Flexible Micro Spring Array Device for High ... - Semantic Scholar

Clinical Chemistry 60:2 323–333 (2014)

Molecular Diagnostics and Genetics

Flexible Micro Spring Array Device for High-Throughput Enrichment of Viable Circulating Tumor Cells Ramdane A. Harouaka,1,2 Ming-Da Zhou1,2, Yin-Ting Yeh,1,2 Waleed J. Khan,1,2 Avisnata Das,3 Xin Liu,3 Christine C. Christ,3 David T. Dicker,3 Tara S. Baney,2 Jussuf T. Kaifi,4 Chandra P. Belani,3 Cristina I. Truica,3 Wafik S. El-Deiry,3 Jeffrey P. Allerton,2 and Si-Yang Zheng1,3*

BACKGROUND: The dissemination of circulating tumor cells (CTCs) that cause metastases in distant organs accounts for the majority of cancer-related deaths. CTCs have been established as a cancer biomarker of known prognostic value. The enrichment of viable CTCs for ex vivo analysis could further improve cancer diagnosis and guide treatment selection. We designed a new flexible micro spring array (FMSA) device for the enrichment of viable CTCs independent of antigen expression. METHODS: Unlike previous microfiltration devices, flexible structures at the micro scale minimize cell damage to preserve viability, while maximizing throughput to allow rapid enrichment directly from whole blood with no need for sample preprocessing. Device performance with respect to capture efficiency, enrichment against leukocytes, viability, and proliferability was characterized. CTCs and CTC microclusters were enriched from clinical samples obtained from breast, lung, and colorectal cancer patients. RESULTS: The FMSA device enriched tumor cells with 90% capture efficiency, higher than 104 enrichment, and better than 80% viability from 7.5-mL whole blood samples in ⬍10 min on a 0.5-cm2 device. The FMSA detected at least 1 CTC in 16 out of 21 clinical samples (approximately 76%) compared to 4 out of 18 (approximately 22%) detected with the commercial CellSearch® system. There was no incidence of clogging in over 100 tested fresh whole blood samples. CONCLUSIONS: The FMSA device provides a versatile platform capable of viable enrichment and analysis

1

Micro & Nano Integrated Biosystem (MINIBio) Laboratory, Department of Biomedical Engineering and Materials Research Institute, Pennsylvania State University, University Park, PA; 2 Penn State Hershey Cancer Institute, State College, PA; 3 Penn State Hershey Cancer Institute, Hershey, PA; 4 Division of Surgical Oncology, Penn State Milton S. Hershey Medical Center, Hershey, PA. * Address correspondence to this author at: N-238 Millennium Science Complex, University Park, PA 16802. Fax 814-863-0490; e-mail [email protected]. Disclaimer: The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions. The content is solely the

of CTCs from clinically relevant volumes of whole blood. © 2013 American Association for Clinical Chemistry

Cancer remains a leading cause of death worldwide, and the ability of malignant tumors to release circulating tumor cells (CTCs)5 that travel through the bloodstream and invade distant organs is responsible for over 90% of these deaths (1 ). Little is understood of the mechanisms by which CTCs spread and settle in metastatic niches, and there is a clear need to obtain viable CTCs from clinical samples for comprehensive analysis. The fundamental challenge with analyzing CTCs is the fact that they typically occur at an extremely low concentration of only a few tumor cells among millions of nucleated cells in peripheral blood (2 ). Several methods have been proposed for CTC isolation from blood. Immunoaffinity-based CTC enrichment techniques use capture antibodies to target specific proteins, such as epithelial cell adhesion molecule (EpCAM) (3– 6 ), cytokeratins (7 ), prostate-specific antibodies (8 ), and selectin-mediated antibody capture (9 ). CellSearch® is currently the only US Food and Drug Administration– cleared instrument for CTC enumeration in breast (3 ), prostate (10 ), and colorectal (11 ) cancers. In breast cancer, CTC analysis by CellSearch has been suggested as a surrogate endpoint superior to current radiologic imaging for predicting overall patient survival (12 ), and CTC monitoring during therapy was reported to predict progression-free survival and overall survival (13 ). The variable expression of cell antigens presents challenges for enrichment by use of immunoaffinitybased methods. In prostate cancer, for example,

responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Received March 25, 2013; accepted September 9, 2013. Previously published online at DOI: 10.1373/clinchem.2013.206805 5 Nonstandard abbreviations: CTC, circulating tumor cell; EpCAM, epithelial cell adhesion molecule; EMT, epithelial–mesenchymal transition; FMSA, flexible micro spring array; PDMS, polydimethylsiloxane; inch WC, inches of water column; DPBS, Dulbecco’s PBS; DAPI, 4⬘,6-diamidino-2-phenylindole; GFP, green fluorescent protein; PDT, population-doubling time; NSCLC, non–small cell lung cancer.

323

EpCAM has been reported to be expressed in 71% of tumor cells (14 ). Expression of EpCAM is variable across other epithelial cancers, with most soft-tissue tumors and all lymphomas being negative. More importantly, clinically relevant subpopulations of CTCs may go through an epithelial–mesenchymal transition (EMT) or exist as cancer stem cells (15–17 ). In each case these cells may lose the epithelial phenotype and could be excluded from immunological enrichment targeting epithelial antigens. Other approaches for CTC enrichment do not rely on specific antigen expression. Density-based gradient centrifugation segregates CTCs in the mononucleocyte fraction away from the more numerous erythrocytic and granulocytic fraction (18 ). CTCs may also be isolated on the basis of size, because they are generally larger than cells of hematopoietic origin in blood (19 ). CTCs have been observed to be larger and morphologically distinct from leukocytes by use of an “enrichmentfree” scanning approach that plates all nucleated cells in blood after erythrocytic lysis (20 –22 ). Cell trap arrays inside microfluidic chambers can enrich CTCs on the basis of size and deformability (23 ). Inertial flow fractionation was demonstrated to enrich tumor cells with a higher throughput than that of conventional microfluidic devices (24 ). Microfiltration allows rapid processing of clinically relevant volumes of blood. Since the invention of track-etched polymer filters (25–27 ) in the 1960s, these devices have been widely used in biological research and clinical practice for cell enrichment. More recently, track-etched polycarbonate filters were used for CTC enrichment and enumeration from fixed blood samples (19, 28 ). To improve clinical CTC enrichment for enumeration, pore-shaped filters were designed and fabricated out of a single 10-␮m–thick layer of parylene by photolithography (29 ). This device was used in a blind comparison study to identify CTCs in 51 of 57 cancer patients compared with only 26 patients detected by the CellSearch method (30 ). Although useful for CTC detection, microfiltration applies concentrated stresses that affect the viability of captured cells (31, 32 ). Efforts have been made toward viable CTC enrichment by designing 3-dimensional structures, changing microfilter geometry, and increasing porosity (i.e., the percentage of open surface area to the total surface area) (32, 33 ). Here we describe a new flexible micro spring array (FMSA) device that uses an innovative design to enable viable enrichment of CTCs directly from whole blood with no requirement for preprocessing (e.g., dilution, erythrocytic lysis, centrifugation). Viable CTCs can be captured from a tube of blood (approximately 7.5 mL) and made available for downstream analysis within 10 min. This rapid enrichment is desirable to minimize 324 Clinical Chemistry 60:2 (2014)

disruption to cells and potential alterations of cell phenotype that could compromise analysis. FMSA performance is achieved by: (a) implementing flexible polymer micro springs as effective microfiltration structures that enrich CTCs on the basis of their size and deformability, while mitigating disruption to cells on initial impact; (b) maximizing device porosity, which increases sample capacity for a given device surface area and allows processing of whole blood without clogging; and (c) minimizing operation pressure and applying a pressure regulation system to reduce mechanical stress experienced by cells during enrichment to maintain their viability. Materials and Methods DEVICE FABRICATION

The FMSA device was microfabricated from parylene polymer with alterations to a previously described process (29 ). First, parylene-C was conformally deposited onto a prime silicon wafer. An aluminum mask was then added by thermal evaporation and patterned by photolithography. The micro spring geometry was etched into the parylene layer by reactive ion etching using O2 plasma. The etched parylene layer was then released and cut into individual devices. A completed device is shown in Fig. 1A with magnified views of the functional micro spring structures. FILTRATION ASSEMBLY AND PRESSURE REGULATION

Blood samples were passed through the FMSA device under precisely regulated pressures by using the setup depicted in Fig. 1B. During enrichment the FMSA device was retained between 2 sealing O-rings formed from polydimethylsiloxane (PDMS) (DOW Corning) and clamped within a plastic housing. The top housing layer incorporated a sample-loading chamber, and the bottom layer included a luer adapter for integration with a waste trap. This housing assembly acted as a reliable support for the FMSA membrane and eliminated the possibility of fluid leakage. Driving pressure measurements were taken with a pressure sensor (ASDXL; Honeywell) with a range of 0 to ⫾10 inches of water column (inch WC). A fine mechanical control valve (McMaster–Carr) was used to set the desired filtration pressure. CELL CULTURE

Human breast cancer cell lines (MCF-7 and MDA-MB 231) and melanoma cell lines (C8161 and WM35) were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 ␮g/mL streptomycin (Invitrogen) in humidified incubators at 5% CO2 and 37 °C ambient temperature.

Viable Enrichment of Circulating Tumor Cells

Fig. 1. Model of an FMSA device. (A), Images of a fabricated FMSA device with magnified top views of the micro spring structures. Blood samples flow directionally through the image plane. Gap width is labeled in the right panel. Scale bars are 20 ␮m. (B), Filtration setup including FMSA device housing and pressure control system.

COLLECTION OF BLOOD SAMPLES

Healthy blood was obtained from consented donors at the Penn State General Clinical Research Center according to an institutional review board–approved protocol. Clinical samples were obtained with consent from advanced breast, lung, and colorectal cancer patients at the Penn State Hershey Medical Group at Benner Pike and the Penn State Hershey Medical Center according to institutional review board–approved protocols. Samples were drawn into 10-mL EDTA (K2) tubes (Vacutainer; Becton Dickinson) from peripheral venipuncture or from a central venous line. Blood in EDTA tubes was processed within 24 h to facilitate optimal filtration conditions (see Fig. 1 in the Data Supplement that accompanies the online version of this report at http://www.clinchem.org/content/vol60/ issue2). Patient samples were concurrently drawn into CellSave tubes (Veridex) for comparative analysis with the CellSearch system. The 7.5-mL blood samples in the CellSave tubes were processed according to the manufacturer’s instructions by a technician trained and certified by the manufacturer to operate the

CellSearch instrument at the Penn State Hershey Medical Center. IMMUNOFLUORESCENT DETECTION

FMSA devices were washed with 5 mL Dulbecco’s PBS (DPBS) (Invitrogen) immediately after enrichment and then fixed with 4% paraformaldehyde (VWR) for 20 min. Cells were permeabilized in 0.3% Triton X-100 (VWR) for 10 min. We added 1 ␮g/mL of 4⬘,6diamidino-2-phenylindole (DAPI) (Invitrogen) as a nucleic acid stain and then blocked the samples with 5% goat serum (Sigma-Aldrich). Cells were subsequently incubated with 1 ␮g/mL mouse monoclonal anticytokeratin 8/18/19 (Abcam, clone 2A4) and then 10 ␮g/mL goat antimouse IgG conjugated to DyLight 488 (Thermo Scientific). Cells were blocked again with 100 ␮g/mL mouse IgG solution (Sigma-Aldrich) and incubated with 0.5 ␮g/mL monoclonal mouse antiCD45 conjugated to Alexa Fluor 647 (Santa Cruz, clone 35-Z6) and 0.5 ␮g/mL monoclonal mouse antivimentin conjugated to Cy3 (Sigma-Aldrich, clone V9). All blocking and antibody incubation steps were carried Clinical Chemistry 60:2 (2014) 325

Fig. 2. Capture efficiency and enrichment factor. Capture efficiency measured (A) under various gap widths and driving pressures (n ⫽ 4) and (B) for simulated 7.5-mL clinical samples (n ⫽ 3). Enrichment factor from whole blood determined (C) under various gap widths and driving pressures (n ⫽ 3) and (D) for simulated 7.5-mL clinical samples (n ⫽ 3). Each data point shows the mean value and SD.

out for 8 –12 h at 4 °C (see online Supplemental Figs. 2 and 3 for antibody control images). Results EFFECT OF GAP WIDTH AND DRIVING PRESSURE ON CAPTURE EFFICIENCY

Gap width is the geometric distance between the micro spring structures (Fig. 1A) and it is the key design parameter for cell capture. It is defined by microfabrication and is uniform on a particular device. The effects of the gap width and driving pressure on cell recovery were investigated. Capture efficiency of the FMSA device was defined as the ratio of tumor cells retained on the device after enrichment to the number of tumor cells present in the sample before enrichment: CE ⫽

共n T 兲 after ⫻ 100% 共n T 兲 before

(Eq. 1)

where CE is the capture efficiency and nT is the number of tumor cells. 326 Clinical Chemistry 60:2 (2014)

Four cancer cell lines (MCF-7, MDA-MB 231, C8161, and WM35) were used separately in model experiments to characterize capture efficiency. We spiked 35–70 cells fluorescently prelabeled with Acridine orange (DYNAL) into 0.5 mL of peripheral blood obtained from a healthy donor. The spiked samples were processed through the FMSA device to investigate the effects of varying gap widths under various set driving pressures for enrichment. The gap widths were 4, 5, 6, and 7 ␮m, and driving pressures were investigated between 1 and 10 inch WC. After enrichment the devices were washed with 1–2 mL of DPBS. Captured cells were observed and enumerated under a fluorescence microscope. Across the 4 tested cell types the mean capture efficiency decreased from ⬎90% to ⬍60% as the gap width was increased from 4 to 7 ␮m (Fig. 2A; also see online Supplemental Fig. 4). Capture efficiency was then evaluated from full tubes of blood to simulate clinical samples. We spiked 50 –75 cells from each cell line into 7.5 mL of healthy donor blood and passed them through a 5-␮m gap width FMSA under 1 inch WC pressure. MCF-7, MDA-MB 231, and C8161

Viable Enrichment of Circulating Tumor Cells

cells were fixed and stained for immunofluorescent detection and enumeration according to the protocol described above, and WM35 cells were fluorescently prelabeled because of a lack of CK 8/18/19 expression (see online Supplemental Fig. 3). The mean (SD) measured capture efficiencies were 92.6% (3.1%) (n ⫽ 12), as shown in Fig. 2B. At least 2 out of 5 spiked cells were recovered when the experiment was repeated with a lower initial tumor cell concentration (n ⫽ 6) (see online Supplemental Table 1). ENRICHMENT AGAINST LEUKOCYTES

Because erythrocytes are small enough to pass through the gaps between the micro springs, given sufficient elution, the concern for CTC purity mainly involves leukocytes. The enrichment factor was defined as the ratio of tumor cells to leukocytes after enrichment divided by the same ratio before enrichment: E⫽

共n L 兲 before 共n L 兲 before 共n T /n L 兲 after ⫽ CE ⫻ ⬵ 共n T /n L 兲 before 共n L 兲 after 共n L 兲 after (Eq. 2)

where E is the enrichment factor and nL is the number of leukocytes. The effect of the range of capture efficiencies observed under tested conditions on the enrichment factor is marginal, thus allowing the enrichment factor to be characterized independently of CTC capture efficiency (Eq. 2). One milliliter of whole blood was passed through FMSA devices of the same gap widths and under the same driving pressure conditions investigated for capture efficiency. Devices were then washed with DPBS, fixed with 4% paraformaldehyde for 20 min, and stained with DAPI to clearly elucidate cell nuclei. Images were acquired at various locations on the device at 4⫻ and 10⫻ magnification and processed using the software ImageJ (NIH) to enumerate cells based on a set threshold for fluorescence intensity. The calculated enrichment factor was plotted as a function of gap width and filtration pressure (Fig. 2C). The mean enrichment factor from the 7.5-mL simulated clinical sample experiments was determined to be approximately 1.4 ⫻ 104 (n ⫽ 12), as shown in Fig. 2D. EFFECT OF DRIVING PRESSURE ON CELL VIABILITY

To assess the benefit of pressure regulation on cell viability, 50 green fluorescent protein (GFP)-labeled MDA-MB 231 cells were spiked into 1 mL of peripheral blood and filtered through the FMSA device at driving pressures of 1, 2, 5, and 10 inch WC. After enrichment the devices were washed with 1–2 mL of DPBS and stained with a LIVE/DEAD cell assay (Invitrogen) consisting of the viable cell indicator Calcein-AM blue (8 ␮mol/L) and the exclusion dye ethidium homodimer-1 (4 ␮mol/L).

The devices were observed under a fluorescence microscope to determine the number of viable GFP-labeled cells that were positive for the Calcein dye (blue) and negative for the ethidium homodimer-1 (red). Corrected viability was determined as the viable cell recovery divided by the previously measured capture efficiency for MDA-MB 231 cells at each pressure condition. Limiting the driving pressure to 1 inch WC allowed a viable cell recovery of ⬎80%, whereas 5 inch WC driving pressure reduced viable recovery to below 50%, and 10 inch WC pressure effectively prohibited any viable cell capture (Fig. 3A). CELL PROLIFERATION AFTER ENRICHMENT

After enrichment, breast and melanoma cancer cell lines were cultured directly on the FMSA device or eluted off the device with reverse pressure into 12-well culture plates. To quantify proliferability, 10 GFPexpressing C8161 cells were spiked into 1 mL of peripheral blood and passed through a 5-␮m gap width FMSA at 5 inch WC driving pressure. The device was washed with 1–2 mL of DPBS and cultured under the conditions described in the Materials and Methods section. GFP labeling facilitated long-term tracking of the individual cells, which were imaged periodically with a fluorescence microscope over 20 days (Fig. 3D; also see online Supplemental Video 1). A plot of the cell number in a particular field of view shows a characteristic logphase growth pattern (Fig. 3C). A population-doubling time (PDT) of 1.6 days was determined from the linear fit of the semilog plot. As positive controls, the PDTs of C8161 cells proliferating on tissue culture polystyrene dishes (Becton Dickinson) with various seeding concentrations were also measured. With the optimal seeding concentration previously determined to be 4000 cells/ cm2, cells were seeded at 6⫻, 12⫻, and 96⫻ diluted concentrations to account for the lower numbers of CTCs on the FMSA device after enrichment. PDTs were calculated to be 1.3, 1.8, and 3.5 days for cell cultures started with 6⫻, 12⫻, and 96⫻ diluted seeding, respectively. CTC DETECTION IN BREAST, COLORECTAL, AND LUNG CANCER

Immunofluorescent detection was established to identify CTCs from clinical samples after viable enrichment with the FMSA device, as described above. CTCs were identified as large nucleated cells that stained positively for cytokeratins 8/18/19 and negatively for CD45. Contaminant leukocytes were noticeably smaller in size and stained positively for CD45. Captured CTCs comprised a wide range of sizes and morphologies (Fig. 4). Table 1 shows the results of blind comparative detection by the CellSearch system and the FMSA device of CTCs enriched from equivalent 7.5-mL clinical samples collected from 12 patients. At least 1 CTC in approximately 76% (16/21) of clinical samples was deClinical Chemistry 60:2 (2014) 327

Fig. 3. (A), Effect of enrichment driving pressure on cell viability. (B), LIVE/DEAD assay with arrows indicating viable (a) and dead (b) tumor cells and viable (c1) and dead (c2) leukocytes. Scale bars are 20 ␮m. (C), Growth curves of FMSA-enriched tumor cells compared with controls. (D), Colony expansion on the FMSA device after enrichment from blood. Scale bars are 50 ␮m.

tected by the FMSA device as opposed to 1 in approximately 22% (4/18) by CellSearch. The FMSA enriched CTC microclusters, which were observed as aggregate masses of 2 or more distinct CTCs that occasionally included leukocytes. Microclusters and multinucleated CTCs were observed in patients with all 3 cancer types, as confirmed with confocal imaging (Fig. 5; also see online Supplemental Video 2). Approximately 35% of all CTCs occurred in microclusters found in approximately 44% (7/16) of CTC-positive samples. Thirteen of 14 tested samples contained CTCs that stained positively for the EMT marker vimentin, which comprised approximately 86% of all assayed CTCs. No morphologically distinct cells with the CTC immunophenotype were detected in any of 10 negative controls obtained from healthy donors of various ages. Discussion We report here the demonstration of a highthroughput versatile platform capable of viable en328 Clinical Chemistry 60:2 (2014)

richment of CTCs independent of antigen expression. The FMSA device allows mechanical separation of CTCs at the microscale, while taking advantage of high throughput and rapid processing speed. An FMSA device with an effective area of 0.5 cm2 can reliably process 7.5 mL whole blood stored in an EDTA tube for up to 24 h with ⬍1 inch WC driving pressure without clogging. The maximal blood volume that can be processed by a single FMSA device is much higher than the standard 7.5-mL blood volume for CTC enumeration (see online Supplemental Fig. 1). This increased sample capacity is attributed to the FMSA high-porosity design of 30%–50%, which is much higher than that of most previous microfiltration devices. CTCs are obtained directly from clinically relevant volumes of whole blood within minutes, which is helpful to maintain cell viability and avert phenotypic changes to cells that may confound analysis. Precise pressure regulation minimizes mechanical stress to avoid damaging cells during enrichment.

Viable Enrichment of Circulating Tumor Cells

Fig. 4. Immunofluorescent detection of FMSA enriched CTCs in clinical blood samples. Cells were stained for cytokeratin 8/18/19 (green), vimentin (red), CD45 (magenta), and DAPI (blue). Examples of differential interference contrast (DIC)/fluorescence and fluorescence composite images of CTCs detected in breast (A and B), NSCLC (C and D) and colorectal (E and F) cancer. Scale bars are 20 ␮m.

The FMSA platform can be easily modified to suit a particular application. Characterization data (Figs. 2 and 3) demonstrate that the device parameters and filtration conditions may be optimized to achieve the desired capture efficiency, enrichment, and preservation of cell viability. Gap width is the most important geometrical design parameter. Increasing the gap width reduces capture efficiency while improving enrichment against leukocytes. Driving pressure is an important operation parameter that may be controlled precisely during enrichment. Increased pressure forces cells through the micro spring gaps, thus reducing capture efficiency at every gap width. The best capture efficiency observed across the 4 cell lines was 93.6% (6.3%) (n ⫽ 9), with a gap width of 4 ␮m under 1 inch

WC driving pressure. On the other hand, increasing pressure from 1 to 10 inch WC improves enrichment by up to 10-fold. However, filtration pressure has a drastic effect on cell viability. Our data suggest that viable CTC enrichment by microfiltration requires the driving pressure to be restricted to an extremely low level. This low driving pressure can potentially reduce flow rates during enrichment and increase the risk of clogging. With the novel high-porosity flexible micro spring design, the FMSA device does not experience clogging with fresh whole blood samples. With one potential goal being the primary culture of CTCs, a representative model system was investigated for cell proliferation. Successful cell proliferation would require that the FMSA device does not cause Clinical Chemistry 60:2 (2014) 329

330 Clinical Chemistry 60:2 (2014)

a

BCA-3

BCA-4

BCA-5

3

4

5

CRC-2

CRC-3

20

21

M, F

M

M

22–68

58

47

40

Not applicable

IV

IV

IV

Not applicable

Metastatic CRC

Metastatic CRC

Metastatic CRC

Not applicable

Chemotherapy

Chemotherapy

Chemotherapy

NA

0

0

NA

BCA, breast cancer; ER, estrogen receptor; PR, progesterone receptor; Her2, human epidermal growth factor receptor 2; NA, not available; CRC, colorectal cancer.

22–31 (n ⫽ 10)

Healthy donor controls

CRC-1

19

M

6

End of treatment

Patients with CRC

18

3

0 Second cycle chemotherapy

0

First cycle chemotherapy

Metastatic lung adenocarcinoma

0

0

0

NA

NA

0

0

17

IV

0 0

16

64

Pretreatment

F

15

NSCLC-4

Fourth cycle chemotherapy

Metastatic lung adenocarcinoma

14

Recurrent

Pretreatment

78

Second cycle chemotherapy

F

12

NSCLC-3

End of treatment

13

Second cycle chemotherapy

Metastatic lung adenocarcinoma

11

IV

10

66

Metastatic lung adenocarcinoma

Pretreatment

M

Chemotherapy

Third cycle chemotherapy

NSCLC-2

0

Pretreatment

BCA (ER⫹/PR⫹/Her2⫺)

Pretreatment

0

Pretreatment

BCA (ER⫹/PR⫹/Her2⫹) Metastatic BCA (ER⫹/PR⫹/Her2⫺)

32

Chemotherapy

Metastatic BCA (ER⫺/PR⫺/Her2⫺) 0

1

CellSearch CTCs

Pretreatment

Metastatic BCA (ER⫺/PR⫺/Her2⫹)

Status

9

IV

IV

III

III

IV

IV

Diagnosis

8

75

51

51

50

46

54

Stage

Second cycle chemotherapy

F

F

F

F

F

F

Sex

Age, years

7

6

NSCLC-1

BCA-2

2

Patients with NSCLC

BCA-1

Patient sample

1

Patients with BCA

a

Sample source and no.

Table 1. Detection of CTCs in clinical samples.

0

3

1

19

97

58

1

36

0

7

5

5

0

29

3

0

0

0

102

10

34

17

FMSA CTCs

0

0

0

0

0

0

0

6 (16.7%)

0

0

5 (100%)

5 (100%)

0

25 (86.2%)

0

0

0

0

72 (70.6%)

0

23 (67.6%)

15 (88.2%)

FMSA CTCs in microclusters, n (%)

0

3 (100%)

1 (100%)

18 (94.7%)

86 (88.7%)

51 (87.9%)

1 (100%)

36 (100%)

0

NA

5 (100%)

0

0

29 (100%)

NA

0

0

0

87 (85.3%)

8 (80%)

27 (79.4%)

7 (41.2%)

FMSA vimentin ⴙ CTCs, n (%)

Viable Enrichment of Circulating Tumor Cells

Fig. 5. FMSA enriched CTC microclusters and multinucleated CTCs obtained from clinical blood samples: cytokeratins 8/18/19 (green), vimentin (red), CD45 (magenta), and DAPI (blue). (A), Microcluster of 20 CTCs that positively expressed vimentin. (B), Six vimentin-negative CTCs and 2 vimentin-positive leukocytes in a microcluster. (C), Vimentin-positive and (D) vimentin-negative multinucleated CTCs (and leukocyte). Scale bars are 20 ␮m.

irreparable damage to cells during the enrichment process, allows sufficient space for cell expansion, and is conducive to cell attachment (for adherent cells). The extreme rarity of CTCs necessitates enrichment onto a small surface area to improve seeding density for cell proliferation (Fig. 3B). CTC culture would also have to overcome the contaminating presence of other cells and factors in blood. Although the optimal culture conditions for the tested cell lines are known, the best conditions for encouraging the growth of CTCs from patient samples must be experimentally determined. Several challenges remain, including the distinction that must be made between viability and proliferability, because not every viable cell will be capable of growth into a colony. Still, viable cells will provide the opportunity for valuable diagnostic and prognostic information through genetic analysis. The development of the FMSA device facilitates efforts toward viable CTC analysis and primary CTC culture.

CTCs were successfully detected from patient blood samples. Although the blind comparison to CellSearch is helpful to evaluate sensitivity of the FMSA technology, distinctions should be made between these 2 systems. CellSave tubes required for CellSearch analysis contain a preservative that prevents degradation of CTCs for up to 96 h (3 ), whereas all clinical blood samples evaluated with FMSA were collected in EDTA tubes to allow viable enrichment. Because the technologies enrich CTCs on the basis of different criteria (physical properties vs surface antigen expression), it is presumable that the enriched CTC subpopulations do not completely overlap. Therefore, clinical studies correlated to patient outcomes must be performed to determine the clinical relevance of CTCs enriched on the basis of size and deformability. Varying trends were observed in CTC counts from non–small cell lung cancer (NSCLC) patients over the course of therapy. Patient NSCLC-4 initially showed a sharp decline in Clinical Chemistry 60:2 (2014) 331

FMSA-detected CTCs after the first cycle of treatment, but then CTC counts increased sharply and the patient died within 2 months of the final blood draw. Patients NSCLC-1 and NSCLC-3, with declining or comparatively low CTC counts over the course of therapy, are alive and have survived at least 6 months from the final blood draw. Aggregate microclusters of CTCs have been previously reported in various cancer types (19, 20, 24, 34, 35 ). Tumor cell aggregates have been associated with enhanced metastatic potential in animal models (36, 37 ), although their precise clinical significance and even their ability to remain intact in circulation are not fully understood. The FMSA device detected CTC microclusters (2–20 cells) from clinical blood samples of breast, lung, and colorectal cancer patients. These microclusters were distinct from other large multinucleated CTCs with no discernible cell boundaries (Fig. 5). The gentle enrichment process using flexible microstructures at low driving pressures with no sample preprocessing may promote the preservation of CTC clusters. We observed that the majority of FMSA-enriched CTCs (approximately 86%) stained positively for the mesenchymal cell marker vimentin. Expression of vimentin intermediate filaments and downregulation of epithelial cell markers is implicated in EMT (38 ), which is considered a prerequisite for CTC dissemination. Vimentin and other EMT markers have been reported in CTCs that may or may not concurrently express epithelial cell markers (35, 39, 40 ). The FMSA does not select for epithelial

antigens during enrichment, making it ideal for characterizing cells that undergo EMT.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: J.P. Allerton, TG Therapeutics. Stock Ownership: None declared. Honoraria: None declared. Research Funding: Penn State Hershey Cancer Institute; C.P. Belani, a grant with the Pennsylvania Department of Health using Tobacco CURE Funds; S.-Y. Zheng, National Cancer Institute of the National Institutes of Health, award numbers R21CA161835 and DP2CA174508. Expert Testimony: None declared. Patents: S.-Y. Zheng, R.A. Harouaka, M.-D. Zhou, and Y.-T. Yeh, patent numbers 13/744,051 and PCT/US2013/021933. Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript. Acknowledgments: We thank the Penn State Materials Research Institute, Nanofabrication laboratory and Microscopy and Cytometry facility. Cell lines were gifted by Dr. Cheng Dong at Penn State University (melanoma), Dr. Henry Lin at the University of Alberta (breast), and Dr. Andrea Mastro of Penn State University through her colleague Dr. Danny Welch at the University of Kansas Cancer Center (GFP-labeled breast).

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