Legends for Supplementary Figures S1-S11. Supplementary Figure S1 - Workflow employed in this study. Supplementary Figure S2 - Photographs of cell lines stained with isotype antibodies. Supplementary Figure S3 - Box-plot representing BM cell numbers before and after immunomagnetic selection, number of screened cells and number of isolated EpCAM+ cells. Supplementary Figure S4 - Photographs of cell lines stained for EpCAM. Supplementary Figure S5 - Photographs and flowcytometric analysis of BM of control patient spiked with DU145 cells. Supplementary Figure S6 - Expression analysis of BM cell pools. Supplementary Figure S7 - QC of WTA and overview of epithelial transcript expression. Supplementary Figure S8 - Results of expression profiling of all samples. Supplementary Figure S9 - Analysis of WTA controls for HBA2 transcript. Supplementary Figure S10 - CGH profiles of analysed single cells. Supplementary Figure S11 - Establishing conditions for specific amplification of selected fragments by PCR and confirmation of identity of amplified fragments.
ARTICLE ABSTRACT
Bone is the most frequent site of metastasis in prostate cancer and patients with bone metastases are deemed incurable. Targeting prostate cancer cells that disseminated to the bone marrow before surgery and before metastatic outgrowth may therefore prevent lethal metastasis. This prompted us to directly analyze the transcriptome of disseminated cancer cells (DCC) isolated from patients with nonmetastatic (UICC stage M0) prostate cancer. We screened 105 bone marrow samples of patients with M0-stage prostate cancer and 18 bone marrow samples of patients without malignancy for the presence of EpCAM+ single cells. In total, we isolated 270 cells from both groups by micromanipulation and globally amplified their mRNA. We used targeted transcriptional profiling to unambiguously identify DCCs for subsequent in-depth analysis. Transcriptomes of all cells were examined for the expression of EPCAM, KRT8, KRT18, KRT19, KRT14, KRT6a, KRT5, KLK3 (PSA), MAGEA2, MAGEA4, PTPRC (CD45), CD33, CD34, CD19, GYPC, SCL4A1 (band 3), and HBA2. Using these transcripts, we found it impossible to reliably identify true DCCs. We then applied combined genome and transcriptome analysis of single cells and found that EpCAM+ cells from controls expressed transcripts thought to be epithelial-specific, whereas true DCCs may express hematopoietic transcripts. These results point to an unexpected transcriptome plasticity of epithelial cancer cells in bone marrow and question common transcriptional criteria to identify DCCs. Cancer Res; 74(24); 7383–94. ©2014 AACR.
