Development of functional models for micrometastasis research: Linking descriptive characteristics of disseminated cancer cells to functional properties
Final Report Abstract
So far it is impossible to investigate functional properties of disseminated tumor cells (DTC) with one or two single cells among millions of bone marrow (BM) cells present in fresh patient specimens. Therefore permanent micrometastatic cell lines are urgently needed. This project was aimed to establish conditionally immortalized DTC cell lines from BM of patients with breast and head and neck cancer by introduction of a temperature-sensitive SV40 large T-antigen. The subsequent investigation of the functional properties of these transiently immortalized DTC should allow new insights into the biology of the metastatic cascade in cancer patients regarding to the resistance to apoptosis, motility and invasiveness. In total, 140 BM aspirates from patients either with primary breast cancer or with head and neck cancer (stage MO) were laken into culture for up to nine passages. Four BM cultures from head and neck squamous cell carcinomas (HNSCC) as well as four BM cultures from breast cancer patients with previously detected DTC were used for the transfection assays. Finally, from 3 patients with head and neck cancer as well as from 1 patient with breast cancer, expanded BM cultures with immortalized cells could be established. The cells will be further propagated to establish stable cell lines and characterized phenotypically and moleculariy in future experiments. Moreover, functional assays will be performed to characterize these cells and to see whether they have an increased potential to form tumors and metastasize in immunsuppressed mice. Since we were not able to establish new permanent cancer cell lines from DTC within the project period, we aimed to analyze the protein expression patterns of three unique micrometastatic breast cancer cell lines established by our group previously. In a proteomic analysis we further characterized the protein expression pattern of the breast cancer DTC cell line BC-M1 by a newly developed efficient protein preparation protocol resulting in high recovery rates and increased display of alkaline proteins. Thereby we found that the cell line BC-M1 shows the rare and highly metastatic vimentin/cytokeratin 5-positive and cytokeratin 8/18-negative breast cancer phenotype and expresses HER2 indicative of a stem/progenitor cell phenotype. This early stage cancer phenotype (vimentin/cytokeratin 5-positive and cytokeratin 8/18-negative) was also identified in two further DTC cell lines, one derived from a lung cancer patient and the other one from a prostate cancer patient. Furthermore, these cell lines have developed a cytoprotective programme of an unfolded protein response including a high expression of the glucose-regulated proteins Grp78 and Grp94. This might enable them to adapt to the hypoxic bone marrow environment and to survive anthracycline-containing chemotherapy by inactivation of caspase-7. In summary, micrometastatic cell lines from carcinoma patients might be suitable models that may contribute to unravel the biology of cancer micrometastasis.
Publications
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Circulating tumor cells and bone marrow micrometastasis. Clin Cancer Res 2008; 14: 5013-21
Alix-Panabières C, Riethdorf S, Pantel K
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Detection, clinical relevance and specific biological properties of disseminating tumour cells. Nature Rev Cancer. 2008 May;8(5):329- 40
Pantel K, Brakenhoff RH, Brandt B.
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Review: Biological relevance of disseminated tumor cells in cancer patients. Int J Cancer 2008; 123:1991-2006
Riethdorf S, Wikman H, Pantel K
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Two-Dimensional Differential Gel Electrophoresis of a Cell Line Derived from a Breast Cancer Micrometastasis Revealed a Stem/Progenitor Cell Protein Profile. J Proteome Res. 2009 April; 8{4), 2004-14
Bartkowiak K, Wieczorek M, Buck F, Harder S, Moldenhauer J, Effenberger KE, Pantel K, Peter-Katalinic J, Brandt BH
