Vanessa Vogel

Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay.

It has been hypothesized that carcinoma metastasis is initiated by a subpopulation of circulating tumor cells (CTCs) found in the blood of patients. However, although the presence of CTCs is an indicator of poor prognosis in several carcinoma entities, the existence and phenotype of metastasis-initiating cells (MICs) among CTCs has not been experimentally demonstrated. Here we developed a xenograft assay and used it to show that primary human luminal breast cancer CTCs contain MICs that give rise to bone, lung and liver metastases in mice. These MIC-containing CTC populations expressed EPCAM, CD44, CD47 and MET. In a small cohort of patients with metastases, the number of EPCAM+CD44+CD47+MET+ CTCs, but not of bulk EPCAM+ CTCs, correlated with lower overall survival and increased number of metastasic sites. These data describe functional circulating MICs and associated markers, which may aid the design of better tools to diagnose and treat metastatic breast cancer.

CYP3A5 mediates basal and acquired therapy resistance in different subtypes of pancreatic ductal adenocarcinoma

Although subtypes of pancreatic ductal adenocarcinoma (PDAC) have been described, this malignancy is clinically still treated as a single disease. Here we present patient-derived models representing the full spectrum of previously identified quasi-mesenchymal (QM-PDA), classical and exocrine-like PDAC subtypes, and identify two markers—HNF1A and KRT81—that enable stratification of tumors into different subtypes by using immunohistochemistry. Individuals with tumors of these subtypes showed substantial differences in overall survival, and their tumors differed in drug sensitivity, with the exocrine-like subtype being resistant to tyrosine kinase inhibitors and paclitaxel. Cytochrome P450 3A5 (CYP3A5) metabolizes these compounds in tumors of the exocrine-like subtype, and pharmacological or short hairpin RNA (shRNA)-mediated CYP3A5 inhibition sensitizes tumor cells to these drugs. Whereas hepatocyte nuclear factor 4, alpha (HNF4A) controls basal expression of CYP3A5, drug-induced CYP3A5 upregulation is mediated by the nuclear receptor NR1I2. CYP3A5 also contributes to acquired drug resistance in QM-PDA and classical PDAC, and it is highly expressed in several additional malignancies. These findings designate CYP3A5 as a predictor of therapy response and as a tumor cell–autonomous detoxification mechanism that must be overcome to prevent drug resistance.

Defined Conditions for the Isolation and Expansion of Basal Prostate Progenitor Cells of Mouse and Human Origin

Summary Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs) have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin−SCA-1+CD49f+TROP2high phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin−CD49f+TROP2high PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin.

Identification and Validation of Novel Subtype-specific Protein Biomarkers in Pancreatic Ductal Adenocarcinoma.

Objectives Pancreatic ductal adenocarcinoma (PDAC) has been subclassified into 3 molecular subtypes: classical, quasi-mesenchymal, and exocrine-like. These subtypes exhibit differences in patient survival and drug resistance to conventional therapies. The aim of the current study is to identify novel subtype-specific protein biomarkers facilitating subtype stratification of patients with PDAC and novel therapy development. Methods A set of 12 human patient-derived primary cell lines was used as a starting material for an advanced label-free proteomics approach leading to the identification of novel cell surface and secreted biomarkers. Cell surface protein identification was achieved by in vitro biotinylation, followed by mass spectrometric analysis of purified biotin-tagged proteins. Proteins secreted into a chemically defined serum-free cell culture medium were analyzed by shotgun proteomics. Results Of 3288 identified proteins, 2 pan-PDAC (protocadherin-1 and lipocalin-2) and 2 exocrine-like-specific (cadherin-17 and galectin-4) biomarker candidates have been validated. Proximity ligation assay analysis of the 2 exocrine-like biomarkers revealed their co-localization on the surface of exocrine-like cells. Conclusions The study reports the identification and validation of novel PDAC biomarkers relevant for the development of patient stratification tools. In addition, cadherin-17 and galectin-4 may serve as targets for bispecific antibodies as novel therapeutics in PDAC.

Abstract A61: Exploring the PDAC-subtype-associated microenvironment in PDX models and patients

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with dismal prognosis (1, 2). Despite extensive research and the discovery of several promising drug candidates, little progress in PDAC treatment has been reported in the last years (3, 4). Two facts can be behind these disappointing results. In one hand, although PDAC is still clinically treated as a single disease, three distinct molecular subtypes (classical, quasi-mesenchymal and exocrine) were recently described based on expression profiling of microdissected epithelial tumor cells of PDAC samples (5). Interestingly, in vitro studies revealed differences in drug response of two of the proposed subtypes for which cell lines were available. These results highlight the importance of considering PDAC as a heterogeneous disease and point to the stratification of patients as a possible way to improve PDAC treatment response. On another hand, an additional reason behind the limited efficacy of PDAC treatment might be the tumor microenvironment. PDAC is the solid tumor with the highest stromal content, which can account for up to 90% of the total tumor mass. The PDAC microenvironment is known to actively affect tumorigenesis (6) and to impair drug delivery (7). Thus, rendering the PDAC microenvironment as an appealing therapeutic target to improve PDAC care (8). We have developed a novel workflow to efficiently generate patient-derived orthotopic xenografts (PDX) and serum-free cell cultures from primary resected PDAC tumors. The established primary cell lines comprise for first time all three described PDAC subtypes. Additionally, when re-injected into immunodeficient mice, these cells generate xenografts with high pathological similarity to the original patient tumor, including a prominent stromal presence. To explore the differences in the microenvironment associated to the individual PDAC subtypes we have now generated gene expression profiles for the stroma of a number of xenografts from our PDX model representing all three subtypes. Besides, RNA sequencing from different sub-populations isolated from fresh primary human PDAC tumors (as separated by fluorescent activated cell sorting according to surface markers) may reveal interesting interactions between the different tumor compartments. We have developed a set of immunohistochemical markers to identify the PDAC-subtypes that can be used in patient paraffin sections. Hence, the RNAseq data of the different tumor populations can be also easily studied in the context of the different subtypes. We believe that these approaches will shed some light on how different stromal expression patterns are interconnected with different epithelial expression profiles and vice versa, and how this information can be ultimately exploited for patient stratification and therapy. 1. Hidalgo M. Pancreatic cancer. N Engl J Med 2010 2. Vincent A et al. Pancreatic cancer. Lancet, 2011 3. Werner J et al. Advanced-stage pancreatic cancer: therapy options. Nature Reviews Clinical Oncology, 2013 4. Hidalgo M et al. Translational therapeutic opportunities in ductal adenocarcinoma of the pancreas. Clin Cancer Res, 2012 5. Collisson EA et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nat Med, 2011 6. Feig C et al. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblastssynergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc Natl Acad Sci USA, 2013 7. Provenzano PP et al. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell, 2012 8. Neesse A et al. Emerging concepts in pancreatic cancer medicine: targeting the tumor stroma. Onco Targets Ther, 2013 Citation Format: Elisa Espinet Espinet, Christian Eisen, Elisa M. Noll, Vanessa Vogel, Corinna Klein, Zuguang Gu, Matthias Schlesner, Tobias Bauer, Nathalia Giese, Roland Eils, Jens Werner, Wilko Weichert, Martin R. Sprick, Andreas Trumpp. Exploring the PDAC-subtype-associated microenvironment in PDX models and patients. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A61.

Abstract A69: A novel mechanism mediates drug resistance in the exocrine-like pancreatic ductal adenocarcinoma (PDAC) subtype

PDAC is a highly aggressive disease with dismal prognosis [1, 2]. Despite extensive research and the discovery of several drug candidates, little progress has been reported since the approval of gemcitabine and erlotinib [1]. Moreover, recent trials with targeted therapies have shown only limited or no benefit [1, 2]. For a number of other carcinomas, tumor subclasses have been uncovered that allow the use of targeted therapies. The mutational landscape of PDAC is complex and heterogeneous, raising the question whether subclasses also exist in PDAC [3]. Collisson et al. described three PDAC subtypes that were identified based on their gene-expression profiles: The classical, the quasi-mesenchymal and the exocrine-like subtype [4]. However, not all subtypes could be identified in the previously available model systems. We have established a novel patient-derived model system that allows the analysis of these three human PDAC subtypes in vitro and in vivo. Hence, we provide a systematic workflow to propagate human PDAC in orthotopic xenografts and to derive tumor-initiating primary cell lines of all three PDAC subtypes. HNF-1 and Keratin 81 were identified as markers for subtype stratification by immunohistochemistry. Application of this two-marker set on a 258 large patient cohort confirmed a predominantly non-overlapping staining and revealed a significant difference in overall survival across the three subtypes. Furthermore, a drug screen uncovered subtype-specific drug sensitivities towards a number of drugs, including gemcitabine and dasatinib. Notably, the exocrine-like subtype was resistant towards all compounds tested. Thus, we aimed to identify the underlying cause of the observed drug resistance. Molecular analysis including gene set enrichment analysis (GSEA) allowed us to identify a putative novel mechanism of drug resistance. Analysis by qRT-PCR and Western blot demonstrated the enhanced expression of several genes mediating this mechanism particularly in the exocrine-like subtype in vitro and in vivo. These findings led to the identification of a novel protein target central to this mechanism. Additionally, retrospective immunohistochemical analysis of a large patient cohort confirmed that this target is predominantly found in those patient tumors classified as exocrine-like. Hence, we hypothesized that the observed strong activation of this mechanism in the exocrine-like PDAC subtype could be responsible for the drug resistance observed in this subclass. In line with this, functional inhibition of this mechanism resulted in increased drug sensitivity in the exocrine-like subtype. Hence, our findings may ultimately advance personalized treatment by applying novel marker-based patient selection strategies in combination with tailored drug use, a strategy which will be presented in more detail at the conference. [1] Hidalgo, M. Pancreatic cancer. The New England journal of medicine. 362, 1605-1617, doi:10.1056/NEJMra0901557 (2010). [2] Vincent, A., Herman, J., Schulick, R., Hruban, R. H. & Goggins, M. Pancreatic cancer. Lancet. 378, 607-620, doi:10.1016/S0140-6736(10)62307-0 (2011). [3] Jones, S. et al. Core signalling pathways in human pancreatic cancers revealed by global genomic analyses. Science. 321, 1801-1806, doi:10.1126/science.1164368 (2008). [4] Collisson, E. A. et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nature medicine. 17, 500-503, doi:10.1038/nm.2344 (2011). Citation Format: Elisa M. Noll, Christian Eisen, Elisa Espinet, Vanessa Vogel, Corinna Klein, Albrecht Stenzinger, Franziska Zickgraf, Peter Neuhaus, Marcus Bahra, Bruno V. Sinn, Christian Lutz, Michael Kulke, Andreas Pahl, Nathalia A. Giese, Oliver Strobel, Jens Werner, Wilko Weichert, Andreas Trumpp, Martin R. Sprick. A novel mechanism mediates drug resistance in the exocrine-like pancreatic ductal adenocarcinoma (PDAC) subtype. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A69.

Abstract B50: A patient-derived renal cell carcinoma model as a platform for the identification of novel diagnostic markers and therapeutic targets

Clear-cell renal cell carcinoma (ccRCC) is highly resistant to conventional therapies and to date effective treatment is restricted to surgical resection. Pre-clinical in vitro and in vivo models, which accurately mimic this disease, are critical for the development of novel effective therapies. Previous attempts to culture ccRCC cells have been proven difficult and thus, only few ccRCC cell lines are currently available. In addition long-term culturing often causes loss of primary tumor characteristics including tumor heterogeneity. This might explain why in contrast to other tumor entities, tumor-initiating cell (TIC) subpopulations have so far not been reported for ccRCC. TICs have been associated with tumor progression, metastasis and drug-resistance. Consequently these cells have also been named cancer stem cells (CSCs). Furthermore, distinct combinations of genetic and epigenetic alterations result in profound inter-patient heterogeneity and can in part promote TIC activity. Therefore, the development and characterization of clinically relevant patient derived ccRCC-models would provide an experimental basis to study the complex mechanisms leading to and maintaining ccRCCs. Hence, we established a patient-derived serum-free spheroid culture system that can be used to propagate primary ccRCC cells. These ccRCC cells retain their tumor-initiating potential and mimic the human malignancy upon orthotopic injection into immunodeficient mice. They not only mirror the histological properties but also the molecular features of the parental tumor. Moreover, these cells show the capacity to form de novo metastasis in lungs, which is the most common metastatic site for ccRCC in patients. The established ccRCC cultures were then used as a screening platform for the identification of surface proteins, which are heterogeneously expressed among tumor cells. Functional in vitro and in vivo assays showed that subpopulations defined by expression of these markers also displayed distinct functional characteristics including TIC activity. Gene Set Enrichment Analysis (GSEA) revealed differences in the signaling networks active in these sub-populations. In addition, molecular and cellular assays addressed the significance of these pathways for the maintenance of ccRCC malignancy. Currently the identified surface markers and underlying pathways are validated as potential novel diagnostic and therapeutic targets. Therefore, our novel patient-derived serum-free spheroid culture system serves as a platform to expand patient derived ccRCCs. It further allows the analysis and characterization of different subpopulations, which may ultimately contribute to the understanding of the molecular basis underlying progression, therapy-resistance and metastasis of this disease. Citation Format: Teresa Rigo-Watermeier, Corinna Klein, Vanessa Vogel, Christian Eisen, Thomas Hoefner, Wilko Weichert, Sascha Pahernik, Markus Hohenfellner, Martin R. Sprick, Andreas Trumpp. A patient-derived renal cell carcinoma model as a platform for the identification of novel diagnostic markers and therapeutic targets. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B50.