Annika Wulf-Goldenberg

Angiotensin-(1–7) stimulates hematopoietic progenitor cells in vitro and in vivo

This study demonstrates that the angiotensin II metabolite Ang-(1-7) stimulates the proliferation of hematopoietic progenitor cells and promotes their engraftment in a xenograft model, suggesting that the renin-angiotensin system is a regulator of blood cell formation. See related perspective article on page 745. Effects of angiotensin (Ang)-(1–7), an AngII metabolite, on bone marrow-derived hematopoietic cells were studied. We identified Ang-(1–7) to stimulate proliferation of human CD34+ and mononuclear cells in vitro. Under in vivo conditions, we monitored proliferation and differentiation of human cord blood mononuclear cells in NOD/SCID mice. Ang-(1–7) stimulated differentially human cells in bone marrow and accumulated them in the spleen. The number of HLA-I+ and CD34+ cells in the bone marrow was increased 42-fold and 600-fold, respectively. These results indicate a decisive impact of Ang-(1–7) on hematopoiesis and its promising therapeutic potential in diseases requiring progenitor stimulation.

A comprehensively characterized large panel of head and neck cancer patient-derived xenografts identifies the mTOR inhibitor everolimus as potential new treatment option

Patient‐derived xenograft (PDX) models have shown to reflect original patient tumors better than any other preclinical model. We embarked in a study establishing a large panel of head and neck squamous cell carcinomas PDX for biomarker analysis and evaluation of established and novel compounds. Out of 115 transplanted specimens 52 models were established of which 29 were characterized for response to docetaxel, cetuximab, methotrexate, carboplatin, 5‐fluorouracil and everolimus. Further, tumors were subjected to sequencing analysis and gene expression profiling of selected mTOR pathway members. Most frequent response was observed for docetaxel and cetuximab. Responses to carboplatin, 5‐fluorouracil and methotrexate were moderate. Everolimus revealed activity in the majority of PDX. Mutational profiling and gene expression analysis did not reveal a predictive biomarker for everolimus even though by trend RPS6KB1 mRNA expression was associated with response. In conclusion we demonstrate a comprehensively characterized panel of head and neck cancer PDX models, which represent a valuable and renewable tissue resource for evaluation of novel compounds and associated biomarkers.

Interwoven Four-Compartment Capillary Membrane Technology for Three-Dimensional Perfusion with Decentralized Mass Exchange to Scale Up Embryonic Stem Cell Culture

We describe hollow fiber-based three-dimensional (3D) dynamic perfusion bioreactor technology for embryonic stem cells (ESC) which is scalable for laboratory and potentially clinical translation applications. We added 2 more compartments to the typical 2-compartment devices, namely an additional media capillary compartment for countercurrent ‘arteriovenous’ flow and an oxygenation capillary compartment. Each capillary membrane compartment can be perfused independently. Interweaving the 3 capillary systems to form repetitive units allows bioreactor scalability by multiplying the capillary units and provides decentralized media perfusion while enhancing mass exchange and reducing gradient distances from decimeters to more physiologic lengths of <1 mm. The exterior of the resulting membrane network, the cell compartment, is used as a physically active scaffold for cell aggregation; adjusting intercapillary distances enables control of the size of cell aggregates. To demonstrate the technology, mouse ESC (mESC) were cultured in 8- or 800-ml cell compartment bioreactors. We were able to confirm the hypothesis that this bioreactor enables mESC expansion qualitatively comparable to that obtained with Petri dishes, but on a larger scale. To test this, we compared the growth of 129/SVEV mESC in static two-dimensional Petri dishes with that in 3D perfusion bioreactors. We then tested the feasibility of scaling up the culture. In an 800-ml prototype, we cultured approximately 5 × 109 cells, replacing up to 800 conventional 100-mm Petri dishes. Teratoma formation studies in mice confirmed protein expression and gene expression results with regard to maintaining ‘stemness’ markers during cell expansion.

Intrahepatically transplanted human cord blood cells reduce SW480 tumor growth in the presence of bispecific EpCAM/CD3 antibody

Humanized mice were generated in order to investigate the anti-tumor efficacy of bispecific antibodies. The engraftment, distribution and differentiation of mononuclear cells (MNC) from cord blood transplanted into the liver of newborn non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were measured. Using a human-specific polymerase chain reaction (PCR), human cells were found to be present in the liver for a time range from 5 min to 5 days. After long-term engraftment of 42 days, the highest level of human cells was measured in mouse thymus, with lower levels in spleen and bone marrow. Engrafted human cells in mouse organs showed T-cell differentiation only, as measured by CD3, CD4 and CD8 expression. The MNC transplanted intrahepatically into newborn mice were tested for T-cell mediated anti-tumor activity in vivo against subcutaneously transplanted human SW480 colon carcinoma in NOD/SCID mice. A delay of SW480 tumor growth in mice in the presence of a bispecific epithelial cell-adhesion molecule (EpCAM)/CD3 antibody was found to be associated with the presence of immunoreactive human CD3 cells within the SW480 tumor. Our data provide evidence that the intrahepatic transplantation of cord blood stem cells into newborn mice represents a valuable model for establishing functionally active human T cells with anti-tumor activity.

Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells

In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction.

Intrahepatic transplantation of CD34+ cord blood stem cells into newborn and adult NOD/SCID mice induce differential organ engraftment

In vivo studies concerning the function of human hematopoietic stem cells (HSC) are limited by relatively low levels of engraftment and the failure of the engrafted HSC preparations to differentiate into functional immune cells after systemic application. In the present paper we describe the effect of intrahepatically transplanted CD34(+) cells from cord blood into the liver of newborn or adult NOD/SCID mice on organ engraftment and differentiation. Analyzing the short and long term time dependency of human cell recruitment into mouse organs after cell transplantation in the liver of newborn and adult NOD/SCID mice by RT-PCR and FACS analysis, a significantly high engraftment was found after transplantation into liver of newborn NOD/SCID mice compared to adult mice, with the highest level of 35% human cells in bone marrow and 4.9% human cells in spleen at day 70. These human cells showed CD19 B-cell, CD34 and CD38 hematopoietic and CD33 myeloid cell differentiation, but lacked any T-cell differentiation. HSC transplantation into liver of adult NOD/SCID mice resulted in minor recruitment of human cells from mouse liver to other mouse organs. The results indicate the usefulness of the intrahepatic application route into the liver of newborn NOD/SCID mice for the investigation of hematopoietic differentiation potential of CD34(+) cord blood stem cell preparations.

Abstract 1697: Humanized immune-oncology mouse models

The recent clinical success of immune checkpoint modulators has stimulated immune-oncology research leading to the identification of new tumor immunology targets. However both, target validation and drug development need better preclinical immune oncology models. Translational research further urgently needs such models for identification of clinically relevant biomarkers and defining rational combination strategies. Human patient-derived xenografts (PDX) from different tumor indications transplanted on immunodeficient mice have demonstrated strong predictive power for many drug development programs in cancer research. However, one caveat of PDX models is, that these mice lack a functional immune system, which allows tumor engraftment in the xenogenic host. To overcome these constraints our aim is the development of PDX models on mice with a functional human immune system. This strategy should allow implementing the highly predictable PDX in a functional human immune environment for better drug efficacy and safety studies. We reconstituted a human immune system in mice by engrafting human hematopoietic stem cells in immunodeficient mice. We demonstrate the engraftment of a full set of human immune cells, including T cells, B cells, NK cells, monocytes and dendritic cells in these mice. Tumor expression of PD-L1 is currently evaluated as first biomarker for immunotherapy with the PD1 checkpoint inhibitors. We screened our tumor cell lines and PDX models for PD-L1 expression to identify such a correlation. PD-L1 positive and negative models were transplanted on humanized mice. Most of them showed no difference in tumor growth compared to non-humanized mice (= fully immune resistant). However other PDX showed a delayed growth on the humanized mice (= partly immune resistant), whereas only one model did not grow at all (= immune sensitive), demonstrating a high sensitivity to the innate immune response of this PDX. To evaluate the function of the human immune cells and checkpoints, mice were treated with checkpoint inhibitors ipilimumab and nivolumab. Checkpoint inhibitors alone or in combination led to a minor tumor growth delay and an increased number of activated T-cells in the blood and in the tumor. Our results from experiments with n=19 models revealed a first correlation: stronger tumor growth inhibition on PD-L1 positive PDX and increased sensitivity of “partly immune resistant” PDX. Furthermore we were able to demonstrate, that treatment effects of checkpoint inhibitors could be increased by combination with radiation. Summary: Our humanized PDX models enable appropriate preclinical studies on tumor immune biology, evaluation of new immune therapies and combinations, as well as the identification and validation of biomarkers for tumor immune therapy. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Iduna Fichtner, Jens Hoffmann. Humanized immune-oncology mouse models [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 1697. doi:10.1158/1538-7445.AM2017-1697

Abstract 1843: Intratumoral heterogeneity of renal cancer is related to differences in drug response and development of therapy resistance

Patients with advanced renal cell cancer (RCC) have a poor prognosis not least because of resistance towards standard drugs. Recently, pronounced intratumoral heterogeneity (ITH) in RCC was shown. We were interested whether this ITH is a potential cause for treatment failure. We developed a large panel of patient-derived xenograft (PDX) models from RCC, including subsets of models from different regions of one individual patient tumor. The PDX models were evaluated for response to targeted standard therapeutics. To better understand correlations between interand intratumoral heterogeneity and treatment response, an explorative analysis of gene expression and panel sequencing data was performed.

Abstract B118: Preclinical model of patient-derived tumor transplant in humanized mice for cancer immunotherapy

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 patient-derived human tumor xenograft models (PDX) are growing on immunodeficient animals. To overcome these constraints our aim is the development of PDX models on mice with a functional human immune system to improve predictability of drug efficacy and safety. We reconstituted a human immune system by engrafting human hematopoietic stem cells isolated from cord blood in immunodeficient mice. A stable expression of lymphoid cell lineages in peripheral blood and secondary lymphoid tissues could be detected by flow cytometry and immunohistochemistry analysis. At the time when the human immune system is developed, established patient-derived tumors were transplanted on these humanized mice. Human tumors developed on humanized mice without evidence of rejection. Tumors from different tumor entities grow on humanized mice with a similar level of engraftment compared to not-humanized mice. We also tested the effect of ipilimumab (anti-CTLA-4 [cytotoxic T-lymphocyte antigen-4] mAB) and the humanized monoclonal antibody herceptin in these tumor-bearing humanized mice. We found ipilimumab could lead to a slight tumor growth delay and an increased percentage of T-cells in the blood and in the tumor. Initially results revealed that our humanized mouse models could enable a more appropriate preclinical assessment of immune-based therapeutic antitumor strategies especially when combining the humanized mouse with patient-derived xenografts. Citation Format: Annika Wulf-Goldenberg, Maria Stecklum, Iduna Fichtner, Jens Hoffmann. Preclinical model of patient-derived tumor transplant in humanized mice for cancer immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B118.

Abstract B126: Correlation of tumor growth inhibition by check point inhibitors with PD-L1 expression in preclinical patient derived xenograft (PDX) models

Objective: The preclinical evaluation of novel immune checkpoint modulators is dependent on tumor models in mice with functional immune cells. In previous experiments, we have demonstrated, that hematopoietic stem cells (HSC) can proliferate and differentiate in vivo to form a functional humanized immune system. Further we have shown engraftment of PDX on these humanized mice. In our translational research project we were interested, whether the PD1 checkpoint inhibitors can block tumor growth in this human model system and if there is a correlation of response and PD-L1 expression. Methods: Hematopoetic stem cells (HSC) from cord blood were transplanted intravenously into 3 week old irradiated nod scid gamma mice. After 12 weeks, blood was collected and screened by FACS for human immune cell engraftment (huCD45 + ). PDX were screened for PD-L1 expression on RNA level by PCR and protein level by FACS, western blot and immunhistochemistry. PD-L1 positiv and negative PDX models were subcutaneously co-transplanted into these humanized mice and followed for growth. Response to the CTLA-4 checkpoint inhibitor ipilimumab and PD-1 checkpoint inhibitor nivolumab was evaluated. Tumor and organs (bone marrow, spleen, thymus) were investigated at the end of the experiments by FACS and exemplary by immunhistochemistry for T cells and other immune cells and the expression of PD-1 and CTLA-4. Results: We confirmed successful engraftment after 12 to 14 weeks, when up to 20% of the human immune cells in the blood were T-cells. Engraftment in different organs has been detected, with up to 15% CD45 + cells in spleen and thymus and 50% in the bone marrow. We engrafted successfully 14 PDX models on the humanized mice. 12 from 14 showed no difference in tumor growth compared to non-humanized mice. These PDX models seem to be tolerated by the human immune cells. Treatment with ipilimumab or nivolumab alone or in combination delayed the tumor growth, accompanied by an increase of “activated” T-cells in blood and tumor. We found a correlation between tumor growth inhibition by the checkpoint inhibitor treatment and tumor expression of PD-L1. Furthermore PDX tumors with a reduced growth on humanized mice (due to innate immune response) showed a stronger response to the checkpoint inhibitors. Conclusion: HSC can be transplanted in immunodeficient mice and establish a “functional” human immune system. PDX models can be successfully engrafted on these humanized mice - generating a fully human preclinical test system for immuno-oncology. Within these models checkpoint inhibitors demonstrate tumor growth inhibition in correlation with tumor PD-L1 expression. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Iduna Fichtner, Jens Hoffmann. Correlation of tumor growth inhibition by check point inhibitors with PD-L1 expression in preclinical patient derived xenograft (PDX) models [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B126.