Abstract 5164: Selectively enriched CD90/Thy-1-positive cells from peripheral blood allow discrimination of patients and further analysis on a single cell level

Abstract

Abstract Aims: The cross-talk between epithelial tumor cells and the microenvironment is of crucial importance for the development of malignancies. The cell-cell communication between epithelial tumor cells and tumor-associated fibroblasts (TAFs) is of interest for the deeper understanding of tumor progression and metastasis formation. We sought to investigate the feasibility of an effective enrichment of peripheral blood CD90-positive cells from cancer patients and their molecular analysis on the single cell level. Methods: CD90/Thy-1 is regarded as a suitable marker for TAFs. CD90/Thy-1-positive cells were detected in leukocyte fractions from peripheral blood from patients suffering from gynecological tumors by laser scanning cytometry. CD90/Thy-1-positive cells were enriched from leukocyte fractions from individual samples as well as from pools of fractions in case of low numbers of CD90/Thy-1 positive cells using immunomagnetic cell separation technology (ROBOSEP®). The CD90/Thy-1-positive fraction and FFPE-tissue samples were further analysed by immunofluorescence and immunohistochemistry. Individual CD90/Thy-1-positive cells were selected with the aureka® system and expression analysis was performed by one-step multiplex RT-PCR. Results: The number of CD90/Thy-1 positive cells in the leukocyte preparation from blood samples from 72 tumor patients and 23 healthy volunteers was estimated. Up to 17 samples per patient were collected over time. The amount of CD90/Thy-1-positive cells ranged from 1 to more than 50,000 cells per ml peripheral blood. 75% of the patients showed CD90/Thy-1-positive cells in at least one sample, one fourth of them in every sample. 25% of the patients were negative. Formalin-fixed tissue sections from representative patients showed a localized expression of CD90/Thy-1 in the tumor stroma, although the intensity and distribution of the staining did not correlate to the number of the CD90/Thy-1-positive cells in the peripheral blood. Comparing the 290 individual samples a CD90/Thy-1 content higher than 1000 cells/ml blood was determined in 65% of the patient samples but only 4% of the samples from healthy volunteers. For molecular analysis CD90/Thy-1-positive cells were enriched and 52 individual vital cells were isolated by cell picking. Multiplex RT-PCR for tumor and stromal markers demonstrated a pronounced expression of SCF (80%) in these cells. For control, CD90 and RPL13A expression was monitored. Conclusion: Remarkable concentrations of CD90/Thy-1-positive cells were detected predominantly in the peripheral blood of tumor patients. Preliminary molecular analyses on a single cell basis demonstrated a robust expression of SCF mRNA, a marker for both tumor-stroma associated fibroblasts as well as endothelial cells. Further studies are needed to elucidate the nature of CD90/Thy-1-positive cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5164. doi:10.1158/1538-7445.AM2011-5164