Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology

Abstract

Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 μL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells.

Keywords: Vortex; circulating tumor cells; immunofluorescent staining; rare cell enrichment; size based cell isolation.

Figure 1. Microfluidic device design and performance

A. The High Throughput Vortex Chip (Vortex HT) is parallelized with 2x more parallel channels and 1.5x more serial reservoirs in each channel than the previous Vortex Chip design. At high flow rates, microvortices develop in each reservoir and trap larger cancer cells while allowing smaller RBCs and WBCs to either pass through or transiently enter vortices. B. With the same processing time, Vortex HT yields ∼1.6x higher capture efficiency of MCF7 breast cancer cells while maintaining comparable purity (n = 3 trials) relative to the Vortex Chip. C. Sample flow-through may be collected and repeatedly processed through multiple cycles to increase cell capture with a tradeoff of slightly diminished sample purity. D. MCF7 cells processed through Vortex HT maintained high relative viability compared with cells not processed through the device. E. MCF7 cells released into a well-plate are able to grow and proliferate over the course of 4 days. Scale bar represents 40 μm.

Figure 2. Immunofluorescent staining characteristics for identifying CTCs

A. Collected cells were classified according to immunostains against CK (green) and CD45 (red), and DNA stained with DAPI (blue). In general, CTCs were defined as either CK+/CD45−/DAPI+ or only DAPI+ with a large nucleus (>9 μm) and N:C ratio (>0.8). Each cell was compared with the table's criteria in the order listed until the characteristics matched. Scale bars represent 10 μm. B. MCF7 cells, used as a staining control, stained strongly for CK and negative for CD45. C. Most WBCs stained for CD45-TRITC but negative for CK. Monocytes and lymphocytes were consistently stained strongly whereas granulocytes exhibited weaker CD45 signals. D. CTCs typically stained weakly or negatively for CK. E. Several cells stained double-positive for both CD45 and CK. Additional staining of the collected cells with CD66b-AF647 confirmed the cells as activated granulocytes. F. Large CK−/CD45- cells with high N:C ratios were present in collected samples and defined as CTCs by our criteria. [All scale bars in (B-F)]represent 20 μm.

Figure 3. Enumeration of patient CTCs

A. More CTCs/mL of whole blood were found in stage IV lung (n = 15) and breast (n = 22) cancer blood samples than in age-matched healthy samples (n = 10). Based on the maximum count for all healthy samples, a minimum threshold of 1.25 CTCs/mL of whole blood (dotted green line) was set to define samples as CTC-positive. Using such a threshold, approximately 80% and 86% of lung and breast cancer samples, respectively, were found positive for CTCs. B. Representative immunofluorescence images of CTCs and WBCs collected from Vortex HT. Scale bars represent 20 μm. C. The purity of collected CTCs varied between samples and averaged ∼20%. Healthy samples exhibited very low purity due to the few collected cells which were classified as CTCs. D. The absolute number of collected WBCs was relatively low as well and was present in all processed samples.

Figure 4. Immunofluorescent profiles of patient CTCs

A. CTCs collected from each patient sample were composed of both CK-positive (green) and CK-negative (DAPI+ only, blue) subpopulations. B. Representative images of CK−/DAPI+ and CK+/DAPI+ stained CTCs. Scale bar represents 20 μm. C. After traditional CK staining (left bar), cells immunostained for epithelial (EpCAM and CK, green) and mesenchymal (VIM, orange) markers (right bar) exhibited diverse variations of all combinations, including expression of both EMT markers or neither. The majority of cells were VIM−/CK+/EpCAM+/DAPI+ or VIM−/CK−/EpCAM−/DAPI+. D. Representative images of cells which express mesenchymal markers, epithelial markers, neither, or both. Scale bar represents 20 μm.

Figure 5. Comparison of Vortex HT with other technologies

A. Vortex HT captures more CTCs than the Vortex Chip in all cases (7 lung and 7 breast). B. Blood tubes from the same patient were split for tests between Vortex HT and CellSearch for 5 lung, 8 breast, and 2 healthy samples. In two breast patients (no. 3 and 8, starred), the test was aborted by the CellSearch machine. C. In 13 of the metastatic cancer patient samples tested, the CellSearch test identified CTCs in 15% of samples, whereas Vortex HT found 85% of samples as positive for CTCs above levels for age-matched healthy controls.