A cancer detection platform which measures telomerase activity from live circulating tumor cells captured on a microfilter

Abstract

Circulating tumor cells (CTC) quantified in cancer patients' blood can predict disease outcome and response to therapy. However, the CTC analysis platforms commonly used cannot capture live CTCs and only apply to tumors of epithelial origin. To address these limitations, we have developed a novel cancer detection platform which measures telomerase activity from live CTCs captured on a parylene-C slot microfilter. Using a constant low-pressure delivery system, the new microfilter platform was capable of cell capture from 1 mL of whole blood in less than 5 minutes, achieving 90% capture efficiency, 90% cell viability, and 200-fold sample enrichment. Importantly, the captured cells retained normal morphology by scanning electron microscopy and could be readily manipulated, further analyzed, or expanded on- or off-filter. Telomerase activity--a well-recognized universal cancer marker--was reliably detected by quantitative PCR from as few as 25 cancer cells added into 7.5 mL of whole blood and captured on the microfilter. Moreover, significant telomerase activity elevation was also measured from patients' blood samples and from single cancer cells lifted off of the microfilter. Live CTC capture and analysis is fast and simple yet highly quantitative, versatile, and applicable to nearly all solid tumor types, making this a highly promising new strategy for cancer detection and characterization.

Figure 1. Microfilter fabrication and constant-pressure fluid delivery system

(A). Microfilter fabrication process. (B) Bright-field micrograph of slot microfilter. (C) Constant-pressure fluid delivery system and filter assembly.

Figure 2. Microfilter optimization and validation

(A). Slot size and filtration pressure optimization. Left: Comparison of cell capture efficiency and viability with different slot sizes. Center: Measurement of enrichment with different slot sizes. Right: Comparison of capture efficiency and viability with various filtration pressures using 6 µm slot filter. (B) Cancer cells captured on microfilter and imaged under bright-field (left) and fluorescence (center) of the same field; yellow arrows indicate live captured cancer cells, red arrows indicate dead cancer cells, and black arrows indicate PBMCs. Right: SEM of captured cancer cell. (C) Validation of cancer cell capture from 7.5 ml whole blood. Shown are capture efficiency (left), cell viability (middle) and enrichment (right). (D) On-filter (top) and off-filter (bottom) cell culture of PC3 cells captured from whole blood after 3 days and 6 days. Yellow arrows denote foci of cancer cell proliferation. All histogram results are means of triplicate independent experiments.

Figure 3. Detection of telomerase activity from live cancer cells captured on slot microfilter

(A) Telomerase activity detected from 7.5ml blood samples spiked with a range of cancer cell numbers or blood only (p=0.01 for each sample compared with blood-only sample). (B) Linear correlation of Ct values with the numbers of spiked cells. All histogram results are means of triplicate independent experiments. (C) Telomerase activity of patient samples versus healthy donor controls. The line in healthy donors indicates the calculated true negative Ct cut-off value of 33; patient samples falling within positive range (Ctp=0.029) (E) Serial filtration on healthy donor samples (p=0.5).

Figure 4. Telomerase activity measurement from single live cancer cells captured on microfilter

(A) Captured cells stained by PE-CD49b. (B) Matched bright field image. (C) Micropipette recovery of single cell. (D) Single cell telomerase activity assays.