Circulating tumor cells (CTCs) are shed by both primary and metastatic cancers and they are thought to mediate the hematogenous spread of cancer to distant sites, including bone, lung, brain, and liver. Because these cells are extremely rare and mixed with normal blood components, technological hurdles have limited their isolation and characterization. Little is known about the timing of CTC release from primary tumors, their heterogeneity, their functional properties, and the degree to which they are representative of either primary or metastatic tumor deposits. However, CTCs are a potential source of cells derived from metastatic cancers that can be analyzed repeatedly and noninvasively (that is, a “liquid biopsy” for epithelial cancers), and their presence in early cancers that have not yet metastasized may indicate vascular invasion; these features could prove useful in the clinical management of cancer.
Multiple approaches have been used to detect CTCs, ranging from cell size–based separation to the use of immunomagnetic beads conjugated with an antibody to EpCAM, a commonly expressed epithelial cell surface marker (1
). Each of these approaches has intrinsic limitations: CTCs are not universally larger than all leukocytes, and cell surface expression of EpCAM is heterogeneous and even absent in some tumor types. Most CTC isolation technologies require cell fixation, which restricts detailed RNA-based molecular assays and precludes functional analyses. Moreover, most approaches have been hampered by low yield and low purity of isolated CTCs, reflecting the fact that these cells are very rare (1 in 109
blood cells) and do not readily survive multistep batch purification. To date, the low number of isolated CTCs (median, ≤1 CTC/ml) has restricted most studies to the use of prognostic endpoints rather than quantitating CTC concentrations to monitor therapeutic responses in individual patients; thus, the detection of CTCs by immunomagnetic enrichment in patients with metastatic prostate cancer is associated with poor overall survival, but monitoring CTCs to guide therapeutic choices has not been established (3
). In localized prostate cancer, immunomagnetic enrichment is less useful because the numbers of CTCs in these patients were similar to numbers of CTCs in men without prostate cancer (10
We recently developed a microfluidic device that provides improved yield and purity of captured CTCs (11
). This CTC-Chip has a surface area of 970 mm2
containing an array of 78,000 microposts, which are made chemically active by coating them with antibodies to EpCAM. The flow kinetics of the device are optimized for minimal shear forces and maximal contact between cells and the functionalized microposts; EpCAM-expressing CTCs bind to the microposts, whereas leukocytes and red blood cells are washed from the chip. In an initial cohort of patients with different metastatic epithelial cancers, CTCs were detected (mean, 86 cells/ml) by EpCAM-mediated capture followed by staining with an antibody to cytokeratin (11
). In lung cancers carrying EGFR
mutations, CTC-based cell isolation by EpCAM capture and cytokeratin staining allowed noninvasive serial genotyping during the course of therapy with targeted kinase inhibitors (12
). Despite these initial demonstrations, microfluidic CTC capture requires highly skilled specimen handling and analysis. To improve and standardize CTC imaging analysis, which is the most variable parameter and limits high-throughput applications, we focused on detection of CTCs in patients with localized and metastatic prostate cancer. This cancer type was attractive for this purpose because of the availability of a unique tumor-specific marker [prostate-specific antigen (PSA)] and potential applications of CTC detection to the management of prostate cancer.
Prostate cancer is the most common malignancy in men, with more than 186,000 new cases in the United States in 2008 and more than 28,000 deaths from metastatic disease (13
). Most patients are diagnosed with localized disease and undergo either surgical resection (prostatectomy) or radiation therapy. Given the indolent nature of many prostate cancers affecting elderly men, however, the benefit of general PSA screening for early detection of prostate cancer has been called into doubt (14
). No standard histological markers reliably distinguish indolent from potentially invasive prostate cancer. About one-third of patients treated for localized disease develop a recurrence of prostate cancer, most frequently with metastases resulting from blood-borne spread to bone (16
). Standard therapy for metastatic disease involves withdrawal of androgenic signals that drive proliferation of prostate cancer cells. Response to such therapy is profound and nearly universal but is invariably followed by the development of castration-resistant disease, which is only modestly responsive to cytotoxic chemotherapy (16
Prostate cancer that is metastatic to bone is not readily biopsied, and only a few immortalized cell lines have been derived from prostate cancer cells, limiting molecular insights into disease progression and drug susceptibility (17
). Chromosomal translocations that fuse the androgen-responsive TMPRSS2
promoter with ERG, an ETS family transcription factor, occur in about half of advanced prostate cancers (18
), but their functional consequences remain to be defined (20
). Primary prostate cancers may harbor multiple cancerous foci with distinct translocations (25
), suggesting that the specific cells that eventually cause metastatic disease may not be readily identifiable at the time of resection.
Here, we apply the unique markers available for prostate cancer to establish an imaging strategy for precise, automated quantification and molecular characterization of CTCs. We were able to estimate their half-life after removal of the primary tumor, as well as the amount of cell proliferation in this poorly understood tumor cell population.