Portable filter-based microdevice for detection and characterization of circulating tumor cells

Abstract

Purpose: Sensitive detection and characterization of circulating tumor cells (CTC) could revolutionize the approach to patients with early-stage and metastatic cancer. The current methodologies have significant limitations, including limited capture efficiency and ability to characterize captured cells. Here, we report the development of a novel parylene membrane filter-based portable microdevice for size-based isolation with high recovery rate and direct on-chip characterization of captured CTC from human peripheral blood.

Experimental design: We evaluated the sensitivity and efficiency of CTC capture in a model system using blood samples from healthy donors spiked with tumor cell lines. Fifty-nine model system samples were tested to determine the recovery rate of the microdevice. Moreover, 10 model system samples and 57 blood samples from cancer patients were subjected to both membrane microfilter device and CellSearch platform enumeration for direct comparison.

Results: Using the model system, the microdevice achieved >90% recovery with probability of 95% recovering at least one cell when five are seeded in 7.5 mL of blood. CTCs were identified in 51 of 57 patients using the microdevice, compared with only 26 patients with the CellSearch method. When CTCs were detected by both methods, greater numbers were recovered by the microfilter device in all but five patients.

Conclusions: This filter-based microdevice is both a capture and analysis platform, capable of multiplexed imaging and genetic analysis. The microdevice presented here has the potential to enable routine CTC analysis in the clinical setting for the effective management of cancer patients.

Figure 1

Illustration of device assembly. a) Schematic drawing of a functional microdevice consists of parylene membrane filter sandwiched between rectangular PDMS slabs and clamped in between acrylic jigs with inlet and outlet for syringes. b) Bright field image of an optically transparent parylene filter with uniformly shaped and spaced 8 μm pores. c) SEM picture of single cultured tumor cell captured on the membrane.

Figure 2

Flow characterization of the microdevices with varying sample composition under constant pressure of 0.5 psi.

1. PBS only (square);

2. PBS with 50k LNCaP cells (circle);

3. PBS with 50k LNCaP cells fixed in 1% acetone (triangle);

4. 50% human blood (triangle);

5. 50% human blood with 25k LNCaP cells (square);

6. 50% blood fixed in 1% NBF (triangle);

7. 50% blood and 25k LNCaP cells fixed in 1% NBF (triangle);

8. 50% blood and 25k LNCaP cells fixed in 2% NBF (hexagon);

9. 50% blood and 25k LNCaP cells fixed in 5% NBF. Dash line part of curve 9 was caused by severe blocking of filter so that the filtration could not be completed (star);

10. 100% human blood (pentagon).

Figure 3

On-Chip capture and immunofluorescent (IF) testing of captured tumor cells. a) Montage of captured tumor cell lines (model system), showing expression of CK (green) by IF (with DAPI nuclear counterstain, blue); note the size heterogeneity within the model system. b) and c) CK positive/PSA positive and d) CK positive/PSA negative CTCs captured from the peripheral blood of a patient with prostate cancer. CK positive cells are green, PSA positive cells are red. When both CK and PSA are expressed, the combined color is yellow.

Figure 4

Histogram demonstrating performance comparison of membrane microfilter vs. CellSearch® assay in clinical samples. Solid and striped bars denote number of CTCs detected using the commercially available CellSearch® assay and microdevice, respectively. The number of CTC positive samples were 27 vs. 14 (microdevice vs. CellSearch®) out of 28 patients for prostate cancer, 10 vs. 4 out of 12 patients for colorectal cancer, 11 vs. 6 out of 11 patients for breast cancer and 4 vs. 3 out of 6 patients for bladder cancer.