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Purpose Pancreatic ductal adenocarcinoma (PDAC) is a molecularly heterogeneous tumor entity with no clinically established imaging biomarkers. We hypothesize that tumor morphology and physiology, including vascularity and perfusion, show variations that can be detected by differences in contrast agent (CA) accumulation measured non-invasively. This work seeks to establish imaging biomarkers for tumor stratification and therapy response monitoring in PDAC, based on this hypothesis. Methods and materials Regional CA accumulation in PDAC was correlated with tumor vascularization, stroma content, and tumor cellularity in murine and human subjects. Changes in CA distribution in response to gemcitabine (GEM) were monitored longitudinally with computed tomography (CT) Hounsfield Units ratio (HUr) of tumor to the aorta or with magnetic resonance imaging (MRI) ΔR1 area under the curve at 60 s tumor-to-muscle ratio (AUC60r). Tissue analyses were performed on co-registered samples, including endothelial cell proliferation and cisplatin tissue deposition as a surrogate of chemotherapy delivery. Results Tumor cell poor, stroma-rich regions exhibited high CA accumulation both in human (meanHUr 0.64 vs. 0.34, p < 0.001) and mouse PDAC (meanAUC60r 2.0 vs. 1.1, p < 0.001). Compared to the baseline, in vivo CA accumulation decreased specifically in response to GEM treatment in a subset of human (HUr −18%) and mouse (AUC60r −36%) tumors. Ex vivo analyses of mPDAC showed reduced cisplatin delivery (GEM: 0.92 ± 0.5 mg/g, vs. vehicle: 3.1 ± 1.5 mg/g, p = 0.004) and diminished endothelial cell proliferation (GEM: 22.3% vs. vehicle: 30.9%, p = 0.002) upon GEM administration. Conclusion In PDAC, CA accumulation, which is related to tumor vascularization and perfusion, inversely correlates with tumor cellularity. The standard of care GEM treatment results in decreased CA accumulation, which impedes drug delivery. Further investigation is warranted into potentially detrimental effects of GEM in combinatorial therapy regimens.

Supplementary tables S1-3 and figure legends. Supplementary Table S1. Antibodies and antibody dilution used for immunohistochemical analyses. Supplementary Table S2. Characterization of human HCC Supplementary Table S3. Number of tumors per analyzed animal of DEN and McA model.

Supplementary Fig. S4. Tumor vascularization and necrosis in human HCC.

Supplementary Figure 1. Summary and survival of murine cohort used in the study. Supplementary Figure 2 Consort diagram of the patient distribution of surgical subcohort 1. Supplementary Figure 3. Consort diagram of the patient distribution of surgical subcohort 2. Supplementary Figure 4. Consort diagram of the patient distribution of surgical subcohort 3. Supplementary Figure 5. Schematic of relative tumor cell and stroma contribution in PDAChigh Supplementary Table 1. Statistics of the patients used in surgical subcohort 1. Supplementary Table 2. Statistics of the patients used in surgical subcohort 2. Supplementary Table 3. Statistics of the patients used in surgical subcohort 3. Supplementary Table 4. Results of a multivariable Cox regression model fit to the data of subcohort 2 that assess whether PDAC is associated with overall survival after adjustment for common predictors. Supplementary Table 5. The permutation test analysis revealing association between overall survival and lowest ADC stratified by each of the relevant categorical variables. Data used for analysis are derived from surgical subcohort 3.

Purpose: Tumor heterogeneity is a hallmark of pancreatic ductal adenocarcinoma (PDAC). It determines tumor biology including tumor cellularity (i.e., amount of neoplastic cells and arrangement into clusters), which is related to the proliferative capacity and differentiation and the degree of desmoplasia among others. Given the close relation of tumor differentiation with differences in progression and therapy response or, e.g., the recently reported protective role of tumor stroma, we aimed at the noninvasive detection of PDAC groups, relevant for future personalized approaches. We hypothesized that histologic differences in PDAC tissue composition are detectable by the noninvasive diffusion weighted- (DW-) MRI-derived apparent diffusion coefficient (ADC) parameter.Experimental design: PDAC cellularity was quantified histologically and correlated with the ADC parameter and survival in genetically engineered mouse models and human patients.Results: Histologic analysis showed an inverse relationship of tumor cellularity and stroma content. Low tumor cellularity correlated with a significantly prolonged mean survival time (PDAClow = 21.93 months vs. PDACmed = 12.7 months; log-rank P < 0.001; HR = 2.23; CI, 1.41–3.53). Multivariate analysis using the Cox regression method confirmed tumor cellularity as an independent prognostic marker (P = 0.034; HR = 1.73; CI, 1.04–2.89). Tumor cellularity showed a strong negative correlation with the ADC parameter in murine (r = −0.84; CI, −0.90– −0.75) and human (r = −0.79; CI, −0.90 to −0.56) PDAC and high preoperative ADC values correlated with prolonged survival (ADChigh = 41.7 months; ADClow = 14.77 months; log rank, P = 0.040) in PDAC patients.Conclusions: This study identifies high tumor cellularity as a negative prognostic factor in PDAC and supports the ADC parameter for the noninvasive identification of PDAC groups. Clin Cancer Res; 23(6); 1461–70. ©2016 AACR.

Rationale and Objectives To investigate the effects of a reduced field-of-view (rFOV) acquisition in diffusion-weighted magnetic resonance imaging of the pancreas. Materials and Methods We enrolled 153 patients who underwent routine clinical MRI work-up including respiratory-triggered diffusion-weighted single-shot echo-planar imaging (DWI) with full field-of-view (fFOV, 3 × 3 × 4 mm3 voxel size) and reduced field-of-view (rFOV, 2.5 × 2.5 × 3 mm3 voxel size) for suspected pancreatic pathology. Two experienced radiologists were asked to subjectively rate (Likert Scale 1–4) image quality (overall image quality, lesion conspicuity, anatomical detail, artifacts). In addition, quantitative image parameters were assessed (apparent diffusion coefficient, apparent signal to noise ratio, apparent contrast to noise ratio [CNR]). Results All subjective metrics of image quality were rated in favor of rFOV DWI images compared to fFOV DWI images with substantial-to-high inter-rater reliability. Calculated ADC values of normal pancreas, pancreatic pathologies and reference tissues revealed no differences between both sequences. Whereas the apparent signal to noise ratio was higher in fFOV images, apparent CNR was higher in rFOV images. Conclusion rFOV DWI provides higher image quality and apparent CNR values, favorable in the analysis of pancreatic pathologies.

Background: Pancreatic Ductal Adenocarcinoma (PDAC) lacks effective treatment options beyond chemotherapy. Although molecular subtypes such as classical and QM (quasi-mesenchymal)/basal-like with transcriptome-based distinct signatures have been identified, deduced therapeutic strategies and targets remain elusive. Gene expression data show enrichment of glycolytic genes in the more aggressive and therapy resistant QM subtype. However, whether the glycolytic transcripts are translated into functional glycolysis that could further be explored for metabolic targeting in QM subtype is still not known. Methods: We used different patient-derived PDAC model systems (conventional and primary patient derived cells, patient derived xenografts (PDX) and patient FFPE samples) and performed transcriptional and functional metabolic analysis. These included: RNAseq and Illumina HT12 bead array, in vitro Seahorse metabolic flux assays and metabolic drug targeting, and in vivo hyperpolarized [1-13C]pyruvate and [1-13C]lactate magnetic resonance spectroscopy (HP-MRS) in PDAC xenografts. Results: We found that glycolytic metabolic dependencies are not unambiguously functionally exposed in all QM PDACs. Metabolic analysis demonstrated functional metabolic heterogeneity in patient-derived primary cells and less so in conventional cell lines independent of molecular subtype. Importantly, we observed that the glycolytic product lactate is actively imported in the PDAC cells and used in mitochondrial oxidation in both classical and QM PDAC cells, although more actively in the QM cell lines. By using hyperpolarized 13C-magnetic resonance spectroscopy (HP-MRS), we were able to non-invasively identify highly glycolytic PDAC xenografts by detecting the last glycolytic enzymatic step and prominent intra-tumoral [1‑13C]pyruvate and [1-13C]lactate interconversion in vivo.Conclusion: Our study adds functional metabolic phenotyping to transcriptome-based analysis and proposes a functional approach to identify highly glycolytic PDACs as candidates for anti-metabolic therapeutic avenues.

The in vivo assessment of tissue metabolism represents a novel strategy for the evaluation of oncologic disease. Hepatocellular carcinoma (HCC) is a high-prevalence, high-mortality tumor entity often discovered at a late stage. Recent evidence indicates that survival differences depend on metabolic alterations in tumor tissue, with particular focus on glucose metabolism and lactate production. Here, we present an in vivo imaging technique for metabolic tumor phenotyping in rat models of HCC. Endogenous HCC was induced in Wistar rats by oral diethyl-nitrosamine administration. Peak lactate-to-alanine signal ratios (L/A) were assessed with hyperpolarized magnetic resonance spectroscopic imaging (HPMRSI) after [1

[Background & Aims]: The existence of different subtypes of pancreatic ductal adenocarcinoma (PDAC) and their correlation with patient outcome have shifted the emphasis on patient classification for better decision-making algorithms and personalized therapy. The contribution of mechanisms regulating the cancer stem cell (CSC) population in different subtypes remains unknown. [Methods]: Using RNA-seq, we identified B-cell CLL/lymphoma 3 (BCL3), an atypical nf-κb signaling member, as differing in pancreatic CSCs. To determine the biological consequences of BCL3 silencing in vivo and in vitro, we generated bcl3-deficient preclinical mouse models as well as murine cell lines and correlated our findings with human cell lines, PDX models, and 2 independent patient cohorts. We assessed the correlation of bcl3 expression pattern with clinical parameters and subtypes. [Results]: Bcl3 was significantly down-regulated in human CSCs. Recapitulating this phenotype in preclinical mouse models of PDAC via BCL3 genetic knockout enhanced tumor burden, metastasis, epithelial to mesenchymal transition, and reduced overall survival. Fluorescence-activated cell sorting analyses, together with oxygen consumption, sphere formation, and tumorigenicity assays, all indicated that BCL3 loss resulted in CSC compartment expansion promoting cellular dedifferentiation. Overexpression of BCL3 in human PDXs diminished tumor growth by significantly reducing the CSC population and promoting differentiation. Human PDACs with low BCL3 expression correlated with increased metastasis, and BCL3-negative tumors correlated with lower survival and nonclassical subtypes. [Conclusions]: We demonstrate that bcl3 impacts pancreatic carcinogenesis by restraining CSC expansion and by curtailing an aggressive and metastatic tumor burden in PDAC across species. Levels of BCL3 expression are a useful stratification marker for predicting subtype characterization in PDAC, thereby allowing for personalized therapeutic approaches., This work was supported by the Deutsche Forschungsgemeinschaft (grants AL 1174/4-1, AL1174/4-2, and Collaborative Research Center 1321 “Modeling and Targeting Pancreatic Cancer” to Hana Algül; SFB824 Z2 to Katja Steiger), the Deutsche Krebshilfe (grant 111646 to Hana Algül), a Ramon y Cajal Merit Award from the Ministerio de Economía y Competitividad, Spain (to Bruno Sainz Jr), a Coordinated Grant from Fundación Asociación Española Contra el Cáncer (GC16173694BARB to Bruno Sainz Jr), funding from The Fero Foundation (to Bruno Sainz Jr), and a Proyecto de Investigacion de Salud, ISCIII, Spain (no. PI18/00757 to Bruno Sainz Jr). Jiaoyu Ai is supported by the “China Scholarship Council” grant program.

Antibodies have become an established treatment modality in cancer therapy during the last decade. However, these treatments often suffer from an insufficient and heterogeneous response despite validated antigen or target receptor expression in the tumor. In fact, therapeutic success depends on both the presence of the tumor antigen and its accessibility by the antibody. In search of a suitable preclinical animal model to evaluate the mechanisms of tumor heterogeneity and hemodynamics, we characterized two exemplary non-Hodgkin lymphoma subtypes with comparable CD20 expression and metabolism, SUDHL-4 and Granta-519, using multimodal imaging techniques. Methods To investigate in vivo biodistribution, two differently modified αCD20 antigen-binding fragments (Fab), prepared by PASylation with a 200-residue polypeptide tag comprising Pro, Ala, and Ser (PAS200) and by fusion with an albumin-binding domain (ABD), were radiolabeled with 125I and intravenously injected into immunocompromised mice bearing corresponding xenografts. Results Validation with 18F-FDG revealed a similar distribution in vital tumor tissue 1 h after injection. However, large differences in tumor uptake were observed when the CD20-specific radiotracers 125I-Fab-ABD and 125I-Fab-PAS200 were applied (respective percentages injected dose per gram at 24 h after injection: 12.3 and 2.4 for Granta-519 vs. 5.8 and 1.2 for SUDHL-4). Three-dimensional light-sheet fluorescence microscopy with Cy5-Fab-PAS200 confirmed better tracer extravasation in the Granta-519 tumors. Moreover, dynamic contrast-enhanced (DCE) MRI revealed significantly reduced perfusion in the SUDHL-4 tumors. Conclusion Tracer uptake was highly dependent on local tumor perfusion and Fab permeation in the SUDHL-4 and Granta-519 tumors. Thus, the SUDHL-4 xenograft offers an excellent model for investigating the influence of therapies affecting tumor angiogenesis.

Maresch, Roman et al., Mouse transgenesis has provided fundamental insights into pancreatic cancer, but is limited by the long duration of allele/model generation. Here we show transfection-based multiplexed delivery of CRISPR/Cas9 to the pancreas of adult mice, allowing simultaneous editing of multiple gene sets in individual cells. We use the method to induce pancreatic cancer and exploit CRISPR/Cas9 mutational signatures for phylogenetic tracking of metastatic disease. Our results demonstrate that CRISPR/Cas9-multiplexing enables key applications, such as combinatorial gene-network analysis, in vivo synthetic lethality screening and chromosome engineering. Negative-selection screening in the pancreas using multiplexed-CRISPR/Cas9 confirms the vulnerability of pancreatic cells to Brca2-inactivation in a Kras-mutant context. We also demonstrate modelling of chromosomal deletions and targeted somatic engineering of inter-chromosomal translocations, offering multifaceted opportunities to study complex structural variation, a hallmark of pancreatic cancer. The low-frequency mosaic pattern of transfection-based CRISPR/Cas9 delivery faithfully recapitulates the stochastic nature of human tumorigenesis, supporting wide applicability for biological/preclinical research., The work was supported by the German Cancer Consortium Joint Funding Program, the Helmholtz Gemeinschaft (PCCC Consortium) and the European Research Council (ERC CoG No. 648521).

Purpose To test combined dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and 18F-FDG positron emission tomography (FDG-PET)-derived parameters for prediction of histopathological grading in a rat Diethyl Nitrosamine (DEN)-induced hepatocellular carcinoma (HCC) model. Methods 15 male Wistar rats, aged 10 weeks were treated with oral DEN 0.01 % in drinking water and monitored until HCCs were detectable. DCE-MRI and PET were performed consecutively on small animal scanners. 38 tumors were identified and manually segmented based on HCC-specific contrast enhancement patterns. Grading (G2/3: 24 tumors, G1:14 tumors) alongside other histopathological parameters, tumor volume, contrast agent and 18F-FDG uptake metrics were noted. Class imbalance was addressed using SMOTE and collinearity was removed using hierarchical clustering and principal component analysis. A logistic regression model was fit separately to the individual parameter groups (DCE-MRI-derived, PET-derived, tumor volume) and the combined parameters. Results The combined model using all imaging-derived parameters achieved a mean ± STD sensitivity of 0.88 ± 0.16, specificity of 0.70 ± 0.20 and AUC of 0.90 ± 0.03. No correlation was found between tumor grading and tumor volume, morphology, necrosis, extracellular matrix, immune cell infiltration or underlying liver fibrosis. Conclusion A combination of DCE-MRI- and 18F-FDG-PET-derived parameters provides high accuracy for histopathological grading of hepatocellular carcinoma in a relevant translational model system.

Recent advances in molecular subtyping of Pancreatic Ductal Adenocarcinoma (PDAC) support individualization of therapeutic strategies in this most aggressive disease. With the emergence of various novel therapeutic strategies and neoadjuvant approaches in this quickly deteriorating disease, robust approaches for fast evaluation of therapy response are urgently needed. To this aim, we designed a preclinical imaging-guided therapy trial where genetically engineered mice harboring endogenous aggressive PDAC were treated with the MEK targeting drug refametinib, which induces rapid and profound tumor regression in this model system. Multi-parametric non-invasive imaging was used for therapy response monitoring. A significant increase in the Diffusion-Weighted Magnetic Resonance Imaging derived Apparent Diffusion Coefficient (ADC) was noted already 24 hours after treatment onset. Histopathological analyses showed increased apoptosis and matrix remodeling at this time point. Our findings suggest the ADC parameter as an early predictor of therapy response in PDAC.

OA gold Surface CD24 has previously been described, together with CD44 and ESA, for the characterization of putative cancer stem cells in pancreatic ductal adenocarcinoma (PDAC), the most fatal of all solid tumors. CD24 has a variety of biological functions including the regulation of invasiveness and cell proliferation, depending on the tumor entity and subcellular localization. Genetically engineered mouse models (GEMM) expressing oncogenic KrasG12D recapitulate the human disease and develop PDAC. In this study we investigate the function of CD24 using GEMM of endogenous PDAC and a model of cerulein-induced acute pancreatitis. We found that (i) CD24 expression was upregulated in murine and human PDAC and during acute pancreatitis (ii) CD24 was expressed exclusively in differentiated PDAC, whereas CD24 absence was associated with undifferentiated tumors and (iii) membranous CD24 expression determines tumor subpopulations with an epithelial phenotype in grafted models. In addition, we show that CD24 protein is stabilized in response to WNT activation and that overexpression of CD24 in pancreatic cancer cells upregulated β-catenin expression augmenting an epithelial, non-metastatic signature. Our results support a positive feedback model according to which (i) WNT activation and subsequent β-catenin dephosphorylation stabilize CD24 protein expression, and (ii) sustained CD24 expression upregulates β-catenin expression. Eventually, membranous CD24 augments the epithelial phenotype of pancreatic tumors. Thus we link the WNT/β-catenin pathway with the regulation of CD24 in the context of PDAC differentiation.

Purpose: Tumor heterogeneity is a hallmark of pancreatic ductal adenocarcinoma (PDAC). It determines tumor biology including tumor cellularity (i.e., amount of neoplastic cells and arrangement into clusters), which is related to the proliferative capacity and differentiation and the degree of desmoplasia among others. Given the close relation of tumor differentiation with differences in progression and therapy response or, e.g., the recently reported protective role of tumor stroma, we aimed at the noninvasive detection of PDAC groups, relevant for future personalized approaches. We hypothesized that histologic differences in PDAC tissue composition are detectable by the noninvasive diffusion weighted- (DW-) MRI-derived apparent diffusion coefficient (ADC) parameter. Experimental design: PDAC cellularity was quantified histologically and correlated with the ADC parameter and survival in genetically engineered mouse models and human patients. Results: Histologic analysis showed an inverse relationship of tumor cellularity and stroma content. Low tumor cellularity correlated with a significantly prolonged mean survival time (PDAClow = 21.93 months vs. PDACmed = 12.7 months; log-rank P < 0.001; HR = 2.23; CI, 1.41–3.53). Multivariate analysis using the Cox regression method confirmed tumor cellularity as an independent prognostic marker (P = 0.034; HR = 1.73; CI, 1.04–2.89). Tumor cellularity showed a strong negative correlation with the ADC parameter in murine (r = −0.84; CI, −0.90– −0.75) and human (r = −0.79; CI, −0.90 to −0.56) PDAC and high preoperative ADC values correlated with prolonged survival (ADChigh = 41.7 months; ADClow = 14.77 months; log rank, P = 0.040) in PDAC patients. Conclusions: This study identifies high tumor cellularity as a negative prognostic factor in PDAC and supports the ADC parameter for the noninvasive identification of PDAC groups. Clin Cancer Res; 23(6); 1461–70. ©2016 AACR.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers and shows resistance to any therapeutic strategy used. Here we tested small-molecule inhibitors targeting chromatin regulators as possible therapeutic agents in PDAC. We show that JQ1, an inhibitor of the bromodomain and extraterminal (BET) family of proteins, suppresses PDAC development in mice by inhibiting both MYC activity and inflammatory signals. The histone deacetylase (HDAC) inhibitor SAHA synergizes with JQ1 to augment cell death and more potently suppress advanced PDAC. Finally, using a CRISPR-Cas9-based method for gene editing directly in the mouse adult pancreas, we show that de-repression of p57 (also known as KIP2 or CDKN1C) upon combined BET and HDAC inhibition is required for the induction of combination therapy-induced cell death in PDAC. SAHA is approved for human use, and molecules similar to JQ1 are being tested in clinical trials. Thus, these studies identify a promising epigenetic-based therapeutic strategy that may be rapidly implemented in fatal human tumors.

Here, we show CRISPR/Cas9-based targeted somatic multiplex-mutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such as Pten or Cdkn2a, and conversely found low frequency of Brca1/2 alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes The work was supported by the German Cancer Consortium Joint Funding Program and the Helmholtz Gemeinschaft (Preclinical Comprehensive Cancer Center).

Purpose: Preclinical model systems should faithfully reflect the complexity of the human pathology. In hepatocellular carcinoma (HCC), the tumor vasculature is of particular interest in diagnosis and therapy. By comparing two commonly applied preclinical model systems, diethylnitrosamine induced (DEN) and orthotopically implanted (McA) rat HCC, we aimed to measure tumor biology noninvasively and identify differences between the models. Experimental Design: DEN and McA tumor development was monitored by MRI and PET. A slice-based correlation of imaging and histopathology was performed. Array CGH analyses were applied to determine genetic heterogeneity. Therapy response to sorafenib was tested in DEN and McA tumors. Results: Histologically and biochemically confirmed liver damage resulted in increased 18F-fluorodeoxyglucose (FDG) PET uptake and perfusion in DEN animals only. DEN tumors exhibited G1–3 grading compared with uniform G3 grading of McA tumors. Array comparative genomic hybridization revealed a highly variable chromosomal aberration pattern in DEN tumors. Heterogeneity of DEN tumors was reflected in more variable imaging parameter values. DEN tumors exhibited lower mean growth rates and FDG uptake and higher diffusion and perfusion values compared with McA tumors. To test the significance of these differences, the multikinase inhibitor sorafenib was administered, resulting in reduced volume growth kinetics and perfusion in the DEN group only. Conclusions: This work depicts the feasibility and importance of in depth preclinical tumor model characterization and suggests the DEN model as a promising model system of multifocal nodular HCC in future therapy studies. Clin Cancer Res; 21(19); 4440–50. ©2015 AACR. See related commentary by Weber et al., p. 4254

Human pancreatic ductal adenocarcinoma (PDAC) displays high heterogeneity in terms of morphology, onset, and therapy response. Genetically engineered mouse models (GEMMs) reflect differences in human PDACs and are valuable, predictive tools for preclinical research. In this study, a comprehensive noninvasive imaging approach using various GEMMs was performed with the aim to identify markers that allow prediction of tumor type and response to therapy in humans. GEMMs with different PDAC characteristics were analyzed for tumor growth kinetics, tissue composition, perfusion and response to chemotherapies using multiparametric MRI and [18F]-FDG-PET imaging. Following genetically engineered mouse models of PDAC were used: Ptf1a wt/Cre ( C ) Kras wt/LSL-G12D ( K ) Ela-Tgfa ( T ), CKp53 ( P ) wt/fl , CKP fl/fl , CKTP wt/R172H and CKTP wt/fl . Beginning at tumor onset animals were subjected to serial T2-weighted (T2w) diffusion weighted (DWI) and dynamic contrast-enhanced (DCE)-MRI employing a 1.5 T Achieva, Philips with a dedicated surface coil. Additionally, metabolic status of tumors was assessed by [18F]-FDG-PET. In a subset of CKP fl/fl mice therapy response to clinical standard of care chemotherapy gemcitabine was monitored. Imaging data was validated by histopathology. Murine PDACs developing from either PanIN or IPMN showed differences in tumor onset, growth kinetics and appearance, mimicking the wide spectrum of human disease. T2w imaging differentiated hypointense mIPMNs and acinus cell carcinomas from hyperintense solid ductal PDACs. Calculated apparent diffusion coefficients (ADC) showed good correlation with tissue cellularity (r2 = 0.73) and collagen content (r2 = 0.65). DWI allowed differentiation of preneoplastic lesions with inflammatory tissue (≥ 1.2 mm2/s), mIPMNs (0.99 ± 0.02 mm2/s), well differentiated mPDAC (1.031 ± 0.02 mm2/s), less differentiated mPDACs (0.80 ± 0.01 mm2/s) and acinus cell carcinomas or anaplastic mPDACs (0.67 ± 0.03 mm2/s). DCE-MRI data was highly heterogeneous and revealed major intraindividual differences between tumor regions. [18F]-FDG tracer uptake tumor to muscle ratios (T/M) enabled differentitation of preneoplastic lesions (T/M 3), independent of the underlying genotype. PDAC in CKP fl/fl mice showed similar resistance to gemcitabine treatment as human PDAC. Only single animals were identified as responders based on an increase in survival and calculated ADC value and a decrease in tumor growth and [18F]-FDG uptake. Here we provide evidence that morphological heterogeneity of PDAC is well modeled with GEMMs and identifiable with multimodal multiparametric imaging. The tissue structure related parameter ADC is identified as a sensitive marker of tumor heterogeneity. The combined evaluation of tumor growth, [18F]-FDG tracer uptake and ADC value identified individual therapy responders. Citation Format: Irina Heid, Marija Trajkovic-Arsic, Irene Esposito, Manuela R. Gretzinger, Andreas Steingoetter, Bernhard Haller, Markus Schwaiger, Ernst J. Rummeny, Jens T. Siveke, Rickmer F. Braren. Imaging heterogeneity of endogenous mouse models of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A5.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies with an alarming resistance to both chemotherapeutics and targeted therapy approaches. Genetically engineered mouse models (GEMM) of PDAC reflect molecularly and pathophysiologically the carcinogenesis and cancer progression observed in humans, thus being excellent tools for preclinical evaluation of new therapies. To successfully predict and characterize the effect of chemotherapies and targeted approaches in PDAC, we use a complex and highly aggressive GEMM of endogenous PDAC, Ptf1a +/Cre Kras +/LSL-G12D p53Lox/Lox (CKP) mice. To do so, we established a highly usable GEMM-based therapy platform including multi-parametric MR imaging using CKP mice, in which tumors develop within the first 6 weeks of age (Bardeesy et al, 2006). Animals receive a T2w scan on a 3T clinical MRI scanner for detection and staging of solid tumors for study enrolment at defined inclusion criteria (tumour volume 200-400 mm3). Mice get a weekly scan throughout the study until endpoint criteria are met. Upon reaching the endpoint criteria, mice are sacrificed and tumor material is processed for histopathological and RNA/Protein analysis, isolation and culturing of primary tumor cells. Tumors are assessed macro- and microscopically and graded. Proliferation, apoptosis and analysis of respective downstream effectors are characterized. Further comprehensive analysis depending on the respective phenotypical features is then carried out. Primary cell lines are generated for further functional and molecular characterization including but not limited to cell viability, drug sensitivity, expression and methylation profiling. Through this platform, we have successfully been able to target key signaling pathways and to identify promising novel chemotherapeutic and targeted combinations for PDAC. Here we want to highlight the vital need to develop improved preclinical tools to characterize individual tumors throughout the course of treatment. We propose that such a platform is highly relevant and usable for predicting responses to chemotherapeutic and targeted agents. Citation Format: Aayush Gupta, Marija Trajkovic-Arsic, Irina Heid, Nicole Teichman, Evdokia Kalederis, Rickmer Braren, Jens Siveke. Predictive value of genetically engineered endogenous mouse models in preclinical therapeutic studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B141.

Background & Aims Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease without effective chemopreventive or therapeutic approaches. Although the role of oncogenic Kras in initiating development of PDAC is well established, downstream targets of aberrant Ras signaling are poorly understood. Acinar-ductal metaplasia (ADM) appears to be an important prerequisite for development of pancreatic intraepithelial neoplasia (PanIN), a common precursor to PDAC. RAS-related C3 botulinum substrate 1 (Rac1), which controls actin reorganization, can be activated by Ras, is up-regulated in several human cancers, and is required for cerulein-induced morphologic changes in acini. We investigated effects of loss of Rac1 in Kras-induced pancreatic carcinogenesis in mice. Methods Using a Cre/lox approach, we deleted Rac1 from pancreatic progenitor cells in different mouse models of PDAC and in mice with cerulein-induced acute pancreatitis. Acinar epithelial explants of mutant mice were used to investigate the role of Rac1 in vitro. Results Rac1 expression increased in mouse and human pancreatic tumors, particularly in the stroma. Deletion of Rac1 in Kras G12D -induced PDAC in mice reduced formation of ADM, PanIN, and tumors and significantly prolonged survival. Pancreatic epithelial metaplasia was accompanied by apical-basolateral redistribution of F-actin, along with basal expression of Rac1. Acinar epithelial explants that lacked Rac1 or that were incubated with inhibitors of actin polymerization had a reduced ability to undergo ADM in 3-dimensional cultures. Conclusions In mice, Rac1 is required for early metaplastic changes and neoplasia-associated actin rearrangements in development of pancreatic cancer. Rac1 might be developed as a diagnostic marker or therapeutic target for PDAC.

Development of strategies for early tumor detection and evaluation of new therapies in preclinical models is one of the main goals of current cancer research. Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with no effective treatment plan. PDAC progression can be very well mimicked by established genetically engineered mouse models (GEMMs), representing an excellent platform for preclinical studies. Therefore, tumor physiology, including growth kinetics, tumor perfusion and tumor composition, were characterized in several GEMM. In addition, tumor response to the standard therapeutic agent gemcitabine was monitored by multiparametric MRI. Ptf1a+/Cre mice were crossed to Kras+/LSL-G12D (K)Tgfα(T), K;p53(P)+/fl, K;Pfl/fl, K;P+/R172H; T and K;P+/fl;T GEMMs to generate conditional endogenous PDAC models. Animals were subjected to serial T2-weighted (T2w) MRI from 4–6 weeks of age onwards using 1,5 T clinical scanner. Upon detection of solid tumor, longitudinal diffusion-weighted imaging (DWI) and dynamic contrast enhanced-MRI (DCE-MRI with Gd-DTPA) protocols were applied. For the evaluation of therapy response monitoring, K;Pfl/fl mice were injected twice a week with 120 mg/kg body weight with gemcitabine or 0.9% NaCl solution. MRI data were correlated with survival analysis, vascular staining, and histopathology of tumor specimens. Volume analysis was performed using semiautomatic segmentation of solid and cystic compartments. K;Pfl/fl, and K;P+/fl; T models showed 100% tumor incidence, detected by MRI and verified by histology. Moreover, K;P+/fl; T animals often presented with metastatic disease to lung and liver, easily identified on T2w scans, while no metastases were observed in K;Pfl/fl mice. The final tumor volume ranged between 800 and 4500 mm3. Characterization of solid tumor tissue composition by DWI correlated well (R=72%) with the histopathology. K;Pfl/fl, K;P+/R172H; T, K;P+/fl; T, and K; T models mostly developed highly fibrotic, well-differentiated PDAC (apparent diffusion coefficient, ADC=1.03-1.14), whereas cancers observed in K;Pfl/+ animals revealed an anaplastic phenotype with significantly lower ADC values (0.8843 ± 0.05056). Spontaneous necrosis was detectable by DWI and DCE analysis. Intraductal mucinous papillary neoplasms (mIPMN) appeared hypointense on T2w MRI and were better perfused compared to hyperintense PDAC. Interestingly, the calculated ADC values of mIPMN lesions were similar to those of solid tumors (1.03 ± 0.012). Gemcitabine-treated K;Pfl/fl animals showed an increased overall survival as well as tumor volume reduction in individual cases. Although selected animals revealed changes in tumor perfusion, no differences in tissue composition, reflected by the ADC values and histology, were noticed in vehicle- or gemcitabine-treated animals. In summary, based on tumor incidence, growth kinetic and overall survival criteria, we propose two very suitable models for preclinical therapy studies: K;Pfl/fl and K;P+/fl; T. The validated multimodal MRI-based methods are feasible for correct interpretation of spontaneous and drug-induced changes in tumor physiology when conducting preclinical therapy studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C8.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a devastating human malignancy that is highly resistant to chemo- and radiation therapy. Lately our research group established a new mouse model of pancreatic cancer which recapitulates two of the most frequent genetic alterations in human PDAC: activation of oncogenic KrasG12D and EGFR signaling. These mice develop invasive and metastatic PDAC from both clinically most relevant preneoplastic lesions, so-called pancreatic intraepithelial neoplasia (PanIN) and intraductal mucinous papillary neoplasm (IPMN). The aim of this study was to characterize Ras-dependent pathways in cell lines isolated from KrasG12D and EGFR activated PDAC in order to develop and test new individual therapeutic strategies for treatment of pancreatic cancer. To evaluate promising candidates in vivo, a multimodal platform for noninvasive imaging was evaluated and used for preclinical studies. Methods: Primary tumor cells isolated from PDAC of Ptf1a+/Cre;KrasG12D and Ptf1a+/Cre;KrasG12D;Ela-Tgf mice were characterized by genetic and biochemical analysis using array-CGH, gene sequencing, protein activation assays and western blotting. Moreover, the cellular response to known chemo therapeutics and selected signal transduction inhibitors were tested in vitro and some of them in vivo using endogenous and transplantation models. Spontaneous tumor progression and therapy response were monitored by multiparametric imaging using a combination of DCE-MRI, DWI and [18F]FDG-PET measurements. Results: Similarly to human PDAC, most of the murine cell lines showed an inactivation of tumor suppressors such as p16Ink4a, p19Arf and Tp53. Furthermore, some of the cell lines showed amplification of Tgf /Egfr and Myc loci. Different levels of MAPK, STAT3 and AKT- phosphorylation as well as activation of Rac1 and cdc42 were observed. We found that, Gemcitabine was the most active agent of all tested in vitro. A combination of the Rac1 inhibitor NSC23766 and Gemcitabine led to a significant additive growth-inhibitory effect in most primary cell lines in vitro, while inhibition of other Ras-dependent pathways in combination with Gemcitabine showed no strong antiproliferative effect. In vivo, we observed a high heterogeneity of endogenous tumors in terms of perfusion, tumor composition and metabolic activity. Multimodal monitoring of Gemcitabine therapy in endogenous PDAC as well as different transplantation models showed resistance of tumors to treatment independent of the model used. Conclusion: Our results reveal that mice with the same genetic background have different genetic and biochemical alterations during cancer progression, emphasizing the necessity for developing new individualized therapeutic strategies for pancreatic carcinoma. Here we developed a robust platform to study PDAC for further preclinical testing in endogenous mouse models, which closely mimics the human situation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A234.

One of the major challenges in using pancreatic cancer patient–derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for endoscopic ultrasound-guided fine-needle aspiration biopsies, typically resulting in specimens with limited tumor cell yield. Here, we tested conditioned media of individual PDOs for cell-free DNA to detect driver mutations already early on during the expansion process to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72 hours after biopsy, recapitulate the mutational profile of the primary tumor, indicating suitability of this approach to subject PDOs to drug testing in a reduced time frame. In addition, we demonstrated that this workflow was practicable, even in patients for whom the amount of tumor material was not sufficient for molecular characterization by established means. Together, our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach, this can be achieved in a rapid and feasible fashion with broad implications in clinical practice. Kein CA