High throughput, label-free isolation of circulating tumor cell clusters in meshed microwells

Extremely rare circulating tumor cell (CTC) clusters are both increasingly appreciated as highly metastatic precursors and virtually unexplored. Technologies are primarily designed to detect single CTCs and often fail to account for the fragility of clusters or to leverage cluster-specific markers for higher sensitivity. Meanwhile, the few technologies targeting CTC clusters lack scalability. Here, we introduce the Cluster-Wells, which combines the speed and practicality of membrane filtration with the sensitive and deterministic screening afforded by microfluidic chips. The >100,000 microwells in the Cluster-Wells physically arrest CTC clusters in unprocessed whole blood, gently isolating virtually all clusters at a throughput of >25 mL/h, and allow viable clusters to be retrieved from the device. Using the Cluster-Wells, we isolated CTC clusters ranging from 2 to 100+ cells from prostate and ovarian cancer patients and analyzed a subset using RNA sequencing. Routine isolation of CTC clusters will democratize research on their utility in managing cancer.


Supplementary
Computer simulation of fluid flow within a microwell.
The plot shows the sample flow speed within an individual meshed microwell simulated with finite element analysis (COMSOL Multiphysics 5.2a). The specific simulated conditions correspond to a sample being processed with the Cluster-Wells at a volumetric flow rate of 25 mL/h. A maximum simulated flow speed of ~65 μm/s, which is still expected to be ~10X lower than physiological free flow speed in human capillaries 1 , is observed at the openings of the micromesh.

Supplementary Figure 2
Microfabrication of the silicon master-mold.
Schematic illustration of the microfabrication process used for manufacturing the silicon mold later used to create polymer-based Cluster-Wells devices. The silicon mold was patterned using a combination of photolithography, thin film deposition and dry/wet etching processes detailed by the top and cross-sectional schematics of the substrate associated with individual microfabrication steps in the figure.

Supplementary Figure 3
Molding of the Cluster-Wells from the micromachined silicon master.
(a) Schematic illustration of the fabrication process involving soft lithography and micromolding-based techniques for the realization of polymer devices from reusable molds in a laboratory environment. The developed method eliminated the continuous need for expensive cleanroom equipment and time-consuming fabrication processes. (b) Scanning electron micrograph of (i) the PDMS mold used for patterning the Cluster-Wells (ii) the top view of fabricated polymer device (iii) the bottom view of the device (iv) the inclined cross-sectional view of an individual microwell. Scale bars, 50 μm.

Supplementary Figure 4
Cluster-Wells capture efficiency obtained by imaging and counting the isolated clusters on the device.
Plot showing Cluster-Wells capture efficiency of spiked LNCaP prostate cell clusters processed at 25 mL/h flow rate as a function of number of cells in the cluster (n=3 independent experiments). Efficiency values from direct counting of clusters directly on the device closely matched with those values obtained with the 2-channel microfluidic interface, confirming the efficiency and reliability of the method used for characterization of the device. Data are presented as mean ± SD.

Supplementary Figure 5
Investigation of the integrity of clusters when captured at different flow rates.
Plots showing the normalized distribution of spiked and processed (captured + missed) clusters' sizes when the Cluster-Wells was operated at (a) 100 mL/h, (b) 250 mL/h, (c) 500 mL/h and (d) 750 mL/h. Matching profiles of spiked and processed cluster populations for flow rates up to 250 mL/h suggests capture of intact clusters, while the mismatch between spiked and processed populations for flow rates >500 mL/h illustrated the dissociation of larger clusters into smaller ones at those higher flow rates.  Fig. 2b, while the capture efficiencies for the Cluster-Chip were taken from reference 2 .

Supplementary Figure 8
Comparison of the measured Cluster-Wells release efficiencies with the published rates of the Cluster-Chip for clusters of MDA-MB-231 human breast cancer cells.
The plot shows the measured release efficiency of clusters of MDA-MB-231 cells from the Cluster-Wells together with the published release efficiency of the Cluster-Chip for MDA-MB-231 cells at 25°C and 4°C obtained from reference 2 . The Cluster-Wells release efficiency was measured under the experimental conditions that mimicked the reported conditions used for characterization of the Cluster-Chip 2 , specifically by releasing MDA-MB-231 cells, initially captured at a forward flow speed of 65 μm/s, at a reverse flow speed of 6.5 mm/s.