Isolation and Analysis of Circulating Tumor Cells in Genitourinary Cancers

Abstract

While accessible by a relatively noninvasive blood draw, circulating tumor cells (CTCs) remain difficult to study because of their rarity and their presence amongst the billions of surrounding normal blood cells. Of particular promise and utility to the in-depth study of CTCs are those technologies making use of microfluidics and nanomaterials, such as the graphene oxide (GO) Chip.

The GO Chip has been applied to a 41-patient metastatic castrate resistant prostate cancer (mCRPC) cohort. CTCs were enumerated from whole blood for all patients (range: 3-166 CTCs/mL, median: 20 CTCs/mL). Clusters of CTCs, defined as two or more directly adjacent CTCs, were observed in 26/41 patients, and ranged in size from 2-8 CTCs/cluster. Within the CTC population, the percentage of CTCs present as clusters ranged from 0-54.8%. Additionally, a parallel device was run for 36 patients to ultimately obtain RNA to use in RT-qPCR to assess levels of 96 genes of interest. Enumeration and RNA expression data were compared with clinical outcomes including overall survival, radioclinical progression, and PSA progression. An eight-gene score was determined to be highly prognostic of overall survival (AUC: 0.88), with the genes comprising the score suggesting the importance of a dedifferentiated expression phenotype in poor prognosis.

Follow-up work in prostate cancer investigated the role of HER2 and EGFR in prostate cancer metastasis. Analysis of tissue microarrays showed HER2 expression in prostate cancer and bone metastases. Primary and secondary prostate sphere formation was dependent on high EGFR expression as determined by FACS, but not on HER2 expression. EGFR was also implicated in survival in transit as shown by the presence of EGFR+ CTCs isolated by the GO Chip in 9/10 mCRPC patients assays, with an average of 35.5% of CTCs showing EGFR expression. Dual inhibition of HER2 and EGFR in mouse xenograft models prevented tumor growth. HER2 and EGFR as well as ADAM15 and CD31 were studied in bladder cancer CTCs as well. In a preliminary study primarily for optimization, antibodies were chosen for higher sensitivity capture as well as to stain bladder CTCs for the markers of interest. Ultimately, CTCs were isolated from five metastatic bladder cancer patients (range: 5-499 CTCs/mL), and a combination of staining antibodies that showed low background in the healthy control was chosen. EGFR+ and CD31+ CTCs were observed, while HER+ and ADAM15+ CTCs were not, and clusters of CTCs were isolated from some patients.

To address drawbacks in the current technology, two strategies were attempted to enable cell release. A layer-by-layer (LbL) substrate enclosed in a microfluidic chamber featured different disadvantages based on film composition, but a thermosensitive polymer substrate enable release when cooled below its lower critical solution temperature of 12-13°C. The polymer-GO composite showed between 84.9 and 95.2% capture efficiency of EpCAM expressing cell lines and released over 91% of cells captured from whole blood. Using this device, CTCs were captured from 2/3 pancreatic cancer patients and 8/10 breast cancer patients. FISH for HER2 was performed on CTCs isolated from one breast cancer patient.

With high performing technologies to separate them from the noise of other cells in the blood, CTCs can provide information about disease spread in genitourinary cancers. The future incorporation of CTC-related information into clinical decision making has the potential to better inform treatment selection and disease prognosis.