Criterio Integridad Institucional

The physical separation of CTCs by size discrimination (Figure 1.9A) is a technique that dates back to the 1960s.184,185 _{Early methods termed isolation by size of tumor cells (ISET; Rarecells, Inc.}45,177,178,186-193 as well as ScreenCell)194 _{filtered blood that was subjected to red blood cell lysis and fixation (to eliminate} CTC deformability and improve recovery)177 _{through poly(carbonate) (PC) membranes, which were track-} etched (i.e., irradiated with charged particles) to produce 8 µm pores.189,190 _{Because the track etching} process is random, porosity of the PC membranes is kept low (<2%)195 _{to avoid cell loss through fused} pores; unfortunately, pore fusion occurs even at low porosity. The common problem associated with filtration are low CTC recoveries (~50%) and clogging of the filters as the pores become occupied by the highly abundant WBCs.191,196 _{To mitigate the issue of filter clogging, the ISET technology uses 10 filters to} process 10 mL of lysed blood (diluted 10 fold), each processing only 1 mL of sample.186,192,193 _{In several} side-by-side comparisons with the CellSearch™ CTC Test, the ISET technology recovered similar CTC levels with variable improvement in clinical sensitivity.186,192,193

Figure 1.9(A) Cell abundance versus cell diameter of blood cells26 _{and CTCs,}83 _{and common size ranges} for CTC discrimination.115,195 _{WBC sizes can be smaller in free solution than when plated for} microscopy.62,63 _{(B) (i) A CK(+)/DAPI(+) CTC amongst CD45(+)/DAPI(+) WBCs on a silicon filter}

membrane.197 _{(ii) SEM of a fixed CTC on a parylene C membrane.}198 _{(iii) Picture of a clogged filter after}

processing 7.5 mL of blood.199 _{(iv) Schematic of a 3D parylene C membrane.}195 _{(v,vi) Images of CTCs}

trapped in a microfluidic filtration device.200 _{(C) The Cluster-Chip, a microfluidic chip for filtering CTC}

micro-emboli.57_{(D) The Vortex Technology hydrodynamically traps large CTCs in side channels at high flow} rates.63 _{(E) Dean Flow Fractionation, a hydrodynamic centrifugation method for size-dependent} separation of CTCs.115_{(F) Dielectrophoretic crossover frequencies for cancer cell lines, leukemia cell lines,} and WBCs.201 _{(inset) Working principle of DEP, showing field lines for positive and negative DEP} experienced by CTCs and WBCs at 65 kHz, respectively.202_{(G) Schematic of the ApoStream™ technology}

Modern filter membranes or microfluidic architectures that are similar to pores have utilized lithographic methods to precisely pattern pores into silicon197,203 _{and polymers such as parylene-} C,117,195,198 _{poly(ethylene glycol diacrylate) (PEGDA),}118 _{and PDMS.}200 _{As long as pores can be reliably} fabricated and the membrane is mechanically stable and biocompatible, the membrane’s material is of little consequence for CTC filtration. However, the workflow and cost for filter fabrication directly impacts the technology’s commercialization potential (

Table 1.2) and has ranged from extremely expensive palladium microfilters204 _{to multi-stage deep} reactive ion etching for each set of silicon or parylene-C microfilters195,197 _{and replication of a} lithographically-patterned master by PEGDA photo-polymerization118 _{or PDMS casting,}200 _{the latter}

methods being more amenable to mass production.

Filtration methods achieve much higher throughput than positive affinity methods (Table 1.1), being limited only by the fluidic force that can be imposed on a trapped CTC without the CTC deforming and passing through the pore118 _{or the CTC breaking apart entirely.}195 _{For example, Lin and coworkers} used a parylene-C microfilter117 _{(16,000 pores, 8 µm diameter; Figure 1.9B(ii))}198 _{to process 7.5 mL of} formalin-fixed blood in only 2 min (see Table 1.1 for clinical results, which showed a 45% increase in clinical sensitivity compared to paired CellSearch™ CTC Tests).117 _{Fixation was critical because under these} fluidic pressures, live CTC cell lines were shredded and lost entirely.195

To reduce the tension and stress on the trapped CTCs that leads to cell lysis, a 3D parylene-C microfilter was designed (Figure 1.9B(iv)). The 3D filter was fabricated by multiple deep reactive ion etching (DRIE) processes and was composed of two surfaces patterned with 7,000 pores (8-9 µm

diameter) that were offset, creating a fluidic conduit through the 6.5 µm spacing between the layers. A CTC that passed through the top pore then rested on the bottom surface, which physically supported the cell and reduced mechanical tension. Smaller and/or more deformable cells squeezed through the 6.5 µm gap between the layers to escape the filter. Unfixed MCF-7 cells could be recovered (~86%) from 1 mL blood (diluted 10 fold) in 3-5 min, but a substantial number of leukocytes (~4,500-11,000/mL) were also retained, causing the filter to clog if more than 1 mL blood was processed195 _{(an example of which is shown} in Figure 1.9B(iii)).199 _{While filtration can process samples rapidly, the maximum blood volume that can} be processed is limited by contaminating WBCs plugging the available pores.

In contrast, a silicon-based membrane (Figure 1.9B(i)) with 100,000 pores (10 µm diameter) per device recovered ~80% of live MCF-7 cells from 1 mL blood (diluted 2 fold) but without any 3D pore structuring.197 _{The technology’s ability to isolate unfixed cells may be attributed to the high number of} pores, a large proportion of which likely remain open as a significantly lower number of WBCs (~200- 6,000/mL) were retained.197 _{The open fluidic paths likely reduced fluidic pressure on trapped cells and} prevented cell lysis. However, more deformable cell lines should be tested with these relatively large 10 µm pores to ensure these results are reproducible across CTC types.

Kim, et al. fabricated a system of micropillars that were “hollowed” with an internal, large open chamber that was connected on either side with two sequential 8 µm channel gaps, i.e., 8 µm “pores”. CTCs passed through the first pore and became trapped in the chamber, where fluidic stress was reduced by 23% at the second gap and reduced the probability that CTCs escape. The authors found that ~85% of un-fixed MCF-7 cells (17 µm diameter) easily squeezed through both 8 µm pore structures,203 _{which is} surprising given retention of live MCF-7 cells using 10 µm pores.197 _{In order to retain these cells in the} micropillar system, Kim, et al. labeled the MCF-7 cells with 3 µm anti-EpCAM beads to increase their diameter to 23 µm, which provided 92% recovery. This method was designed to improve recovery and

micropillars203 _{where shear stress was likely too weak to disrupt nonspecific interactions. Further, the} method appears to require very high EpCAM expression to reliably amplify CTC size.

Common to the above filtration technologies, low purity of the CTC isolate has been the most persistent obstacle. Yet one filtration method detailed in 2009205 _{and 2010}200 _{by Tan, et al. achieved} purities that rival the MagSweeper,72 _GEDI,47,56 _{and sinusoidal}28,75,76 _{positive-affinity technologies. By} PDMS casting, the authors fabricated a 20 µm deep microfluidic channel filled with cell traps: sets of three 3-4 µm posts spaced by 5 µm in an arc shape (Figure 1.9B(v,vi)). Blood samples (diluted 3 fold) from five cancer patients were processed, and the larger CTCs were trapped (10-42 CTCs/mL; median 18). WBCs were effectively cleared through the traps, and extremely high purities were achieved (89%, 2-6 leukocytes/mL). Unfortunately, further reports to expand on this small cohort have not been published. For example, it would be advantageous to release clinical CTCs for subsequent molecular analysis by reversing the flow direction, as the authors have demonstrated for cell lines.205 _{Also, some technical} aspects remain to be resolved. (i) Due to the microchannel’s small dimensions, the technology has low sample throughput (0.23 mL/h)205 _{that required >8 h to process each diluted 2 mL blood sample, although} this could be resolved in a similar manner as developed by the Ephesia technology.65 _{(ii) The recovery of} cell lines spiked into blood or clinical CTCs should be determined as well to add to the recoveries from phosphate buffer (~80% recovery).200,205

Lastly, a filtration-based method for specifically isolating micro-emboli or clusters of CTCs (defined as ≥2 joined CTCs) was developed by Sarioglu and coworkers (Cluster-Chip; Figure 1.9C). CTC clusters have been associated with increased metastatic potential and poor patient prognosis but are even rarer than single CTCs (~0.1-0.5/mL blood).57 _{CTC clusters have been identified by membrane filtration technologies} as well.177,197 _{The Cluster-Chip created a 12 µm gap between the bases of two triangular micropillars,} where the passing fluid then split around the top of another triangular micropillar. A cluster of CTCs attempting to split around the micropillar would be retained by cell-cell junctions; the efficiency of this

mechanism increased with as the number of CTCs in a cluster increased (Table 1.1). The authors demonstrated improved recovery of CTC clusters compared to 5 µm PC membranes,57 _{but it was likely} that clusters were not lost but broken apart by fluidic pressures on the membrane so they could not be differentiated from single CTCs. The rarity of CTC clusters was evident as the Cluster-Chip detected them in only 30-41% of 58 cancer patients (4 mL blood), which showed no correlation to the number of individual CTCs in the samples. Further, CTC clusters could be released by reversing the flow direction and increasing flow rate by 10-fold to 250 mL/h. The authors noted that release efficiency was temperature dependent and best at 4°C, which supposedly reduced nonspecific cell adhesion.57 _{However, absolute} purity of the cluster isolate was not discussed, and it is not clear to what degree the stagnant flow regions behind the triangular pillars retained contaminating WBCs. As in other reports,86 _{the group used} micromanipulator peripheral instrumentation to physically select CTC clusters for further molecular transcriptional analysis.57