Julia Schueler

Human Vδ1 γδ T cells expanded from peripheral blood exhibit specific cytotoxicity against B-cell chronic lymphocytic leukemia-derived cells

BACKGROUND AIMS There is increasing interest in using γδ T cells (GDTC) for cancer immunotherapy. Most studies have been concerned with the Vδ2 subset in blood, for which several expansion protocols exist. We have developed a protocol to expand Vδ1 and Vδ2 preferentially from human blood. We have characterized these subsets and their specificities for leukemic targets. METHODS GDTC were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors via positive magnetic cell sorting; their proliferation in vitro was induced by exposure to the mitogen concanavalin A (Con A). CD107 and cytotoxicity (Cr(51)-release and flow cytometric) assays were performed. GDTC clones and target cells were immunophenotyped via flow cytometry. RESULTS Longer initial exposure to Con A typically resulted in higher Vδ1 prevalence. Vδ1 were activated by and cytotoxic to B-cell chronic lymphocytic leukemia (B-CLL)-derived MEC1 cells, whereas Vδ2 also responded to MEC1 but more so to the Philadelphia chromosome-positive [Ph+] leukemia cell line EM-enhanced green fluorescent protein (2eGFPluc). Vδ2 clone cytotoxicity against EM-2eGFPluc correlated with Vδ2 T-cell antigen receptor (TCR) and receptor found on Natural Killer cells and many T-cells (NKG2D), whereas Vδ1 clone cytotoxicity versus MEC1 correlated with Vδ1 TCR, CD56 and CD95 expression. Vδ1 also killed Epstein-Barr Virus (EBV)-negative B-CLL-derived TMD2 cells. Immunophenotyping revealed reduced HLA-ABC expression on EM-2eGFPluc, whereas MEC1 and TMD2 exhibited higher Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAILR1). CONCLUSIONS Our ability to expand peripheral Vδ1 cells and show their cytotoxicity to B-CLL-derived cell lines suggests that this novel approach to the cellular treatment of B-CLL may be feasible.

Intratibial Injection of Human Multiple Myeloma Cells in NOD/SCID IL-2Rγ(Null) Mice Mimics Human Myeloma and Serves as a Valuable Tool for the Development of Anticancer Strategies

Background We systematically analyzed multiple myeloma (MM) cell lines and patient bone marrow cells for their engraftment capacity in immunodeficient mice and validated the response of the resulting xenografts to antimyeloma agents. Design and Methods Using flow cytometry and near infrared fluorescence in-vivo-imaging, growth kinetics of MM cell lines L363 and RPMI8226 and patient bone marrow cells were investigated with use of a murine subcutaneous bone implant, intratibial and intravenous approach in NOD/SCID, NOD/SCID treated with CD122 antibody and NOD/SCID IL-2Rγ(null) mice (NSG). Results Myeloma growth was significantly increased in the absence of natural killer cell activity (NSG or αCD122-treated NOD/SCID). Comparison of NSG and αCD122-treated NOD/SCID revealed enhanced growth kinetics in the former, especially with respect to metastatic tumor sites which were exclusively observed therein. In NSG, MM cells were more tumorigenic when injected intratibially than intravenously. In NOD/SCID in contrast, the use of juvenile long bone implants was superior to intratibial or intravenous cancer cell injection. Using the intratibial NSG model, mice developed typical disease symptoms exclusively when implanted with human MM cell lines or patient-derived bone marrow cells, but not with healthy bone marrow cells nor in mock-injected animals. Bortezomib and dexamethasone delayed myeloma progression in L363- as well as patient-derived MM cell bearing NSG. Antitumor activity could be quantified via flow cytometry and in vivo imaging analyses. Conclusions Our results suggest that the intratibial NSG MM model mimics the clinical situation of the disseminated disease and serves as a valuable tool in the development of novel anticancer strategies.

Abstract 1026: Novel Tie2 inhibitor with in vivo efficacy in disseminated hematological tumor models in mice

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The receptor tyrosine kinase Tie2 is predominantly expressed in the endothelium but has also been identified on primitive hematopoietic stem cells, monocyte and macrophage subclasses, as well as on glioma or hematological tumor cells. Based on its expression in many patient-derived leukemic blasts inhibition of the Tie2 pathway may provide an attractive opportunity for therapeutic intervention in leukemias. In this study we report the pharmacological profile of a novel, highly potent and orally available Tie2 inhibitor (BAY-Tie2). The discovery and design process leading to BAY-Tie2 was performed with the goal of sparing other angiogenic RTKs, such as VEGFRs, FGFRs or PDGFRs. BAY-Tie2 is based on a novel imidazopyrazole core, combined with a SF5-substituted phenyl ring that fills the deep DFG-out pocket. BAY-Tie2 binds to Tie2 with a Kd value of 1.6 nM and is selective against VEGFR2 (Kd of 1600 nM), FGFR1 (<30% inhibition at 1 µM), FGFR2/3/4 (<10% inhibition at 1 µM) and PDGFRα/β (<30% inhibition at 100 nM). BAY-Tie2 potently inhibits Tie2 autophosphorylation in recombinant CHO-Tie2 and primary human umbilical vein endothelial cells (HUVEC) with IC50 values of 0.7 and 1.3 nM. Consistently, BAY-Tie2 was shown to inhibit Tie2 phosphorylation in vivo by analyzing angiopoietin-1 induced Tie2 phosphorylation status in extracts of murine lungs from BAY-Tie2-treated mice. In subcutaneous xenograft models of highly angiogenic tumors, BAY-Tie2 reduced tumor growth and showed evidence for potential combination benefit with anti-VEGF therapy. In order to explore the potential of a Tie2 inhibitor beyond affecting angiogenesis, we established disseminated leukemia models, using Tie2-expressing cell lines, such as the CML cell lines MEG-01 and EM-2. Both cell lines engrafted predominantly in bone marrow and spleen. Treatment started 3 days after i.v. cell implantation with either BAY-Tie2 or cytarabine and was well tolerated. Efficacy was monitored by a) inhibition of disease progression, b) weekly fluorescence-based in vivo imaging (IVI) using an Alexa750-labeled anti-human CD33 antibody, and c) q-RT-PCR specific for BCR-ABL and hCD45 in murine peripheral blood. BAY-Tie2 inhibited disease progression comparable to cytarabine. Tumor load measured by IVI was reduced in BAY-Tie2 treated groups by 45% in the MEG-01 and by 65% in the EM-2 model compared with the untreated control, very similar to the cytotoxic treatment with cytarabine. Quantitative RT-PCR on peripheral blood revealed that BAY-Tie2 and cytarabine delayed the appearance of circulating tumor cells in both CML models. These data demonstrate that BAY-Tie2 is an orally active Tie2 inhibitor that may have therapeutic benefit not only in angiogenic tumors but also in hematological, Tie2-expressing malignancies. Citation Format: Sylvia Gruenewald, Julia Schueler, Michael Haerter, Frank Suessmeier, Kerstin Klingner, Ulf Boemer, Stefan Kaulfuss, Alexander Walter, Mario Lobell, Ingo V. Hartung, Bernd Buchmann, Dieter Heldmann, Holger Hess-Stumpp, Karl Ziegelbauer. Novel Tie2 inhibitor with in vivo efficacy in disseminated hematological tumor models in mice. [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 1026. doi:10.1158/1538-7445.AM2014-1026

Abstract 5770:in vitroPDX models: 3D cultured patient-derived tumors for compound evaluation

Background. Patient-derived xenograft (PDX) models in immune-compromised mice allow propagation of and compound testing in human-derived tumors in vivo. To expand the potential of these human-relevant PDX models, we sought to develop 3D in vitro culture methods for PDX-derived tumor cells that show in vivo-like growth characteristics, invasion and responses to therapeutics. In combination with advanced 3D image analysis methods, we created a unique high throughput in vitro PDX screening platform that not only allows efficient identification of active and selective molecules but also enables selection of the optimal PDX tumor models for subsequent validation of candidates in vivo. Results. Each PDX model has its own unique growth characteristics. Hydrogel and growth media composition were optimized to support growth of tumor tissues in vitro from cells derived from bladder, stomach, breast, pancreas, colon and lung cancer PDX tumors. Tumor tissues were cultured in a 384-well format and used to test chemotherapeutics (e.g. 5-FU, doxorubicin, paclitaxel, cisplatin), small molecules (e.g. erlotinib, lapatinib, trametinib, everolimus), antibodies (e.g. cetuximab, trastuzumab) and antibody-drug-conjugate (ADC, T-DM1) dose ranges. Using OcellO’s 3D image analysis platform, Ominer, tumoroid growth, cell proliferation, apoptosis, invasion, cell polarity, differentiation and other aspects of cell and tissue architecture were analyzed and the effects of compound exposure on tumoroids was determined. By performing feature training based on reference compounds, we selected ±10 morphological features (out of more than 500) to generate a phenotypic signature that described the unique phenotypic change induced by each compound. Different compounds that target the same molecule were found to induce a similar morphological change whereas compounds with off-target effects could be discriminated. This approach enabled a high resolution evaluation and comparison of compound activity in an automated manner. Conclusions. We established several PDX model-derived 3D tumor cultures in which standard-of-care and novel therapeutic agents (small molecules, antibodies and ADCs) can efficiently be screened, based on therapeutically relevant parameters and their changing morphological profile. This method enables both the in vitro selection of promising compounds in a pre-clinically relevant setting and the selection of optimum PDX tumor models for follow-up in vivo studies. This highly translational in vitro-in vivo PDX pipeline is expected to reduce attrition and increase efficiency in early drug-discovery. Citation Format: Sander Basten, Bram Herpers, Julia Schueler, Torsten Giesemann, Leo S. Price. in vitro PDX models: 3D cultured patient-derived tumors for compound evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5770. doi:10.1158/1538-7445.AM2017-5770

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 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 C8: Functional and molecular characteristics of patient-derived xenografts of colon cancer.

Introduction: Colorectal carcinoma (CRC) is the second leading cause of cancer-related deaths worldwide. Patient-derived xenografts (PDX) subcutaneously implanted in nude mice are highly suitable tools to aid in the development of novel anti-cancer therapeutics as their response is a good predictor of clinical outcome. In the present study, a panel of colon cancer PDXs (CXFs) was characterized for their molecular profile and sensitivity to standard of care (SoC) drugs. Material and Methods: CRC samples from patients undergoing surgery were implanted subcutaneously in nude mice and sequentially passaged. Tumor material was collected for molecular profiling, including mutational analysis by Sanger sequencing and Sequenom OncoCarta panels I, II, III. The efficacy of the SoC drugs 5-fluoruracil, oxaliplatin, irinotecan, bevacizumab and cetuximab was tested in vivo. All compounds were administered at clinically relevant dose levels and schedules. The tumor load was determined by caliper measurements twice a week. Anti-tumor activity was determined by comparing the relative tumor load of test groups vs. the vehicle-treated control group. Tumors were considered sensitive if the minimal test / control (T/C [%]) value recorded during an experiment was smaller than 30%. For cytotoxic SoC drugs, sensitivity of CXFs were also analyzed in an ex vivo tumor colony assay (TCA). Results: 73 CRC patient samples were implanted within the framework of this study. Up to now, 65 of them were established as CXF corresponding to a take rate of 89%. Mutational analysis was performed for 58 CXFs. Regarding the RAS/RAF pathway, mutations were found in K-RAS (26 out of 58 CXFs, 45%), NRAS (3%) and BRAF (7%). Mutations in the AKT/PI3K pathway were also found (PTEN, 9% and PIK3CA, 17%). In addition, one CXF had an IDH1 mutation (2%). For all SoC drugs both sensitive and resistant CXFs were identified and given CXFs differed in their sensitivities to different SoC drugs. In vivo sensitivity data will be correlated with molecular characteristics of the tested CXFs and with their sensitivities determined in the TCA. Conclusion and Outlook: Due to the high take rate, our CXF panel represents a large proportion of clinical variants of CRC. We will describe mutation profiles and sensitivity profiles to SoC drugs used for colon cancer. Correlation of sensitivity data and molecular data may reveal interesting correlations and identify biomarkers predictive of response to SoC drugs and experimental compounds. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C8. Citation Format: Jianing M. Guo, Kerstin Klingner, Rebekka Krumbach, Armin Maier, Silvia Naus, Vincent Vuaroqueaux, Evelyn Lamy, Heiner Fiebig, Julia B. Schueler. Functional and molecular characteristics of patient-derived xenografts of colon cancer. [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 C8.

Abstract 5254: Xenograft-derived 3D cultures of hematological maligancies ex vivo and in vivo as a model system to investigate efficacy of antitumor compounds

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Hematological malignancies account for a notable proportion of cancers worldwide, and the heterogeneity and biological characteristics induce unique therapeutic challenges. A panel of 20 hematological cell lines comprising different entities like leukemia (acute lymphoblastic ALL; acute myeloid AML; chronic myeloid CML), Hodgkin-, and non-Hodgkin-lymphoma (NHL), as well as multiple myeloma (MM) was established for ex vivo anti-tumor efficacy profiling using a clonogenic assay. These 3D cell culture assays often mimic the in vivo scenario better than 2D cell culture assays. Tumor cells were injected into the flanks of NOD/SCID mice to obtain subcutaneous tumor xenografts, which were kept at low passages (n <3). These xenografts served as starting material to prepare single cell suspensions for ex vivo analysis, or to carry out in vivo efficacy tests with either subcutaneous or disseminated growing tumors. Twenty four cytotoxic and targeted drugs were tested for their ex vivo chemosensitivity. The drugs showed diverse patterns of selectivity and potency: vincristine, doxorubicin, cytarabine, and bortezomib exhibited activity with IC50 values in the nanomolar range (mean IC50 = 1 - 100nM), not limited to their respective clinical application, but also in other tumor entities, such as in ALL and AML in case of bortezomib. IC50 values of prednisolone and dexamethasone were in the micromolar range (mean IC50 = 22 - 58µM) in cell lines of ALL (CCRF-CEM, MOLT-4), AML (NOMO-1), NHL (DAUDI, U-937), and MM (IM-9). All-trans-retinoic acid (mean IC50 = 1.3µM) as well as interferon-gamma-1b (mean IC50 = 0.43µM) showed specific activity patterns with pronounced growth inhibition in cell lines of AML (KG-1, NOMO-1, OCI-AML2), CML (EM-2), and MM (L-363). IC50 values of tyrosine kinase inhibitors imatinib and nilotinib showed strong correlation (spearman coefficient: 0.76, p <0.001) with selectivity mainly for bcr-abl-positive cells. In vivo follow-up testing confirmed the ex vivo results. E.g. cyclophosphamide that showed strong antitumor activity against the NHL cell line DAUDI ex vivo (IC50 = 0.3µM), also induced tumor remissions of 80% in xenografts. Moreover, antitumor activity of sorafenib in AML cells assessed via clonogenic assay (mean IC50 0.84µM in AML cells vs. mean IC50 4.0µM over all tested entities) could be confirmed in the disseminated in vivo model using HL-60 cells (reduction of 99% vs. untreated control). The presented ex vivo panel screen is of great value for time and cost effective profiling of cytotoxic and targeted anti-cancer agents, which can be confirmed in in vivo tumor models of hematological malignancies, and can thereby guide to more effectively designed in vivo experiments. Diverse activity and resistance patterns ex vivo and in vivo also contribute to create clinical development strategies of standard and novel compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5254. doi:1538-7445.AM2012-5254

Abstract C110: Mutation and chemosensitivity profile of a panel of 25 patient-derived melanoma xenografts.

Melanomas account for a notable proportion of cancers worldwide. The heterogeneity and biological characteristics of melanomas present unique therapeutic challenges. Recently a number of somatic mutations were determined, which influence the prognosis and thus therapeutic needs markedly. Here we present a panel of patient-derived melanoma xenografts, established by Oncotest from primary patient material. From 80 patient explants, growth in serial passage was observed in 43% of the cases. Molecular profiling included mutational analysis for BRAF, PI3Kalpha, KRAS, NRAS, TP53, CTNNB1, MET and JAK3 using OncoCarta ™ and exon sequencing. The chemosensitivity profile was determined in vitro using 2D and 3D culture assays. Clinically relevant BRAF mutations were the most prevalent mutation and were found in almost two thirds of the melanoma models. The second most common gene mutated was NRAS, with about one quarter of the models harbouring activating mutations of NRAS. PI3Kalpha mutations were rare, and for KRAS no mutations were detected. Most of the melanomas showed multiple mutations of the investigated genes. Chemosensitivity profiling included cytotoxic (e.g. cisplatin, carboplatin, vinblastine, vincristine, ifosfamide, paclitaxel, and docetaxel), as well as targeted drugs (e.g. sorafenib and PLX-4720). The compounds showed diverse patterns of selectivity and potency: Differential activity was determined e.g. for vinblastine, vincristine, paclitaxel, and docetaxel, but also for the BRAF inhibitor PLX-4720. PLX-4720 was most active in melanoma models carrying the BRAF mutation as determined in monolayer and soft agar assays. Activity of vincristine revealed a correlation of sensitivity with a BRAF wild type genotype and resistance with BRAF mutation. The latter result was also described recently for melanoma patients with BRAF mutations, showing a diminished duration of response to treatment with vincristine, dacarbazine, bleomycin, lomustine, and human leukocyte interferon. A collection of 25 patient-derived melanoma models is presented. Their mutational status as well as their responsiveness towards many cytotoxic and targeted agents was analyzed. This extensive genotypic and phenotypic characterization makes them a valuable tool for hypothesis generation and testing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C110.