Colliding CTCs with Ab-Coated Microposts

The CTC Chip (Figure 1.6A) was the first microfluidic device for positive-affinity CTC selection and was comprised of 78,000 micropillars (100 µm diameter, 50 µm spacing) that were etched in silicon and

Figure 1.6 Microfluidic technologies for the affinity selection of CTCs. (A) Assembly of the silicon CTC chip, SEM of a pseudo-colored cancer cell isolated on the Ab-coated micropillars, and simulation of fluid velocity field through micropillars.44_{(B) A schematic of the GEDI device that hydrodynamically induces a strong}

bias towards recovering cells >18 µm (blue dot) and minimizing WBC (yellow dot) interaction.56 _{(C) Picture} a PDMS herringbone chip, which uses convective mixing to encourage CTCs to interact with Ab-coated surfaces.68_{(D) A schematic of the silicon nanopillar chip, where a convective mixing chamber is attached}

to a silicon substrate that is etched to produce nanotexturing prior to Ab-functionalization.100_{(E) Another}

nano-textured device, where standard polyurethane tubing is coated with naturally occurring halloysite nanotubes and adsorbed Abs and selectins.145_{(F) The thermoplastic-based sinusoidal chip uses narrow,}

Ab-coated microchannels to isolate CTCs. CTC release enables off-chip enumeration and viability testing by impedance sensing and phenotyping in a microfluidic imaging module, which are combined in an integrated microfluidic system.146

enclosed with a PDMS substrate. Every third row of microposts was staggered to form a triangular array that encouraged the collision of CTCs with anti-EpCAM polyclonal Abs immobilized on the micropost surfaces. The device was tested against several cell lines and showed consistently high recovery (74-80%) regardless of the cell’s EpCAM expression (Table 1.1).44 _{In contrast, others have found that cell line} recovery decreased along with decreasing antigen expression,65 _{which has been theoretically predicted} by the Chang-Hammer model. This model describes a cell rolling along an Ab-coated surface and details that the probability of Ab-CTC binding and CTC recovery (𝑃𝑅) scales as:147

𝑃𝑅 = 1 − 1/𝑒 𝐶∞ 𝐿 𝑘𝑓

𝑉 (1.2)

Thus, recovery should decrease with the cell’s velocity (𝑉) and increase with the density of antigens on a cell’s surface (𝐶∞), the length of the rolling interaction (𝐿), and the forward binding constant (𝑘𝑓), which is a complex function of how often the Ab-antigen interactions occur and how probable a given binding event is considering the balance of binding kinetics with interaction time.147 _{As the CTC Chip’s} recovery was independent of antigen expression, either rolling distances would need to be very long, which is unlikely as the micropillar design limits CTC-Ab interactions to ~75 µm per collision (one quarter of a pillar),28,87 _{or a reduced velocity would be needed to lengthen the CTC-Ab interactions. The latter} explanation is more likely as the CTC Chip’s flow rate was limited to 1-2 mL/h (0.5-0.9 mm/s velocity), above which recovery dropped precipitously.44

Clinical samples were tested for several cancers (Table 1.1) with median CTC yields that were much higher than those reported for the CellSearch™ CTC Test, which was a very promising result for microfluidic-based CTC analysis. Of note, patients with localized prostate cancer had higher CTC counts than for metastatic prostate cancer,44 _{indicating that the screening for early cancer detection may be} feasible.17 _{Relative to other micropillar technologies,}47,56 _{the CTC Chip had a relatively low purity of only} 34 ± 8% (~233 WBCs/mL).44 _{In microfluidics, two primary factors can cause such nonspecific WBC}

retention. The low fluid shear stress during blood infusion (maximally 0.4-0.8 dynes/cm2_{), which was} limited by recovery,44 _{may have been too weak to disrupt nonspecific interactions; or more likely, low} shear regions behind the micropillars (Figure 1.6A), which had flow velocities of <0.05 mm/s due to the equilateral arrangement,44 _{could have acted as stagnate zones and reservoirs for the WBCs.}28,87

A second micropillar device, termed the geometrically enhanced differential immunocapture (GEDI) device56 _{(Figure 1.6B), used 5,000 silicon micropillars of similar dimensions to the CTC Chip (80 µm} diameter, 100 µm spacing). While the device was operated at lower shear stress (~0.1 dynes/cm2₎79 _than the CTC chip, the GEDI device achieved very high purities (62 ± 2%, ~10 leukocytes/mL) by staggering each row of microposts in a manner that developed hydrodynamic lift forces, which strongly encouraged pillar collisions for cells >15-18 µm and discouraged interactions with cells <15-18 µm.47,56 _{The size threshold} for the GEDI technology is arguable large and a drawback for the GEDI technology, because CTC below this targeted size range, which have been noted in prostate cancer,99 _{were recovered with very low} efficiency (~30% recovery for 13 ± 3 µm BxPC-3 cells; ~60-70% for cells >15 µm ).47

Early clinical research with the GEDI device used the highly specific J591 mAb to target PSMA(+) CTCs for prostate cancer,56,79 _{and the authors demonstrated a 2-400 fold increase in CTC recoveries} relative to the CellSearch™ CTC Test79 _{but with an undisclosed clinical sensitivity.}56,79 _{It should be noted} that PSMA(+) CTCs had variable EpCAM expression with only ~60% of CTCs being PSMA(+)/EpCAM(+);79 after our adjustment to exclude PSMA(+)/EpCAM(-) CTCs based on these results, the GEDI device’s yields would still be approximately 10-fold greater than the CellSearch™ CTC Test. More recently, EpCAM(+) CTCs have been isolated from 33% of patients with pre-cancerous pancreatic lesions, which could be used to identify patients at risk for the development of PDAC.95 _{Also, Abs targeting both EpCAM and} hypoglycosylated mucin 1 (hMUC1),47 _{also an epithelial CTC marker,}148 _{were immobilized within the GEDI} device, but expression of the markers was correlative in pancreatic cancer cell lines; there was no

improvement in cell line recovery;47 _{and there was too limited a clinical data set to draw substantial} conclusions regarding differences in CTC phenotype by including the hMUC1 target.