Purpose: Metastases in distant organs are the major cause of death for cancer patients, and bone marrow is a prominent homing organ for early disseminated cancer cells. However, it remains still unclear which of these cells evolve into overt metastases. We therefore established a new approach based on the analysis of viable and proliferating cancer cells by single-cell comparative genomic hybridization.Experimental Design: The bone marrow of early-stage breast tumor patients (pN 0 M 0 ) was screened for tumor cells by immunostaining. By applying special short-term culturing, we selected for viable and proliferative tumor cells. The short-term culturing allowed us to evaluate the proliferative potential of micrometastatic cells, which we had previously shown to represent an independent prognostic marker. We assessed genomic changes in single disseminated cancer cells by single-cell comparative genomic hybridization.Results: We found that these viable disseminated cancer cells already had a plethora of copy number changes in their genome. All of these cells showed chromosomal copy number changes with a substantial intercellular heterogeneity and differences to the matching primary tumors.Conclusions: The established experimental strategy might pave the way for the identification of metastatic stem cells in cancer patients. Our preliminary results support the new concept that early disseminated cancer cells evolve independently from their primary tumor.

To investigate interrelations of human obligate airway pathogens, such as Bordetella pertussis, and their hosts test systems with high in vitro/in vivo correlation are of urgent need. Using a tissue engineering approach, we generated a 3D test system of the airway mucosa with human tracheobronchial epithelial cells (hTEC) and fibroblasts seeded on a clinically implemented biological scaffold. To investigate if hTEC display tumour-specific characteristics we analysed Raman spectra of hTEC and the adenocarcinoma cell line Calu-3. To establish optimal conditions for infection studies, we treated human native airway mucosa segments with B. pertussis. Samples were processed for morphologic analysis. Whereas our test system consisting of differentiated epithelial cells and migrating fibroblasts shows high in vitro/in vivo correlation, hTEC seeded on the scaffold as monocultures did not resemble the in vivo situation. Differences in Raman spectra of hTEC and Calu-3 were identified in distinct wave number ranges between 720 and 1662 cm(-1) indicating that hTEC do not display tumour-specific characteristics. Infection of native tissue with B. pertussis led to cytoplasmic vacuoles, damaged mitochondria and destroyed epithelial cells. Our test system is suitable for infection studies with human obligate airway pathogens by mimicking the physiological microenvironment of the human airway mucosa.

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