Circulating tumor cells (CTCs) are prognostic in all stages of breast cancer. However, since they are extremely rare, little is known about the molecular nature of these cells. We report a novel strategy for the isolation and expression profiling of pure populations of CTCs derived from peripheral blood. We developed a method to isolate CTCs based on immunomagnetic capture followed by fluorescence-activated cell sorting (IE/FACS). After assay validation using the BT474 cell line spiked into blood samples in vitro, RNA from CTCs isolated from the blood of five metastatic breast cancer (MBC) patients was linearly amplified and subjected to gene expression profiling via cDNA microarrays. We isolated a range of 9-993 captured CTCs from five MBC patients’ blood and profiled their RNA in comparison to a diverse panel of primary breast tumors (n = 55). Unsupervised hierarchical clustering revealed that CTC profiles clustered with more aggressive subtypes of primary breast tumors and were readily distinguishable from peripheral blood (PB) and normal epithelium. Differential expression analysis revealed CTCs to have downregulated apoptosis, and they were distinguishable from PB by the relative absence of immune-related signals. As expected, CTCs from MBC had significantly higher risk of recurrence scores than primary tumors (p = 0.0073). This study demonstrates that it is feasible to isolate CTCs from PB with high purity through IE/FACS and profile them via gene expression analysis. Our approach may inform the discovery of therapeutic predictors and be useful for real-time identification of emerging resistance mechanisms in MBC patients.
It is feasible to use RNA-seq of CTCs in non-metastatic patients to discover novel tumor biology characteristics.
PurposeThe potential utility of circulating tumor cells (CTCs) as liquid biopsies is of great interest. We hypothesized that CTC capture using EpCAM based gating is feasible for most breast cancer subtypes.ResultsCancer cells could be recovered from all intrinsic subtypes of breast cancer with IE/FACS, however, claudin-low cell lines showed very low capture rates compared to the four other groups (p = 0.03). IE/FACS detection of CTC mimic cells was time sensitive, emphasizing controlling for pre-analytic variables in CTC studies. Median fluorescent intensity for flow cytometry and RNA flow cell type characterization were highly correlated, predicting for CTC isolation across molecular subtypes. RNA-Seq of IE/FACS sorted single cell equivalents showed high correlation compared to bulk cell lines, and distinct gene expression signatures compared to PB.Materials and MethodsTen cell lines representing all major subtypes of breast cancer were spiked (as CTC mimics) into and recovered from peripheral blood (PB) using immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). Flow cytometry and RNA flow were used to quantify the expression of multiple breast cancer related markers of interest. Two different RNA-Seq technologies were used to analyze global gene expression of recovered sorted cells compared to bulk cell lines and PB.ConclusionsEpCAM based IE/FACS detected and captured a portion of spiked cells from each of the 10 cell lines representing all breast cancer subtypes, including basal-like but not claudin-low cancers. The assay allows for the isolation of high quality RNA suitable for accurate RNA-Seq of heterogeneous rare cell populations.
Background Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models exist to facilitate studies of RIS. Methods We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a RIS. Results Short tandem repeat (STR) profiling of UACC-SARC1 was virtually identical to its parental tumor. Immunohistochemistry (IHC) analysis of the tumor and immunocytochemistry (ICC) analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers α1-antitrypsin and α1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. Comparative genomic hybridization (CGH) provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. NOD/SCID gamma mouse xenografts demonstrated tumor formation and metastasis. Clonogenicity assays demonstrated that 90% of single cells produced viable colonies. NOD/SCID gamma mice produced useful patient-derived xenografts for orthotopic or metastatic models. Conclusion Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. UACC-SARC1 is an aneuploid cell line with complex genomics lacking common oncogenes or tumor suppressor genes as drivers of its biology. The UACC-SARC1 cell line will enable further studies of the drivers of RIS. Synopsis We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a radiation-induced sarcoma (RIS). Our novel RIS cell line constitutes a useful tool for pre-clinical studies of this rare, aggressive disease.
Background: Circulating tumor cells (CTCs) have been demonstrated to be prognostic in all stages of breast cancer. Immunomagnetic enrichment followed by fluorescence activated cell sorting (IE/FACS) isolates enriched populations of CTCs derived from peripheral blood without the need for background subtraction of leukocytes. We previously reported IE/FACS as a strategy for gene expression profiling of CTCs. We hypothesized that variable expression of the epithelial cell adhesion marker (EpCAM) may bias IE/FACS recovery rates and composition. Patients and Methods: 10 breast cancer cell lines were acquired from the ATCC, authenticated by short tandem repeat profiling, and stratified according to subtype: HER2 positive (SKBR3, MDA-MB-453, SUM190), luminal A (T47D, MCF7, ZR751), luminal B (BT474) and basal-like (SUM149, MDA-MB-231, Hs578T). Unknown quantities of cell lines were spiked into PBS or peripheral blood (PB) from healthy female donors. IE/FACS was performed using EpCAM (MJ37) ferrofluid particles via a magnetic separator followed by incubation with Thioflavin T, CD45 PE-Cy7, and EpCAM (EBA-1) mAb conjugated to phycoerythrin. FACS sorting was performed using a FACS Aria II (BD Biosciences) and a gating strategy devised based on negative controls (n = 23). Absolute cell counts and recovery rates were determined using the TruCOUNT method (BD Biosciences) with acquisition of 35,000 beads. Two-way ANOVA was used to analyze variation in recovery rates between groups (Prism GraphPad 6.0). Results: Overall mean recovery rates for the 10 cell lines were 51.4% from PBS and 39.5% from PB. The specific cell type being analyzed was a more significant source of variation (p = 0.03) than was whether measurements were made from PBS or PB (p = 0.2). However, analysis by molecular subtype did not show differences between intrinsic groups (p = 0.23) nor did it show differences in recovery rates from PBS or PB (p = 0.26). The two inflammatory breast cancer cell lines (SUM 149 and SUM190) showed no differences in recovery rates compared to other cell lines (p = 0.41) nor in recovery rates from PBS versus PB (p = 0.75). Recovery rates for the 10 cell lines are shown in Table 1. Recovery Rate Recovery from PBS (%)Recovery from PB (%)SKBR368.051MDA-MB-45361.270.6T47D28.911.4MCF794.024.4BT47497.147.6ZR75152.850.8SUM1496372.9SUM19048.358.3MDA-MB-2310.927.04Hs578T0.250.84Recovery rate of immunomagnetic enrichment/fluorescence activated cell sorting of cell lines spiked into saline (PBS) or peripheral blood (PB) Conclusions: Significant variation occurred in recovery rates of spiked, sorted cells depending on cell line type. Negative control PB specimens from healthy individuals could successfully define a consistent gating strategy that captured zero CTCs from healthy individuals and permitted acquiring a portion of CTCs from each of the 10 cell lines. Further experiments will characterize the gene expression of the sorted cells compared to bulk RNA for each cell line. Better biomarkers are needed to improve upon the recovery rates of CTCs while minimizing selection bias. Future studies are required to determine if expression profiling of CTCs is an informative biomarker that may be applied clinically as a prognostic or predictive tool. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-04-11.
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