Models of treatment and metastasis formation

The concept of personalized tumor therapy is based on the use of specific, custom-tailored medication to treat an individual’s cancer. Besides comprehensive knowledge about the tumor’s molecular properties, development of such treatment strategies benefits considerably from the use of cell models representing the actual target cells of the individualized therapy. Such cell models are needed to test novel therapeutics and to study the biology of specific tumor cell populations. Tumor cells that have already spread in the body – e.g. disseminated tumor cells (DTC), which are metastatic progenitor cells, and circulating tumor cells (CTC) in the patient’s blood – are important target cells for specific therapy approaches in patients with metastatic disease.

Due to the very low abundance of DTC and CTC and the resulting difficulty to detect and enrich them, it is nearly impossible at present to expand these cells and thereby establish representative models for preclinical drug testing. The use of cell models based on these rare cells, however, will enable a deeper understanding of metastasis formation and improved testing of novel therapeutics.

Preclinical in-vitro/in-vivo models are essential for anticancer drug development. It is becoming increasingly evident, however, that cell models based on the primary tumor provide only insufficient information for treatment of already disseminated tumor cells. In contrast, DTC and CTC are the target cells of such therapies. Fraunhofer ITEM in Regensburg is developing workflows for rare-cell expansion and has already established first in-vitro-/in-vivo models based on DTC or CTC. We are using these models to gain a better functional understanding of DTC and CTC biology and of their response to therapy.

DTC and CTC are very rare tumor cells that can be found in bone marrow and/or lymph nodes or blood of cancer patients. Isolation and expansion of these rare cells pose considerable challenges. In a first step, the few tumor cells have to be separated from healthy cells in the tissue sample. This requires a phenotypic difference (e.g. surface markers or cell size) between tumor cells and healthy cells. In a second step, the tumor cells are cultured under highly specific culturing conditions (in vitro and/or in vivo) that allow unrestricted growth of DTC and CTC. In both these steps, the phenotypic differences between tumor cells and healthy cells, but also the enormous variations in optimal growth conditions between different types of tumors pose substantial challenges. A focus of our work, therefore, is on the development of customized protocols for expansion of DTC and CTC from different organs. In addition, we use the expanded cells to establish preclinical models (in vitro/in vivo) mimicking the specific conditions of the tumor cells’ tissue of origin (e.g. human immune cells). Such models put us in a position to test how these cells will respond to therapy in patients.

With 1-10 detectable cells per 10⁶ bone marrow cells, disseminated tumor cells (DTC) are among the rarest cell populations in the human organism. Nevertheless, isolated DTC can yield valuable information by direct ex-vivo analyses, such as identification of therapeutic targets including antigens, T-cell epitopes, and signaling pathways. Validation of target structures and their functional analysis, however, as well as epigenetic, protein-chemical, metabolomic, and biochemical analyses require additional cellular models.

This is why a focus of research at Fraunhofer ITEM in Regensburg is on establishing DTC cell lines, in particular from patients without manifest metastases at the time point of sampling. This includes the definition of tumor-specific and tissue-specific in-vitro culturing conditions and times which closely map the situation in patients. The generated cell lines are compared with the ex vivo isolated DTC regarding their genotypic and phenotypic properties and are planned to be used as representative models of the functional and molecular properties of disseminated tumor cells in patients.

Translating complex biomarkers into molecular diagnostics

Introducing mutational profiles and/or gene expression signatures into a biomarker development approach is coupled with translating the clinical outcome-related biomarker into a robust assay applicable to widespread analysis platforms. Via the certified and accredited single-cell laboratory (Link zu Molekulare Diagnostik, B. Polzer), the Fraunhofer ITEM team in Regensburg has access to large sample numbers allowing evaluation of whether a molecular test has sufficient power to actually predict clinical outcome. Moreover, access to a biobank with sufficient quantities of high-quality single-cell samples also allows adaptation of molecular tests to constantly changing technologies.

Frequently used in-vivo tumor mouse models comprise immunocompetent murine (mostly oncogene-driven) models and human xenografts without immune system. Both models do not sufficiently mimic the situation in patients and do not allow representative testing of immunomodulatory approaches. Fraunhofer ITEM in Regensburg is developing optimized patient-derived xenograft (PDX) models which enable us to follow tumor development as well as dissemination of cancer cells in different organs based on patient-derived DTC or CTC. In addition, we concomitantly generate a human immune system in such mice which infiltrates human PDX tumors and develops immune cell phenotypes (e.g. tumor-associated macrophages) as found in patient samples. Our optimized mouse models allow investigation of a much broader spectrum of immune-tumor interactions, as several human immune populations are present. Such double-humanized mouse models present an opportunity to analyze the influence of human immune cells on tumor development, metastasis formation as well as drug therapy responses of human patient-derived tumor cells.