Tumor Stem Cells

The objective of the unit is to develop therapeutic strategies for the treatment of neoplastic disease which are based both on living cells as well as on pharmaceutically active ingredients. The foundation for these approaches is the elimination or modification of tumor stem cells (TSCs) in the targeted malignancy. Using the TSC concept, further TSC should be described and therapeutic innovations in the field of internal oncology will be made possible.

Colony forming cell assay (CFCA) of tumor cells and stem cells

Time-dependent formation of tumor stem cell spheres in a novel collagen-based colony-forming cell assay (CFCA).
© Photo Fraunhofer IZI

Time-dependent formation of tumor stem cell spheres in a novel collagen-based colony-forming cell assay (CFCA).

The generation of tumor spheres is one of the most common methods for isolating, expanding and characterizing tumor stem cells. This serum-free culture system has also been used for expanding stem cells and determining their frequency in a plurality of normal tissues. It could be shown recently that there is a direct correlation between tumor sphere formation and the occurrence of malignoma.

We use a collagen-based semi-solid assay, the so-called tumor colony-forming cell assay (T-CFCA), by means of which the discrimination of tumor cells having stem cell characteristics is facilitated by virtue of their long-term proliferative potential.

Immunophenotyping and FACS – Separation of tumor stem cells

Flow-cytometric characterization and reanalysis of the tumor stem cell fraction of a primary human mammary Ca cell line.
© Photo Fraunhofer IZI

Flow-cytometric characterization and reanalysis of the tumor stem cell fraction of a primary human mammary Ca cell line.

Validating the identification of stem cells in malignoma is effected via FACS analysis by an immunophenotypic characterization of stem cell characteristics of the tumor cells in the respective sample. In this method, the cells are labeled with different monoclonal antibodies against tumor antigens, stem cell antigens and tissue-associated antigens. Each of these antibodies is conjugated to a particular fluorescent dye. The result is one tumor stem cell fraction and one residual tumor cell fraction derived from the initial parental tumor cell suspension (single cell suspension).

Equipment

  • FACS »FC500« (Beckmann Coulter)
  • Cell Sorter »MoFlo« (Beckmann Coulter)
  • MACS Separator (Miltenyi)
  • EliSpot Reader (AID)
  • Light Cycler (Roche)
  • 96-Well Cell Harvester (Wallac)
  • ß-Counter (Applied Biosystems)
  • Bioluminescence Imaging System (Caliper Xenogen IVIS Spectrum Imaging System)
  • Animal Models (luciferase-transgenic NOD/SCID murine models [RCC, Panc-CA, PCA, Ov-CA, Breast-CA] are available for tumor induction)

Realtime monitoring of tumor engraftment and of remission after adjuvant therapy in the luciferase-transgene-based NOD / SCID murine model

Monitoring of tumor initiation and remission after cisplatin combination therapy.
© Photo Fraunhofer IZI

Monitoring of tumor initiation and remission after cisplatin combination therapy.

For investigating a survival advantage in case of adjuvant antitumor therapy (e.g. a cytostatic + small molecules), a remission of pancreas carcinoma could be represented in the NOD / SCID murine model.

On day 1 (d1) each animal received 1x10E5 luciferase-transduced cells of the pancreatic carcinoma line DAN-G into the nuchal fold. Engraftment and tumor progression were monitored for the first 21 days. On day 22 (d22) post application the adjuvant therapy was initiated (cisplatin [8 mg/ml] + adjuvant). From this day on, a tumor remission could be represented in the treated animal. On day 29 (d29), the second therapeutic application was performed, which resulted in further tumor remission until day 36 (d36). The follow-up investigation was discontinued on day 29 (d29) because of the increased tumor load.

It is expected that the survival advantage provided by such an adjuvant combination therapy significantly exceeds that of specific tumor stem cell inhibition.

Development of a new, epigenetic cancer therapy based on newly synthesized, non-covalent DNMT inhibitors

The degeneration of healthy somatic cells into malignant cancer cells is closely connected to epigenetic changes in the cells‘ genome. This includes the hypermethylation of so-called tumor suppressor genes through enzymes which are referred to as DNA methyltransferases (DNMT). Unlike genetic mutations, this type of change, however, can, in principle, be reversed and therefore presents a promising approach to the development of new drugs.

The project therefore looks at the design, synthesis and the pharmaceutical development of so-called DNA methyl­transferase inhibitors, which intervene in the metabolism of cells and are intended to specifically prevent the hyper­methylation of tumor suppressor genes. Besides a specially developed animal model and modern imaging processes, cancer stem cells (CSC) are also used as part of the development.

Cancer stem cells are regarded as germ cells for the formation and growth of tumors. They are equipped with the characteristics typical of stem cells, such as the ability to self-regenerate, or a high differentiation potential. Recent studies allow the assumption that the CSC are particularly resistant to common types of therapy (chemotherapy, radiotherapy) and are therefore responsible for relapses and metastasis. The development of therapy concepts which specifically aim to eliminate CSC is therefore of utmost urgency. The cancer stem cell lines established at the Fraunhofer IZI thus represent an ideal platform for pharmacological development.

As part of the project, the DNMT-relevant target molecules within the CSC-specific signaling pathways are first to be identified and characterized. The DNMT inhibitors identified as being optimal will then be evaluated in a GLP trial on the basis of selected CSC compartments in the animal model. The tumor initiation derived from CSC and the DNMT-based remission of a malignancy can thereby be monitored using bioluminescence imaging. At the same time, the modern imaging procedure allows the entire progress of the disease and therapy within the organism to be observed.

In vitro testing of cytostatics in tumor stem cells

Spheroid formation of TSCs in mammary carcinoma.
© Photo Fraunhofer IZI

Spheroid formation of TSCs in mammary carcinoma.

Tumor stem cells (TSCs) have been indicated to play an important role in the evolution of different types of cancer. They have the typical properties that are characteristic for stem cells, such as the potential for self-renewal and differentiation. It is currently assumed that this cell type is resistant to various forms of therapy which leads to both relapses and metastasis. A test platform was developed at the Fraunhofer IZI that allows for a rapid and application-oriented investigation of multiple candidate agents for intervening against tumor stem cell entities. Specific tumor stem cells are subjected to tests with respect to their sensitivity to novel candidate agents (e.g. cytostatics). In detail, this is about dosage-dependent kinetics in connection with specific irradiation regimes. In the first instance, this test platform provides growth curves of tumor stem cells upon drug exposure that could be complemented by a follow-up in vivo approach subsequently to tumorigenesis in a murine model.

  • Red Hill Ltd.
  • RESprotect GmbH
  • University Hospital Leipzig, Department of Ophthalmology
  • University of Leipzig, Faculty of Medicine
  • University Teaching Hospital St. Georg Leipzig, Robert Koch Clinic
  • University Teaching Hospital St. Elisabeth Leipzig, Breast Center
  • University Teaching Hospital St. Elisabeth Leipzig, Urology
  • Urological Practice Dr. Schulze
  • Quimatrix SME

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  • Vochem R, Einenkel J, Horn LC, Ruschpler P. Die Bedeutung der Tumorstammzellhypothese für das Verständnis des Ovarialkarzinoms. Der Pathologe. 2014 Jul;35(4):361-70. DOI dx.doi.org/10.1007/s00292-014-1910-6.
  • Gessner C, Rechner B, Hammerschmidt S, Kuhn H, Hoheisel G, Sack U, Ruschpler P, Wirtz H. Angiogenic markers in breath condensate identify non-small cell lung cancer. Lung Cancer, 68 (2010), 2, S. 177-184.
  • Fricke S, Ackermann M, Stolzing A, Schimmelpfennig C, Hilger N, Jahns J, Hildebrandt G, Emmrich F, Ruschpler P, Pösel C, Kamprad M, Sack U. Allogeneic non-adherent bone marrow cells facilitate hematopoietic recovery but do not lead to allogeneic engraftment. PLoS One (2009), Jul 7, 4(7), e6157.