Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection

<div><p>Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) “smart coating” to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as <i>ex vivo</i> culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.</p></div

Overview of the CMx platform.

<p>(A) Strategies to capture, purify and release CTCs of the CMx platform. In the top row, blood flows through a microfluidic channel coated with anti-EpCAM conjugated to NeutrAvidin which is adhered to a SLB layer on substrate. Selective binding of CTCs to anti-EpCAM is reinforce by the antibody clustering effects through the mobility of the fluidic SLB layer, while other blood cells are easily flush away from the fluidic surface. The bottom row showed the release process, in which introduced air bubbles disrupt the weakest links between substrate and the SLB layer allowing elute intact CTCs for the collection outside of microfluidic chip. (B) Overview of CMx platform and summary of the CMx platform workflow. About 2 mL of the whole blood samples obtained freshly from the CRC patients were loaded equally into each CTC capturing devices. All chips went through cell capture, purification, and released for various downstream applications, including immunofluorescence staining, cell counting, molecular analysis, <i>ex vivo</i> cell culture and/or cryobanking.</p

Capture performance and purification by Ab-SLB coated microfluidics.

<p>(A) The geometry and patterns of 6 different microfluidic channel designs (left) and the capture efficiency of these microfluidic platforms (right) as defined by dividing captured cells over total spiked cells. (B) The cropped fluorescent images (5.5 mm x 5.5mm) inside the flow channel and the enumeration of HCT116 (green) and WBCs (blue) on Ab-SLB or Ab-silane coated Type E chips. (C) Cell detachment efficiency (%, Y-Axis) vs. flow rates (ml/h, lower X-axis) and the corresponding shear stress (upper X-axis). Flow rates are generally maintained below 4 ml/h to avoid any potential loss of captured CTCs. (D) Highly CTM capture and recovery efficiency of the Ab-SLB coated chip. The capture efficiency and recovery rate of HCT116-RFP generated tumor microemboli were showed in right panel. The released HCT116-RFP CTC clusters with DAPI staining were showed in left panel. *: <i>p</i> < 0.05; **: <i>p</i> < 0.01.</p

Release of captured cells by air foams from CMx platform.

<p>(A) The florescent images of the HCT116 cancer cell released from the Texas Red-SLB coated chip. The cell was wrapped with Texas Red-SLB lipid molecules around the membrane (blue: DAPI; green: pre-stained CellTracker Green CMFDA; red: Texas Red conjugated lipid molecule). (B and C) Overlaid fluorescent images of the released single CTC and CTM for cell characterization. Eluted cells from clinical samples were categorized as CTC by size, morphology and immunostainning (DAPI+/CK20+/CD45-). WBCs were identified by DAPI+/CK20-/CD45+ immunostaining (blue: DAPI; green: CD45; red: CK20).</p

Enumeration and correlation of single CTCs and cluster CTCs with disease progression.

<p>(A) Correlation and statistical analysis of single CTC enumeration and clinical stage of CRC patients and healthy donors. (B) Correlation and statistical analysis of CTM enumeration and clinical stage of CRC patients and healthy donors. Correlation between (C) CTCs and (D) CTM and lymphnode metastasis of CRC patients were shown with mean±SEM. (* <i>p</i> < 0.05, ** <i>p</i> < 0.01)</p

Cultivation of viable eluted CTCs.

<p>Capture and release of HCT116 cancer cell from CMx platform for further cultivation under (A) complete DMEM medium, (B) SPH culture medium, (C) suspension culture with SPH medium. (D) CTCs isolated from a stage III CRC patient. The cells gathered and attached to the bottom of culture plate at day 7 and became more spread out at day 9. Cells exhibited CK20+ (red) and nuclei+ (blue) with a size around 25μm. To prove these cells have a propensity to re-attach, they were treated with 0.1% trypsin and re-seeded at day 9. After 1 day, these cells re-attached to the substrate firmly (day 10). (E and F) Immunocytochemistry stain for the confirmation of colorectal origin of patient-derived CTC primary cultured cells. The skin-derived fibroblast cell line HS68 was used as negative staining control (blue: DAPI; red: CK20; green: α-SMA or FGFR).</p

Genetic mutation analyses of eluted CTCs.

<p>Hotspot analyses of randomly selected 3 healthy (H1~H3) and 10 colorectal cancer patient samples (P1~P10) by castPCR. The mutation loci detected include 3 loci for p53, 2 loci for APC, and 2 loci for KRAS. (Loci with ΔCt value < 9.96 indicate positive and were represented in bold format).</p