High sensitive detection of circulating tumor cell by multimarker lipid magnetic nanoparticles and clinical verifications
Tumor cells with heterogeneity and diversity can express different markers. At present, positive separation of circulating tumor cells (CTC) taking EpCAM as the marker was used in most cases which could be one-sided, while this study successfully prepared four antibody-modified magnetic immunoliposomes (MIL) by using the self-assembled liposome with antibody derivatives. This study aims to explore the separation efficiency and clinical detection feasibility of single or combined use of MIL with multi-tumor markers on different tumors. Captured CTC were stained with CK-FITC, CD45-PE and DAPI, and fluorescence microscope was used for the observation, analysis and calculation. The result indicated that the CTC number positive rate in blood samples of four different magnetic balls on the same patient could be up to 87.5% in 32 patients with 14 different kinds tumors. While the effect of directly mixed separation by four kinds of magnetic balls was not satisfying. It suggested that the MIL of multi-tumor markers could be a powerful tool for CTC separation in application of tumor screening and prognosis.
KeywordsCirculating tumor cell Magnetic immunoliposomes Epithelial cell adhesion molecule Clinical verifications
Proliferation and metastasis of malignant tumor cells are the key factors resulting in tumor patients' death [1, 2]. At present, the curative effect of tumors is not ideal, mainly due to post-operative recurrence caused by tumor adjacent and distant metastasis [3, 4]. Tumor has been perceived as a systematic disease, and early cancer should be considered as systematic disease even there's no clinical and influential evidence . Considering biological characteristics of tumors, early diagnosis had roused physicians' attention [6, 7]. Physicians and patients had gradually attached great importance to the concept of early screen, which could be helpful for the improvement of curative effect on tumors [8, 9]. It's difficult for traditional tumor diagnosis methods, like imaging monitoring and biopsy which have a certain degree of lagging effect, to achieve early screen [10, 11].
In recent years, circulating tumor cell (CTC) monitoring has become one of the most active fields in cancer research and been applied to the early screen of multiple tumors [8, 12, 13]. CTC examination plays an important role in prognosis prediction, curative effect verification and recurrence monitoring of multiple tumors [14, 15]. So far, CTC examination has been widely applied to multiple malignant tumors [16-18]. For the past decade, researchers from all round the world developed several CTC examination methods and separation techniques, but most of them mainly depend on the surface markers (such as epithelial cell adhesion molecule, EpCAM) of epithelial cells [19, 20]. CTC separation and counting focusing only on positive EpCAM could be one-sided, which could lead to a large amount of tumor cells with other positive markers (such as EGFR positive cells, EMT inverted cells) being ignored, and the sensitivity could be low as well.
Polypeptide magnetic lipid system constructed by lipid materials with similar bilayer structure as the cell membrane could increase the separation efficiency of liver cancer CTC by a wide margin. Based on previous studies [21, 22, 23], and focusing on the limitation of the above magnetic immunization positive separation of single EpCAM, this study successfully prepared four antibody-modified magnetic immunoliposomes (MIL), i.e. EpCAM, EGFR, HER-2 and MUC-1, using liposome technique. This study aims to explore the separation efficiency of single use or combined use of MIL with multi-tumor markers on CTC of patients with different tumors so as to find out a more sensitive scheme for the detection of CTC in different tumors.
All different tumor cells used in this study were purchased from American Type Culture Collection(ATCC) cell bank. Dulbecco's Modified Eagle Media(DMEM), RPIM-1640 culture solution, fetal bovine serum and trypsin were purchased from Gibco. CD45-PE was purchased from eBioscience; CK-FITC, magnetic grate, dimethyl octadecyl epoxypropyl ammonium chloride(GHDC), Fe3O4 hydrophobic magnetic nanoparticles (Fe3O4-HMN) were purchased from Shanghai Shengna Industrial Co., Ltd. DAPI staining fluid was purchased from Beyotime Biotechnology Co., Ltd. EpCAM antibodies were purchased from Shanghai Raygene Biotechnology Co., Ltd. Molecular weight 8000–1400 Da Dialysis bag Purchased from Shanghai Yuanye Biotechnology Co., Ltd. Cholesterol, dichloromethane and other common reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.
Preparation of antibody derivatives
Take the preparation of Anti-EpCAM antibody derivative as an example. A total of 57.1 μg EpCAM antibody and 100 μg GHDC were dissolved in 3.0 mL phosphate buffered saline (PBS, pH = 7.4), and reacted in the magnetic stirrer at 4 ℃ overnight. The next day, a dialysis bag with a molecular weight of 8000–1400 Da was used for dialysis for 12 h, and the dialysate(ddH2O) was changed once every two hours, and it’s freeze-dried after dialysis and antibody derivative EpCAM-GHDC was obtained and weighed. The same method was used to obtain Anti-EGFR-GHDC, Anti-HER-2-GHDC and Anti-MUC-1-GHDC antibodies.
Preparation of MIL
Weigh 5 mg of DOPC and 5 mg of Cholesterol into two 50 mL three-necked flasks, measure 1.0 mL of Fe3O4-HMN to ethanol, dissolve in 3.0 mL of CH2Cl2, and transfer Fe3O4-HMN/CH2Cl2 to a three-necked flask. A probe ultrasound equipment was used to conduct emulsification on the round-bottom flask in an ice bath for 6 min. Meanwhile, dissolve 2 mg EpCAM-GHDC in 6 mL ddH2O and add to the 3-mouth flasks slowly. After ultrasonic emulsification, a rotary evaporator was used to eliminate the remaining CH2Cl2. After magnetic separation, the solution was washed for 3 times to obtain magnetic nanoparticles. The same method was used to obtain EGFR, HER-2 and MUC-1 antibody-modified MIL.
Characteristics and performance of MIL
Polyacrylamide gel electrophoresis (PAGE) was used to detect the antibody content on the surface of magnetic immunoliposomes and confirm the presence of antibodies. Atomic force microscopy (AFM) was used to observe the microstructure of different MIL. PPMS-9 (QUANTUM DESIGN, USA) was used to detect the hysteresis loop of magnetic nanoparticles. An ultraviolet spectrophotometer was used to scan the absorption peak of MIL solution to further confirm the presence of antibodies on the surface of magnetic nanoparticles. The BCA protein quantification method was used to detect the antibody content on the surface of MIL. Zetasizer Nano-ZS 90 (Malvern Instruments Ltd.,UK) was used to detect the diameter and potential of MIL. Nanosight (Malvern Instruments Ltd., UK) was used to verify the diameter of MIL, and diameter change after the combination of MIL and tumor cells was analyzed. A fluorescence microscope (OLYMPUS B × 61, Japan) was used to observe immunofluorescence.
The experiment on the separation effect of MIL on different tumor cells
Add 30 μL MIL into 7.5 mL PBS solutions containing 100 tumor cells respectively, mix evenly, and conduct magnetic separation for 15 min, discard the supernatant; then add 30 μL of DAPI, 30 μL of CK8, 18, 19-FITC and 10 μL of CD45-PE and mix evenly, stain avoiding light for 15 min; Add to the magnetic separation grate for separation, add 1 mL PBS solution to wash uncombined antibodies, repeat for twice; finally, add 30 μL ddH2O and suspend again, when mixed, smear evenly to the center of APES glass slide, observed by fluorescence microscope, take photos, count, and analyze the recovery rate of tumor cells.
Separation and verification methods of CTC
CTC separation and identification steps of tumor peripheral blood including: Collect 7.5 mL whole blood from tumor patients using anti-coagulation blood collection tube, 1500 rpm centrifuge for 10 min; take pelagic liquid and put it in a centrifuge tube, add isometric PBS buffer(pH = 7.4) and mix uniformly; equally divide into five blood samples: A-E. For blood sample A-D, add 30 μL of four different magnetic nanoparticles respectively, incubate in room temperature for 30 min, blend once for every 5 min; insert EP tube to magnetic separation grate for 15 min of absorption, discard supernatant, and take out the EP tube; conduct magnetic separation wash on the captured CTC for one time using PBS; next, add 30 μL of DAPI, 30 μL of CK19-FITC, 10 μL of CD45-PE, mix uniformly, stain for 15 min avoiding light; after staining, add 1 mL ddH2O and conduct magnetic separation for 15 min on the magnetic separation grate, discard supernatant; at last, add 30 μL dd H2O in the EP tube for suspending, smear evenly to the center of APES glass slide when mixed, observed by fluorescence microscope, take photos and count. For blood sample E, add 30 μL equal-proportion mixture of four different MIL first, following by the same steps as described above.
Collection of clinical samples
A total of 32 multi-tumor patients who accepted treatment in our hospital from 2016 to 2017 were collected as subjects, and all the patients were confirmed in clinical diagnosis and pathological examination. The following patients were excluded: those who were allergic to medications; those with other primary tumors; those who were not willing to participate in the experiment; those who didn’t accept radiotherapy and chemotherapy. At the same time, 20 healthy volunteers were enrolled in blood samples. All the subjects signed the informed consent, and this study was approved by the institutional review board of our hospital. Tumor patients and control patients were requested to rest on time the night before blood collection, and 7.5 mL of blood was collected from median cubital vein in the morning of the second day and stored in anti-coagulation blood collection tubes.
SPSS 19.0 was used to analyze the data. Chi-squared test was used to analyze count variables, statistical significance was set at P < 0.05.
Results and discussion
Preparation and characteristics of MIL
The ultraviolet absorption spectrum of MIL was shown in Fig. 2c. It could be seen from the figure that EpCAM, EGFR, HER-2, MUC-1 magnetic nanoparticles presented obvious ultraviolet absorption peak at 280 nm, magnetic nanoparticles have no peaks. However, as the denaturation of antibodies and influenced by ultraviolet absorption, the absorption peaks of antibody derivatives and immunization nanoparticles at 280 nm were weakened and broadened and shifted a little bit. It’s proved by protein electrophoresis and ultraviolet absorption results that antibodies had been established on the surface of magnetic nanoparticles, and the antibody content on the surface of nanoparticles was 0.1 mg/mg per nanoparticle(BCA quantification method). Observation result of MIL by atomic force microscopy(AFM) was shown in Fig. 2d. As seen in the figure, formed immunization nanoparticles presented an irregular ball structure, with the size of around 200 nm (231 nm), and were not distributed uniformly, and vesicle features of liposome was shown.
Diameter and other surface characteristics of MIL
Comparative analysis on the interaction of MIL and tumor cells
The recovery rate of MIL to different tumor cells
The separation performance of MIL to target cells was analyzed by analyzing the recovery rate of antibody lipid magnetic nanoparticles to 100 counted tumor cells. As shown in Fig. 4b, after mixing with four different magnetic nanoparticles, EpCAM, HER-2, MUC-1 and EGFR could capture four kinds of cells at the same time with a capture rate of up to 80.00% and a maximum capture rate of 97.50%. However, the mixture of four different magnetic nanoparticles didn’t present obviously increased capture rate to cells, which was not statistically different from single kind of magnetic nanoparticles. In conclusion, the antibody lipid magnetic nanoparticles prepared in this study had high affinity to multiple kinds of tumor cells, which can combine tumor cells rapidly and possess a high recovery rate of tumor cells. The separation efficiency of magnetic spheres was also detected by using a flow cytometer (Fig. 4c–g). As shown in Fig. 4c–f, the recovery efficiency of SKBR3 cells by the EpCAM, EGFR, HER-2, and MUC-1 magnetic nanoparticles is 85%, 90%, 86% and 88%, respectively. The combined use of the four magnetic spheres has a capture efficiency of 82% and does not significantly improve (Fig. 4g), this result is consistent with the conclusion of Fig. 4b.
Morphological observation of circulating tumour cells in clinical blood samples
Calculation of circulating tumour cells in clinical blood samples
Different CTC counts in different tumors and their clinical significance
In recent years, more and more studies have shown that circulating tumor cells (CTC) are associated with metastatic recurrence and increased mortality of tumors. The detection, statistics and quantitative studies of CTC have become a research boom. At present, the EpCAM-based CTC detection method is one of the most common CTC detection methods. It is worth noting that many recent studies have shown that CTCs are heterogeneous, including epithelial tumor cells, epithelial–mesenchymal transition (EMT) cells, mixed (epithelial and EMT positive) tumor cells, circulating cancer stem cells (CTSC), and irreversible EMT-positive tumor cells; in addition, the expression of CTC surface protein epithelial cell adhesion molecule (EpCAM) is dynamic. EpCAM-based assays were unable to detect CTC, EpCAM-negative cells, CTSCs, and EMT-positive cells with low EpCAM expression. Therefore, EpCAM-based enrichment in tumor spread may underestimate the importance of CTC, CTSC, and EMT-positive tumor cells, and pure EpCAM may not be a perfect marker for detecting CTC [24, 25].
Adequate evidence suggests that other epithelial CTC markers include epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER-2), and mucin 1 (mucin). 1, MUC-1), etc., CTSC surface markers include CD26, CD44, CD133 and CXC chemokine receptor 4 (CXCR4), etc., circulating EMT positive tumor cell surface markers are vimentin, fibronectin, calcium adhesion protein-N and calcium adhesion protein-O  and so on. Therefore, future research should be to combine EpCAM antibodies with antibodies to other positive tumor cell surface markers to achieve the best results.
This study prepared four kinds of MIL at the same time, and through the analysis of the preparation and structure properties of MIL, proved that an immunization magnetic lipid nanoparticle system with high recovery rate to target tumor cells could be obtain using direct preparation of antibody liposome by antibody derivatives. By analyzing the number of CTC in blood samples from 32 patients with different tumors collected in each group, and comparing the sensitivity of different CTC separation schemes with different tumor-markers, it’s shown in this study that single use of EpCAM, EGFR, Her-2 and MUC-1 could realize a higher CTC separation positive rate than that of combination use.
This study provides a feasible plan for high sensitive detection of CTC in tumor patients, It suggested that the MIL of multi-tumor markers could be a powerful tool for CTC separation in application of tumor screening and prognosis, and the improvement of application method could be useful for the precise separation and acute calculation of CTC, which also provides a scientific evidence for early broad screening of tumor patients and for the application of curative monitoring after treatment.
Conceptualization, JC; Data curation, SD; Investigation, JW; Methodology, LC; Project administration, XL and HJ; Resources, HJ; Software, MQ; Supervision, HY; Validation, YW; Visualization, XL; Writing—original draft, JC; Writing—review and editing, JW. All authors read and approved the final manuscript.
This work was supported by The Natural Science Foundation of Shanghai Health Bureau (201740038), Shanghai’s Three-Year Action Plan for the Development of Chinese Medicine (2018-2020) (ZY(2018-2020)-CCCX-4013) and Shanghai Young Physician Training Program  NO. 147. We thank JuKang (Shanghai) Bio-Sci & Tech Co., Ltd. (song ping) for help with the CTC detection.
Ethics approval and consent to participate
Consent for publication
All authors agree to be published.
The authors declare that they have no competing interests.
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