Cancer cells metastasize through the bloodstream either as single migratory circulating tumor cells (CTCs) or as multicellular groupings (CTC clusters). Existing technologies for CTC enrichment are designed to isolate single CTCs, and although CTC clusters are detectable in some cases, their true prevalence and significance remain to be determined. Here we developed a microchip technology (the Cluster-Chip) to capture CTC clusters independently of tumor-specific markers from unprocessed blood. CTC clusters are isolated through specialized bifurcating traps under low–shear stress conditions that preserve their integrity, and even two-cell clusters are captured efficiently. Using the Cluster-Chip, we identified CTC clusters in 30–40% of patients with metastatic breast or prostate cancer or with melanoma. RNA sequencing of CTC clusters confirmed their tumor origin and identified tissue-derived macrophages within the clusters. Efficient capture of CTC clusters will enable the detailed characterization of their biological properties and role in metastasis.
At a glance
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- Supplementary Figure 1: Finite-element analysis of cell cluster dynamics in different cluster trap architectures. (243 KB)
(a) Cluster-Chip (b) Filter (c) A structure identical to the Cluster-Chip except that one of the openings is closed d) A filter formed by two triangular pillars. In all cases except the Cluster-Chip, cluster larger than the opening size passes due to its elastic behavior. The cluster being missed points to the importance of split flow fields simultaneously effecting the cluster in the Cluster-Chip and proves that the capture in the Cluster-Chip is not simply due to filtering. In all simulations, the opening widths and diameters of individual cells are 14 μm.
- Supplementary Figure 2: Probability of chip-mediated artifact cluster formation from single cells on the Cluster-Chip. (82 KB)
The calculated probabilities are based on simplifying assumptions on chip geometry and cell adhesion. We assume a blood sample with 1000 CTCs. In addition, each of the 4096 parallel cluster traps on the Cluster-Chip is assumed to have equal probability of receiving CTCs and whenever multiple CTCs go into the same trap, a CTC-cluster is formed. We used Poisson approximation to calculate probability of forming a CTC-cluster composed of a specific number of cells. Our model confirms that it is extremely unlikely to form artifact CTC clusters on the Cluster-Chip.
- Supplementary Figure 3: Experimental setup used to quantify the capture efficiency of the Cluster-Chip. (287 KB)
A smaller version of the Cluster-Chip is connected to a large microfluidic waste chamber, which permits to accurately identify and analyze non-captured cell clusters in whole blood. Both the chip and waste chamber are imaged using fluorescence microscopy to calculate the cell cluster capture efficiency.
- Supplementary Figure 4: Distribution of captured small and large clusters on the Cluster-Chip. (198 KB)
Normalized distribution of (a) 2-cell clusters (b) 3-cell clusters (c) clusters containing 4 or more cells across the Cluster-Chip. We used simulated whole blood samples spiked with artificial clusters of MDA-MB-231 breast cancer cell line. The Cluster-Chip was operated at 2.5 ml/hr and was on a thermoelectric cooler set at 4°C.
- Supplementary Figure 5: Characterization of MDA-MB-231 cell cluster size distribution before spiking. (133 KB)
We prepare clusters of fluorescently labeled MDA-MB-231 cancer cells and deposit the suspension on an ultra-low attachment culture dish. Using fluorescence microscopy, we count the number of cells within each cluster and then spike into whole blood in order to prepare a simulated sample containing characterized cluster population for quantitative device testing.
- Supplementary Figure 6: Intact and damaged cell clusters captured using a membrane filter. (413 KB)
Fluorescent and brightftield images of intact and damaged MDA-MB-231 cell clusters when captured using a polycarbonate track-etched membrane filter operated under 1.5 psi.
- Supplementary Figure 7: Viability of cell clusters released from the Cluster-Chip. (104 KB)
The Cluster-Chip is operated at 4°C and the clusters were released at 250 ml/hr reverse flow rate (A) Fluorescent images of MDA-MB-231 clusters treated with two-color viability (green)/cytotoxicity (red) assay. The cells are divided in two batches: Control and cell clusters captured and released from Cluster-Chip (B) Percentage of viable clusters captured and released using Cluster-Chip against the control. Scale bar 100 μm.
- Supplementary Figure 8: Images of CTC -clusters isolated from a breast cancer patient and released in solution. (377 KB)
CTC-clusters isolated from a patient with metastatic breast cancer using the Cluster-Chip operated at 4°C and released with reverse flow. CTC clusters are not fixed and live stained for common breast cancer surface markers. Scale bar 20 μm.
- Supplementary Figure 9: Correlation between the number of CTCs and CTC clusters in patients. (85 KB)
Comparison of number of CTC clusters isolated using the Cluster-Chip and number of single CTCs isolated using CTC-iChip from the same patient at the same timepoint. Among the 19 cases studied, we found no correlation between the numbers of CTC-clusters and that of single CTCs.
- Supplementary Figure 10: Proliferative index of single CTCs isolated from a breast cancer patient. (294 KB)
Representative images of a Ki67-negative (-) and a Ki67-positive (+) CTC stained with antibodies against wide spectrum cytokeratin (CK, red), Ki67 (yellow) and CD45 (green). Nuclei are stained with DAPI (blue). The bar graph shows the mean percentage of Ki67-positive cells isolated from a breast cancer patient across multiple time points. All together, 439 single CTCs were stained and 162 resulted positive for Ki67.
- Supplementary Figure 11: Heat map showing expression levels of CTC clusters isolated from a patient. (197 KB)
Heatmap showing expression levels of representative epithelial-to-mesenchymal transition (EMT) genes in released CTC-clusters and healthy donor white blood cells (WBCs). Epithelial markers E-cadherin (CDH1), EpCAM and Mucin1 (MUC1), as well as mesenchymal markers Vimentin (VIM), Fibronectin1 (FN1), N-cadherin (CDH2) and TWIST1 are shown.
- Video 1: Real-time capture of cell clusters in whole blood on the Cluster-Chip (7.33 MB, Download)
- Real-time video of the Cluster-Chip processing a simulated sample at 2.5 ml/hr. The sample is prepared by spiking artificially formed clusters of fluorescently labeled MDA-MB-231 cancer cells into whole blood samples collected from a healthy donor. Unlabeled blood cells flowing through the chip are not visible in the fluorescent channel but can be noticed as they obscure the fluorescence emission from captured clusters.
- Supplementary Text and Figures (797 KB)
Supplementary Figures 1–11 and Supplementary Table 1