Enhanced and High-Purity Enrichment of Circulating Tumor Cells

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Enhanced and High-Purity Enrichment of Circulating Tumor Cells Based on Immunomagnetic Nanospheres Xu-Yan Ma, Ling-Ling Wu, Lan Chen, Yin-Hui Qin, Jiao Hu, Man Tang, Chun-Miao Xu, Chu-Bo Qi, Zhi-Ling Zhang, and Dai-Wen Pang ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00802 • Publication Date (Web): 18 Jul 2018 Downloaded from http://pubs.acs.org on July 24, 2018

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ACS Applied Nano Materials

Enhanced and High-Purity Enrichment of Circulating Tumor Cells Based on Immunomagnetic Nanospheres Xu-Yan Ma,†,§ Ling-Ling Wu,†,§ Lan Chen,† Yin-Hui Qin,† Jiao Hu,† Man Tang,† Chun-Miao Xu,† Chu-bo Qi,†,‡ Zhi-Ling Zhang,† Dai-Wen Pang*,† †

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, P.R. China.

‡

Hubei Cancer Hospital, Wuhan, 430079, P.R. China.

§

Xu-Yan Ma and Ling-Ling Wu contributed equally to this work.

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ABSTRACT: Enrichment and purification of circulating tumor cells (CTCs) from peripheral blood is very critical for their detection and downstream analyses, in which many technologies have achieved good effects. However, efficient and high-purity enrichment of CTCs is still challenging because of their rarity and heterogeneity. Herein, anti-CD45 antibody modified immunomagnetic nanospheres (IMNs (CD45)) and anti-EpCAM and anti-EGFR antibodies cocktail modified IMNs (IMNs (EpCAM & EGFR)) were employed to address the two challenges. The cocktail of anti-EpCAM and anti-EGFR antibodies enables enhance the capture of tumor cells more efficiently than single antibody, offsetting the loss of CTCs with EpCAM-negative expression or undergoing epithelial to mesenchymal transition (EMT) potentially. Notably, using IMNs (CD45) to remove vast majority of white blood cells and IMNs (EpCAM & EGFR) to capture tumor cells successively, our method exhibits much excellent enrichment and purification capacity. The purity of the enriched tumor cells can reach 97.6% in mixed cell samples, while only 57.3% and 35.5% by IMNs (CD45)-based isolation and IMNs (EpCAM & EGFR)-based isolation, and all the enrichment efficiencies reached about 90% by these three methods. Moreover, the rare tumor cells achieved up to 4368-fold enrichment in whole blood samples. Besides, (90.6±0.9)% of the enriched tumor cells keep viability and can be re-cultured without release step. Furthermore, the CTCs are detected successfully in 12 blood samples from cancer patients coupled with immunocytochemistry (ICC) identification. Therefore, by our strategy, the CTCs can be efficiently enriched with high-purity, which is helpful for downstream analyses in cancer diagnoses and personalized treatments.


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KEYWORDS: immunomagnetic nanosphere, enhanced, high-purity, enrichment, circulating tumor cell



Circulating tumor cells (CTCs) are cancer cells circulated through the bloodstream to other tissues of the body after being shed from primary or metastatic lesions.1 Efficient purification and enrichment of CTCs are highly demanded by the downstream analyses for providing valuable information about cancer metastasis and personalized medicine.2-5 Nevertheless, their high heterogeneity and inherent rarity (a few to hundreds among 106–109 hematologic cells in 1 mL of peripheral blood)

6,7

bring tremendous challenges to efficiently enrich CTCs with

high-purity. Numerous technologies have been developed to efficiently isolate and enrich CTCs, they are mainly based on their biological characteristics (antibody-antigen,8-11 aptamer12,13 and E-selectin14,15) or physical characteristics (size,16,17 density,18,19 deformability20 and adhesion preference21,22). However, few strategies actually give consideration to the heterogeneity of CTCs. The most widely employed EpCAM-based isolation may significantly undercount the number of CTCs that were EpCAM-negative or undergo epithelial to mesenchymal transition (EMT), which will reduce the expression of epithelial markers (e.g. EpCAM) and raise the expression of mesenchymal markers (e.g. EGFR).23-27 Besides, the enriched CTCs by most of the isolation strategies have relatively low purity caused by the very large number of hematologic cells (106–109 per mL of complex blood), interfering the downstream analyses, such as gene expression, mutation, polymorphism and sequencing,23,28,29 In order to better enrich CTCs, additional antibodies or aptamers have been combinatively used to enhance and differentiate capture of CTCs,30,31 and some other technologies have also been explored to improve the CTC enrichment purity.32-34 And these have mostly preferred microfluidic technology, with some limitations. In general, the fluid channel needs complexly designing


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and manufacturing, and the enriched CTCs require a release step from capture substrate for downstream analyses, which is harmful to the cells. Moreover, most of them depend only on one set of criteria, so it is still challenging for efficient and high-purity enrichment of CTCs. Thus, it is very urgent to develop a facile strategy to enrich CTCs with a minimum CTC loss and negligible interference from hematologic cells for downstream analyses. Immunomagnetic separation is one of the most commonly used technologies to enrich CTCs from complex peripheral blood, due to its easy magnetic manipulation, specific and efficient binding to target cells, and convenient coupling with subsequent identification and analyses.35-37 In our previously work, magnetic nanospheres (MNs) with convenient functionalization, quick-response to external magnetic field and fast binding kinetics were synthesized by layer-by-layer (LBL) assembly method.38-41 They have exhibited excellent superiorities in pathogen isolation and detection.42-45 Especially, the antibody modified immunomagnetic nanospheres (IMNs) have been successfully applied to efficiently isolate and enrich CTCs from peripheral blood in cancer patients.46-49 However, the EpCAM-based immunomagnetic separation was unable to effectively isolate EpCAM-negative CTCs. In addition, the purity of the enriched CTCs by all these strategies was not so high even if a high capture efficiency of CTCs or a high depletion efficiency of white blood cells (WBCs) could be obtained. Herein, a cell sorting strategy based on two kinds of IMNs was developed for enhanced and high-purity enrichment of CTCs (schematic diagram in Figure 1A). The unique advantages of our strategy are below: (1) The anti-EpCAM and anti-EGFR antibodies cocktail coupled with MNs can enhance the recognition of tumor cells more efficiently than single antibody, showing good general applicability, which has the potential to offset the loss of



CTCs with low EpCAM expression or EMT. (2) Employing IMNs (CD45) to specifically remove vast majority of WBCs and IMNs (EpCAM & EGFR) to efficiently capture tumor cells successively can be of more excellent capacity for purification and enrichment of tumor cells than those by conventional techniques. In mixed cell samples, the purity of the enriched tumor cells can reach 97.6%, while only 57.3% and 35.5% by IMNs (CD45)-based isolation and IMNs (EpCAM & EGFR)-based isolation, and all the enrichment efficiencies are about 90% by these three methods. (3) Due to the excellent efficiency and specificity of IMNs, the rare tumor cells achieve up to 4368-fold enrichment in whole blood samples, providing a sound basis of high-purity enrichment of CTCs for downstream analyses. (4) IMNs with easy manipulation and hardly any influence on enriched tumor cells can facilitate the downstream combination with analysis and identification techniques. For example, the enriched cells can be directly used for immunocytochemistry (ICC) identification and re-culture without any release step.


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Figure 1. (A) Schematic diagram of CTC detection based on IMNs (CD45) and IMNs (EpCAM & EGFR). (B) TEM image of MNs. (C) Magnetic hysteresis loop of MNs measured at RT. (D) Capture efficiencies of MNs by a commercial magnetic scaffold with different attraction time. (E) Hydrodynamic diameters and PDI of MNs with different storage time. (F) Microscopic images of MNs (a), IMNs (CD45) (b) and IMNs (EpCAM & EGFR) (c) incubated with Dylight488-labeled goat anti-mouse IgG.

EXPERIMENTAL SECTION Reagents and Instruments. Bovine serum albumin (BSA) was purchased from Biosharp. Anti-CD45

mAb,

anti-EpCAM

N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

mAb,

anti-EGFR

hydrochloride

(EDC)

mAb, and

Nhydroxysuccinimide (NHS) were obtained from Sigma-Aldrich. FITC-labeled anti-CK19



mAb and PE-labeled anti-CD45 mAb were got from Abcam. Hoechst 33342 was bought from Invitrogen Corp. All cell lines were acquired from China Center for Type Culture Collection. Blood samples were obtained from Hubei Cancer Hospital and Hospital of Stomatology Wuhan University. TEM images were obtained by electron microscope (FEI Tecnai G2 20 TWIN). Microscopic images were achieved by inverted fluorescence microscope (Ti, Nikon), equipped with a CCD camera (Nikon, DS-Ri1). Fabrication of IMNs. MNs were synthesized by our reported LBL assembly method.41 Then, MNs were coupled with antibody by carbodiimide chemistry. Firstly, 25 mM EDC and 25 mM NHS were mixed with 2 mg of MNs for 25 min, then the activated MNs were reacted with 10 µg of antibody at room temperature (RT) for 4 h. After washing, the resultant IMNs were dispersed in 10 mM pH 7.2 PBS at 4 °C for later use. IMNs (CD45) and IMNs (EpCAM & EGFR) were the abbreviation for IMNs coupled with anti-CD45 antibody, the cocktail of anti-EpCAM and anti-EGFR antibodies (anti-EpCAM mAb : anti-EGFR mAb=1:1) respectively. Capture of Target Cells With IMNs. Jurkat T cells (5 x 106) were incubated with IMNs (CD45) in 1 x PBS at RT for 30 min with gentle shaking, then separated by a magnetic scaffold. The captured and uncaptured cells by IMNs (CD45) were all counted by a hemocytometer to calculate the depletion efficiency. As controls, MCF-7 and MDA-MB-231 cells were treated with IMNs (CD45) respectively, and Jurkat T cells were incubated with unmodified MNs to investigate the specificity of IMNs (CD45). Similarly, to evaluate the efficiency and specificity of IMNs (EpCAM & EGFR) to capture tumor cells, MCF-7 cells (1 x 105 ) and MDA-MB-231 cells (1 x 105 ) were incubated with IMNs (EpCAM & EGFR)


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respectively, as comparisons, IMNs (EpCAM & EGFR) were treated with Jurkat T cells, and unmodified MNs were reacted with MCF-7 and MDA-MB-231 cells respectively. Besides, five groups of the mixed tumor cells (total number is 1 x 105) with different ratios of MCF-7 cells to MDA-MB-231 cells were incubated with IMNs (EpCAM), IMNs (EGFR) and IMNs (EpCAM & EGFR) respectively to research the capability of IMNs (EpCAM & EGFR) to enhance the capture of tumor cells. For test the general applicability of IMNs (EpCAM & EGFR) to capture tumor cells, six kinds of tumor cells were also reacted with IMNs (EpCAM & EGFR). Enrichment of Tumor Cells in Mixed Cell Samples. The cell sorting strategy based on IMNs (CD45) and IMNs (EpCAM & EGFR) was used to enrich tumor cells in the mixed cell samples, prepared by adding Hoechst 33342-prestained 5 x 104 MCF-7 cells and 5 x 104 MDA-MB-231 cells to DiI-prestained Jurkat T cell suspension (5 x 106 cells mL-1 in 1 x PBS). Firstly, IMNs (CD45) were add to the mixed cell samples at RT for 15 min with gentle shaking. After removing the Jurkat T cells bound to IMNs (CD45) by a magnetic scaffold., IMNs (CD45) were mixed with the retained cell suspension with a new round. After the two rounds of Jurkat T cells depletion consecutively, IMNs (EpCAM & EGFR) were mixed with the retained cell suspension for tumor cells capture, and isolated by a magnetic scaffold. The nonspecific adsorbed Jurkat T cells, the captured and uncaptured tumor cells with IMNs (EpCAM & EGFR) were all counted respectively by a hemocytometer to count the enrichment efficiency and the purity of the enriched tumor cells. As controls, IMNs (CD45) and IMNs (EpCAM & EGFR) were also used to treat the mixed cell samples respectively. Enrichment and ICC Identification of Rare Tumor Cells From Mimic Clinical



Samples. In order to research the capability of enriching rare tumor cells by our strategy, three groups of rare MCF-7 cell-spiked mimic clinical samples were prepared: about 10-300 Hoechst 33342-prestained MCF-7 cells were respectively spiked into 1 mL of 1 x PBS suspension contained Jurkat T cells (5 x 106), blood without plasma, and whole blood. After the erythrocytes lysis in blood, IMNs (CD45) were mixed with the samples to remove vast majority of WBCs with two consecutive rounds. Then, IMNs (EpCAM & EGFR) were add to the retained cell suspension to capture and isolate MCF-7 cells. The enriched and unenriched MCF-7 cells, as well as the WBCs among the enriched MCF-7 cells were all recorded respectively by the inverted fluorescence microscope. For ICC identification, the enriched cells were incubated with 4% paraformaldehyde and 0.1% Triton-X 100 for 10 min respectively for fixation and permeabilization. Then stained with FITC-labeled anti-CK19 mAb, Hoechst 33342 and PE-labeled anti-CD45 mAb at 37 °C for 30 min. Finally, they were observed by an inverted fluorescence microscope, only cells had round morphology and met these criteria were tumor cells: positive for Hoechst 33342 and CK19, and negative for CD45. Cell Viability Analyses. The enriched cells were incubated with 4.5 µM PI and 2 µM calcein AM at 37 °C for 30 min. After washing, they were observed and counted by an inverted fluorescence microscope to quantitative analyse the viability rate. As control, the cultured MCF-7 cells without incubation with IMNs were also examined. In order to investigate whether the IMNs could go with the proliferated cells, fluorescent-magnetic nanospheres (FMNs) were also prepared with the layer-by-layer assembly method (three layers of hydrophobic CdSe/ZnS quantum dots, and five layers of nano-γ-Fe2O3). After modified with antibodies, the obtained IFMNs (EpCAM & EGFR) were substituted for IMNs


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(EpCAM & EGFR) in the experiment of enriching tumor cells. The enriched cells binding with IFMNs (EpCAM & EGFR) were re-cultured in a 24-well plate at 37 °C with Dulbecco's modified Eagle's medium under sterile conditions. Detection of CTCs in Cancer Patient Blood Samples. The CTCs in blood samples of 12 cancer patients and 4 healthy donors were detected with our cell sorting strategy coupled with ICC identification as the above procedures. RESULTS AND DISCUSSION Characterization of IMNs. The MNs were uniform in size (382.4 ± 21.4 nm) and exhibited excellent dispersibility (Figure. 1B). The large magnetic saturation value at RT (23.9 emu/g) (Figure 1C) showed that MNs had so well superparamagnetic property, and most of MNs could be captured using a commercial magnetic scaffold (325 ± 25 mT) within 1 min (Figure 1D). As shown in Figure 1E, MNs’s hydrodynamic diameter and the polydispersity index (PDI) which refers to the monodispersity of the MNs (The smaller the PDI, the better the monodispersity) had almost no change during the six months of storage time, indicating MNs kept good stability and monodispersibility with a long time. With these above characteristics, IMNs modified with anti-CD45 antibody, anti-EpCAM and anti-EGFR antibodies cocktail were fabricated respectively, which could specifically react with FITC-labeled goat anti-mouse IgG (Figure 1F), indicating that the successful fabrication of IMNs (CD45) and IMNs (EpCAM & EGFR). Besides, the number of the effective binding sites on each IMN is an important factor for the capture efficiency of the IMNs to target cells. We evaluated that there are about 100 effective binding sites on each IMN (Figure S1). Furthermore, we monitored the bioactivity of IMNs over a long time. As shown in Figure



S2(A-C), the IMNs could reacted Dylight 488-labeled goat anti-mouse IgG efficiently and capture more than 90% of target cells even after 6 months’ storage, confirming that the antibodies modified on the IMNs still have the biological activity for a long storage time. Efficiency and Specificity of IMNs. As shown in Figure 2A, 93.6% of the Jurkat T cells were depleted with IMNs (CD45), which hardly depleted any tumor cells (

Enhanced and High-Purity Enrichment of Circulating Tumor Cells

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