Sven Christian

Spheroid-based engineering of a human vasculature in mice.

The complexity of the angiogenic cascade limits cellular approaches to studying angiogenic endothelial cells (ECs). In turn, in vivo assays do not allow the analysis of the distinct cellular behavior of ECs during angiogenesis. Here we show that ECs can be grafted as spheroids into a matrix to give rise to a complex three-dimensional network of human neovessels in mice. The grafted vasculature matures and is connected to the mouse circulation. The assay is highly versatile and facilitates numerous applications including studies of the effects of different cytokines on angiogenesis. Modifications make it possible to study human lymphangiogenic processes in vivo. EC spheroids can also be coimplanted with other cell types for tissue engineering purposes.

Angiopoietin-2 Levels Are Associated with Disease Progression in Metastatic Malignant Melanoma

Purpose: The blood vessel-destabilizing Tie2 ligand angiopoietin-2 (Ang-2) acts in concert with the vascular endothelial growth factor/vascular endothelial growth factor receptor system to control vessel assembly during tumor progression. We hypothesized that circulating soluble Ang-2 (sAng-2) may be involved in melanoma progression. Experimental Design: Serum samples (n = 98) from melanoma patients (American Joint Committee on Cancer stages I-IV), biopsies of corresponding patients, and human melanoma cell lines were analyzed for expression of Ang-2 and S100β. Multiple sera of a subcohort of 33 patients were tested during progression from stage III to IV. Small interfering RNA-based loss-of-function experiments were done to assess effects of Ang-2 on melanoma cells. Results: Circulating levels of sAng-2 correlate with tumor progression in melanoma patients (P < 0.0001) and patient survival (P = 0.007). Analysis of serum samples during the transition from stage III to IV identified an increase of sAng-2 up to 400%. Comparative analyses revealed a 56% superiority of sAng-2 as predictive marker over the established marker S100β. Immunohistochemistry and reverse transcription-PCR confirmed the prominent expression of Ang-2 by tumor-associated endothelial cells but identified Ang-2 also as a secreted product of melanoma cells themselves. Corresponding cellular experiments revealed that human melanoma-isolated tumor cells were Tie2 positive and that Ang-2 acted as an autocrine regulator of melanoma cell migration and invasion. Conclusions: The experiments establish sAng-2 as a biomarker of melanoma progression and metastasis correlating with tumor load and overall survival. The identification of an autocrine angiopoietin/Tie loop controlling melanoma migration and invasion warrants further functional experiments and validate the angiopoietin/Tie system as a promising therapeutic target for human melanomas.

Endosialin (Tem1) Is a Marker of Tumor-Associated Myofibroblasts and Tumor Vessel-Associated Mural Cells

Endosialin (Tem1) has been identified by two independent experimental approaches as an antigen of tumor-associated endothelial cells, and it has been claimed to be the most abundantly expressed tumor endothelial antigen, making it a prime candidate for vascular targeting purposes. Recent experiments have challenged the endothelial expression of endosialin and suggested an expression by activated fibroblasts and pericytes. Thus, clarification of the controversial cellular expression of endosialin is critically important for an understanding of its role during tumor progression and its validation as a potential therapeutic target. We have therefore performed extensive expression profiling analyses of endosialin. The experiments unambiguously demonstrate that endosialin is expressed by tumor-associated myofibroblasts and mural cells and not by endothelial cells. Endosialin expression is barely detectable in normal human tissues with moderate expression only detectable in the stroma of the colon and the prostate. Corresponding cellular experiments confirmed endosialin expression by mesenchymal cells and indicated that it may in fact be a marker of mesenchymal stem cells. Silencing endosialin expression in fibroblasts strongly inhibited migration and proliferation. Collectively, the experiments validate endosialin as a marker of tumor-associated myofibroblasts and tumor vessel-associated mural cells. The data warrant further functional analysis of endosialin during tumor progression and its exploitation as marker of tumor vessel-associated mural cells, expression of which may reflect the non-normalized phenotype of the tumor vasculature.

Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis-related protein Mac-2 BP/90K

Tumor development involves complex bidirectional interactions between tumor cells and host stromal cells. Endosialin (Tem1) has been identified as a highly O‐glycosylated transmembrane glycoprotein, which is specifically expressed by tumor vessel‐associated pericytes and stromal fibroblasts of a wide range of human tumors. Recent experiments in endosialin‐deficient mice have unraveled a critical role of endosialin in site‐specific tumor progression and metastasis. To molecularly understand the mechanisms of endosialin function, we aimed to identify extracellular endosialin ligands and identified Mac‐2 BP/90K as a specific interaction partner. Detailed biochemical analyses identified a C‐terminal fragment of Mac‐2 BP/90K, which was shown to contain binding sites for galectin‐3, and collagens as the structures responsible for endosialin binding. Subsequent expression analysis of Mac‐2 BP/90K in vivo revealed weak or no expression in most normal tissues and strong up‐regulation in tumor cells of human neoplastic tissues. Intriguingly, the expression patterns of Mac‐2 BP/90K and endosialin were mutually exclusive in all human tissues. Correspondingly, loss‐of‐function adhesion experiments of Mac‐2 BP/90K‐expressing tumor cells on endosialin‐expressing fibroblasts revealed a repulsive outcome of the Mac‐2 BP/90K interaction. Taken together, the experiments identify a novel repulsive interaction between endosialin on stromal fibroblasts and Mac‐2 BP/90K on tumor cells.—Becker, R., Lenter, M. C., Vollkommer, T., Boos, A. M., Pfaff, D., Augustin, H. G., Christian, S. Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis‐related protein Mac‐2 BP/90K. FASEB J. 22, 3059–3067 (2008)

Inhibition of Multiple Vascular Endothelial Growth Factor Receptors (VEGFR) Blocks Lymph Node Metastases but Inhibition of VEGFR-2 Is Sufficient to Sensitize Tumor Cells to Platinum-Based Chemotherapeutics

Vascular endothelial growth factor receptors (VEGFR) have important roles in cancer, affecting blood and lymphatic vessel functionality as well as tumor cells themselves. We compared the efficacy of a VEGFR tyrosine kinase inhibitor, PTK787/ZK222584 (PTK/ZK), which targets the three VEGFRs, with blocking antibodies directed against VEGFR-2 (DC101) or VEGF-A (Pab85618) in a metastatic melanoma model. Although all inhibitors exerted comparable effects on primary tumor growth, only PTK/ZK significantly reduced lymph node metastasis formation. A comparable decrease in lymphatic vessel density following blockade of VEGFR-2 (DC101) or the three VEGFRs (PTK/ZK) was observed in the metastases. However, the functionality of lymphatics surrounding the primary tumor was more significantly disrupted by PTK/ZK, indicating the importance of multiple VEGFRs in the metastatic process. The antimetastatic properties of PTK/ZK were confirmed in a breast carcinoma model. B16/BL6 tumor cells express VEGF ligands and their receptors. Blockade of a VEGFR-1 autocrine loop with PTK/ZK inhibited tumor cell migration. Furthermore, the tumor cells also showed enhanced sensitivity to platinum-based chemotherapy in combination with PTK/ZK, indicating that autocrine VEGFRs are promoting tumor cell migration and survival. In summary, our results suggest that, in addition to blocking angiogenesis, combined inhibition of the three VEGFRs may more efficiently target other aspects of tumor pathophysiology, including lymphatic vessel functionality, tumor cell dissemination, survival pathways, and response to chemotherapeutic compounds.

Functional inhibition of acid sphingomyelinase by Fluphenazine triggers hypoxia-specific tumor cell death

Owing to lagging or insufficient neo-angiogenesis, hypoxia is a feature of most solid tumors. Hypoxic tumor regions contribute to resistance against antiproliferative chemotherapeutics, radiotherapy and immunotherapy. Targeting cells in hypoxic tumor areas is therefore an important strategy for cancer treatment. Most approaches for targeting hypoxic cells focus on the inhibition of hypoxia adaption pathways but only a limited number of compounds with the potential to specifically target hypoxic tumor regions have been identified. By using tumor spheroids in hypoxic conditions as screening system, we identified a set of compounds, including the phenothiazine antipsychotic Fluphenazine, as hits with novel mode of action. Fluphenazine functionally inhibits acid sphingomyelinase and causes cellular sphingomyelin accumulation, which induces cancer cell death specifically in hypoxic tumor spheroids. Moreover, we found that functional inhibition of acid sphingomyelinase leads to overactivation of hypoxia stress-response pathways and that hypoxia-specific cell death is mediated by the stress-responsive transcription factor ATF4. Taken together, the here presented data suggest a novel, yet unexplored mechanism in which induction of sphingolipid stress leads to the overactivation of hypoxia stress-response pathways and thereby promotes their pro-apoptotic tumor-suppressor functions to specifically kill cells in hypoxic tumor areas.

Abstract 3086: Inhibitors of the enzyme dihydroorotate dehydrogenase, overcome the differentiation blockade in acute myeloid leukemia

The prognosis for adults diagnosed with acute myeloid leukemia (AML) remains poor, with a five-year survival of only 25%. This prognosis is even more dismal in older patients who are not well enough to receive standard induction chemotherapy. Speaking to the need for new therapies is the fact that our therapeutic backbone - a combination of cytarabine and an anthracycline - remains unchanged since 1973. The promise of differentiation therapy was realized in the small subset of patients diagnosed with acute promyelocytic leukemia (APL). Here, treatment in the form of all-trans retinoic acid (ATRA) and arsenic trioxide inverted the survival curve; where APL was once the worst form of myeloid leukemia, it now carries the best prognosis, with a five-year survival exceeding 85%. The goal of this study was thus to develop differentiation therapy for patients with non-promyelocytic AML with the question: “Can we identify small molecules that overcome myeloid differentiation arrest?” A phenotypic differentiation screen in a HOXA9 driven leukemia model followed by target deconvolution, identified DHODH as an unexpected target for overcoming differentiation arrest in AML. We used 2 potent small molecule inhibitors of DHODH to validate this initial finding: Brequinar, a known DHODH inhibitor and an in house compound BAY DHODHi. In several in vitro experiments we demonstrated induction of AML differentiation in a dose dependent fashion. Interestingly, these effects could be completely rescued by addition of uridine, confirming target specificity. Treating mice in multiple genetically diverse AML in vivo models with a DHODH inhibitor led to tumor growth reduction and AML differentiation. Expression analysis of leukemia cells explanted from mice xenografts treated with a DHODH inhibitor demonstrate an early onset of differentiation markers indicating a direct role of DHODH with the onset of differentiation in vivo. The mechanism for selective vulnerability of leukemia cells to DHODH inhibition remains under investigation. Despite the observation that DHODH is expressed in all cells, normal and malignant, mice can tolerate DHODH inhibitor therapy for more than 100 days without weight-loss or other concerning side-effects. Thus, our pre-clinical studies point towards DHODH as a new metabolic target in the differentiation treatment of AML. Hopefully, small molecule DHODH inhibitors will provide a much-needed differentiation therapy for patients with acute myeloid leukemia. Citation Format: Andreas Janzer, David Sykes, Stefan Gradl, Steven Ferrara, Sven Christian, Claudia Merz, Henrik Seidel, Andreas Bernthaler, Ralf Lesche, Mathias Wawer, David T. Scadden. Inhibitors of the enzyme dihydroorotate dehydrogenase, overcome the differentiation blockade in acute myeloid leukemia [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 3086. doi:10.1158/1538-7445.AM2017-3086

Abstract 4989: 3D spheroid screen yields SCD1 pathway inhibitors for the treatment of cancer

With three-dimensional growth conditions, multicellular tumor spheroids reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients, characteristic of poorly vascularized tumor regions. 3D high content screening (HCS) identified compounds that selectively kill tumor cells in the inner core of tumor cell spheroids by targeting the Stearoyl CoA Desaturase 1 (SCD1) pathway. SCD1 catalyzes the rate-limiting step in the production of mono-unsaturated fatty acids (MUFAs). Cancer cells are dependent on higher levels of MUFAs compared to normal cells and SCD1 is highly expressed in multiple tumor types. Changes in the MUFA / SFA (saturated fatty acid) ratio alters lipid biosynthesis and thus triggers cellular (ER) stress and induces the Unfolded Protein Response. Although the lead compound was very effective in vitro, it had unfavorable PK and physical chemistry properties, including low permeability and solubility and very high lipophilicity. This led to insufficient oral bioavailability, which could be overcome by optimization of PK and physical chemistry properties. Here, we report on the in vitro/in vivo effects of our 3D HCS compounds which showed high potency in the 3D spheroid inner core death assay with T47D breast cancer cells. In this in vitro model compound-induced inner core cell death is enhanced by SCD1 substrates palmitic or stearic acid and rescued by the SCD1 products palmitoleic or oleic acid. Furthermore, the effects can be reproduced in 2D cultures, which become increasingly sensitive to inhibition by our 3D HCS compounds with decreasing FBS concentration in the culture medium and this effect can also be rescued by addition of MUFAs but not of palmitic acid. Mode of action analysis showed that our compounds reduced palmitoleoyl- or oleoyl-CoA levels and simultaneously increased saturated fatty acyl-CoAs of palmitate or stearate in several cell lines as well as in vivo. In the sensitive T47D cells, the compounds induced expression of stress response genes and genes related to lipid metabolism. While these results support the SCD1 pathway as target for our 3D HCS compounds, we also observed striking differences to published SCD1 inhibitors suggesting a new cancer target beyond SCD1. Thus, further validation of our inhibitors in vitro and in vivo will be required, but these results suggest that 3D spheroid cultures may be a valuable tool for elucidation of new drug targets for cancer therapy. Citation Format: Sylvia Gruenewald, Carolyn Sperl, Patrick Steigemann, Alexander Walter, Sylvia Zacharias, Uwe Eberspaecher, Roland Neuhaus, Ludwig Zorn, Wolfgang Schwede, Kai Thede, Sven Christian. 3D spheroid screen yields SCD1 pathway inhibitors for the treatment of cancer [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 4989. doi:10.1158/1538-7445.AM2017-4989

Abstract 3248: Identification and optimization of a highly active, cross reactive Complex-1 inhibitor

Mitochondria are key regulators of both energy supply and apoptotic cell death. The mitochondrial electron transport chain (ETC) consists of four enzyme complexes that transfer electrons from NADH to oxygen. During electron transfer, the ETC pumps protons into the inter-membrane space, generating a gradient across the inner mitochondrial membrane that is used by Complex V to drive ATP synthesis. Recent publications have shown that tumor cells harboring specific mutations (LKB1, mIDH and others) are more sensitive to Complex I inhibition, potentially providing an opportunity for selectively targeting tumor cells. Based on a high throughput screen (HTS), we identified new, albeit moderately active, lead structures with cross reactivity between mouse and human Complex 1. SAR elaboration of the lead structure allowed for optimization of the potency, although compounds still suffered from low metabolic stability. Further improvement of the in vitro and in vivo PK properties finally permitted in vivo animal studies. Herein, we report for the first time the preclinical profile and structure of a highly active, optimized, human/mouse cross-reactive Complex I inhibitor that allowed for the further investigation into the therapeutic potential of Complex I inhibition in cancer. Citation Format: Jeffrey Mowat, Sven Christian, Carolyn Sperl, Alexander Ehrmann, Stephan Menz, Judith Guenther, Roman Hillig, Marcus Bauser, Andrea Haegebarth, Wolfgang Schwede. Identification and optimization of a highly active, cross reactive Complex-1 inhibitor [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 3248. doi:10.1158/1538-7445.AM2017-3248

Abstract 539: Endosialin (TEM1) is a tumor stroma marker and receptor for the metastasis-related Mac-2 binding protein

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Endosialin is a heavily sialylated C-type lectin. It is a single transmembrane molecule that was identified by two independent studies as an angiogenic endothelial specific marker. However, recent high resolution confocal expression studies by our and other laboratories have revealed that Endosialin is not expressed by endothelial cells but instead by angiogenic blood vessel associated pericytes. Detailed tissue array-based expression profiling validated Endosialin as an oncofetal gene product of the mesenchymal lineage that is hardly detectable in normal tissues but abundantly expressed by tumor vessel-associated pericytes and tumor stromal fibroblasts (myofibroblasts). As such, Endosialin was not just identified as a marker of activated pericytes but based on its exclusive tumor-associated expression also as a novel therapeutic tumor stroma target. Cytokine induction experiments identified Endosialin as a major downstream target of TGFs stimulation. Cellular siRNA-based loss-of-function experiments unravelled a role of Endosialin in the regulation of mesenchymal cell adhesion, migration and proliferation. These findings prompted experiments aimed at identifying the extracellular ligand of Endosialin. Using Endosialin-Fc as an affinity probe, the metastasis associated molecule Mac-2BP/90K was identified as high affinity extracellular Endosialin ligand. As Endosialin, Mac-2BP/90K displays an exclusive tumor progression associated expression pattern. However, in contrast to the stromal expression of Endosialin, Mac-2BP/90K is strictly expressed by the tumor cell compartment. The mutually exclusive expression pattern suggests a repulsive interaction between Endosialin and Mac-2BP/90K which was confirmed in cellular experiments. Ongoing experiments are aimed at mechanistically studying the role of the Endosialin - Mac-2BP/90K interaction in controlling the crosstalk between the tumor cell and the stromal cell compartments during tumor progression and metastasis. 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 539.