Olaf Hardt

NeuroD1 induces terminal neuronal differentiation in olfactory neurogenesis

After their generation and specification in periventricular regions, neuronal precursors maintain an immature and migratory state until their arrival in the respective target structures. Only here are terminal differentiation and synaptic integration induced. Although the molecular control of neuronal specification has started to be elucidated, little is known about the factors that control the latest maturation steps. We aimed at identifying factors that induce terminal differentiation during postnatal and adult neurogenesis, thereby focusing on the generation of periglomerular interneurons in the olfactory bulb. We isolated neuronal precursors and mature neurons from the periglomerular neuron lineage and analyzed their gene expression by microarray. We found that expression of the bHLH transcription factor NeuroD1 strikingly coincides with terminal differentiation. Using brain electroporation, we show that overexpression of NeuroD1 in the periventricular region in vivo leads to the rapid appearance of cells with morphological and molecular characteristics of mature neurons in the subventricular zone and rostral migratory stream. Conversely, shRNA-induced knockdown of NeuroD1 inhibits terminal neuronal differentiation. Thus, expression of a single transcription factor is sufficient to induce neuronal differentiation of neural progenitors in regions that normally do not show addition of new neurons. These results suggest a considerable potential of NeuroD1 for use in cell-therapeutic approaches in the nervous system.

Highly sensitive profiling of CD44+/CD24− breast cancer stem cells by combining global mRNA amplification and next generation sequencing: Evidence for a hyperactive PI3K pathway

We performed next generation sequencing- and microarray-based gene expression profiling of CD44(+)/CD24(-)/CD45(-) breast CSCs (cancer stem cells) isolated from primary ERα-positive breast cancer. By combining semi-automated dissociation of human tumor tissue, magnetic cell sorting and cDNA amplification less than 500 CSCs were required for transcriptome analyses. Besides overexpressing genes involved in maintenance of stemness, the CSCs showed higher levels of genes that drive the PI3K pathway, including EGFR, HB-EGF, PDGFRA/B, PDGF, MET, PIK3CA, PIK3R1 and PIK3R2. This suggests that, in CSCs of ERα-positive breast cancer, the PI3K pathway which is involved in endocrine resistance is hyperactive.

Comparison between cold plasma, electrochemotherapy and combined therapy in a melanoma mouse model.

The study was undertaken to compare antitumor efficacy of electrochemotherapy (ECT) with cold plasma therapy (CP) in a melanoma mouse model. After melanoma implantation into the flank of C57BL/6N mice, CP by two different plasma sources (APPJ and DBD) was applied directly to the tumor surface. ECT was performed with bleomycin intravenously at a field strength of 1000 V/cm without or combined with CP. Primary endpoints were tumor growth acceleration (TGA), daily volume progression (DVP) and survival after treatment. Both plasma sources as single treatment showed a significant TGA delay, which proved less effective than ECT. CP (APPJ) combined with ECT (ECJ) significantly improved per cent mouse survival, with significant superiority compared with ECT. Plasma therapy alone albeit less effective seems a potential alternative to ECT in patients with melanoma and can be applied manifold in a session without general anaesthesia. Accordingly, CP alone and combined with ECT may serve as new option in palliative skin melanoma therapy.

Gene expression analysis defines differences between region-specific GABAergic neurons

Gamma-aminobutyric acid (GABA)ergic neurons are a diverse group of inhibitory neurons playing crucial roles in information processing. We analyzed the gene expression of regionally defined GABAergic neurons from the cortex, olfactory bulb, striatum, and cerebellum of glutamate decarboxylase 67-green fluorescence protein (GAD67-GFP) knock-in mice. We introduce a generally applicable method for singularization of brain cells, flow cytometric enrichment, and global mRNA amplification for sensitive gene expression profiling. Systematic quantification elicited a high dynamic range of GABAergic cell numbers in different brain regions. Clustering of our gene expression results revealed major differences between hind and forebrain GABAergic neurons indicating that the development of GABAergic neurons depends on their regional location. While GABAergic neurons of the forebrain are characterized by three main groups of transcription factors of the Distal-less-family, the POU-family, and ETS/FOX family, specific members of the ZIC- and LHX-family of transcription factors appear to define hindbrain inhibitory neurons.

The sialyl-glycolipid stage-specific embryonic antigen 4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

IntroductionChemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells.MethodsWe performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations.ResultsHigh expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial–mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-β-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy.ConclusionsIn this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.

Identification, isolation, and characterization of human LGR5-positive colon adenoma cells

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells, whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas, adenocarcinomas and normal colon, which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage, and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids, we found correlations between LGR5 and CRC-specific genes, including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively, this work provides resources, methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis. Summary: Immunohistochemical and transcriptomic analyses of organoids generated from precancerous adenoma, colon adenocarcinoma and normal human tissue shows that the intestinal stem cell marker LGR5 is a colon cancer prognostic factor.

The sialyl-glycolipid SSEA4 marks a subpopulation of chemotherapy resistant breast cancer cells with mesenchymal features

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype associated with high risk of early relapse and metastasis [1]. At the moment chemotherapy remains the main option for systemic therapy of TNBC patients but complete remission occurs only in 20% of the patients [2]. In order to identify biomarker for chemotherapy-resistant TNBC cells, we performed a cell surface marker screen in 4 TNBC patient-derived xenograft (PDX) models that respond well to adriamycin/cyclophosphamide-based (A/C) chemotherapy but fail to reach complete pathological response. We used multi-parameter flow cytometry to screen the expression of 45 cell surface markers during the course of chemotherapy. We identified the sialyl-glycolipid SSEA4 as a marker of chemotherapy-resistant cancer cells in all four models. In addition, 3 out of 4 TNBC PDXs showed higher percentage of SSEA4-positive cells compared to all A/C-sensitive TNBC PDXs analysed. Gene expression comparison between SSEA4-positive and SSEA4-negative tumor cells from 3 TNBC PDXs highlighted an overexpression of mesenchymal-associated genes and a deregulation of drug resistance pathway-associated genes and miRNAs in SSEA4+ breast cancer cells. In addition, high expression of ST3 beta-galactoside alpha-2,3-sialyltransferase 2 (ST3Gal2), the enzyme catalyzing the last step of SSEA4 synthesis, was found associated with poor outcome in ER-, PR- breast cancer patients treated with chemotherapy (p < 0.01, HR 3.08). Thus, we propose SSEA4 as a novel marker of mesenchymal and chemoresistant breast cancer cells, and ST3GAL2 expression as a predictive marker for chemoresistance associated with poor outcome in breast cancer patients.

Abstract 3757: Novel monoclonal antibodies for the analysis and isolation of Lgr5 positive cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Cancer stem cells (CSCs), also called tumor initiating cells, have gained substantial interest in the research field over the past few years. CSCs have been isolated from multiple tumor entities and were shown to play a crucial role during tumor growth and metastasis. However, there is still a major debate about specific cell surface markers capable of identifying CSCs in most tumor entities. The leucine-rich repeat-containing G-protein-coupled receptor (LGR) Lgr5 and its close homologues Lgr4 and Lgr6 associate with Wnt-receptors and act as R-spondin receptors thereby playing a central role in the modulation of Wnt/β-catenin signaling in normal and neoplastic stem cells (de Lau et al., 2011, Carmon et al., 2011). Initially described as a highly specific marker for stem cells in the small intestine, colon, hair follicle, stomach, and during kidney development (Barker et al., 2007, Jaks et al., 2008, Barker et al., 2010, Barker et al., 2012), Lgr5 positive cells were also shown to be crucial during the development and progression of cancer. It was shown that Lgr5 positive crypt stem cells are the cells-of-origin in intestinal cancer and that CSCs in human colorectal cancer can be identified and isolated based on Lgr5 expression (Barker et al., 2009, Kemper et al., 2012). However, the analysis of Lgr5 expressing cells is hampered by the lack of highly specific monoclonal antibodies. We have developed rabbit monoclonal antibodies against peptide sequences of Lgr5. After generating cell lines stably expressing Lgr5, we screened for high affinity antibodies by flow cytometry. Using stable transfectants for Lgr4 and Lgr6, we could prove that there is no cross reactivity with its close homologues. This is of particular importance for Lgr4, as this protein is also expressed on more differentiated progenitor cells. To allow for identification, analysis, and isolation of Lgr5 positive cells from solid tissues, automated and therefore user-independent methods for tissue dissociation were developed. This resulted in enzyme mixtures which allow dissociating different tissues without harming the target epitopes. To detect cells expressing Lgr5 at low levels in flow cytometry based assays we combined our novel high affinity antibodies with fluorescence amplification systems. In addition, we generated a new magnetic cell separation (MACS) protocol that can be used as an easy and fast method to isolate Lgr5 positive cells from primary human tissue. Taken together, we have developed highly specific monoclonal antibodies allowing for the analysis and isolation of Lgr5 positive cells from cell lines and primary tissues. Citation Format: David Agorku, Greg Parker, Nadine Chelius, Andreas Bosio, Olaf Hardt. Novel monoclonal antibodies for the analysis and isolation of Lgr5 positive cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3757. doi:10.1158/1538-7445.AM2013-3757

Abstract 3357: Isolation of tumor cell subpopulations using semi-automated tissue dissociation and magnetic cell separation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The analysis of tumor resident cell and stem cell populations requires proper methods for tissue dissociation and cell purification. Over the past few years, there has been a particular focus on the study of tumor infiltrating lymphocytes (TIL) and cancer stem cells (CSC). TIL can be isolated and characterized using conventional lymphocyte markers whereas CSC-specific cell surface markers are currently established (1). Two important markers, namely CD24 and CD44, have been shown to define CSC in various neoplasms such as breast cancer (2) or colon cancer (3). We have established methods for the enzymatic and mechanical dissociation of solid tumors and optimized them according to the specific needs of a given tissue or cell type. Solid tumors are built up of a mixture of cell types, which are interconnected to each other and surrounded by an extracellular matrix composed of a variety of proteins and polysaccharides. The major goal was to disrupt the extracellular matrix and cell adhesion components without harming the integrity of the cell membrane. This was achieved by a combination of varying enzyme mixes, mechanical forces, incubation periods, and temperatures. The automation of all mechanical steps (gentleMACS™ dissociator) led to reproducible and moreover user independent results with reduced overall processing times. Viable tumor infiltrating lymphocytes (TIL) as well as tumor cells were obtained from melanoma metastases in high numbers. The isolated TIL expanded significantly faster than those from manually dissociated tumors and showed functionality in vitro and in vivo. As for cancer stem cells, we present novel tools for the immunomagnetic isolation of CD44+ and CD44+/CD24- cells. Using magnetic cell sorting (MACS®), a subpopulation of human teratocarcinoma cells could be isolated to a purity of 92% based on their expression of CD44. Furthermore, a combined depletion and positive selection approach allowed for the enrichment of a CD44+/CD24- human CML subpopulation from a starting frequency of 34% to final purity of 95%. The separation of pure tumor cell populations will be necessary for their further characterization and also targeted drug screening approaches. The methods presented here are broadly applicable as they can be easily adapted to other cancer tissues and cell types and should help to optimize and standardize the future basic and clinical cancer research. 1. Trumpp, A. and Wiestler, O. D. (2008); Nature Clin. Prac. Oncology.; Vol. 5 No. 6 2. Al-Hajj, M. et al. (2003); Proc. Natl. Acad. Sci. USA; Vol. 100 No. 7 3. Du, L. et al. (2008); Clin. Cancer Res.; 6751 14 (21) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3357.

653. Sialyl Glycolipid Stage-Specific Embryonic Antigen 4 (SSEA4) - A Novel Target for CAR T Cell Therapy of Solid Cancers

Owing to their capacity to eradicate tumors, T cells represent an attractive means for immunomodulation in cancer immunotherapy. In this context, chimeric antigen receptor (CAR) - based therapies are receiving increasing attention. The combination of antibody-derived specificity with T cell effector function renders the immune cells MHC-independent and even enables targeting of antigens for which there is immunological tolerance. T cells, genetically modified with CAR, have shown impressive success in the treatment of leukemia. However, the application of CAR T cells to the treatment of solid tumors remains challenging due to the lack of truly cancer-specific targets and an immunosuppressive tumor microenvironment hostile to T cells.We have identified the sialyl glycolipid stage-specific embryonic antigen 4 (SSEA4) as an epitope whose expression strongly correlates with metastasis and chemoresistence in triple negative breast cancer cells (TNBC). Single chain antibody fragments (scFv) were derived from an antibody that specifically recognizes this sialyl-glycolipid and were cloned into a lentiviral expression vector encoding a CAR containing an IgG1 spacer domain with CD137 and CD3z signaling domains. Healthy donor T cells were enriched by magnetic cell sorting and activated with TransActTM Reagent, a colloidal nanomatrix-based activation reagent, before lentiviral transduction of the anti-SSEA4 CAR expression construct. Engagement of SSEA4 by CAR expressing T cells induced T cell degranulation, secretion of inflammatory cytokines and resulted in an effective killing of SSEA4 expressing target cells.As TNBC patients are exposed to multiple rounds of chemotherapy and SSEA4 expression is found enriched in residual tumor cells surviving chemotherapy, a combinatorial approach using chemotherapy followed with CAR T cell therapy holds great promise to improve treatment outcome and overall survival of TNBC patients. Having assessed the performance of different anti-SSEA4 CAR constructs in vitro, current studies are focusing on in vivo functionality using mouse models.