, Nicole Taylor, Regina Nieu, Juliet Markeson, Hanna Rammoth, Archana Dilip, Kayla Fasano, Emily Janke, Erica Anderson, Holger Karsunky, Scott Dylla. Comparison of orthotopic and subcutaneous implantation of pancreatic cancer patient-derived xenograft models for drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4937. doi:10.1158/1538-7445.AM2017-4937

Abstract 4943: Orthotopic transplantation of pancreatic cancer PDX models increases murine stroma content, but does not influence therapeutic response to standard of care

Abstract Background: Reasons for the poor prognosis of pancreatic cancer are advanced and inoperable tumor stages at time of diagnosis and resistance to conventional therapies. Patient-derived xenografts (PDX) represent a valuable tool for the prediction of therapy response, the identification of new biomarkers and therapeutic targets or pancreatic cancer specific activated pathways (MAPK, hedgehog). However, PDX tumors differ from patient tumors as their surrounding tissue is replaced by murine stroma within 3 to 9 weeks after primary transplantation. Since the desmoplastic stroma has an impact on the progression and treatment of pancreatic cancer, we investigated the characteristics and function of murine stroma components in PDX models of pancreatic ductal adenocarcinoma (PDAC). Methods: We analyzed the relevance of murine cancer-associated fibroblasts (CAFs) for therapeutic response by comparing orthotopic with s.c. transplantation in PDX. We selected a cohort of 3 PDAC PDX with different growth rates (slow, median and fast growth). Human pancreatic tumor material was implanted s.c. or orthotopically into immunodeficient mice. After successful engraftment, the chemosensitivity to standard of care (SoC) drugs (focus on 2 to 3 drugs) was determined according to clinically relevant and optimized schedules. Growth of orthotopic PDX models was monitored via high-resolution ultrasound. Cryo- and formalin-preserved tumor tissues from the chemosensitivity studies were collected and analyzed for specific stroma markers (collagen I and αSMA). Results: In general, the response profiles in our experiments have closely reflected patient’s response in the clinic for the drug combination gemcitabine and nab-paclitaxel. There were no differences in response when comparing s.c. with orthotopic growing tumors. Semi-quantitative analysis of tumor sections showed that orthotopic tumors contain more murine stroma compared to s.c. tumors. Comparison of treated and untreated tumors revealed a strong reduction of the tumor cell content by the SoC treatment, whereas CAFs (collagen I and αSMA positive fraction) were not affected. Conclusion: In summary, this study revealed that orthotopic transplantation results in the growth of tumors with an improved and functional tumor microenvironment. Therapy is effective on the pancreatic cancer cells, however, tumor stroma seems apparently to be inherent resistant towards conventional therapy. The remaining tumor stroma might provide a survival nice for some tumor cells - leading to the frequently observed disease relapse. Therefore, orthotopic PDX are considered to be the more clinical relevant models in comparison to s.c. PDX because they mimic closely the human pancreatic cancer microenvironment and also favor the development of metastasis. Further, targeting the tumor stroma should be implicated into clinical treatment strategies. Citation Format: Ulrike Pfohl, Diana Behrens, Iduna Fichtner, Jens Hoffmann, Britta Büttner, Wolfgang Walther. Orthotopic transplantation of pancreatic cancer PDX models increases murine stroma content, but does not influence therapeutic response to standard of care [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4943. doi:10.1158/1538-7445.AM2017-4943

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.

Effects of Combined Treatment with Ionizing Radiation and the PARP Inhibitor Olaparib in BRCA Mutant and Wild Type Patient-Derived Pancreatic Cancer Xenografts.

BackgroundThe BRCA2 gene product plays an important role in DNA double strand break repair. Therefore, we asked whether radiation sensitivity of pancreatic cancers developing in individuals with germline BRCA2 mutations can be enhanced by agents that inhibit poly (ADP-ribose) polymerase (PARP).MethodsWe compared the sensitivity of two patient-derived pancreatic cancer xenografts, expressing a truncated or wild type BRCA 2, to ionizing radiation alone or in combination with olaparib (AZD-2281). Animals were treated with either a single dose of 12Gy, 7 days of olaparib or 7 days of olaparib followed by a single dose of 12Gy. Response was assessed by tumour growth delay and the activation of damage response pathways.ResultsThe BRCA2 mutated and wild type tumours showed similar radiation sensitivity, and treatment with olaparib did not further sensitize either model when compared to IR alone.ConclusionsWhile PARP inhibition has been shown to be effective in BRCA-mutated breast and ovarian cancers, it is less well established in pancreatic cancer patients. Our results show no radiosensitization in a germline BRCA 2 mutant and suggest that combining PARP inhibition and IR may not be beneficial in BRCA 2 related pancreatic tumors.

Activity of the novel polo-like kinase 4 inhibitor CFI-400945 in pancreatic cancer patient-derived xenografts.

BackgroundPolo-like kinase 4 PLK4 plays a key role in centriole replication. Hence PLK4 inhibition disrupts mitosis, and offers a novel approach to treating chromosomally unstable cancers, including pancreatic cancer. CFI-400945 is a first in class small molecule PLK4 inhibitor, currently undergoing early phase clinical trials.ResultsTreatment with CFI-400945 significantly reduced tumor growth and increased survival in four out of the six models tested. Consistent with PLK4 inhibition, we observed reduced expression of the proliferation marker Ki-67 associated with an increase in nuclear diameter during treatment with CFI-400945. Additionally, treatment with CFI-400945 resulted in a significant reduction of tumor-initiating cells.DiscussionThese results support the further investigation of PLK4 as a drug target in pancreatic cancer.MethodsSensitivity to CFI-400945 was tested in a series of six patient-derived pancreatic cancer xenografts, selected to represent the range of growth characteristics, genetic features, and hypoxia found in pancreatic cancer patients.

Acetyl-CoA carboxylase inhibitors attenuate WNT and Hedgehog signaling and suppress pancreatic tumor growth.

Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in de novo fatty acid synthesis, and its ACC1 isoform is overexpressed in pancreatic and various other cancers. The activity of many oncogenic signaling molecules, including WNT and Hedgehog (HH), is post-translationally modified by lipidation. Here, we report that inhibition of ACC by a small molecule inhibitor, BAY ACC002, blocked WNT3A lipidation, secretion, and signaling. In pancreatic cancer cells, where WNT and HH are key oncogenic drivers, ACC inhibition simultaneously suppressed WNT and HH signaling, and led to anti-proliferative effects. Treatment with ACC inhibitors blocked tumor growth and converted the poorly differentiated histological phenotype to epithelial phenotype in multiple cell line-based and patient-derived pancreatic cancer xenograft models. Together, our data highlight the potential utility of ACC inhibitors for pancreatic cancer treatment, and provide novel insight into the link between upregulated de novo fatty acid synthesis in cancer cells, protein lipidation, and oncogenic signaling.

Generation and molecular characterization of pancreatic cancer patient-derived xenografts reveals their heterologous nature.

Pancreatic ductal adenocarcinoma (PDAC) is the most challenging type of cancer to treat, with a 5-year survival rate of <10%. Furthermore, because of the large portion of the inoperable cases, it is difficult to obtain specimens to study the biology of the tumors. Therefore, a patient-derived xenograft (PDX) model is an attractive option for preserving and expanding these tumors for translational research. Here we report the generation and characterization of 20 PDX models of PDAC. The success rate of the initial graft was 74% and most tumors were re-transplantable. Histological analysis of the PDXs and primary tumors revealed a conserved expression pattern of p53 and SMAD4; an exome single nucleotide polymorphism (SNP) array and Comprehensive Cancer Panel showed that PDXs retained over 94% of cancer-associated variants. In addition, Polyphen2 and the Sorting Intolerant from Tolerant (SIFT) prediction identified 623 variants among the functional SNPs, highlighting the heterologous nature of pancreatic PDXs; an analysis of 409 tumor suppressor genes and oncogenes in Comprehensive Cancer Panel revealed heterologous cancer gene mutation profiles for each PDX-primary tumor pair. Altogether, we expect these PDX models are a promising platform for screening novel therapeutic agents and diagnostic markers for the detection and eradication of PDAC.

Abstract A24: Early-stage pancreatic cancer patient derived xenograft (PDX) models established from endoscopic ultrasound (EUS) fine needle aspiration (FNA) biopsies

Abstract Background: We developed and characterized PDX models generated from FNA endoscopic biopsies at the time of diagnosis of pancreatic ductal adenocarcinoma (PDAC). Our goal was to determine if an FNA-PDX model could be established within a clinically relevant time frame to inform treatment decisions prior to disease recurrence in patients with early stage, localized disease. Methods: Patients presenting with a localized mass in the pancreas and no metastatic disease were considered for EUS/FNA biopsy. Under sonographic guidance FNA biopsies were obtained. On-site cytopathology confirmed a histologic diagnosis of PDAC. The diagnostic FNA specimen was sent to pathology for formalin fixed paraffin embedded tissue block analysis per standard institutional pathologic protocols. Additional endoscopic FNA passes from the primary tumor were obtained during the index procedure by EUS guidance. The cell specimen was washed in cold DMEM and centrifuged. The cell pellet was rapidly transplanted into a subcutaneous pocket within the flank of a single NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD scid gamma, NSG) mouse. Tumor growth was monitored with calipers and engrafted tumors were passaged to subsequent mice when they reached a diameter of 10mm2. Growth kinetics and immunohistochemical staining for human cells with antibody against the human HLA Class 1 ABC antigen were obtained. Treatment groups were established from the F3 passage and randomized for in vivo chemosensitivity testing using FDA-approved chemotherapeutics: gemcitabine, cisplatin, docetaxel, irinotecan and fluorouracil. Results: The warm ischemia time was negligible. The average cold ischemia time, defined as the time from the endoscope to transplantation in the mouse, was 39 minutes. Engraftment was successful in 9 of 24 (37.5%) individual FNA patient specimens. Engraftment however increased with the number of FNA biopsy passes: 3/12 (25%) engraftment from one pass and 6/12 (50%) from two passes. Tumor size and surgical resectability status by CT imaging did not correlate with engraftment success. Procedural variables such as needle gauge and cytopathologic features such as degree of differentiation and percent tumor cellularity also did not correlate with rate of engraftment. PDX tumor morphology was maintained between F0 and F4 mice by H&amp;E and human HLA immunohistochemical staining. The initial engrafted PDX tumors took 12 weeks (average) to grow to a diameter of 5 mm. The rate of engraftment for subsequent passaged mice was faster, averaging 6 weeks for the F1 development and stable for passaged tumors to the F5 generation. As expected, in vivo chemosensitivity was variable between FNA-PDX tumors from different patients as depicted in patient model #048 which was resistant to oxaliplatin, intermediate to docetaxel and fluorouracil but sensitive to cisplatin, irinotecan and gemcitabine. Conclusions: Pancreatic cancer PDX models can be generated from the diagnostic FNA biopsy specimen obtained during a patient's EUS procedure. The rate of engraftment doubles with the number of endoscopic passes suggesting that the amount of biopsy specimen tissue is important for engraftment while cytopathologic features associated with aggressive tumor growth such as tumor cellularity and tumor cell differentiation did not. Chemosensitivity was variable between FNA-PDX models. In patients with early stage disease the FNA-PDX model is stable and results from chemosensitivity assays can be obtained in a clinically meaningful window. Citation Format: Kerrington Smith, Dawn Fischer, Timothy Gardner, Stuart Gordon, Vijayalakshmi Padmanabhan, Thomas Colacchio, Richard Barth, Arief Suriawinata. Early-stage pancreatic cancer patient derived xenograft (PDX) models established from endoscopic ultrasound (EUS) fine needle aspiration (FNA) biopsies. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A24.

Abstract 4215: Hyperpolarized magnetic resonance metabolic imaging and NMR metabolomics to assess the progression and aggressiveness of patient-derived pancreatic cancer xenografts

Abstract Pancreatic cancer, the most lethal of solid tumors is an aggressive disease that develops relatively symptom-free. The absence of early symptoms has created a critical need for identifying and developing new noninvasive biomarkers and therapeutic intervention to improve the survival rate of pancreatic cancer patients. Our goal is to identify metabolic biomarkers for early detection with an understanding of pancreatic cancer progression from benign lesions to a malignant state. Hyperpolarized magnetic resonance imaging (HP-MRI) provides a &amp;gt;10,000-fold signal enhancement for detecting of endogenous metabolic substrates to monitor metabolic fluxes through the multiple key biochemical pathways including glycolysis and citric acid cycle. In particular, conversion between hyperpolarized 13C-labeled pyruvate and lactate, catalyzed by lactate dehydrogenase (LDH), has been shown to have a number of potential applications such as diagnosis, staging tumor grade and monitoring therapy response. Genetic mutation is evident in causing tumorigenesis and often time these mutations trigger the signaling pathways that are associated with “metabolic transformations. There is currently tremendous research interest in dissecting the mechanisms of metabolic transformation as the cancers progress. In this effort, we are applying high-resolution NMR (nuclear magnetic resonance) metabolomics and the real-time hyperpolarized metabolic imaging of patient-derived xenograft tumors for probing the underlying mechanism of altered-metabolism and correlate with pancreatic cancer progression and its aggressiveness. The protocol detailing heterotopic engraftment of pancreatic cancer patient tumors into immunodeficient mice and expansion of direct xenograft tumors was recently reported elsewhere. These patient-derived xenografts (PDX) were well annotated with original patient tumors. For the NMR metabolomics study, the fresh-frozen tumor samples were homogenized, lyophilized, and resuspended in D2O and 1-D proton NMR spectroscopy was employed to characterize the water-soluble portion of the metabolome. These data suggest that elevated glycolysis (production of high lactate) and alterations in choline metabolism may arise in pancreatic cancer development. Also, the metabolite concentrations were significantly higher in aggressive tumors compare to non-aggressive one. The real-time metabolic imaging shows the immediate conversion of lactate after injection (i.v.) of hyperpolarized 13C pyruvate and probe the upregulated LDH activity as the cancer progress. The basic genomic profiling of these tumors is in progress. This model may provide an excellent platform where mutational status of these patient tumors can be correlated with both high resolution and hyperpolarized dynamical metabolomics data for characterizing individual tumor phenotypes. Citation Format: Prasanta Dutta, Mayrim Rios Perez, Travis Cole Salzillo, Michael Pratt, Yaan Kang, Niki Zacharias, Anirban Maitra, Jason B. Fleming, Pratip Bhattacharya. Hyperpolarized magnetic resonance metabolic imaging and NMR metabolomics to assess the progression and aggressiveness of patient-derived pancreatic cancer xenografts. [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 4215.

Abstract 639: Subtyping of pancreatic cancer patient-derived xenograft tumors and implications for anticancer agent testing

Abstract Despite improvements in treatment, pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers, with a continuous increase in incidence emphasizing the need for further research and therapeutic development. In recent years, we have developed a collection of &amp;gt;40 patient-derived xenograft (PDX) models from PDAC. In order to determine their relevance for anticancer agent testing, we extensively characterized our models for histology features, whole exome mutations (Hiseq 2000), chromosome rearrangements, gene copy number variations (Affymetrix SNP6) and gene expression (Affymetrix U133 Plus2.0). PDAC from 65 patients were implanted into immuno-compromised mice, resulting in the development of 42 PDX models (success rate 65%). The PDAC from which models were established included moderate and poorly differentiated tumors and were heterogeneous for stroma content. In patient tumors, we showed fibroblast activation but not stroma content predicted poor patient outcome (p&amp;lt;0.0001) and success of PDX establishment (p&amp;lt;0.01). Correlating with those of the parental tumors, the resulting PDAC_PDX also showed heterogeneity for stroma content and for murine fibroblast activation. As seen in patient tumors, the PDAC_PDX were characterized by frequent chromosomal instability, with 38% of the models presenting a moderate hypoploidy while the 62% remaining ones showed a hyperploidy. Two models showed hypermutation due to mismatch repair deficiency while the others had on average a lower mutation load when compared to other histotypes such as colon, lung or melanoma tumors. In total, we identified more than 9000 genes altered by homozygous deletions, high gene amplifications and/or mutations, most of the models showed alterations in KRAS (81%), TP53 (67%), CDKN2A (64%) and TGFBR2/SMAD4 (64%) genes. Next, PDAC_PDX profiles were merged with those of patient tumors to determine PDAC_PDX subtypes and were found to be of the classical (61%), quasi mesenchymal (QM) (34%) and exocrine like subtype (5%) according to the 62 gene expression signature established by Collisson et al. 2011. Preliminary biomarker analysis revealed certain associations between genomic alterations and transcriptome subtypes, such as more frequent alterations in the TGFB pathway in classical PDX (80%) compared to QM (47%). Further detailed biomarkers analyses also addressing questions of molecular determinants of stroma content, of fibroblast activation and of sensitivity toward anticancer agents will be presented. Extensive characterization of our PDAC_PDX collection revealed similarities with patient tumors with regards of histology features including stroma content and fibroblast activation. At molecular level, the models showed similar genomic and transcriptomic patterns as those reported for patient PDAC, altogether, proving the value of this collection for drug development investigations. Citation Format: Peter Bronsert, Tim Kees, Bruno Zeitouni, Anne-Lise Peille, Manuel Landesfeind, Heinz-Herbert Fiebig, Simon Kuesters, Vincent Vuaroqueaux. Subtyping of pancreatic cancer patient-derived xenograft tumors and implications for anticancer agent testing. [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 639.

Nicotine Reduces Survival via Augmentation of Paracrine HGF-MET Signaling in the Pancreatic Cancer Microenvironment.

The relationship between smoking and pancreatic cancer biology, particularly in the context of the heterogeneous microenvironment, remains incompletely defined. We hypothesized that nicotine exposure would lead to the augmentation of paracrine growth factor signaling between tumor-associated stroma (TAS) and pancreatic cancer cells, ultimately resulting in accelerated tumor growth and metastasis. The effect of tobacco use on overall survival was analyzed using a prospectively maintained database of surgically resected patients with pancreatic cancer. Nicotine exposure was evaluated in vitro using primary patient-derived TAS and pancreatic cancer cells independently and in coculture. Nicotine administration was then assessed in vivo using a patient-derived pancreatic cancer xenograft model. Continued smoking was associated with reduced overall survival after surgical resection. In culture, nicotine-stimulated hepatocyte growth factor (HGF) secretion in primary patient-derived TAS and nicotine stimulation was required for persistent pancreatic cancer cell c-Met activation in a coculture model. c-Met activation in this manner led to the induction of inhibitor of differentiation-1 (Id1) in pancreatic cancer cells, previously established as a mediator of growth, invasion and chemoresistance. HGF-induced Id1 expression was abrogated by both epigenetic and pharmacologic c-Met inhibition. In patient-derived pancreatic cancer xenografts, nicotine treatment augmented tumor growth and metastasis; tumor lysates from nicotine-treated mice demonstrated elevated HGF expression by qRT-PCR and phospho-Met levels by ELISA. Similarly, elevated levels of phospho-Met in surgically resected pancreatic cancer specimens correlated with reduced overall survival. Taken together, these data demonstrate a novel, microenvironment-dependent paracrine signaling mechanism by which nicotine exposure promotes the growth and metastasis of pancreatic cancer.

Nab-Paclitaxel Plus S-1 Shows Increased Antitumor Activity in Patient-Derived Pancreatic Cancer Xenograft Mouse Models.

To investigate the antitumor activity of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) plus S-1 in patient-derived pancreatic cancer xenograft mouse models and to explore biomarkers that could predict drug efficacy. Ten patient-derived xenograft models were established. The third-generation tumor-bearing mice were randomized into 4 treatment groups: (1) control; (2) S-1; (3) nab-paclitaxel; (4) S-1 plus nab-paclitaxel. Resected tumors were tested by immunohistochemistry for the expression of thymidylate synthase, orotate phosphoribosyltransferase (OPRT), dihydropyrimidine dehydrogenase (DPD), secreted protein that is acidic and rich in cysteine, human epidermal growth factor receptor 2 (HER2), collagen-1, and CD31. Tumor growth inhibition of the S-1 group, nab-paclitaxel group, and combination group was 69.52%, 86.63%, 103.56%, respectively (P < 0.05). The efficacy of S-1 is better in thymidylate synthase-negative, OPRT-positive, and DPD-negative tumors. The efficacy of nab-paclitaxel is better in HER2-positive tumors. Collagen-1 was decreased and CD31 was increased in tumors treated with nab-paclitaxel and S-1 plus nab-paclitaxel compared with control or S-1. This preclinical study showed that S-1 plus nab-paclitaxel exerted significantly better antitumor activity than S-1 or nab-paclitaxel alone. Thymidylate synthase, OPRT, and DPD were possibly biomarkers of S-1 and HER2 of nab-paclitaxel.

Metformin Treatment Does Not Inhibit Growth of Pancreatic Cancer Patient-Derived Xenografts.

There is currently tremendous interest in developing anti-cancer therapeutics targeting cell signaling pathways important for both cancer cell metabolism and growth. Several epidemiological studies have shown that diabetic patients taking metformin have a decreased incidence of pancreatic cancer. This has prompted efforts to evaluate metformin, a drug with negligible toxicity, as a therapeutic modality in pancreatic cancer. Preclinical studies in cell line xenografts and one study in patient-derived xenograft (PDX) models were promising, while recently published clinical trials showed no benefit to adding metformin to combination therapy regimens for locally advanced and metastatic pancreatic cancer. PDX models in which patient tumors are directly engrafted into immunocompromised mice have been shown to be excellent preclinical models for biomarker discovery and therapeutic development. We evaluated the response of four PDX tumor lines to metformin treatment and found that all four of our PDX lines were resistant to metformin. We found that the mechanisms of resistance may occur through lack of sustained activation of adenosine monophosphate-activated protein kinase (AMPK) or downstream reactivation of the mammalian target of rapamycin (mTOR). Moreover, combined treatment with metformin and mTOR inhibitors failed to improve responses in cell lines, which further indicates that metformin alone or in combination with mTOR inhibitors will be ineffective in patients, and that resistance to metformin may occur through multiple pathways. Further studies are required to better understand these mechanisms of resistance and inform potential combination therapies with metformin and existing or novel therapeutics.

Tumor-Priming Smoothened Inhibitor Enhances Deposition and Efficacy of Cytotoxic Nanoparticles in a Pancreatic Cancer Model.

Most pancreatic adenocarcinoma patients present with unresectable disease and benefit little from chemotherapy. Poor tumor perfusion and vascular permeability limit drug deposition. Previous work showed that Smoothened inhibitors of hedgehog signaling (sHHI) promote neovascularization in spontaneous mouse models of pancreatic cancer (PaCA) and enhance tumor permeability to low-molecular weight compounds. Here, we tested the hypothesis that sHHI can enhance tumor deposition and efficacy of drug-containing nanoparticles consisting of 80 to 100 nm sterically-stabilized liposomes (SSL) containing doxorubicin (SSL-DXR). SCID mice bearing low-passage patient-derived PaCA xenografts (PDX) were pretreated p.o. for 10 days with 40 mg/kg/d NVP-LDE225 (erismodegib), followed by i.v. SSL-DXR. Microvessel density, permeability, perfusion, and morphology were compared with untreated controls, as was SSL deposition and therapeutic efficacy. The sHHI alone affected tumor growth minimally, but markedly increased extravasation of nanoparticles into adenocarcinoma cell-enriched regions of the tumor. Immunostaining showed that sHHI treatment decreased pericyte coverage (α-SMA(+)) of CD31(+) vascular endothelium structures, and increased the abundance of endothelium-poor (CD31(-)) basement membrane structures (collagen IV(+)), suggesting increased immature microvessels. SSL-DXR (15 mg/kg) administered after sHHI pretreatment arrested tumor volume progression and decreased tumor perfusion/permeability, suggesting an initial vascular pruning response. Compared with controls, one cycle of 10-day sHHI pretreatment followed by 6 mg/kg SSL-DXR doubled median tumor progression time. Three cycles of treatment with sHHI and SSL-DXR, with a 10-day between-cycle drug holiday, nearly tripled median tumor progression time. Based upon these data, short-term sHHI treatment sequenced with nanoparticulate drug carriers constitutes a potential strategy to enhance efficacy of pancreatic cancer therapy.

Metformin Treatment Does Not Inhibit Growth of Pancreatic Cancer Patient-Derived Xenografts

Metformin Treatment Does Not Inhibit Growth of Pancreatic Cancer Patient-Derived Xenografts

Patient-Derived Tumor Xenografts Are Susceptible to Formation of Human Lymphocytic Tumors

Patient-derived xenograft (PDX) tumor models have emerged as a new approach to evaluate the effects of cancer drugs on patients’ personalized tumor grafts enabling to select the best treatment for the cancer patient and providing a new tool for oncology drug developers. Here, we report that human tumors engrafted in immunodeficient mice are susceptible to formation of B-and T-cell PDX tumors. We xenografted human primary and metastatic tumor samples into immunodeficient mice and found that a fraction of PDX tumors generated from patients’ samples of breast, colon, pancreatic, bladder and renal cancer were histologically similar to lymphocytic neoplasms. Moreover, we found that the first passage of breast and pancreatic cancer PDX tumors after initial transplantation of the tumor pieces from the same human tumor graft could grow as a lymphocytic tumor in one mouse and as an adenocarcinoma in another mouse. Whereas subcutaneous PDX tumors resembling human adenocarcinoma histology were slow growing and non-metastatic, we found that subcutaneous PDX lymphocytic tumors were fast growing and formed large metastatic lesions in mouse lymph nodes, liver, lungs, and spleen. PDX lymphocytic tumors were comprised of B-cells which were Epstein-Barr virus positive and expressed CD45 and CD20. Because B-cells are typically present in malignant solid tumors, formation of B-cell tumor may evolve in a wide range of PDX tumor models. Although PDX tumor models show great promise in the development of personalized therapy for cancer patients, our results suggest that confidence in any given PDX tumor model requires careful screening of lymphocytic markers.

BRCA1 and BRCA2 mutations sensitize to chemotherapy in patient-derived pancreatic cancer xenografts.

Background:Germline mutations of the BRCA tumour suppressors have been associated with increased risk of pancreatic cancer. Clinical evidence suggests that these patients may be more sensitive to treatment with cisplatin. As the frequency of germline BRCA mutations is low, definitive experimental data to support the clinical observations are still missing.Methods:We tested gemcitabine and cisplatin sensitivity of four BRCA1 and BRCA2 mutant and three BRCA1 and BRCA2 wild-type (WT) patient-derived pancreatic cancer xenografts.Results:We observed treatment sensitivity to gemcitabine and cisplatin in the BRCA WT and mutant models. The BRCA1 and BRCA2 mutant xenografts were significantly more sensitive to cisplatin although these models also showed sensitivity to gemcitabine. The BRCA1 and BRCA2 WT models showed sensitivity to gemcitabine but not cisplatin. Treatment sensitivity in the xenograft models closely resembled treatment response in the corresponding patients.Discussion:We have characterised a panel of xenografts derived from pancreatic cancer patients carrying germline BRCA mutations, and shown that their genetic features resemble the patient donor. Our results support further clinical testing of treatment regimens combining gemcitabine and platinum drugs in this patient population, as well as preclinical research aiming to identify mechanisms of cisplatin resistance in BRCA mutant pancreatic cancers.