BioProject

The ability to interrogate circulating tumor cells (CTC) and disseminated tumor cells (DTC) is restricted by the small number detected and isolated (typically <10). We wanted to determine if a commercially available technology could provide a transcriptomic profile of a single prostate cancer (PCa) cell. We clonally selected and cultured a single passage of cell cycle synchronized C4-2B PCa cells. Ten sets of single, 5-, or 10-cells were isolated using a micromanipulator under direct visualization with an inverted microscope. Additionally, we analyzed 10 individual DTC isolated from each of 2 patients with metastatic PCa. We have shown that a transcriptomic profile can be obtained from a single cell using commercially available technology. As expected, fewer amplified genes are detected from a single-cell sample than from pooled cell samples, but this method can be used to obtain a transcriptomic profile from DTC isolated from the bone marrow of patients with PCa. Overall design: Custom Agilent 44K whole human genome expression oligonucleotide microarrays were used to profile clonally selected and cultured single passage of cell cycle synchronized C4-2B PCa cells isolated using a micromanipulator under direct visualization with an inverted microscope into ten sets of single, 5-, or 10-cells. Single disseminated tumor cells were isolated from bone marrow (BM) samples of two advanced prostate cancer patients. Essentially, a two-step selection process was employed, in which anti-CD45 and anti-CD61 conjugated to immunomagnetic beads were used for negative selection, and anti-HEA was used for positive selection. Cells were then fluorescently stained for BerEP4, counter stained with RPE anti-CD45, and individually selected (10 single cells each per patient) under fluorescent light using a micropipette system for further analysis. RNA was amplified using the WT-Oviation one-direct system and hybridization against a common reference pool of prostate tumor cell lines. Data from C42B cell data and data from single cells isolated from the bone marrow of patients were normalized and analyzed separately.