Optimization and Evaluation of a Novel Size Based Circulating Tumor Cell Isolation System

Abstract

Isolation of circulating tumor cells (CTCs) from peripheral blood has the potential to provide a far easier “liquid biopsy” than tumor tissue biopsies, to monitor tumor cell populations during disease progression and in response to therapies. Many CTC isolation technologies have been developed. We optimized the Parsortix system, an epitope independent, size and compressibility-based platform for CTCs isolation, making it possible to harvest CTCs at the speed and sample volume comparable to standard CellSearch system. We captured more than half of cancer cells from different cancer cell lines spiked in blood samples from healthy donors using this system. Cell loss during immunostaining of cells transferred and fixed on the slides is a major problem for analyzing rare cell samples. We developed a novel cell transfer and fixation method to retain >90% of cells on the slide after the immunofluorescence process without affecting signal strength and specificity. Using this optimized method, we evaluated the Parsortix system for CTC harvest in prostate cancer patients in comparison to immunobead based CTC isolation systems IsoFlux and CellSearch. We harvested a similar number (p = 0.33) of cytokeratin (CK) positive CTCs using Parsortix and IsoFlux from 7.5 mL blood samples of 10 prostate cancer patients (an average of 33.8 and 37.6 respectively). The purity of the CTCs harvested by Parsortix at 3.1% was significantly higher than IsoFlux at 1.0% (p = 0.02). Parsortix harvested significantly more CK positive CTCs than CellSearch (p = 0.04) in seven prostate cancer patient samples, where both systems were utilized (an average of 32.1 and 10.1 respectively). We also captured CTC clusters using Parsortix. Using four-color immunofluorescence we found that 85.8% of PC3 cells expressed EpCAM, 91.7% expressed CK and 2.5% cells lacked both epithelial markers. Interestingly, 95.6% of PC3 cells expressed Vimentin, including those cells that lacked both epithelial marker expression, indicating epithelial-to-mesenchymal transition. CK-positive/Vimentin-positive/CD45-negative, and CK-negative/Vimentin-positive/CD45-negative cells were also observed in four of five prostate cancer patients but rarely in three healthy controls, indicating that Parsortix harvests CTCs with both epithelial and mesenchymal features. We also demonstrated using PC3 and DU145 spiking experiment that Parsortix harvested cells were viable for cell culture.

The Novel Association of Circulating Tumor Cells and Circulating Megakaryocytes with Prostate Cancer Prognosis

“…We have previously shown that CD45 À cells with both epithelial and mesenchymal features can be detected in prostate cancer patients using Parsortix (9). Here, we developed a repeated FISH analysis approach following immunofluorescence analysis on the same slide to investigate multiple genomic regions for alterations and, consequently, establish the malignancy of VIM þ /CD45 À cell populations, which were associated with disease burden and poor prognosis and have the potential to serve as an additional marker to traditional epithelial CTCs for clinical application.…”

“…Isolation of CTCs from whole blood using a size-and deformability-based system Parsortix was performed as described previously (9). Sample harvest and the process of immunostaining are detailed in Supplementary Materials and Methods.…”

“…Hence, CTCs undergoing EMT as part of the metastatic process may be missed when isolated on the basis of their epithelial characteristics only. To address this challenge, we have optimized a novel epitopeindependent CTC isolation system, Parsortix, to capture CTCs based on the much larger size and less deformability nature of tumor cells compared with normal blood cells (9). The efficiency of Parsortix in capturing CTCs has been independently validated by other research groups in comparison with CellSearch in patients with small-cell lung cancer (10), and other types of cancers, including breast, colon, and lung (11).…”

Circulating Tumor Cells: A Review of Non–EpCAM-Based Approaches for Cell Enrichment and Isolation

“…Moreover, the processing time of 7.5 mL of whole blood is 2 h in contrast to the 4 h reported for the Cell Search (Cell Search, Jansen Diagnostic). It is worth noting that with the second version of Parsortix 10 mL can be processed in 2.5 h. The main drawback of this technique is the difficulty of eliminating all leukocytes owing to size overlap with CTCs (39 ). In addition to the previous devices described above, ClearCell FX (Clearbridge Biomedics) recovers viable cells in small sample volumes and in a short period of time (e.g., 1 mL of blood in 10 min) (Fig.…”

Improving the CellSearch® system

“…This definition has shown to be quite robust in several ring studies that have been conducted [107,108]. A variety of new technologies have been introduced after the introduction of CellSearch, and most compare the results obtained with those from CellSearch [109][110][111]. A difficulty here is that the definitions used to define a CTC are not the same, thus comparing apples with oranges.…”