Diana Behrens

The nerve growth factor receptor CD271 is crucial to maintain tumorigenicity and stem-like properties of melanoma cells

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.

The Antiangiogenic and Antitumoral Activity of Titanocene Y* In Vivo

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.

Preclinical study on combined chemo- and nonviral gene therapy for sensitization of melanoma using a human TNF-alpha expressing MIDGE DNA vector

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.

The mTOR pathway inhibitor RAD001 (everolimus) is highly efficacious in tamoxifen-sensitive and -resistant breast cancer xenografts

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.

Predictive In Vivo Models for Oncology

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.

Pancreatic cancer models for translational research

&NA; 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.

Abstract 4080: Analysis of murine stromal components in patient-derived xenograft (PDX) models of pancreatic cancer

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA 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 patients 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 Buttner, 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.

Abstract 1463: Engraftment of patient derived xenografts on mice with a humanized immune system

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA 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

Abstract A100: In vivo models of pancreatic cancer for translational medicine

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.