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Supplemental Figure 4. mRNA abundance in triptolide-treated PDAC 001 cells

Purpose: Even when diagnosed prior to metastasis, pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with almost 90% lethality, emphasizing the need for new therapies optimally targeting the tumors of individual patients.Experimental Design: We first developed a panel of new physiologic models for study of PDAC, expanding surgical PDAC tumor samples in culture using short-term culture and conditional reprogramming with the Rho kinase inhibitor Y-27632, and creating matched patient-derived xenografts (PDX). These were evaluated for sensitivity to a large panel of clinical agents, and promising leads further evaluated mechanistically.Results: Only a small minority of tested agents was cytotoxic in minimally passaged PDAC cultures in vitro. Drugs interfering with protein turnover and transcription were among most cytotoxic. Among transcriptional repressors, triptolide, a covalent inhibitor of ERCC3, was most consistently effective in vitro and in vivo causing prolonged complete regression in multiple PDX models resistant to standard PDAC therapies. Importantly, triptolide showed superior activity in MYC-amplified PDX models and elicited rapid and profound depletion of the oncoprotein MYC, a transcriptional regulator. Expression of ERCC3 and MYC was interdependent in PDACs, and acquired resistance to triptolide depended on elevated ERCC3 and MYC expression. The Cancer Genome Atlas analysis indicates ERCC3 expression predicts poor prognosis, particularly in CDKN2A-null, highly proliferative tumors.Conclusions: This provides initial preclinical evidence for an essential role of MYC–ERCC3 interactions in PDAC, and suggests a new mechanistic approach for disruption of critical survival signaling in MYC-dependent cancers. Clin Cancer Res; 22(24); 6153–63. ©2016 AACR.

AUC results of the drug sensitivity screens in conditionally reprogrammed PDAC cells.

Table S1. Genetic characterization of patient-derived xenografts using the Illumina Trusight panel capturing frequent mutations in 94 genes and 284 SNPs associated with cancer Table S3. Triptolide-induced effects on the mRNA abundance was determined by Illumina HighSeq RNA sequencing of 001 cells treated with 100 nM triptolide in vitro for 6 hours.

Patient-derived in vivo models of human cancer have become a reality, yet their turnaround time is inadequate for clinical applications. Therefore, tailored ex vivo models that faithfully recapitulate in vivo tumour biology are urgently needed. These may especially benefit the management of pancreatic ductal adenocarcinoma (PDAC), where therapy failure has been ascribed to its high cancer stem cell (CSC) content and high density of stromal cells and extracellular matrix (ECM). To date, these features are only partially reproduced ex vivo using organoid and sphere cultures. We have now developed a more comprehensive and highly tuneable ex vivo model of PDAC based on the 3D co-assembly of peptide amphiphiles (PAs) with custom ECM components (PA-ECM). These cultures maintain patient-specific transcriptional profiles and exhibit CSC functionality, including strong in vivo tumourigenicity. User-defined modification of the system enables control over niche-dependent phenotypes such as epithelial-to-mesenchymal transition and matrix deposition. Indeed, proteomic analysis of these cultures reveals improved matrisome recapitulation compared to organoids. Most importantly, patient-specific in vivo drug responses are better reproduced in self-assembled cultures than in other models. These findings support the use of tuneable self-assembling platforms in cancer research and pave the way for future precision medicine approaches., Biotechnology and Biological Sciences Research Council; LIDo Grant; Medical Research Council; UK Regenerative Medicine Platform Acellular/Smart Materials-3D Architecture; Fondazione Italiana Malattie Pancreas; Italian Ministry of Health; Fondazione Cariverona: Oncology Biobank Project “Antonio Schiavi”; European Union (EU); Horizon 2020; European Community Seventh Framework Programme; FP7/2007-2013; CAM-PaC Consortium; BIOMORPH; Marie Curie Integration Grant; STROFUNSCAFF; ERC Starting Grant; Associazione Italiana Ricerca Cancro; Barts Cancer Institute Catalyst; IMPETUS Awards

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. Drug resistance is the major cause for therapeutic failure in PDAC patients with progressive disease. The mechanisms underlying resistance formation are complex and remain poorly understood.To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data.We found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. The effector caspase CASP3 is one of the 114 genes for which transcriptional downregulation inversely correlated with the methylation status of the associated DNA region. CASP3 inactivation in resistant cells led to attenuation of drug-induced apoptosis which could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells.Overall, our data provide a context for characterization and targeting of epigenetically mediated resistance mechanisms in PDAC.

Simple Summary Current therapies for pancreas carcinoma (PC) are of limited efficacy due to tumor aggressiveness and therapy resistance. Bacterial toxins with pore-forming (oncoleaking) potential are promising tools in cancer therapy. We have developed a novel, suicide gene therapy treatment, based on Clostridium perfringens enterotoxin (CPE)-mediated oncoleaking. This is achieved by CPE suicide gene therapy to treat PC, which overexpresses the claudin-3 and -4 (Cldn3/4) tight junction proteins, which are targets of CPE action. This targeted gene therapy causes rapid eradication of Cldn3/4 overexpressing PC cells via oncoleaking and initiation of apoptotic/necrotic signaling. We demonstrate efficacy of this approach in vitro and after nonviral in vivo gene transfer in cell lines and in patient derived xenograft PC models. This therapy approach has translational potential for treatment of pancreas carcinomas and could also be translated into new combination settings with conventional chemotherapy. Abstract Pancreatic cancer (PC) is one of the most lethal cancers worldwide, associated with poor prognosis and restricted therapeutic options. Clostridium perfringens enterotoxin (CPE), is a pore-forming (oncoleaking) toxin, which binds to claudin-3 and -4 (Cldn3/4) causing selective cytotoxicity. Cldn3/4 are highly upregulated in PC and represent an effective target for oncoleaking therapy. We utilized a translation-optimized CPE vector (optCPE) for new suicide approach of PC in vitro and in cell lines (CDX) and patient-derived pancreatic cancer xenografts (PDX) in vivo. The study demonstrates selective toxicity in Cldn3/4 overexpressing PC cells by optCPE gene transfer, mediated by pore formation, activation of apoptotic/necrotic signaling in vitro, induction of necrosis and of bystander tumor cell killing in vivo. The optCPE non-viral intratumoral in vivo jet-injection gene therapy shows targeted antitumoral efficacy in different CDX and PDX PC models, leading to reduced tumor viability and induction of tumor necrosis, which is further enhanced if combined with chemotherapy. This selective oncoleaking suicide gene therapy improves therapeutic efficacy in pancreas carcinoma and will be of value for better local control, particularly of unresectable or therapy refractory PC.

// Brajesh P. Kaistha 1, * , Anja Krattenmacher 1, * , Johannes Fredebohm 2, * , Harald Schmidt 1 , Diana Behrens 3 , Miriam Widder 4 , Thilo Hackert 5 , Oliver Strobel 5 , Jorg D. Hoheisel 2 , Thomas M. Gress 1 and Malte Buchholz 1 1 Department of Medicine, Division of Gastroenterology, Endocrinology and Metabolism, Philipps-University Marburg, Marburg, Germany 2 Division of Functional Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Experimantal Pharmacology and Oncology (EPO Berlin-Buch), Berlin, Germany 4 Institute for Bioprocessing and Analytical Measurement Techniques (IBA-Heiligenstadt), Heilbad Heiligenstadt, Germany 5 Department of Surgery, University Clinic Heidelberg, Heidelberg, Germany * These authors have contributed equally to this work Correspondence to: Malte Buchholz, email: malte.buchholz@staff.uni-marburg.de Keywords: pancreatic cancer, shRNA barcode library screen, ubiquitin specific peptidase, deubiquitinases, molecular pathogenesis Received: March 31, 2017 Accepted: July 11, 2017 Published: August 03, 2017 ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid tumors. With an overall five-year survival rate remaining below 6%, there is an explicit need to search for new molecular targets for therapeutic interventions. We undertook a barcode labelled short-hairpin (shRNA) library screen in pancreatic cancer cells in order to identify novel genes promoting cancer survival and progression. Among the candidate genes identified in this screen was the deubiquitinase USP5, which subsequent gene expression analyses demonstrated to be significantly upregulated in primary human pancreatic cancer tissues. Using different knockdown approaches, we show that expression of USP5 is essential for the proliferation and survival of pancreatic cancer cells, tested under different 2D and 3D cell culture conditions as well as in in vivo experiments. These growth inhibition effects upon knockdown of USP5 are mediated primarily by the attenuation of G1/S phase transition in the cells, which is accompanied by accumulation of DNA damage, upregulation of p27, and increased apoptosis rates. Since USP5 is overexpressed in cancer tissues, it can thus potentially serve as a new target for therapeutic interventions, especially given the fact that deubiquitinases are currently emerging as new class of attractive drug targets in cancer.

Oncogenic KRAS mutations are encountered in more than 90% of pancreatic ductal adenocarcinomas. MEK inhibition has failed to procure any clinical benefits in mutant RAS-driven cancers including pancreatic ductal adenocarcinoma (PDAC). To identify potential resistance mechanisms underlying MEK inhibitor (MEKi) resistance in PDAC, we investigated lysosomal drug accumulation in PDAC models both in vitro and in vivo. Mouse PDAC models and human PDAC cell lines as well as human PDAC xenografts treated with the MEK inhibitor trametinib or refametinib led to an enhanced expression of lysosomal markers and enrichment of lysosomal gene sets. A time-dependent, increase in lysosomal content was observed upon MEK inhibition. Strikingly, there was a strong activation of lysosomal biogenesis in cell lines of the classical compared to the basal-like molecular subtype. Increase in lysosomal content was associated with nuclear translocation of the Transcription Factor EB (TFEB) and upregulation of TFEB target genes. siRNA-mediated depletion of TFEB led to a decreased lysosomal biogenesis upon MEK inhibition and potentiated sensitivity. Using LC-MS, we show accumulation of MEKi in the lysosomes of treated cells. Therefore, MEK inhibition triggers lysosomal biogenesis and subsequent drug sequestration. Combined targeting of MEK and lysosomal function may improve sensitivity to MEK inhibition in PDAC.

Poster Abstract

Melanoma is a leading cause of high mortality that frequently spreads to the brain and is associated with deterioration in quality and quantity of life. Treatment opportunities have been restricted until now and new therapy options are urgently required. Our focus was to reveal the potential heterogeneity of melanoma brain metastasis. We succeeded to establish a brain melanoma metastasis cell line, namely MUG-Mel1 and two resulting clones D5 and C8 by morphological variety, differences in lipidome, growth behavior, surface, and stem cell markers. Mutation analysis by next-generation sequencing, copy number profiling, and cytogenetics demonstrated the different genetic profile of MUG-Mel1 and clones. Tumorigenicity was unsuccessfully tested in various mouse systems and finally established in a zebra fish model. As innovative treatment option, with high potential to pass the blood-brain barrier a peptide isolated from lactoferricin was studied in potential toxicity. Brain metastases are a major clinical challenge, therefore the development of relevant in vitro and in vivo models derived from brain melanoma metastases provides valuable information about tumor biology and offers great potential to screen for new innovative therapies.

Nonviral gene therapy represents a realistic option for clinical application in cancer treatment. This preclinical study demonstrates the advantage of using the small-size MIDGE(®) DNA vector for improved transgene expression and therapeutic application. This is caused by significant increase in transcription efficiency, but not by increased intracellular vector copy numbers or gene transfer efficiency. We used the MIDGE-hTNF-alpha vector for high-level expression of hTNF-alpha in vitro and in vivo for a combined gene therapy and vindesine treatment in human melanoma models. The MIDGE vector mediated high-level hTNF-alpha expression leads to sensitization of melanoma cells towards vindesine. The increased efficacy of this combination is mediated by remarkable acceleration and increase of initiator caspase 8 and 9 and effector caspase 3 and 7 activation. In the therapeutic approach, the nonviral intratumoral in vivo jet-injection gene transfer of MIDGE-hTNF-alpha in combination with vindesine causes melanoma growth inhibition in association with increased apoptosis in A375 cell line or patient derived human melanoma xenotransplant (PDX) models. This study represents a proof-of-concept for an anticipated phase I clinical gene therapy trial, in which the MIDGE-hTNF-alpha vector will be used for efficient combined chemo- and nonviral gene therapy of malignant melanoma.

Pancreatic cancer is a cruel, progressive disease that is highly metastatic and barely treatable, a situation that is devastating for patients, family members, oncologists, clinicians and scientists. Open questions that need to be resolved by research into pancreatic cancer relate to its aggressiveness, the underlying molecular causes, the factors that promote tumor progression, the ways cancer cells interact with their environments, and whether more effective therapeutic options can be developed. Studies over the last 15 years have provided some partial answers, but in the absence of a real cure the main agenda remains: to identify new therapeutic targets, predictive markers and novel treatment strategies that would help the disease under control. These goals can be advanced by translational research based on clinically relevant and standardized protocols and more reliable disease models. This review gives an overview of the preclinical in vitro and in vivo models for pancreatic cancer that are currently available. The restrictions on applicability, strengths and limitations of various experimental platforms including 3D organoids, syngeneic xenografts and genetically engineered mice are considered with respect to the complexity of pancreating cancer. Patient-derived xenografts (PDX) presently offer the most promise for translational research, so a particular emphasis is placed on key features as preclinical models for pancreatic cancer and their advancement toward precise simulations of clinical problems.

The 4-diethylaminomethylbenzyl-substituted titanocene dichloride (Titanocene Y*), which is completely water- soluble and showed nanomolar activity against the human renal cancer cells CAKI-1, was tested in vitro in an antiangiogenesis assay against human umbilical vein endothelial cells, HUVEC, delivering an IC50 value of 23 +/- 17 {Mu}M. Titanocene Y* was then given at 25, 50 and 75 mg/kg/d, on five consecutive days per week for up to three weeks to one cohort of six CAKI-1 tumor-bearing female NMRI:nu/nu mice, while a further cohort was treated with solvent only. At the two higher dosages Titanocene Y* showed high toxicity leading to mortality, while the titanocene-treated mouse cohort treated with the lowest dosage showed a moderate but statistically significant tumor growth reduction with respect to the solvent-treated control group, with an optimal T/C value of 76% at the end of the experiment. Immunohistological analysis revealed that the expression of the proliferation marker Ki-67 was reduced by 21%. Furthermore, anti-angiogenic activity was identified by CD31 staining; the number of micro vessels in a defined tumor area decreased by 23% due to Titanocene Y* treatment. The substance caused dose-dependent body weight loss but did not reduce the number of white blood cells at doses of 25 and 50 mg/kg/d.

Pancreatic cancer (PC) remains a lethal disease with only 3 - 8% of patients surviving 5 years after diagnosis of the tumor (WHO, 2012). Within the EU project “CAM-PaC” a comprehensive panel of thirty patient-derived PC xenografts (PDX) was established and used for the efficacy screening of new therapeutic options. Within this study, responders to the MPS-1 inhibitor BAY1161909, the Super Enhancer disrupting agent Minnelide and the MEK inhibitor Trametinib were identified and analyzed for potential biomarkers. Patient tumors were collected during surgery and circulating tumorigenic cancer stem cells were isolated from the peripheral blood using VAR2CSA-coated magnetic beads. Both were transplanted subcutaneously into NOD/SCID/IL2y- mice and propagated in NMRI:nu/nu mice after engraftment. These were morphologically and molecularly characterized by histopathological revision and with NGS panels, designed based on pathway aggregated genes identified with the International Cancer consortium (described by Bailey et al., Nature 531, 2016). Standard drugs were applied using clinically relevant dosages and schedule. MPS-1 inhibitor BAY1161909 was given in monotherapy and in combination with Abraxane. Minnelide (MTD) was applied second line after three cycles of chemotherapy (Cisplatin, Abraxane, Gemcitabine) and Trametinib was tested as monotherapy. All PDX correlated with histopathological and molecular characteristics of patient tumours. BAY1161909 monotherapy showed moderate anti-tumor efficacy with an average tumor growth inhibition of 30% (p > 0.05). However, tumor relapse after the end of chemotherapy was delayed in mice treated with the combination of BAY1161909 and Abraxane compared to Abraxane alone. 13 out of 22 PDX models tested to date were identified as responders (tumor growth inhibition > 50%) to Minnelide and 5 out of 11 to Trametinib. While Minnelide induced tumor growth inhibition above 80% in 32% of the models, Trametinib achieved the same efficacy in only 9% of the tested PDX models. The described PDX panel clearly reflects clinical situation of pancreatic cancer due to their histologic growth and detection of inherent and acquired treatment resistance as well as recurrent disease. In a few cases, the tested drugs induced complete remissions. We are currently analyzing the molecular data to determine response markers. Our approach may offer personalized treatment options for PC patients. Citation Format: Diana Behrens, Britta Büttner, Rita L. Lawlor, Christopher Heeschen, Malte Buchholz, Jens Siveke, Antje M. Wengner, Ashok Saluja, Jens Hoffmann. Preclinical evaluation of novel treatment strategies in patient-derived xenograft (PDX) models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4093.

The rapamycin derivative RAD001 (everolimus) is presently in clinical trials. Preclinical studies have suggested preferential activity in antiestrogen resistant breast cancer cells. We investigated the response of breast cancer xenografts with different tamoxifen (TAM) sensitivity towards RAD001 and analyzed the regulatory machinery as well as the cross-talk between different signaling pathways. The ERα-positive and TAM-sensitive patient-derived breast carcinoma model 3366, its TAM-resistant counterpart 3366/TAM and 4049, a breast cancer with inherent TAM-resistance, were transplanted to immunodeficient nude mice and treated with RAD001 or TAM or the combination of both compounds. Shock frozen tumors were prepared for Western Blot and immunohistochemical analysis to semi-quantitatively evaluate the expression of the ERα and the ERα regulated IGF-IR as well as PTEN, pAkt, mTOR, (phospho)-p70S6K, (phospho)-4E-BP1 and cyclin D1. RAD001 significantly inhibited the growth of tamoxifen responding and non-responding xenografts. The highest efficacy was found for the combined treatment with TAM and RAD001. RAD001 modified the protein expression of mTOR and its downstream molecule 4E-BP1 as well as the level of PTEN and ERα, but independent of the tumors sensitivity towards TAM. The protein kinase Akt was found in the active phosphorylated form (pAkt) only in TAM-resistant xenografts, but not detectable in the TAM-responding 3366 line. All treatment modalities down-regulated pAkt expression in the TAM-resistant tumors. p70S6K and IGF-IR proteins were not significantly influenced by RAD001 treatment. Our findings document the linkage between different growth-controlling pathways. Due to its capability to be active in a TAM-resistant in vivo model, RAD001 could potentially serve as a promising second-line therapy in breast cancer.

Experimental oncology research and preclinical drug development both substantially require specific, clinically relevant in vitro and in vivo tumor models. The increasing knowledge about the heterogeneity of cancer requested a substantial restructuring of the test systems for the different stages of development. To be able to cope with the complexity of the disease, larger panels of patient-derived tumor models have to be implemented and extensively characterized. Together with individual genetically engineered tumor models and supported by core functions for expression profiling and data analysis, an integrated discovery process has been generated for predictive and personalized drug development.Improved “humanized” mouse models should help to overcome current limitations given by xenogeneic barrier between humans and mice. Establishment of a functional human immune system and a corresponding human microenvironment in laboratory animals will strongly support further research.Drug discovery, systems biology, and translational research are moving closer together to address all the new hallmarks of cancer, increase the success rate of drug development, and increase the predictive value of preclinical models.

// Giuseppina Sannino 1, 6 , Nicole Armbruster 1, 7 , Mona Bodenhofer 1 , Ursula Haerle 1 , Diana Behrens 2 , Malte Buchholz 3 , Ulrich Rothbauer 4 , Bence Sipos 5 , Christian Schmees 1 1 Natural and Medical Sciences Institute (NMI) at the University of Tuebingen, Tumor Biology Group, Reutlingen, Germany 2 Experimental Pharmacology and Oncology GmbH, Berlin, Germany 3 Department of Medicine, Division of Gastroenterology, Endocrinology and Metabolism, Philipps University Marburg, Marburg, Germany 4 Pharmaceutical Biotechnology, University of Tuebingen, Tuebingen, Germany 5 Institute of Pathology, University of Tuebingen, Tuebingen, Germany 6 Current address: Institute of Pathology, Laboratory of Pediatric Sarcoma Biology, Ludwig-Maximilians-Universitat Munich, Munich, Germany 7 Current address: Department of Internal Medicine II, University of Tuebingen, Tuebingen, Germany Correspondence to: Christian Schmees, email: christian.schmees@nmi.de Keywords: pancreatic cancer, BCL9L, β-catenin, E-cadherin, EMT Received: August 20, 2015 Accepted: September 24, 2016 Published: October 04, 2016 ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) has a low overall survival rate, which is approximately 20% during the first year and decreases to less than 6% within five years of the disease. This is due to premature dissemination accompanied by a lack of disease-specific symptoms during the initial stages. Additionally, to date there are no biomarkers for an early prognosis available. A growing number of studies indicate that epithelial to mesenchymal transition (EMT), triggered by WNT-, TGF-β- and other signaling pathways is crucial for the initiation of the metastatic process in PDAC. Here we show, that BCL9L is up-regulated in PDAC cell lines and patient tissue compared to non-cancer controls. RNAi-induced BCL9L knockdown negatively affected proliferation, migration and invasion of pancreatic cancer cells. On a molecular basis, BCL9L depletion provoked an increment of E-cadherin protein levels, with concomitant increase of β-catenin retention at the plasma membrane. This is linked to the induction of a strong epithelial phenotype in pancreatic cancer cells upon BCL9L knockdown even in the presence of the EMT-inducer TGF-β. Finally, xenograft mouse models of pancreatic cancer revealed a highly significant reduction in the number of liver metastases upon BCL9L knockdown. Taken together, our findings underline the key importance of BCL9L for EMT and thus progression and metastasis of pancreatic cancer cells. Direct targeting of this protein might be a valuable approach to effectively antagonize invasion and metastasis of PDAC.

The study was performed to investigate the potency of liposomal formulations to reduce or to overcome tamoxifen resistance. Therefore, estrogen receptor (ER) positive MCF-7 and ER deficient NCI/ADR cells with different sensitivity to tamoxifen were used. Instead of the parent compound, 4-hydroxytamoxifen (HT) was used as drug, because this metabolite is the most cytotoxic derivative in vitro. Liposomes further contained the membrane-active alkylphospholipid octadecyl-(1,1-dimethyl-piperidino-4-yl)-phosphate (OPP). Cellular uptake of HT during 3-24 h was determined by high performance thin layer chromatography technique (HPTLC). Free HT was taken up time independently by the two cell lines at 1.5-2.1 g HT/g cellular phosphate. Liposomal HT was taken up at a significantly higher degree than free HT in both cell lines, but the uptake was delayed in the sensitive MCF-7 cell line with the highest concentration detected after 24 h (3.5 g/g). Oppositely, the highest amount in the 'resistant' line (2.3 g/g) was already measured after 3 h in NCI/ADR cells. It successively decreased with incubation time. The faster uptake of liposomal HT by the NCI/ADR cells correlated with a stronger and earlier destruction of resistant NCI/ADR cells whereas the sensitive MCF-7 cells were mainly inhibited in their proliferation. Cytolytic effects were observed in both cell lines after extended incubation periods. The combination of HT with an alkylphospholipid further enhanced the cytotoxicity of the formulation. The IC50 in the NCI/ADR cells could be significantly reduced by liposomes combining both drugs to 15.1 microM compared with the IC50 of the free drugs (HT:28.9 microM; OPP: 36.8 microM). It is assumed that the enhanced and accelerated uptake of liposomal HT in the cell line with relative drug resistance can increase the intracellular bioavailability of HT. The results of this study demonstrated that liposomes with encapsulated antiestrogen have a superior cytotoxic effect in resistant breast cancer cells. That coincided with the enhanced therapeutic effect of these vesicles observed in vivo.

Experimental oncology research and preclinical drug development both substantially require specific, clinically relevant in vitro and in vivo tumor models. The increasing knowledge about the heterogeneity of cancer requested a substantial restructuring of the test systems for the different stages of development. To be able to cope with the complexity of the disease, larger panels of patient-derived tumor models have to be implemented and extensively characterized. Together with individual genetically engineered tumor models and supported by core functions for expression profiling and data analysis, an integrated discovery process has been generated for predictive and personalized drug development.

BACKGROUND: Large-scale genomic analyses of patient cohorts have revealed extensive heterogeneity between individual tumors, contributing to treatment failure and drug resistance. In malignant melanoma, heterogeneity is thought to arise as a consequence of the differentiation of melanoma-initiating cells that are defined by cell-surface markers like CD271 or CD133. RESULTS: Here we confirmed that the nerve growth factor receptor (CD271) is a crucial determinant of tumorigenicity, stem-like properties, heterogeneity and plasticity in melanoma cells. Stable shRNA mediated knock-down of CD271 in patient-derived melanoma cells abrogated their tumor-initiating and colony-forming capacity. A genome-wide expression profiling and gene-set enrichment analysis revealed novel connections of CD271 with melanoma-associated genes like CD133 and points to a neural crest stem cell (NCSC) signature lost upon CD271 knock-down. In a meta-analysis we have determined a shared set of 271 differentially regulated genes, linking CD271 to SOX10, a marker that specifies the neural crest. To dissect the connection of CD271 and CD133 we have analyzed 10 patient-derived melanoma-cell strains for cell-surface expression of both markers compared to established cell lines MeWo and A375. We found CD271+ cells in the majority of cell strains analyzed as well as in a set of 16 different patient-derived melanoma metastases. Strikingly, only 2/12 cell strains harbored a CD133+ sub-set that in addition comprised a fraction of cells of a CD271+/CD133+ phenotype. Those cells were found in the label-retaining fraction and in vitro deduced from CD271+ but not CD271 knock-down cells. CONCLUSIONS: Our present study provides a deeper insight into the regulation of melanoma cell properties and points CD271 out as a regulator of several melanoma-associated genes. Further, our data strongly suggest that CD271 is a crucial determinant of stem-like properties of melanoma cells like colony-formation and tumorigenicity.

Pancreatic cancer remains a lethal disease with only 3 - 8% of patients surviving 5 years after initial diagnosis (WHO, 2012). Reasons for this poor situation are advanced and inoperable tumor stages at time of diagnosis and resistance to conventional therapies. One bottleneck in the development of novel therapies is the restricted availability of preclinical models of high clinical relevance. Since the desmoplastic stroma has impact on the progression and treatment of pancreatic cancer, we investigated the attributes of the murine stroma in patient-derived xenografts that completely replaced the human surrounding tissue within a few months after primary transplantation. We elucidated the functionality of murine tumor microenvironment for growth and therapeutic response in a cohort of well-characterized pancreatic cancer (PDAC) PDX. PDX are a valuable tool for the prediction of therapy response, the identification of new biomarkers and therapeutic targets or pancreatic cancer specific pathways. In this study, 57 patient tumors were collected and immediately transplanted into immunodeficient mice. So far, 14 out of 57 samples were established as passageable pancreatic cancer xenografts (PDX). All engrafted PDX are poorly or moderately differentiated adenocarcinomas. Global gene expression analysis and determination of cancer associated mutations revealed K-ras mutations in 13 and additionally p53 mutations in 9 out of 14 PDX. Furthermore, chemosensitivity to standard of care (SoC) drugs was determined by using clinically relevant and optimized schedules and doses. The testing revealed that the response to Gemcitabine (1/10 responder) was moderate within the PDX panel, while the most efficient drug was Abraxane with 5 out of 10 responders. In general, the response profile of all PDX closely reflected patient's situation in the clinic. Cryo- and formalin-preserved tumor tissues of these chemosensitivity studies were investigated for markers of desmoplastic stroma (SPARC, alpha-SMA, FAP and collagen I). Immunohistochemistry and real-time PCR revealed, that even the replacing murine stroma is characterized by a distinct reactivate nature. Semi-quantitative analysis of stromal components showed that the tumor surrounding tissue mass was not significantly reduced due to therapeutic intervention. Though the tumor burden was diminished under SoC, the mRNA expression level of SPARC and FAP was unaffected in corresponding samples of the treatment groups compared to vehicle-treated control. The same effect was found for alpha-SMA and collagen I in immunohistochemically stained specimens. In summary, this study revealed a functional tumor environment of murine origin in patient-derived xenografts of pancreatic cancer and furthermore an apparently inherent resistance of this stromal tissue towards conventional therapy. Thus, targeting the tumor microenvironment should be implicated into clinical decisions. Citation Format: Diana Behrens, Ulrike Pfohl, Britta Büttner, Jens Hoffmann, Wolfgang Walther, Iduna Fichtner. Analysis of murine stromal components in patient-derived xenograft (PDX) models of pancreatic cancer. [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 4080.

In Germany pancreatic cancer remains a lethal disease with only 3 - 8% of patients surviving 5 years after diagnosis of the tumor (2005). Reasons for this poor situation are advanced and inoperable tumor stages at time of diagnosis and resistance to conventional therapies. One bottleneck in the development of novel therapies is the restricted availability of preclinical models with a high clinical relevance. The aim of our study was to develop well-defined in vivo xenografts derived from patient pancreatic carcinomas to address translational questions. These xenografts can be used for identification of biomarkers and cancer related pathways as well as for the evaluation of targeted therapies. The genesis of pancreatic neoplasm, the progression of tumor growth as wells as the development of resistance can be elucidated with animal models of patient-derived material. And last but not least preclinical data can be used prospectively for efficient stratification of patients. 57 patient tumors were collected from the clinical cooperation partner and transplanted immediately into immune-deficient mice. 14 out of 57 samples could be established so far as passagable pancreatic cancer xenografts (PDX), 5 were identified as inflammations of the pancreas. Interestingly, we observed 4 out of 57 as post-transplant lymphoproliferative disorders (PLTD); 29 failed to grow in mice. All of the engrafted PDX are poor or moderate differentiated adenocarcinomas. Global gene expression analysis and determination of cancer associated mutations were performed from engrafted tumor models. According to clinical data we found K-ras mutations in 13 and additionally p53 mutations in 9 out of 14 PDX. Chemosensitivity was evaluated by transplanting tumor material into cohorts of immune-deficient mice. Beginning from palpable tumor sizes mice were treated with clinically relevant therapies (Gemcitabine, Abraxane, 5FU, Oxaliplatin, Erlotinib as monotherapy or combinations) as well as PDAC untypical drugs like Irinotecan, Vincristine, Avastin and Sorafenib at optimized schedules and doses. The response to Gemcitabine was moderate within the PDX panel. The most efficient therapeutic was Abraxane. Additionally, we could establish a luciferase expressing PDX for in vivo imaging of orthotopic tumor grafts. Patient-derived xenografts are a crucial tool for the prediction of therapy and potentially relevant for the implication into clinical decisions. We have successfully developed a panel of 14 pancreatic cancer PDX for translational research projects. Citation Format: Diana Behrens, Diana Anders, Cora Hallas, Jessica Pahle, Iduna Fichtner. In vivo models of pancreatic cancer for translational medicine. [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 A100.

The stimulation of the endogenous antitumor immunity has the potential to achieve clinically significant tumor regression. The recent clinical success with antibodies interfering with immune checkpoints on T-cells (PD1 and CTLA-4) has motivated oncology research world wide to focuses on new immunotherapy approaches. Further immune regulatory molecules are currently in target validation and together with cancer vaccines, therapeutic antibodies, immunoconjugates, and tumor reactive T-cells (CARTs) they all might contribute to an improved cancer therapy in the next years. The identification and validation of new targets for antitumor immune therapy is still a challenge for the preclinical research as the classical syngeneic tumor models are of limited translational value and the patient-derived human tumor xenograft models (PDX) are growing on immunodeficient animals. In first studies using human mononuclear cells (MNC) we demonstrated the engraftment of human T cells on immunodeficient mice. The transplanted human mononuclear cells (MNC) differentiated in T cell as measured by CD3, CD4 and CD8 expression. The inoculation and tumor growth of SW480 colon cancer cells on humanized mice was possible concurrent with accumulation of human T cells in the tumor. Our aim was the further improvement of this test system and the demonstration of a functional reconstitution of a human immune system by engrafting human hematopoietic stem cells in immunodeficient mice. Humanized mice were transplanted with patient derived melanoma fragments without evidence for rejection correlated with an increase of human T cells in the peripheral blood. Drug sensitivity testing with various immunoconjugates will follow. Our humanized mouse models will enable a more appropriate preclinical assessment of immune-based therapeutic antitumor strategies especially when combining the humanized mouse with patient-derived tumor xenografts. Citation Format: Annika Wulf-Goldenberg, Maria Stecklum, Klaus Eckert, Diana Behrens, Iduna Fichtner, Jens Hoffmann. Engraftment of patient derived xenografts on mice with a humanized immune system. [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 1463. doi:10.1158/1538-7445.AM2015-1463

The proteasome inhibitor bortezomib (Velcade) was recently approved for the treatment of therapy-refractive multiple myeloma and is under investigation for numerous other types of cancer. A phase I clinical trial in paediatric patients resulted in tolerable toxicity. Since the emergence of chemoresistance represents one of the major drawbacks in cancer therapy, we investigated the influence of bortezomib on multi-drug resistant human neuroblastoma cell lines characterised by P-glycoprotein expression and p53 mutation. Nanomolar concentrations of bortezomib inhibited the cell cycle and induced apoptosis in chemosensitive as well as in chemoresistant cell lines. In vivo growth of chemosensitive and chemoresistant neuroblastoma cell lines was inhibited to a similar extent. In addition, bortezomib inhibited vessel formation in neuroblastoma xenografts. These findings and the favourable toxicity profile of bortezomib in children make it reasonable to further pursue additional development of the drug for the treatment of neuroblastoma and other paediatric solid tumours.

In Germany pancreatic cancer remains a lethal disease with only 3-8% of patients surviving 5 years after diagnosis of the tumor (2005). Reasons for this poor situation are advanced and inoperable tumor stages at time of diagnosis and resistance to conventional therapies. One bottleneck in the development of novel therapies is the restricted availability of preclinical models with a high clinical relevance. The aim of our study was to develop well-defined in vivo xenografts derived from patient pancreatic carcinomas to address translational questions. These xenografts can be used for identification of biomarkers and cancer related pathways as well as for the evaluation of targeted therapies. The genesis of pancreatic neoplasm, the progression of tumor growth as wells as the development of resistance can be elucidated with animal models of patient-derived material. And last but not least, preclinical data can be used prospectively for efficient stratification of patients. Fifty-two patient tumors were collected from the clinical cooperation partner and transplanted immediately into immune-deficient mice. Nine out of 52 samples could be established so far as passagable pancreatic cancer xenografts (PDX), 5 were identified as inflammations of the pancreas. Interestingly, we observed 4 out of 52 as post-transplant lymphoproliferative disorders (PLTD). Twenty-nine failed to grow in mice and 5 are still under observation and potential candidates for the model library. All of the engrafted PDX are poor or moderate differentiated adenocarcinomas. Global gene expression analysis and determination of cancer associated mutations were performed from engrafted tumor models. According to clinical data we found K-ras mutations in 8 and additionally p53 mutations in 4 out of 9 PDX. Chemosensitivity was evaluated by transplanting tumor material into cohorts of immune-deficient mice. Beginning from palpable tumor sizes mice were treated with 5-fluorouracil, Irinotecan, Vincristine, Avastin, Gemcitabine and Sorafenib at optimized schedules and doses. The most efficient therapeutics were Gemcitabine and Irinotecan, whereas the growth of PDX was only marginally inhibited by Vincristine and Avastin. Patient-derived xenografts are a crucial tool for the prediction of therapy and potentially relevant for the implication into clinical decisions. We have successfully developed a panel of 9 pancreatic cancer PDX for translational research projects. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A10. Citation Format: Diana Behrens, Cora Hallas, Diana Anders, Iduna Fichtner. In vivo models of pancreatic cancer for translational medicine. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A10.

The aim of this study was to evaluate the potential of different types of sialyl Lewis X-conjugated liposomes as competitive inhibitors for tumour cell adhesion to endothelial E-selectin. Sterically stabilised liposomes with the sLeX ligand at the terminal end of the polyethyleneglycol (PEG) chain, as well as vesicles that had the ligand embedded within the PEG-layer, were compared to ligand-bearing liposomes without sterical stabilisation. First, 14 different tumour cell lines were characterised for their expression of sialyl Lewis X and/or A. Tumour cell adhesion was characterised in three static assays in vitro using: (i) immobilised E-selectin, (ii) CHO cells, transfected to express E-selectin and (iii) human umbilical vein endothelial cells (HUVEC). Sterically stabilised liposomes with the ligand at the terminal end of the polyethylene chain were the most effective inhibitors in all three assays and inhibited the adhesion of HT29 colon- and Lewis lung (LL) carcinoma cells by about 60–80%. The binding was not affected by a PEG-coating of the liposomes. Sterical stabilisation, on the other hand, completely prevented macrophage uptake (J774 cell line) independently of the presence of the ligand, while plain liposomes were taken up in an amount of 5.4 nmol liposomal lipids/106 macrophages.