Precision microfilters as an all in one system for multiplex analysis of circulating tumor cells

Creatv MicroTech, Inc., 1 Deer Park Dr., Mo dan@creatvmicrotech.com Fox Chase Cancer Center, Protocol Su Philadelphia, PA 19111, USA University of Maryland Baltimore Greeneba Baltimore, MD 21136, USA Mayo Clinic Cancer Center, 4500 San Pabl Robert H Lurie Comprehensive Cancer C Michigan Avenue, Chicago, IL 60611, USA Rutgers, the State University of New Jersey, USA Creatv MicroTech, Inc., 2242 West Harriso Creatv MicroTech, Inc., 9900 Belward Cam Hememics Biotechnologies, 12111 Parklawn † Electronic supplementary informa 10.1039/c5ra21524b Cite this: RSC Adv., 2016, 6, 6405


Introduction
The primary mechanism of metastasis is believed to be the extravasation, or shedding, of cancerous epithelial cells into circulation.These circulating tumor cells (CTCs) can travel throughout the body, adhere to vascular beds of organs, inltrate, grow and impair organ function.2][3] This lack of viability could be due to apoptosis through anoikis, actions of natural killer cells of the host, or shear stress from capillary ow. 4,56][7][8][9] Therefore, technologies that can capture and analyze these rare cells from patient blood samples are being vigorously pursued for diagnostic purposes, and to determine whether prescribed therapies are effective.
Strategies for isolating CTCs from whole blood samples generally fall into two broad categories (1) affinity based isolation and (2) label-free isolation.The only clinically accepted method for enumerating CTCs from cancer patients is the CellSearch® CTC test, which is an affinity based isolation of CTCs using magnetic nanoparticles coated with antibodies against the epithelial cell adhesion molecule (EpCAM). 6,8,10,11he captured cells are then further characterized by the staining of anti-cytokeratin (CK) antibodies and the non-staining with anti-CD45 (leukocyte common antigen) antibodies.Although the CellSearch® CTC test is clinically validated, it is not designed for CTC analysis beyond enumeration of basic biomarker expression and therefore has limited personalized medicine applications. 5,9,124][15][16] These label-free CTC isolation techniques (i.e.8][19] Although the greater number of CK + cells isolated by these techniques can be attributed to greater isolation efficiency, the clinically validated data provided by CellSearch® has only been recently reproduced by ltration methodology. 6,12,2015][16][17][20][21][22] It has been postulated that more detailed examinations of CTCs will yield clinically important data.However, currently, there is no single commercially available lab on a chip platform that can isolate and sequentially analyze CTCs in situ using multiple methods.To date, the only label-free CTC assay that shows both increased sensitivity as well as correlation to the clinically validated CellSearch® test is the CellSieve™ system. 5,6,12,20Since CellSieve™ lters can identify the clinically relevant CTCs, without using magnetic particles that can obscure cellular details; we have been actively assessing techniques that can further characterize CTCs immobilized on the microlters.The ability to expand CTC analysis beyond single plex enumeration would greatly extend utility of CTCs to include more than basic prognostication.The ability to expand CTC analysis beyond single plex enumeration would greatly extend utility of CTCs to include more than basic prognostication. CellSieve™ lters are not autouorescent and are biologically inert, allowing for staining of captured cells using multiple uorescent antibodies and for the potential growth of growing captured cells on the lters. 18,23,24Here we suggest that cells captured on this platform can be cultured in situ or harvested using a backwash procedure, and subsequently analyzed using numerous downstream applications (Fig. 1).We show that the platform allows for multiple downstream techniques, such as culture, histopathological subtyping, uorescent in situ hybridization (FISH), scanning electron microscopy (SEM) and single cell capture using micromanipulation.Our data supports the view that CTC capture, culture and clinically applicable testing are possible using a size based all-in-one lab-on-chip platform capable of analyzing and characterizing CTC biology.

Healthy and patient blood samples used for these studies
Sixteen anonymized cancer patient peripheral blood samples, breast (n ¼ 10) or prostate (n ¼ 6), collected in CellSave tubes were supplied through a collaborative agreement with Fox Chase Cancer Center (FCCC) and University of Maryland Baltimore (UMB).Samples were collected with written informed consent and according to their local Institutional Review Board (IRB) approval at each institution.In addition, healthy volunteer blood samples were collected in CellSave preservative tubes™ or K 2 EDTA vacutainers with subject signed informed consent and IRB approval by Western IRB.All blood samples were kept at room temperature before ltration (storage at 4 C causes formation of microclots in whole blood that can clog the lters).

Cell lines
Tumor cell lines used in this study were purchased from ATCC (Manassas, VA).These include MCF-7, MDA-MB-231 and SK-BR-3 human breast cancer cell lines and PANC-1 pancreas epithelioid carcinoma.All cell lines were grown in their cell line specied media containing fetal bovine serum (FBS) as recommended by ATCC.Cell lines were maintained in T-25 or T-75 asks using prescribed cell culture conditions (5% CO 2 , 37 C) with media changes every 3-4 days, with the exception of the MDA-MB-231 cell line, which were grown at 37 C with no Fig. 1 Overview of the work flow and methodologies described in this study.(A) Assays are developed and optimized with cancer cell lines spiked into normal blood samples (green).Cancer cell lines are spiked into blood samples collected in CellSave® tubes.The sample is filtered and CTCs are identified using presence of anti-cytokeratin and anti-EpCAM, with absence of anti-CD45.CTCs are quantified, then stained by FISH, H&E, etc. (B) Assays are run on patient samples (purple).Blood from cancer patients were collected in CellSave® tubes, filtered and CTCs were identified.CTCs were counted and the clinically useful subtypes were quantified (i.e.CTCs in division, apoptotic CTCs, etc.). 6any of these cells were further subtyped by FISH or H&E stain (Fig. 2).(C) Proof of principal assay for expansion of CTCs.Viable cancer cell lines were spiked into normal blood collected in either EDTA and isolated by filtration.The filter bound cells were then expanded in culture media for eventual use in other models. 24,25Dotted arrow indicates that cell lines were used.Solid lines indicate the assay was developed with cell lines and has proceeded onto patient samples.
added CO 2 .When xed cells were used, cells were harvested using a trypsin-EDTA solution (ATCC Manassas, VA), spun at 125 Â g for 5 min in 10% serum containing media to neutralize the trypsin, resuspended in PBS containing 1-4% paraformaldehyde (PFA) and incubated for 20 min at room temperature.Aer incubation, cells were diluted in 10Â volume of PBS, centrifuged and resuspended in fresh PBS.When live cells were used, cells were harvested on the same day using trypsin-EDTA, neutralized as described above and resuspended in their specied serum-free media and stored for no more than 1 hour at room temperature before being spiked into normal blood and isolated using CellSieve™ microlters within 5 min.CellSieve™ microlters have been previously described as a 10 mm thick modied SU-8 polymer lm with an array patterned lter with 7 mm diameter pores. 6,17

Capture and culture of live tumor cells spiked into normal blood
To evaluate cell viability by ltration, we spiked $100-1000 live MCF-7, PANC-1 SKBR3, and MB231, cells, into normal blood samples collected in K 2 EDTA vacutainers.Immediately aer spike in (<1 min), the blood was drawn through the lters at 5 ml min À1 using a low pressure ltration system 17 contained within an enclosed device (Fig. 2).Aer ltration and wash with PBS, as previously described, 6,17,25 lters with the captured cells were placed into 12-well plates containing their specied serum-containing culture media.Aer 1-3 weeks the cell colonies were imaged on the lters.In addition, viability and cytotoxicity of the assay was evaluated using Calcein AM and POPO-3 (Life Technologies) according to manufacturer's protocols (ESI Fig. 1 †).

CTC staining procedures performed on CellSieve™ lters
Blood samples were ltered, xed, permeabilized and washed, CTC identication by uorescent enumeration was done as previously described. 6,17Filters were washed with PBS to remove unbound antibody, placed onto a microscope slide with Fluoromount-G/DAPI (Southern Biotech) and sealed with a glass cover slip.An Olympus BX54WI Fluorescent microscope with Carl Zeiss AxioCam was used to image the samples.Exposures were preset as 2-5 s (Cyanine5), 2 s (PE), 100-750 ms (FITC), and 10-50 ms (DAPI) for equal signal comparisons between cells.A Zen2011 Blue (Carl Zeiss) was used to process the images.

Fluorescent in situ hybridization (FISH) analysis of cells captured by CellSieve™ lters
Following ltration and CTC immunostaining lters/cells can be probed using HER-2/CR-17 FISH probes performed as previously described 25,26 with PathVysion HER-2 DNA Probe Kits, supplied by Abbott Molecular Inc.The identied CTCs x/y placement on the lter was marked on the lter substrate, and cell placement was recorded using Zen2011 Blue soware (Carl Zeiss).Samples were demounted in a 2Â SSC solution for 10 min and dried by air.The protease solution was added to each sample for 20 min in a 37 C incubator.Slides were washed twice in 2Â SSC for 5 min and were dried on a 45 C warmer.Slides were placed in the denaturing solution at 72 C for 5 min and were sequentially washed with 70%, 85%, and 100% ethanol for 1 min each, then dried on a 45 C warmer, followed by the addition of 10 ml of probe to the slides, a coverslip was added, and sealed with rubber cement.The slide was incubated for 22 h in a 37 C hybridization chamber.Coverslips were then removed in post-hybridization wash buffer at room temperature, washed with post-hybridization wash buffer at 72 C for 2 min, rewashed in 2Â SSC for 10 min, and dried at room temperature.Samples were mounted with Fluoromount-DAPI (Southern Biotech) and imaged on an Olympus BX54WI Fluorescent microscope with a Carl Zeiss AxioCam.Images were overlaid using Zen2011 Blue soware (Carl Zeiss) as described. 25

H&E staining of cells on CellSieve™ microlters
Following ltration and CTC immunostaining, cells/lters were further characterized with Hematoxylin and Eosin Y (Sigma), as previously described. 26Aer CTCs were imaged, lters were demounted in PBS and washed in PBS for 15 minutes.Filters were transferred to a microscope slide and lters were placed into a hematoxylin solution (Sigma) for 2 minutes.The slides were then rinsed 3 times in DI water then placed into an Eosin Y solution (Sigma) for 2 minutes, followed by briey dipping in DI water until the desired color was achieved.Pre-imaged CTCs were then reimaged under white light.

Isolating individual cells from CellSieve™ lters using micropipettes
Following ltration from blood, captured cells/lters were immediately incubated with permeabilization buffer for 15 minutes and then incubated with the CTC stain solution for 1 hour.Filters were washed with 5 ml 1Â PBS/0.1% Tween-20.All steps were done without drying the sample.Cells identi-ed as EpCAM positive, FITC positive and CD45 negative were removed using a micropipette with capillary pipette tips (Thermo Fisher) under an inverted dissecting microscope.

Scanning electron microscopy (SEM) of CellSieve™ captured cells
For SEM, following ltration and CTC immunostaining, cells/ lters were analyzed by SEM.Aer CTCs were imaged, lters were demounted in PBS and washed in PBS for 15 minutes.A solution of 2.5% glutaraldehyde in PBS was placed onto the lter and placed at 4 C for 1 hour.Following incubation, lters were washed in DI water at room temp for 30 minutes.By holding the lter down on a microscope slide with tweezers, the lters were sequentially dehydrated through graded percentage of increasing ethanol -70%, 80%, 90% and 100% ethanol for 2 minutes per each solution.Samples were dried, positioned onto a sample pin stub, and placed in a desiccator until imaged using a Phenom ProX Desktop Scanning Electron Microscope (NanoScience Instruments).
For TEM, following ltration and CTC immunostaining, cells/lters were sliced by microtome for imaging. 27Aer CTCs were imaged, lters were demounted and washed in PBS for 5 minutes.Filters were transferred to a Swinnex lter holder (Thermo Fisher) and then sequentially dehydrated in ethanol -70%, 80%, 90% and 100% ethanol for 30 seconds per each solution.The lter holder, with lter, was placed into a 37 C incubator and pre-warmed liquid PEG 1000 (Sigma) was added to the lter holder and incubated for 30 minutes. 27The ltered holder was placed at À80 C freezer for 1 hour.The lter was removed from the lter holder and placed onto a microtome specimen holder.The PEG block was cut cold on a rotary microtome, at 5-10 mm slices, and slices were transferred to poly-L-lysine coated slides.

Photobleaching and restaining of cells captured on CellSieve™ lters
The ltered and immunostained CTCs were further subtyped using additional immunomarkers.CTCs x/y placement on the lter was marked on the lter substrate and the cell placement was recorded using Zen2011 Blue soware (Carl Zeiss).Samples were then archived and placed in storage at 4 C for $2 years.Samples were removed from storage and PE uorescence was photobleached by exposure to the excitation uorescence (565 nm) for $10 seconds.Samples were demounted and placed into a lter holder.Cells on lters were again permeabilized for 20 min at RT and restained using an antibody panel of CXCR4 and vimentin, in the PE channel and eour 660 channel, respectively.Filters were washed, placed onto a microscope slide with Fluoromount-G/DAPI (Southern Biotech) and sealed with a glass cover slip.An Olympus BX54WI Fluorescent microscope with Carl Zeiss AxioCam was used to re-image all bleached CTC.Exposures were preset as 500 ms (euor 660) and 2 s (PE), and 10-50 ms (DAPI) for equal signal comparisons between cells.A Zen2011 Blue (Carl Zeiss) was used to process the images.

Capture and backwash recovery of tumor cells and CTCs from patient samples using CellSieve™ lters
Prior to cell elution (backwash) of cells captured by CellSieve™, the lters were rst blocked with 100% FBS for $10 min at room temperature, to prevent non-specic cell adherence.Capture efficiencies and contamination rates were calculated by spiking a known number of MCF-7 cells spiked into CellSave collected blood.A syringe pump (KD scientic) was used to provide low pressure vacuum ltration in a controlled ow format preventing accidental dehydration of the lter.Filtration was performed using CellSieve™ lters enclosed in a specialized lter holder, reducing blood cell retention (Fig. 2).Aer ltration and wash, the used syringe was removed and a clean syringe with 10 ml PBS was placed onto the bottom of the lter holder.The lter holder was placed upside down over a 15 ml conical tube and the PBS was gently pushed through the lter by hand, $10 ml min À1 for evaluating capture efficiency (ESI Fig. 2 †).The eluted cells were then re-isolated on a fresh lter mounted on a normal lter holder.Both lters were then counted for the presence of CTCs and contaminating blood cells using a Zen2011 Blue (Carl Zeiss) soware to count the cells, to determine the capture efficiency and determine the total backwash efficiency.
The backwash procedure was also used to recover CTCs captured from patient samples.Sixteen patient samples drawn into 2 duplicate CellSave tubes, one tube was tested for capture efficiency and the duplicate tube was tested for backwash release.The rst patient blood tube sample was run and CTCs were counted according to the standard CellSieve™ micro-ltration assay as described. 6,17In conjunction, the second patient blood tube sample was run in same manner, but lter captured cells were eluted from the lters using the backwash method.CTC and contamination counts were then compared between the 2 sets of ltered samples (ESI Fig. 3 †).

Results and discussion
The CellSieve™ microltration assay isolates CTCs using size based separation and is applicable to numerous clinical assays when used in singleplex CTC assessment. 17,25Interestingly, CTCs isolated by this system were described as clinically correlated to the CellSearch® assay, 6 and thus allows for more detailed analysis of a specic and prognostically valuable CTC population.In the present study we expand on these initial singleplex assays, describing the evaluation of multiple biological techniques on rare CTCs directly isolated from whole blood patient samples.

Summary of experimental protocols used in this study
We rst developed the ltration and multiplex processes using cancer cell lines spiked into whole peripheral blood (Fig. 1A).We then validated our ltration and multiplex assays on patient samples (Fig. 1B).Finally as a proof of principle concept, we ltered cell lines, cultured the cells on the lters and ran sequential multiplex analysis on the cultured cells (Fig. 1C).
As in a real clinical setting there are two possible starting points for utilizing CTCs, (1) shown in green/purple, blood collected in CellSave tubes for direct clinical evaluation of cells, without culture (Fig. 1A and B); or (2) shown in red are the protocols used for cell lines spiked into whole blood collected in EDTA vacutainers for indirect clinical evaluation aer the captured cells are cultured and expanded (Fig. 1C).This system could provide isolation, culture expansion, and/or clinical relevant testing of the CTCs (Fig. 1C) and lead to broad study of cancer in real time by providing a supply of cancer cells.Unfortunately, while numerous groups are optimizing the specic environmental components for CTC expansion in tissue culture, 24,28,29 the exact factors needed for consistent CTC culture are not known.Therefore, in this study we focused solely on isolation and in situ studies of CTCs using a microuidic platform, knowing that the CTC culture is a separate study.

Capture and viability of live tumor cells in whole blood
1][32][33][34][35] To examine the effects of ltration on cell viability and cell growth patterns, we spiked a breast cancer cell line (MCF-7), which grows in clustered domes, and a pancreatic cell line (PANC-1), which grows as a monolayer sheet.Cells were spiked into EDTA blood vacutainers, isolated by ltration, and grown in the ltration unit (Fig. 2).We found the cell lines adhered and grew on CellSieve™ lters in their preferred growth pattern, i.e. a domed clustered structure for MCF-7 (Fig. 3A) or a monolayer sheet as seen with PANC-1 cells (Fig. 3B).Both cell lines were grown for 7 days in culture, and stained with CTC antibody cocktail including DAPI.Using these model cell lines, our results show that cells spiked into blood can be isolated on and subsequently grown within a lter based microuidic holder.Additionally, using cell lines with two different growth properties, monolayer and clustered island, it was possible to track cancer growth behavior, suggesting that the lter polymer does not negatively affect cell viability or growth properties of the cells (Fig. 3 and ESI Fig. 1 †).
Despite our attempts, we have been unable to grow captured CTCs from cancer patient blood samples using published methods. 24,29,36,375,37 As we observed no inhibitory effect on cell growth on the lters, we can only theorize that a lack of optimized culture parameters for propagating CTCs from cancer patients is the likely issue.As such, once CTC culture is possible, additional studies should be performed using the optimized media.

Multiplex CTC assays with a single device
Sequential clinical assays can be performed on cells isolated from whole peripheral blood.The entire proof of concept of multiplex in situ assaying is shown in Fig. 3. Aer cells are isolated, they can be cultured (Fig. 3A and B), and identied by chromogenic or uorescent stains (Fig. 3A-E) and then evaluated for other subtyping assays (Fig. 3E-H).In theory the entire process could include CTCs in blood isolated by ltration and then either grown in tissue culture media; and/or stained; and/ or molecularly characterized by FISH; and/or removed for single cell analysis; and/or histopathologically characterized by H&E stain.
Aer optimizing the isolation and analytical procedures using cell lines (Fig. 1A) we transitioned to CTCs isolated from patient samples (Fig. 1B).Sixteen breast and prostate cancer patient samples were ltered and stained for classical CTC markers.Since the CellSieve™ low shear stress has been shown to preserve ne intracellular structures for cytological analysis; 6,25 we assessed the clinical ramications of cytological subtyping.In patient derived CTCs, we observed cells in various stages of the cell cycle, including apoptosis, (Fig. 4A and B) and mitosis (Fig. 4C and D).These CTC subtypes have been show as clinically relevant as the presence of apoptotic CTCs are associated with better prognosis than the presence of mitotic CTCs. 6,23n standard tissue biopsies, cancer cells are rst identied and graded and then cells are subtyped using a multitude of additional biomarkers (i.e.Ki-67, ER, PR, HER2, vimentin, etc.).In most other liquid biopsy techniques, CTCs are identied, enumerated, and might be subtyped, for a total of 2-3 biomarkers. 8,38For CTCs to yield similar clinical information as tissue biopsies, multiple additional markers must be used for subtyping CTCs. 5,11,23,25,26,28To establish a multi-biomarker panel, we took advantage of the anti-CD45 antibody, which is Cyanine5 labeled and is negative in CTCs; and therefore is open to staining with a CTC reactive Cyanine5 uorescent antibody.Additionally, the uorophore R-phycoerythrin (PE) attached to the EpCAM marker is bright but it is not photostable and prone to bleaching.Aer imaging the EpCAM, we used the photo instability to bleach the PE uorescence signal by illuminating the cells for $10 seconds at 560 nm, thus facilitating the use of a new PE-labeled antibody.For illustration purposes, aer bleaching, the samples were re-stained and subtyped with an antibody mixture of CXC chemokine receptor type 4 (CXCR4)-PE and vimentin-euor 660 (Fig. 5).
The ability to continue with further molecular characterization of CTCs using additional proling greatly enhances their clinical utility.Thus far, we have described the ability to isolate, identify, proteomically subtype, genomically subtype and nally archive patient derive CTCs.We accomplish this all from a single sample in a sequential multiplexing manner to probe each CTC as an individual cell, or as a population of cells, all in relation to real world clinical utility.

Capture and backwash of tumor cells spiked into whole blood using CellSieve™ microlters
Previously we demonstrated that tumor cells spiked into whole blood can be efficiently captured ($90%) by CellSieve™ lters. 17ince many analytical methods require CTCs to be in solution Fig. 3 Isolation, culture and expansion of cells isolated on CellSieve™.(A) Live MCF-7 cells spiked into vacutainers, isolated by filtration and grown on the filter for 2-3 weeks.The 3 dimensional clustering attributed to this cell line can be seen on the filter.(green ¼ anti-cytokeratin, blue ¼ DAPI) (B) live PANC-1 cells spiked into vacutainers, isolated by filtration and grown on the filter for 2-3 weeks.This cell line can be seen growing as a monolayer on the filter.(C) SKBR3 cancer cell line is spiked into blood collected by CellSieve™.The CTCs are identified using presence of anti-cytokeratin and anti-EpCAM, with absence of anti-CD45.After CTCs are counted the cells are subtyped by HER2 FISH.(D) Live SKBR3 cells spiked into vacutainers, isolated by filtration and grown on the filter for 2-3 weeks.The expanded colonies can be directly analyzed as a whole colony, or as individual cells, molecularly by HER2/CR17 FISH analysis.(E) After filtration, a single CTC can be identified and harvested using a micropipette.(F) Removal of a single cell for downstream analysis (i.e.whole genome amplification, mRNA analysis).(G) After filtration, cells can be identified with histopathological stains (e.g.H&E) for cytological analysis or (H) after H&E, external cell structures can be analyzed by SEM.phase (i.e.mRNA, single cell WGA, etc.), we examined whether cancer cells could be released from the lter aer capture.For proof of concept, we determined the release capability of the assay by rst spiking a known number of xed cancer cells into whole blood and capturing them by ltration, as described (Fig. 1A). 17The captured cells are not covalently xed onto the CellSieve™ lter and can therefore be easily eluted from the lter by injecting buffer through the lter holder in a retrogressive manner.Aer capture and wash, the cells were eluted from the lter using a backwash procedure, which consist of pushing buffer through the lter outlet and collecting the eluted cells into a 15 ml conical tube (ESI Fig. 2 †).The eluted cells were then applied to a second lter to calculate both the capture efficiency and release rate of the cancer cells.
Using MCF-7 tumor cells we nd that cell capture efficiency is $90%, consistent with our previous results, 17 and 97% AE 2% of the MCF-7s were released from the rst lter (Fig. 6).We then enumerated white blood cells (WBC) that were captured along with the tumor cells to determine the rates of contaminating WBCs.Using 24 hour old blood, collected in CellSave tubes, we nd that that $30 000 WBCs were captured along with the tumor cells on the second lter with a calculated release rate of $97% (Fig. 6).However, being that human blood contains $10 Â 10 7 WBCs per sample 39 this is a 10 000-fold purication, or a >99.9% removal of the WBCs from the blood sample, while retaining 90% of the spiked cancer cells.As patient derived CTCs are subjected to shear stress in the circulation and are considered "fragile" we looked to test patient isolated CTCS. 40o examine whether this backwash procedure can be performed Fig. 4 Cytological analysis and subtyping of CTCs from patients (A) CTC from a breast cancer patient categorized as early apoptotic with punctate cytokeratin, and an nucleus that appears as malignant with an abnormal salt-and-pepper pattern.This CTC subtype is associated with a favorable outcome. 6,19(B) CTC from a breast cancer patient categorized as late apoptotic with punctate cytokeratin and a nucleus which also appears punctate, or blebbing.This CTC subtype is associated with a favorable outcome.(C and D) CTCs from 2 breast cancer patients in the final stages of division (i.e.telophase/cytokinesis).This CTC subtype is associated with poor prognosis.Scale ¼ 30 mm box.on CTCs from cancer patient blood samples we used duplicate 7.5 ml blood samples collected from 16 cancer patients.One 7.5 ml tube was used to capture and analyze CTCs using our standard staining protocol, 17 while in parallel, a second 7.5 ml blood sample from the same patient was used to evaluate the capture and release rates.CTCs on both lters were then identied by uorescent enumeration as previously described. 17y running an interassay comparative study we nd a CTC correlation of R 2 ¼ 0.97, p < 0.001, between the two assays indicating high concordance.Additionally, by purifying patient derived CTC, this data suggests that the CellSieve™ material is not reactive with circulating cells and that CTCs can be isolated and resuspended for additional downstream experimentation.

Conclusions
While many technologies have been developed to capture CTCs from cancer patients, the rarity and fragility of CTCs coupled with the lack of commercially available platform technologies have limited the broad scale CTC analysis. 5,9,12Additionally, most CTC isolation platforms, are not conducive to multiple downstream clinical assays (i.e.Blood smears do not retain viability, microuidics are slow, and density gradients require additional purication, etc.). 5,9,12,18,21,35These facts make detailed clinical testing and replication of CTC analysis difficult.We have previously described the ability to isolate, identify and test clinical assays on single CTCs from patients using commercial microlters.Here we describe the next step in a clinical system, a disposable device that can be used as an all in one multiplexing platform with commercially available components (Fig. 2).We used this device to show initial proof of principle.We rst described an optimal CTC workow (Fig. 1) which would allow: (1) CTC isolation, (2) CTC culture, (3) CTC identication and (4) multiplex subtyping on a molecular and proteomic level.Cancer cells spiked into blood samples showed that microlters were both conducive to culture, and the ltration process did not appear to alter cell growth behavior (Fig. 3).We then stained the cultured cells with CTC identication markers (i.e.cytokeratin and EPCAM) followed by FISH analysis, H&E histopathological stains, single cell separation, and numerous additional cell-based analysis (Fig. 3).To further elaborate on the possible downstream processing possibilities, we tested cancer cell elution from the lter to provide CTCs in suspension as required (i.e.single cell WGA, mRNA analysis, pharmacokinetics, etc.), and successfully applied this procedure to cancer patient CTCs.We showed that patient derived CTCs easily release from the lters, and thus would be a simple method to rapidly and efficiently purify CTCs for assays requiring suspended cells (Fig. 6).
The main advantage of tissue biopsies over liquid biopsies is the plethora of clinically validated proteomic and genomic subtyping assays that can be performed on the samples.Our proof of principle study demonstrates that precision micro-lters are ideal lab-on-a-chip platforms for studying CTCs, applicable to downstream analytical and staining techniques.Although staining CTCs with basic epithelial and tumor specic cell markers can determine their tissue origin, expanded molecular analysis can reveal specic tumor expressed mutations/amplications. We have illustrated the rst uidic platform which isolates CTCs applicable for analysis using standard tissue protocols.Histopathological cell characteristics, commonly applied to cytology samples, can be applied to these CTCs, e.g.apoptotic and mitotic events present in CTCs (Fig. 4). 6,23Considering that mitosis in tumor cells is used as a predictive factor to inform therapeutic decisions, the ability to analyze CTCs using a mitotic index is likely to similarly inform therapy decisions during the life time of treatment.Furthermore, we suggest that histopathological assessment of CTCs can be followed by sequential proteomic and genomic proling of cells, allowing sequential testing in regards to predictive medicine.
Despite the harsh environment of whole blood, the shear stress of isolation and the fragility of CTCs, we showed that that it is possible to purify patient CTCs while retaining the clinically important cytological information.We used this cytological based assessment in the same manner as classical histopathology to determine prognostic signicance. 6,23We then went further, showing the ability to additionally subtype patient CTCs using a number of biomarkers within the same device.Again, as a proof of principle we added the mesenchymal marker vimentin, and the motility marker CXCR4, to show that proteomic multiplexing of CTCs is possible.While cell line propagation was successful, our initial attempts to culture CTCs from patients did not succeed.However, being that no group has consistently cultured CTCs, likely do to the high mortality of CTCs, 24,28,29 we did prove that lters can be used as a culture surface.We showed that cells lines grew equivalent to standard culture techniques, unaffected by the lters or the ltration process.This is in contrast to other CTC isolation technologies which do not culture directly within their systems, but require additional steps to remove cells prior to culture.Using a single platform for all applicable assays allows for a more streamlined all-in-one assay which includes isolation, culture, proteomic testing and genomic testing without cell loss.Here we describe that an all-in-one system is feasible and that our initial testing using clinical patient samples already allows for in depth subtyping on a histopathological basis, which has not been described previously.While these data clearly call for additional studies, we present a simple, commercially applicable method, to purify CTCs, identify the cells with numerous biomarkers, while retaining the ability to characterize the CTCs with a variety of downstream techniques.

Fig. 2
Fig. 2 Flow through device with all-in-one reaction chamber.(A) The microfilter chip device consists of a holder and removable microfilter.(B) The device is designed with a reaction chamber which can be used run assays without then need to transfer the cells.(C and D) The entire device connects to sterile disposable syringes and a medical pump which allows uniform flow through the filter.

Fig. 5
Fig.5Bleaching and restaining CTCs After identifying and imaging patient derived CTCs using epithelial cell markers (e.g.cytokeratin + , EpCAM + and CD45 À ), the PE fluorescence from EPCAM was bleached, freeing the channel for an additional marker.The CD45 was negative, allowing the channel to remain open for an additional marker.The DAPI channel and the cytokeratin-FITC channel remained unchanged, and can be used to identify the CTCs after restaining.The sample was then restained with the mesenchymal marker vimentin with efluor 660 and a stromal regulation marker CXCR4 with PE.Scale ¼ 72 mm box.

Fig. 6
Fig. 6 CTC release efficiency from microfilters for downstream analysis.To determine the ability of the CellSieve™ filters to backwash cells off the filters.MCF-7 cells ($50 cells) were spiked into 7.5 ml normal blood and filtered.Cells were then backwashed off the filters and counted, as was the number of both CTCs and WBCs remaining on the filter.Y-axis shows the percentage of cells captured on the second filters versus cells still remaining on the first filters.Black bar, MCF-7 cells backwashed off the first filter and captured on the second filter versus cells remaining on the first filter.Striped bar ¼ normal WBCs backwashed off the first filter and captured on the second filter versus cells remaining on the first filter.White bar, 16 patient samples of CTCs processed as described for the MCF-7 cells.Striped bar, same 16 patient samples of WBCs backwashed off the filter versus remaining on the filter as described above.