David Agorku

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. n nWe 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). n nThus, 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, DCnnCancer 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.nnCitation 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

Cellular Prion Protein PrPC and Ecto-5′-Nucleotidase Are Markers of the Cellular Stress Response to Aneuploidy

Aneuploidy is a hallmark of most human tumors, but the molecular physiology of aneuploid cells is not well characterized. In this study, we screened cell surface biomarkers of approximately 300 proteins by multiparameter flow cytometry using multiple aneuploid model systems such as cell lines, patient samples, and mouse models. Several new biomarkers were identified with altered expression in aneuploid cells, including overexpression of the cellular prion protein CD230/PrPC and the immunosuppressive cell surface enzyme ecto-5-nucleotidase CD73. Functional analyses associated these alterations with increased cellular stress. An increased number of CD73+ cells was observed in confluent cultures in aneuploid cells relative to their diploid counterparts. An elevated expression in CD230/PrPC was observed in serum-deprived cells in association with increased generation of reactive oxygen species. Overall, our work identified biomarkers of aneuploid karyotypes, which suggest insights into the underlying molecular physiology of aneuploid cells. Cancer Res; 77(11); 2914-26. ©2017 AACR.

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays(1). Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research (2-4). Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors(4) or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable. Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis (5). To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Next-generation sequencing of human tumor xenografts is significantly improved by prior depletion of mouse cell

Human tumor xenografts represent the gold standard method for many research areas, including drug discovery, cancer stem cell biology, and metastasis prediction. When compared to in vitro cell culture models, human tumor xenografts show a higher validity for most assays1. During the growth phase in vivo, xenografted tissue is vascularized and infiltrated by cells of murine origin. The level of infiltration is highly dependent on multiple factors like tumor subtype, growth rate, and region of transplantation. However, even when these factors are kept constant, the amount and composition of infiltrating mouse cells is highly variable. Due to this, molecular downstream analyses such as microarray-based expression profiling are biased by cross-hybridization of mouse-derived molecules to human probes. In addition, a reduction of sensitivity caused by measuring undesired mouse signals during nextgeneration sequencing analysis can be expected. To overcome these limitations, we developed a fast and easy method allowing for the effective depletion of all cells of mouse origin by using automated tissue dissociation and magnetic cell sorting (MACS® Technology). We performed whole exome sequencing (WES) of bulk human tumor xenografts from lung, bladder, and kidney cancer, and compared the results to samples depleted of mouse cells.

Abstract 4317: The sialyl-glycolipid SSEA4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnCurrently, the main option for systemic therapy of high-risk breast cancer is chemotherapy, with an overall poor efficacy and severe side effects. Chemotherapy-resistance and incomplete pathologic response associate with risk of metastasis and early relapse in breast cancer.nnIn order to characterize treatment-resistant tumor cells, we performed a cell surface marker screen in 4 triple-negative breast cancer patient-derived xenograft (PDX) models that respond well to adriamycin/cyclophosphamide-based chemotherapy but fail to reach complete pathological response. We used multi-parameter flow cytometry to screen for the expression of a set of 45 cell surface markers during the course of chemotherapy. This set of markers represented both proteins involved in stem cell function and proteins known to be over-expressed in stem cells or cancer stem cell sub-populations.nnWe identified the sialyl-glycolipid SSEA4 as a constant marker of chemotherapy-resistant cancer cells in all four models. In addition, SSEA4 expression was found higher in 3 out of 4 TNBC PDXs that are de novo resistant to neo-adjuvant chemotherapy compared to sensitive TNBC PDXs. Two cell populations with different percentage of SSEA4-positive (SSEA4+) cells and with different growth characteristics were identified in a PDX model. When treated with genotoxic compounds, the cell population with higher SSEA4+ expression showed increased resistance to chemotherapy, indicating this post-translational modification as potential marker of tumor resistance. Comparison of SSEA4+ and SSEA4-negative (SSEA4-) tumor cells from TNBC PDX models by global gene expression profiling showed overexpression of mesenchymal-associated genes in SSEA4+ tumor cells and a deregulation of drug resistance pathway-associated genes and miRNAs. 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 breast and ovarian cancer patients treated with chemotherapy.nnThus, we propose SSEA4 as a novel marker of epithelial-mesenchymal transition associated with chemoresistance, and ST3GAL2 expression as a predictive marker for tumor chemoresistance associated with poor outcome in breast and ovarian cancer patients. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance and to develop alternative treatment regimens for breast and ovarian cancer patients.nnCitation Format: Andrea Aloia, Evgeniya Petrova, Stefan Tomiuk, Ute Bissels, Sophie Banis, Olivier Deas, Silvia Ruberg, Bernhard Gerstmayer, David Agorku, Jean-Gabriel Judde, Andreas Bosio, Stefano Cairo, Olaf T. Hardt. The sialyl-glycolipid SSEA4 marks a subpopulation of chemotherapy-resistant breast cancer cells with mesenchymal features. [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 4317. doi:10.1158/1538-7445.AM2015-4317

Abstract 1455: Next generation sequencing of human tumor xenografts is significantly improved by prior depletion of mouse cells

Human tumor xenografts represent the gold standard method for many research areas, including drug discovery, cancer stem cell biology, and metastasis prediction. When compared to in vitro cell culture models, human tumor xenografts show a higher validity for most assays1. During the growth phase in vivo, xenografted tissue is vascularized and infiltrated by cells of murine origin. The level of infiltration is highly dependent on multiple factors like tumor subtype, growth rate, and region of transplantation. However, even when these factors are kept constant, the amount and composition of infiltrating mouse cells is highly variable. Due to this, molecular downstream analyses such as microarray-based expression profiling are biased by cross-hybridization of mouse-derived molecules to human probes. In addition, a reduction of sensitivity caused by measuring undesired mouse signals during nextgeneration sequencing analysis can be expected. To overcome these limitations, we developed a fast and easy method allowing for the effective depletion of all cells of mouse origin by using automated tissue dissociation and magnetic cell sorting (MACS® Technology). We performed whole exome sequencing (WES) of bulk human tumor xenografts from lung, bladder, and kidney cancer, and compared the results to samples depleted of mouse cells.

Abstract 95: Depletion of mouse cells from human tumor xenografts significantly reduces bias in molecular analysis and improves culture of target cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnHuman tumor xenografts represent the gold standard method for research areas like drug discovery, cancer stem cell biology, and metastasis prediction. They can be derived from primary human tumor material, serially transplanted tumor tissue, or cultured cells. When compared to in vitro cell culture models, human tumor xenografts show a higher validity for most assays (Rubio-Viqueira et al., 2009).nnDuring the growth phase in vivo, xenografted tissue is vascularized and infiltrated by cells of murine origin including heterogeneous lymphocyte subpopulations, fibroblasts, and endothelial cells. The level of infiltration is highly dependent on multiple factors like tumor subtype, growth rate, and region of transplantation. However, even when these factors are kept constant, the amount and composition of infiltrating mouse cells is highly variable. Due to this, some molecular downstream analyses are challenged. The contaminating mouse cells lead to cross hybridization of mouse derived molecules to human probes on microarrays and a significant reduction of sensitivity caused by measuring mouse signals during next-generation sequencing or proteome analysis. In addition, the culture of human tumor cells is frequently hampered by murine fibroblasts efficiently plating and overgrowing the target cells.nnTo overcome these limitations, we have developed a fast and easy method allowing for the comprehensive depletion of all cells of murine origin. We have dissociated human tumor xenografts as well as normal murine tissue of multiple origins including skin, brain, kidney and lung followed by screening panels of cell surface markers to define an optimized combination of antibodies capable of binding all cells from all murine origins. Subsequently, we generated conjugates of suitable antibodies with paramagnetic nanoparticles (MicroBeads) and titrated all reagents to optimize the depletion efficiency by using magnetic cell sorting (MACS). Even tumors that contain high numbers of mouse cells (> 60%) can be cleaned up to purities of human tumor cells higher than 96% in less than 25 minutes. This depletion step substantially increased sensitivity during empiric cell surface marker screenings as well as during the isolation of tumor cell subpopulations. Finally, we show that downstream molecular analysis and culture of human tumor cells is significantly standardized upon removal of mouse contamination.nnTaken together, this novel method prevents inaccurate human tumor xenograft analysis caused by contamination with cells of murine origin. As antibodies specific only for mouse cells are used, the target cells stay “untouched” and the procedure can be used for all kinds of xenografted material without the need for a positive marker expressed on the human cells.nnCitation Format: David Agorku, Andreas Bosio, Olaf Hardt. Depletion of mouse cells from human tumor xenografts significantly reduces bias in molecular analysis and improves culture of target cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 95. doi:10.1158/1538-7445.AM2014-95