Molecular Profiling of Pooled Circulating Tumor Cells from Prostate

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Molecular profiling of pooled circulating tumor cells from prostate cancer patients using dual-antibody-functionalized microfluidic device Changqing Yin, Yuhui Wang, Jia Ji, Bo Cai, Hao Chen, Zhonghua Yang, Kun Wang, Changliang Luo, Wu-wen Zhang, Chunhui Yuan, and Fubing Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03536 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 2018

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Analytical Chemistry

Molecular profiling of pooled circulating tumor cells from prostate cancer patients using dual-antibody-functionalized microfluidic device Changqing Yin1#, Yuhui Wang1#, Jia Ji1, Bo Cai2, Hao Chen3, Zhonghua Yang4, Kun Wang5, Changliang Luo1, Wuwen Zhang1, Chunhui Yuan6*, Fubing Wang1*

1

Department of Laboratory Medicine, Zhongnan Hospital of Wuhan University,

Wuhan 430071, P.R. China 2

School of Physics and Technology, Wuhan University, Wuhan 430072, P.R. China

3

Department of Pathology, Zhongnan Hospital of Wuhan University, Wuhan 430071,

P.R. China 4

Department of Urology, Zhongnan Hospital of Wuhan University, Wuhan 430071,

P.R. China 5

Department of Laboratory Medicine, Hubei Cancer Hospital, Wuhan 430079, P.R.

China 6

Department of Laboratory Medicine, Wuhan Children's Hospital, Huazhong

University of Science and Technology, Wuhan 430016, P.R. China

#

These authors contributed equally to this work.

*

Corresponding author: Fubing Wang, Department of Laboratory Medicine,

Zhongnan Hospital of Wuhan University, No 169 Donghu Road, Wuchang District, Wuhan 430071, P.R. China. Email address: [email protected]; Tel: +86-27-67813517; Fax: +86-27-67813128; Chunhui Yuan, Department of Laboratory Medicine, Wuhan Children's Hospital, Huazhong University of Science and Technology, Wuhan 430016, P.R. China. Email address: [email protected]

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Abstract Here, we constructed a novel dual-antibody-functionalized microfluidic device by employing antibodies against PSMA and EpCAM to capture CTCs from PCa patients in order to capture multiple subpopulations of CTCs at different metastatic stages. In vitro experiments with dual capture system for capturing both LnCAP and LnCAP-EMT cells have shown significantly enhanced capture efficiency as compared to that of EpCAM single capture system. Furthermore, dual capture system could successfully identify CTCs in 20 out of 24 (83.3%) PCa patients and the CTCs counts from dual capture system were statistically correlated with TNM stage of the patients (P＜0.05), while conventional diagnostic methods, such as serum PSA level and Gleason score, failed to correlate to patient TNM stages. To further explore potential clinical application of our dual capture system, captured CTCs were recovered and subjected to qRT-PCR to quantify known factors involved in PCa development and therapy. The results demonstrated that the combined detection of SChLAP1 and PSA in CTCs is potential marker for identifying patients with metastatic PCa, while detection of AR and PD-L1 in CTCs may have the potential to determine the sensitivity of PCa patients to androgen deprivation therapy and immunotherapy, respectively. Taken together, the dual-antibody-functionalized microfluidic device established in our study overcomes the limitations of some CTCs capture platforms that only detect epithelial or mesenchymal CTCs in PCa patients, and detection of PCa-related RNA signature from purified CTCs hold great promise to offer warnings for early metastasis of PCa and may provide guidance for therapy decision. Keywords: Prostate cancer; Anti-EpCAM/PSMA-functionalized microfluidic device; circulating tumor cells; RNA signature


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Prostate cancer (PCa) is the most frequently diagnosed malignancy and the third leading cause of cancer-related death of men in the United States 1. Although the 5-year relative survival rates of patients with localized and regional PCa can reach 99%, for metastatic PCa patients, especially among the metastatic castration-resistant prostate cancer (mCRPC) patients, the 5-year survival rates can drop to lower than 30% 1. To date, early screening of PCa is predominantly depended on prostate specific antigen (PSA) test. However, it is no longer recommended not only because of growing concerns about high rates of over-diagnosis, only 25% are found to have prostate cancer as verified by prostate biopsy 2,3, but also due to the low efficiency in differentiating low-risk and aggressive types of PCa 4. In a randomized controlled trial including over 70,000 men, Andriole et. al. 5 had demonstrated that the incorporation of PSA test didn’t significantly change PCa-related mortality. Thus, the development of alternative strategies to complement PSA levels to assess disease progression status is imperative, such as the monitoring of circulating tumor cells (CTCs). The formation of cancer metastasis is mainly attributed to the presence of CTCs, which invade into the blood circulation from a primary or metastatic tumor deposits 6. Currently, the epithelial cell adhesion molecule (EpCAM) based immunocapture has emerged as the most commonly used techniques for isolating CTCs, particularly represented by the FDA-approved CellSearch system 7. Depending on Cellsearch system, CTCs have been validated as prognostic biomarker of PCa in different clinical trials

8,9

. However, about 26.2% patient with castration-resistant PCa were

negative for EpCAM-based CTCs isolation, which may be attributed to the epithelial-mesenchymal transition (EMT) during the process of tumor progression and thus a metastatic subpopulation of CTCs are negative for EpCAM expression

10,11

.

Therefore, a more robust system with capacity to capture multiple subpopulations of CTCs is an unmet medical need in the field of PCa CTCs research and clinical application. Prostate-specific membrane antigen (PSMA) is a type II transmembrane protein specific to prostate-derived epithelial cells and highly expressed by malignant prostate epithelial cells

12

. Moreover, unlike EpCAM, it has been demonstrated that PSMA ACS Paragon Plus Environment


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expression increases progressively in PCa patients with higher tumor stages, Gleason scores, metastasis status and a higher risk of relapse 13,14. Miyamoto et al 15,16 used the four immunofluorescence imaging parameters (DAPI, CD45, PSA and PSMA) had confirmed that EpCAM-captured CTCs of PCa patients showed PSMA- phenotype in the vast majority (75%-100%). Thus, Kirby et al. had substituted EpCAM with PSMA to capture CTCs from whole blood using a geometrically-enhanced differential immunocapture (GEDI) microfluidic device 17,18. However, PSMA-based capture may miss the EpCAM+ epithelial subset (only about 50% showed PSMA+) of CTCs. A high

efficiency

CTCs

capture

microfluidic

device

that

consisted

of

a

polydimethylsiloxane (PDMS) herringbone structure and a substrate of Ni micropillar arrays has been constructed in our previous study 19,20, we therefore evaluated whether the microfluidic device decorated with a combination of anti-EpCAM and anti-PSMA antibodies has the potential to enhance capture of CTCs with epithelial or mesenchymal phenotypes in PCa patients. In addition, CTCs have been demonstrated to be genetically representative of the main tumor site 21. Successful interrogation of the molecular signatures of CTCs may provide a real-time assessment of cancer metastasis biology, treatment response and avoid the necessity of repeated invasive biopsies. A number of assays have been developed to analyze RNA signatures of PCa patient derived CTCs to predict their responses to hormonal therapies, such as abiraterone and enzalutamide. Like the EpCAM-based AdnaTest, enumerated EpCAM+ CTCs are then lysed and RNA is extracted for downstream analysis with qRT-PCR. The AdnaTest includes primers against PSA, PSMA, androgen-receptor splice variant 7 (AR-V7) and the epidermal growth factor receptor (EGFR)

22

. Hormonal therapies were best for CTC- patients

with castration-resistant PCa, intermediate for CTC+/AR-V7-patients, and worse for CTC+/AR-V7+ patients 11. However, the applications of these assays have been greatly limited due to the low efficiency of current CTC capture technologies. Thus, the feasibility and sensitivity of the dual-antibody-functionalized capture system for identification of RNA signatures was further evaluated to predict the patients’ response to various therapeis in the current study. We found that the combined ACS Paragon Plus Environment

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detection of SChLAP1 and PSA in CTCs is potential marker for identifying patients with metastatic PCa, while detection of AR and PD-L1 in CTCs may have the potential to determine the sensitivity of PCa patients to androgen deprivation therapy and immunotherapy, respectively. In summary, our method has shown a higher efficacy to capture CTCs for diagnosis and development of personalized therapy for treatment of PCa. EXPERIMENTAL SECTION Fabrication of the microfluidic device and functionalization of the MBs. The microfluidic device fabrication and modification was conducted according to our previous reports

19,20

. Briefly, the microfluidic device was mainly composed of two

components: a chaotic mixer made of PDMS herringbone structure and a patterned Ni micropillar substrate. Furthermore, to functionalize the Ni micropillars, biotinylated antibodies (prepared in 1% BSA in PBS) were first conjugated to the previously prepared graphite oxide (GO)-coated Fe3O4@MBs

19

, which was coupled with

streptavidin (10 µg/mL in PBS) through the activated carboxyl groups on GO surface. The antibody-functionalized Fe3O4@MBs then can be immobilized on the micropillars under an external magnetic condition and then enabled the effective capture of cancer cells. Cells and blood sample. The prostate cancer cell lines LnCAP, PC3 and DU145 were obtained from American Type Culture Collection (ATCC) and maintained in RPMI 1640

medium

supplemented

with

10%

fetal

bovine

serum

and

1%

penicillin/streptomycin solution at 37 °C in a 5% CO2 incubator. The immortalized normal prostate epithelial cell line RWPE1 were obtained from China Center for Type Culture Collection (CCTCC) and cultured in keratinocyte serum-free media in an incubator with 5% CO2 at 37 °C. To mimic mesenchymal subset of CTCs, LnCAP prostate cancer cells were seeded in 6 cm-dishes at a density of 3 × 105 cells per dish and then stimulated with 10 ng/mL TGF-β1 (PeproTech, USA) for three days. These TGF-β1 treated LnCAP cells showing mesenchymal phenotype were designated as LnCAP-EMT. Blood samples of healthy donors and PCa patients were obtained from Zhongnan Hospital of Wuhan University under approval of Institutional Review ACS Paragon Plus Environment


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Board. Immunofluorescent microscopy. LnCAP cells were seeded in 6-cm dishes at a density of 3×105 cells per dish containing sterile glass coverslips and then were divided into control and treatment group. The LnCAP cells in the treatment group were then stimulated by the TGF-β1 as previously described. The adherent cells were next fixed in 4% paraformaldehyde for 15 min, followed by the permeabilization using 0.1% Triton X-100 at room temperature. After washing three times with PBS for 5 min each, cells were then stained with FITC-labeled anti-EpCAM (10 µg/mL in PBS) and PE-labeled anti-PSMA (10 µg/mL in BSA) for 30 min in the dark. After washing, DAPI was added to the cells to stain the nuclei for another 15 min, the cells were rinsed and photographed under an Olympus IX81 microscope (Olympus, Tokyo, Japan). Immunohistochemical

(IHC)

staining.

Formalin-fixed

paraffin-embedded

specimens were subjected to IHC staining according to our previously published protocol 23. For IHC, tissue sections (4 µm thick) was blocked with goat serum for 30 minutes, and then incubated with mouse anti-human PSMA monoclonal antibody (MXB, China) in binding buffer at 4°C overnight in a moist chamber. After being treated with corresponding secondary antibody for 30 minutes, the slides were stained with diaminobenzidine and counterstained with hematoxylin. All the images were taken under an Olympus IX81 microscope. CTCs capture and identification. The process of cell capture was conducted according to our previous literature

20

. The prepared Fe3O4@MBs were firstly

functionalized with either biotinylated anti-EpCAM antibody (R&D Systems, 10µg/mL in BSA), anti-PSMA antibody (BioLegend, clone LNI-17, 10µg/mL in BSA) or a mixture of anti-EpCAM (5 µg/mL in BSA) and anti-PSMA antibody (5 µg/mL in BSA) using streptavidin-biotin chemistry. Meanwhile, the microfluidic devices were washed three times with PBS, and then 200 µL antibody-functionalized Fe3O4@MBs was injected into the microchannel using syringe pump (Longer, China). At the same time, two NdFeB permanent magnets were put at the two sides of the PDMS for immobilizing antibody-functionalized Fe3O4@MBs onto the micropillars. Then, the ACS Paragon Plus Environment

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cell suspensions was injected into the device at a flow rate of 0.8 ml/h. After the cell capture assays, the cells that attached the micropillar device were then fixed with 4% paraformaldehyde for 15 min. Afterwards the device was refilled with Triton X-100 (0.1% in PBS) for cell permeation and blocked with BSA (3% in PBS) for 30 min. Finally, the attached cells were stained with FITC-labeled anti-CD45 (BD Bioscience, USA), PE-labelled anti-CK (BD Bioscience, USA) and DAPI (Beyotime Biotechnology, China) and then released from the Ni micropillar to count under an inverted microscope IX-81 (Olympus, Japan). Isolation of CTCs from blood with spiking cells and clinical samples. Cell capture efficiency of the optimal capture condition was validated using anti-EpCAM and/or anti-PSMA-functionalized microfluidic devices to capture target cells (LnCAP and LnCAP-EMT) spiked in 1 mL blood sample of healthy donors. After enrichment, followed by staining of anti-CK, anti-CD45, and DAPI, specifically captured LnCAP and LnCAP-EMT cells were identified and counted. For CTCs of PCa patients detection, 2 mL peripheral blood sample was injected into our microfluidic devices according to the procedure described above 20. RNA isolation, cDNA synthesis and qRT-PCR. Total RNAs were extracted from prostate cell lines and enriched CTCs using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. The isolated RNA samples were then quantified by Nanodrop 2000 spectrophotometer (Thermo Scientific Inc., USA) and reversed transcribed using a cDNA reverse transcription kit (Toyobo, Osaka, Japan). Then, the synthesized cDNA was quantified by the real-time RT-PCR (qRT-PCR) with SYBR Green Real-time PCR Master Mix (Bio-Rad, Hercules, CA, USA) on an ABI StepOnePlus real-time PCR System (Applied Biosystems, Foster, CA, USA). The Primer sequences for target genes were listed in Table S1. GAPDH was used as an internal control. RESULTS AND DISCUSSION PSMA is an ideal surface marker for PCa cells undergoing EMT and metastasis. We first evaluated the expression level of PSMA in the tumor specimens collected from the PCa patients by IHC analysis. The results showed that the expression level ACS Paragon Plus Environment


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of PSMA in PCa patients was significantly higher than that of patients with benign prostatic hyperplasia (BPH), and increased in patients with higher Gleason score and lymph nodes or bone metastases (Figure 1A). Since EMT plays a critical role in tumor metastasis, we then evaluated the expression of PSMA in PCa cells undergo EMT. TGF-β is one of key molecules that induce EMT, so we used TGF-β for EMT induction in our in vitro analysis of potential markers for EMT including ZEB1, E-cadherin, TWIST, and SNAIL in addition to PSMA and EpCAM. After treatment of TGF-β1 at 10 ng/ml for 24, 48 and 72 hours, expression of PSMA was most significantly induced as compared to other tested factors in LnCAP cells, as validated by qRT-PCR, western blotting, cell immunofluorescent staining and flow cytometry, respectively (Figure 1B-1E). Interestingly, the expression of EpCAM was decreased upon TGF-β treatment suggesting EpCAM is not a good marker for EMT induced metastatic cells (Figure 1B-1E). Thus, the dynamic expression of EpCAM and PSMA during TGF-β induced EMT suggested the combination of anti-EpCAM and anti-PSMA has potential to enhance CTCs capture efficiency in PCa patients. Enhanced capture of CTCs from artificial blood by dual-antibody-functionalized microfluidic

device.

To

evaluate

the

cell

capture

efficiency

of

the

dual-antibody-functionalized microfluidic device (Figure 2A), we performed LnCAP and LnCAP-EMT cells spiking assay under four scenarios: (a) without any surface modification, (b) with only anti-EpCAM modification, (c) with only anti-PSMA modification, and (d) with both anti-EpCAM and anti-PSMA modification. Firstly, LnCAP and LnCAP-EMT cells were spiked into 1 mL healthy human blood to generate cell suspensions at 10, 50, 100, 200, 300 and 400 cells/mL. After treatment with the red blood cell lysis buffer, the artificial blood was injected into the microfluidic device, the captured cells were imaged and counted under a fluorescence microscope (Figure 2B). As demonstrated in Figure 2C, as for single anti-EpCAM based method, the capture efficiency decreased from 73.8% to 38.1% (mean) when the target changed from LnCAP to LnCAP-EMT cells. However, the combination of anti-EpCAM and anti-PSMA significantly enhanced capture efficiency (higher than 85.0%) as compared to their single antibody counterparts even when the EpCAM ACS Paragon Plus Environment

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expression is decreased as a result of EMT. EpCAM-based assays capture only sub-populations expressing sufficient levels of EpCAM

24,25

, thus, the microfluidic

device limitation (a few to hundreds CTCs in 1 mL blood) is enough for the identification of EpCAM+/EpCAM- CTCs in almost clinical samples of PCa patients. Capture and identification of CTCs from clinical samples. To test the technical capability of our dual-antibodies based platform for capturing CTCs from clinical samples, we collected peripheral blood samples from 24 patients with pathologically confirmed PCa (spanning four different clinical stages). The clinical characteristics of 24 PCa patients were summarized in Table 1. For each individual, 4 mL peripheral blood sample was equally divided into two parts: 2 mL of blood was processed with our technology using anti-EpCAM/PSMA capture and the rest blood using anti-EpCAM capture alone for comparison. Three-color immunocytochemistry method was then used to distinguish the cancer cell, leukocyte and nucleus. In this study, CTCs have a cellular diameter between 10~30 µm and coated by a large number of Fe3O4@MBs (Figure 3A). Immunofluorescence staining further confirmed that CTCs showed two subtypes: DAPI+/CD45−/CK+ and DAPI+/CD45−/CK− (Figure 3A). More interesting is that apart from capturing the single cancer cell in whole blood, the cell cluster also could be isolated by our chip (DAPI+/CD45−/CK+, sticking together and number ≥ 2) in a subset of patients (Figure 3A). As expected, our device captured a significantly higher number of CTCs than single anti-EpCAM counterparts (6.6 ± 5.0 vs. 3.6 ± 2.4 per 2 mL；P=0.0088) and 20 PCa patients (83.3%) were positive for CTCs (Figure 3B). Although CTCs with mesenchymal phenotype (captured with 84-1 mAb against cell-surface vimentin) was positively correlated with disease progression in metastatic PCa patients 26, the efficiency of this immunocapture system was not tested in early stage of PCa patients. In this study, the positive rate of CTCs captured by our device was highly correlated with TNM stage from 66% in early stage (stage I/II, 2.4 ± 2.4/2 mL) to 100% in advanced stage (stage III/IV, 9.6 ± 4.0/2 mL) (P100 521.504

5+4=9 4+5=9 3+3=6 3+3=6 4+5=9 3+4=7 5+5=10 5+4=9 3+4=7 4+5=9 4+5=9 4+5=9 4+4=8 3+4=7 4+4=8

T2N0M0 T2N0M0 T2N0M0 T2N0M0 T2N0M0 T4N0M0 T4N0M0 T4N0M0 T4N0M0 T3N0M1 T4N0M1 T4N0M1 T3N1M1 T4N1M1 T4N1M1


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Figure legends Figure 1 PSMA and EpCAM expression in tissues of PCa patients and TGF-β1-induced EMT in PCa cells. (A) IHC staining of PSMA in representative PCa and BPH patient tissues. (B) The expression levels of ZEB1，E-cadherin，EpCAM， PSMA，Snail and Twist mRNA in LnCAP cells treated with TGF-β1 were determined by qRT-PCR. The protein expression levels of EpCAM and PSMA were analyzed by western blotting (C) and immunofluorescence (D) in TGF-β1-induced LnCAP cells. (E) FACS analysis for the expression of EpCAM and PSMA in LnCAP cells with or without TGF-β1 treatment. Figure

2

Evaluation

of

the

cell

capture

efficiency

of

dual

antibody

dual-antibody-functionalized microfluidic device. (A) Schematic workflow of the dual antibody dual-antibody-functionalized microfluidic device. (B) The fluorescent micrographs of CTCs captured from the LnCAP cell spiked normal blood samples. (C) Cell-capture efficiency for different cell concentrations (100, 200, 300 and 400 cells/mL in blood from healthy donors), and for different cell populations (LnCAP and

LnCAP-EMT) were

compared between

anti-EpCAM and anti-PSMA

functionalized microfluidic device and the other three different controls (no antibody functionalized, only anti-EpCAM functionalized, and only anti-PSMA functionalized). (D) The determination of PCa-related RNA signatures in three PCa cell lines compared to a normal prostate cell line RWPE-1 using cell spiking assay. Error bars represent the standard deviations (n=3). Figure 3 Identification and enumeration of circulating tumor cells in PCa patients and analysis of their clinical characteristics. (A) Representative images for CTCs isolated from PCa patients. (B) CTC enumeration results obtained from 24 PCa patients based on anti-EpCAM/PSMA or single anti-EpCAM functionalized microfluidic device. (C) Scatter plot for CTCs numbers in PCa patients with different Gleason score, PSA level and TNM stage, respectively. Each dot represents one PCa patient. Figure 4 Expressive detection of four PCa-related RNA signatures in enriched CTCs



from 15 PCa patients. The expression level of SChLAP1 (A), PSA (B), PD-L1 (C) and AR (D) in the enriched CTCs from 15 PCa patients. For TOC only


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Figure 1 PSMA and EpCAM expression in tissues of PCa patients and TGF-β1-induced EMT in PCa cells. (A) IHC staining of PSMA in representative PCa and BPH patient tissues. (B) The expression levels of ZEB1，E-cadherin，EpCAM， PSMA，Snail and Twist mRNA in LnCAP cells treated with TGF-β1 were determined by qRT-PCR. The protein expression levels of EpCAM and PSMA were analyzed by western blotting (C) and immunofluorescence (D) in TGF-β1-induced LnCAP cells. (E) FACS analysis for the expression of EpCAM and PSMA in LnCAP cells with or without TGF-β1 treatment. 124x90mm (300 x 300 DPI)



Evaluation of the cell capture efficiency of dual antibody dual-antibody-functionalized microfluidic device. (A) Schematic workflow of the dual antibody dual-antibody-functionalized microfluidic device. (B) The fluorescent micrographs of CTCs captured from the LnCAP cell spiked normal blood samples. (C) Cellcapture efficiency for different cell concentrations (100, 200, 300 and 400 cells/mL in blood from healthy donors), and for different cell populations (LnCAP and LnCAP-EMT) were compared between anti-EpCAM and anti-PSMA functionalized microfluidic device and the other three different controls (no antibody functionalized, only anti-EpCAM functionalized, and only anti-PSMA functionalized). (D) The determination of PCa-related RNA signatures in three PCa cell lines compared to a normal prostate cell line RWPE-1 using cell spiking assay. Error bars represent the standard deviations (n=3). 110x71mm (300 x 300 DPI)


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Figure 3 Identification and enumeration of circulating tumor cells in PCa patients and analysis of their clinical characteristics. (A) Representative images for CTCs isolated from PCa patients. (B) CTC enumeration results obtained from 24 PCa patients based on anti-EpCAM/PSMA or single anti-EpCAM functionalized microfluidic device. (C) Scatter plot for CTCs numbers in PCa patients with different Gleason score, PSA level and TNM stage, respectively. Each dot represents one PCa patient. 121x86mm (300 x 300 DPI)



Figure 4 Expressive detection of four PCa-related RNA signatures in enriched CTCs from 15 PCa patients. The expression level of SChLAP1 (A), PSA (B), PD-L1 (C) and AR (D) in the enriched CTCs from 15 PCa patients. 75x33mm (300 x 300 DPI)


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Molecular Profiling of Pooled Circulating Tumor Cells from Prostate

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