Nanoroughened adhesion-based capture of circulating tumor cells with heterogeneous expression and metastatic characteristics

Abstract

Background: Circulating tumor cells (CTCs) have shown prognostic relevance in many cancer types. However, the majority of current CTC capture methods rely on positive selection techniques that require a priori knowledge about the surface protein expression of disseminated CTCs, which are known to be a dynamic population.

Methods: We developed a microfluidic CTC capture chip that incorporated a nanoroughened glass substrate for capturing CTCs from blood samples. Our CTC capture chip utilized the differential adhesion preference of cancer cells to nanoroughened etched glass surfaces as compared to normal blood cells and thus did not depend on the physical size or surface protein expression of CTCs.

Results: The microfluidic CTC capture chip was able to achieve a superior capture yield for both epithelial cell adhesion molecule positive (EpCAM+) and EpCAM- cancer cells in blood samples. Additionally, the microfluidic CTC chip captured CTCs undergoing transforming growth factor beta-induced epithelial-to-mesenchymal transition (TGF-β-induced EMT) with dynamically down-regulated EpCAM expression. In a mouse model of human breast cancer using EpCAM positive and negative cell lines, the number of CTCs captured correlated positively with the size of the primary tumor and was independent of their EpCAM expression. Furthermore, in a syngeneic mouse model of lung cancer using cell lines with differential metastasis capability, CTCs were captured from all mice with detectable primary tumors independent of the cell lines' metastatic ability.

Conclusions: The microfluidic CTC capture chip using a novel nanoroughened glass substrate is broadly applicable to capturing heterogeneous CTC populations of clinical interest independent of their surface marker expression and metastatic propensity. We were able to capture CTCs from a non-metastatic lung cancer model, demonstrating the potential of the chip to collect the entirety of CTC populations including subgroups of distinct biological and phenotypical properties. Further exploration of the biological potential of metastatic and presumably non-metastatic CTCs captured using the microfluidic chip will yield insights into their relevant differences and their effects on tumor progression and cancer outcomes.

Keywords: Adhesion; Breast cancer; Circulating tumor cells; Lung cancer; Metastasis; Microfluidics.

Fig. 1

Nanotopography-based microfluidic chip for CTC capture. a Photo of the microfluidic CTC capture chip (left) and SEM images (right) showing the nanorough glass surface (top right, R _q = 150 nm) and a cancer cell adhered to the surface (bottom right). b Bar graph showing 30 min capture yield for breast cancer cells (MCF-7, MBA-MB-231, and SUM-149) and lung cancer cells (A549) using the capture chip with smooth (R _q = 1 nm) and nanorough (R _q = 150 nm) glass surfaces as indicated. For each cell type, 1,000 cells were spiked in 1 mL lysed human blood. EpCAM expression of each cell line is denoted below the graph. Error bars, s.e.m. (n = 4). **, p < 0.01

Fig. 2

Capture of pre- and post-EMT lung cancer cells using the nanotopography-based microfluidic CTC capture chip. a Representative staining images showing pre- (top) and post-EMT (bottom) A549 cells captured on nanorough glass surfaces (R _q = 150 nm) 1 h after cell seeding. 10,000 pre- and post-EMT A549 cells labeled with CellTracker Green were spiked in 500 μL lysed blood that was pre-stained with DiI to label peripheral blood mononuclear cells (PBMCs). b & c Regression analysis of 1 h capture efficiency for pre- and post-EMT A549 cells (n = 40–900 spiked in 500 μL lysed blood) using the microfluidic CTC capture chip. The number of A549 cells captured (b) and the capture yield (c) is plotted as a function of the total number of A549 cells spiked in blood samples. d Ratio of pre- and post-EMT A549 cells captured 1 h after cell seeding as a function of their ratio when spiked in blood samples. 1,000 post-EMT A549 cells were mixed with 500–4,000 pre-EMT cells in 500 μL lysed blood to achieve ratios from 2 : 1 to 1 : 4. Solid lines in b & d represent linear fitting. Error bars, s.e.m. (n > 4)

Fig. 3

CTCs captured using the microfluidic CTC capture chip from mice with breast cancer orthotopic xenografts. a Photos of MDA-MB-231 xenografts, 1 cm scale bar. The arrow indicates the small tumor at 3 weeks. b Representative staining images showing CTCs captured on nanorough glass surfaces from mice with MDA-MB-231 tumor xenografts. Cells were co-stained for nuclei (DAPI; blue), cytokeratin (green), and CD45 (red). c-e Temporal changes in CTC number and tumor weight during tumor progression. Tumor weight (c) from mice with MDA-MB-231 and SUM-149 tumor xenografts as a function of xenograft time. Scatter plot (d) of CTC number per 100 μL blood vs. tumor weight. Bar plot (e) showing number of CTCs captured by the microfluidic CTC chip as a function of xenograft time. For each CTC capture assay, 300–800 μL blood samples were obtained via cardiac puncture. Error bars, s.e.m

Fig. 4

Capture of CTCs from metastatic and non-metastatic syngeneic mouse models of lung cancer. a Photos of lung metastases from 344SQ (top) and 393P (bottom) implants. Mouse 344SQ lung cancer cells are highly metastatic, while mouse 393P lung cancer cells are metastasis-incompetent. b Representative staining images showing CTCs captured on nanorough glass surfaces from mice implanted with 344SQ cells. Cells were co-stained for nuclei (DAPI; blue), cytokeratin (green), and CD45 (red). c-g Analysis of CTC number and tumor volume for mice with 344SQ and 393P tumor allografts. Bar plots show tumor volume (c) and CTC number per 100 μL blood (d) for individual mice. Bar plots showing average tumor volume (e) and average CTC number per 100 μL blood (f) of all mice. Scatter plot (g) of CTC number per 100 μL blood vs. tumor volume for mice with 344SQ and 393P tumor allografts. Mice were subcutaneously implanted with tumor allografts of 344SQ and 393P lung cancer cells. For each CTC capture assay, 350–600 μL blood samples were obtained via cardiac puncture. Error bars, s.e.m. *, p < 0.05