Efficient Capture and High Activity Release of Circulating Tumor Cells

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Biological and Medical Applications of Materials and Interfaces

Efficient Capture and High Activity Release of Circulating Tumor Cells by Using TiO2 Nanorod Arrays Coated with Soluble MnO2 Nanoparticles Rui Li, Fangfang Chen, Huiqin Liu, Zixiang Wang, Zitong Zhang, Yuan Wang, Heng Cui, Wei Liu, Xingzhong Zhao, Zhi-Jun Sun, and Shi-Shang Guo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04683 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 23, 2018

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ACS Applied Materials & Interfaces

Efficient Capture and High Activity Release of Circulating Tumor Cells by Using TiO2 Nanorod Arrays Coated with Soluble MnO2 Nanoparticles R. Lia, F.F.Chenb, H.Q. Liua, Z.X. Wanga, Z.T.Zhanga, Y.Wanga, H. Cuia, W.Liua, X.Z. Zhaoa, Z.J. Sunc* and S.S. Guoa* a

Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education,

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

Department of Oncology, Zhongnan Hospital of Wuhan University, Hubei Cancer Clinical Study Center, Hubei Key Laboratory of Tumor Biological Behaviors, Wuhan, Hubei, 430072, P. R. China. c

State Key Laboratory Breeding Base of Basic Science of Stomatology, Key

Laboratory of Oral Biomedicine of Ministry of Education, Department of Oral Maxillofacial Head Neck Oncology, School and Hospital of Stomatology, Wuhan University, Wuhan, Hubei 430079, China

KEYWORDS: MnO2 nanoparticles; TiO2 nanorod arrays; self-assembly; efficient capture; high activity release; circulating tumor cells;

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ABSTRACT: Effective capture and release of circulating tumor cells(CTCs) with high viability is still a challenge in medical research. We design a novel approach with efficient yield, high cell activity to capture and release of CTCs. Our platform is based on TiO2 nanorod arrays coated with transparent MnO2 nanoparticles. We use hydrothermal synthesis to prepare TiO2 nanorod arrays, the MnO2 nanoparticles are fabricated through in situ self-assembly on the substrate to form a monolayer and etched by oxalic acid with low concentration at room temperature. Up to 92.9% of target cells are isolated from samples using our capture system and the captured cells can be released from the platform, the saturated released efficiency is 89.9%. Employing lower than 2*10−3M concentration of oxalic acid to dissolve MnO2, the viability of MCF-7cancer cells exceed 90%. Such a combination of the two-dimensional and three-dimensional platform provides a new approach isolate CTCs from patient blood samples.


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Introduction In 1869, compared to the morphology of different tumor cells in the blood, Ashworth, T. R.1 first described circulating tumor cells (CTCs). A lot of scientific research has proved that at the early stage of malignant progression, the tumor cells were broken away from the original or metastatic tumors and then invaded a distal site in different tissues of the body, which is the main route of cancer metastasis2,3. Data displays that about 90 % of the cancer patients died from cancer metastasis4,5.Unlike bone marrow biopsies, blood draws are very low invasive and it can be well provided with the effectiveness of chemotherapy, many people set off the climax of the study of CTCs at that time6. Due to the ultra-low amount of CTCs (less than hundreds per milliliter)7 against the high background of blood cells (10^7 white blood cells (WBCs) per mL and 10^9 red blood cells(RBCs) per mL), the isolation and characterization of CTCs meet a big challenge8. Recently, numerous technologies have been exploited to separate CTCs from cancer patients. In line with physical attributes and affinity, physical attributes mainly include size9, density10, deformability11, and adhesion reference12, while affinity contains antibody- antigen13, E-selectin14, and aptamer15-17. Each capture method has dual personality. For instance, capture-agent-labelled magnetic beads or immunomagnetic separation is widely used, due to its high capture efficiency and easy manipulation18,19, but how to release the magnetic beads from the captured cells has become a difficult obstacle to overcome. According to the size difference of cells for isolate circulating tumor cells from whole blood cells, which is the most direct and effective method20. However, because that size of the white blood cell and the circulating tumor cell are partially overlaped, during the sorting process, so the white blood cells can be mixed into the circulating tumor cells, thereby reducing the purity of isolation.



Compared with flat substrate, nanostructured substrate can increase the interactions between targeted cell surface and substrate, thus obtain a high capture efficiency. A number of researchers have engaged into the study of increasing the roughness of the substrate21-23. Two-dimensional nanostructure substrates were fabricated, such as TiO2 and hydroxyapatite/chitosan (HA/CTS) nanoparticles, a biocompatible and surface roughness controllable nanofilm on the substrates for isolating the CTCs24,25. Many people have found that three-dimensional (3D) structure can increase the contact area between the substrate and the cell22-24. Wang, S. T.26 firstly reported that using hydrofluoric acid etching process to create a silicon nanopillar substrate for capture CTCs, the capture yield ranged from 45% to 65%. Zhang, N. G.27 used the electrospinning to fabricate the horizontally oriented titanium nanofibers on silicon substrates, this apparatus was applied to separate CTCs from the whole blood sample, the capture efficiency is up to 70%. Moreover, bionic TiO2 inverse opal photonic crystal was designed to capture CTCs, three-dimensional hierarchical nanostructured graphene28 and ZnO nanorod arrays29 were exploited to isolate CTCs, which greatly contribute to detecting the rare CTCs. CTCs analysis as a new type of liquid biopsy, the first critical step is to obtain a highly capture and release efficiency, in the process of release, the activity of cells are not affected30,31. Although the nanostructured surfaces can enhance the capture yield, few studies has done in efficient capture can effectively release at the same time. We developed a combination of two-dimensional and three-dimensional capture platform just meet this application requirement. Firstly, by hydrothermal synthesis method, we prepare the TiO2 nanorod arrays on FTO glass. TiO2 has the characteristics of good stability, biocompatibility, light transmittance, easy modification and so on, hence, titanium dioxide is widely applied in capture platform. Secondly, TiO2 nanorod arrays


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coated with MnO2 nanoparticles. After preparing the MnO2/TiO2/FTO substrates, we use 3-mercaptopropyl trimethoxysilane to modify the substrates (incubate for 1h at room temperature). Next step is to use the coupling agent N-maleimidobutyryloxy succinimide ester (GMBS), standing at normal temperature for 45 minutes, after that streptavidin (SA) were introduced onto the surfaces of the substrates. Finally, we conjugated the Biotinylated anti-EpCAM onto the streptavidin-coated substrates (incubate for 1h at room temperature). Cell capture experiments can be performed. Using oxalic acid to dissolve MnO2 nanoparticles, during the dissolution of the MnO2 particles, the captured cells can be released from the capture platform. A scheme demonstrate that the modification of substrate to capture and release CTCs (Figure 1). We fabricated a combination of two-dimensional and three-dimensional capture platform using TiO2 nanorod arrays coated with soluble MnO2 nanoparticles. Owing to the strong combination cell- TiO2 or MnO2/TiO2 nanorod arrays affinity and interaction, in addition to MnO2/TiO2/FTO substrate can afford much antibody modification sites for target cell contact, the capture and release efficiency can be significantly improved. This capture device offer mew ways to isolate and release CTCs from the patient blood samples.

Results and Discussion Our group has consistantly demonstrated that the roughness of substrate and the EpCAM expression can affect cell capture efficiency 27,32,33. TiO2 nanorod arrays and MnO2 nanoparticles preparation and characterization (Figure S1, S2, S3) are displayed in supporting information. In this work, SW480 and MCF-7 as EpCAM-positive cancer-cell lines are the first choice for our experiment. As shown in Figure 2, the capture efficiency of SW480 cell line was significantly affected by the



different substrates, while for MCF-7 cell line, capture efficiency was achieved on the other three substrates except for the lower capture efficiency on FTO substrate. Although the FTO glass and MnO2 nanoparticles have the same surface roughness, the FTO glass does not contain hydroxyl, it cannot be modified with antibodies, resulting in low trapping efficiency. The SW480 and MCF-7 captured-cells on different substrates (a)FTO, (b)MnO2/FTO, (c)TiO2/FTO, (d) MnO2/TiO2/FTO) are shown in Figure S4. The maximum capture efficiency of SW480 and MCF-7 are 82.7% and 92.2%, respectively. It suggests that the expression of EpCAM, the roughness of the substrates, the difficulty of modifying antibodies on the base surface are contribute to high capture efficiency. Figure 3 displays the relationship between capture efficiency and incubation time at MnO2/TiO2/FTO substrate. From the picture we can see, the capture yield of the MCF-7 cells line is increased originally and it reaches the saturation point at 120minutes, the maximum capture efficiency up to 92.9%, meanwhile, for the SW480 cell line, the capture efficiency was initially increased rapidly as the incubation time increased and after reaching 1h, the capture efficiency was still increasing slightly. The SW480 and MCF-7 captured-cells at different incubation times (a) 30min, (b)60min, (c)120min, (d) 180min are shown in the Figure S3. So in our experiments, the optimal capture time was 2h. It is known that cellular surface is composed of filopodia, if we increase the topographical interaction between substrates and cancer cells, it can enhance the cell-capture efficiency, in order to further study the relationship of them, SEM was used to characterize. From the Figure 4, combined with the basal characterization of surface roughness (Figure S2), we could find that on the rough interface (TiO2/FTO and MnO2/TiO2/FTO), the cancer cells will protrude more filopodia than that on flat


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substrate (FTO and MnO2/FTO). On MnO2 /FTO substrate, large pseudopods were found around the cells, meanwhile, on TiO2/FTO and MnO2/TiO2/FTO substrates, the cancer cells presented spread, a lot of filopodia attached to TiO2 nanorod arrays. The results indicated that on TiO2/FTO and MnO2/TiO2/FTO substrates, there exist strong combination cell-TiO2 or MnO2/TiO2 nanorod arrays affinity and interaction. Moreover, compared with FTO and MnO2/FTO substrates, TiO2/FTO and MnO2/TiO2/FTO substrates can offer added antibody modification sites for target cell contact. In order to test the effectiveness of optimal cell-capture and release conditions (MnO2/TiO2/FTO substrate), we use this device to isolate CTC from the artificial CTC samples containing MCF-7 cells (Figure 5a and Figure 5b). Firstly, we prepared Green-Dye-Labeling MCF-7 cells. Secondly, we take out different numbers of cells (approximately 250, 500, 750, and 1000 cells mL-1, respectively) and spiked these cells into artificial CTCs DMEM medium and whole blood samples. Finally, we calculate the capture efficiency of cells from the simulated cancer patient peripheral blood. Under optimal cell-capture conditions, in artificial CTCs DMEM medium, more than 80% recovery efficiencies of MCF-7 at different cell numbers can be obtained. Meanwhile, overlap 60% of cells spiked in the blood sample can be captured. In the blood sample, the nonspecific adsorption of a large number of white cells and platelets occupies the sites of the captured cells, resulting in lower recovery efficiency in artificial CTCs DMEM medium than that in the blood sample. The recovery efficiencies of MCF-7 at different cell numbers from artificial CTCs blood samples on MnO2/TiO2/FTO substrate are shown in Figure S4. No matter in simulation experiments or from patients’ blood samples to capture CTC, there exist a lot of interference from leukocyte adsorption. We know that the surface



of the cell membrane is negatively charged. Because the attraction of positive and negative charge, it will cause cell adsorption. According to the Zeta potential test( Figure 6), we found that the surface charge of white cells (the concentration of 10^6/ml), SW480 cells (the concentration of 10^4/ml) and MCF-7 cells (the concentration of 10^4/ml) are -7.08ev, -7.98ev and -7.8ev, respectively. The surface charge difference between white cells and CTCs is not significant. This is also the main reason for the adsorption of white blood cells, suggesting that we should try to decrease the surface charge of WBCs, which will help to cut down the adsorption of WBCs. CTCs are widely used in clinical research. How to capture and release them efficiently has become the focus of many people's research. In our experiments, High capture and release efficiency can be obtained at the same time. On the basis of the previous capture experiments on the different substrates (FTO, MnO2/FTO, TiO2/FTO, MnO2/TiO2/FTO), we released the captured cells by further. Previous articles have studied that MnO2 can be dissolved by oxalic acid and the cell viability can be effected by the concentration of oxalic acid

33

, so in our experiment, 2 mM

concentration of the oxalic acid solution was used to dissolve the substrate. As shown in Figure 7a, compared to the FTO and TiO2/FTO substrates (the release efficiency ~0% and ~60%), the release efficiency of MnO2/FTO and MnO2/TiO2/FTO substrates presented a dramatically increased ~97.2% and ~86.7 %, respectively. For the MnO2/FTO substrate, there is only a combination cell- MnO2 affinity and interaction, but for MnO2/TiO2/FTO substrate, there is not only a combination cell- MnO2, but also exist a strong combination cell- TiO2 nanorod arrays affinity and interaction, the combination of cells and titanium dioxide cannot be interrupted by dissolving MnO2 nanoparticles, so the release efficiency on the MnO2/TiO2/FTO substrate is lower than


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the capture efficiency on the MnO2 /FTO substrate. Figure 7b and Figure 7c demonstrate that the release efficiency is only related to the material of the substrate and regardless of the cancer cell types. Bright images of released SW480 and MCF-7 cell lines on the different substrates are shown in Figure S5. If we want to use the circulating tumor cells to conduct molecular characterization and cell analysis, it is crucial to guarantee the released cells maintain high viable. In our experiment, according to our group previous work33, the optimal concentration of oxalic acid to dissolve the MnO2 is 2*10−3M, the released efficiency up to 90% and the released cells could be further cultured(6h,12h,24h,48h) ( Figure 8). At the same time, we want to verify whether nanorod arrays can cause the damage to cells, we capture the cells at different times (6h,12h,24h,48h), the proportion of viable cells approximately 95% ( Figure 9). To avoid the influence of based background and the process of cultured-cells waste, a live/dead cell test was employed to assess the viability of collected cells and used oxalic acid to dissolve MnO2 nanoparticles, all the cells were released from the substrates. We have used the cancer cell line to confirm the CTC-capture and release device, and now, we will use this platform to test cancer patient samples. The samples were drawn from different stage MCF-7 patients (early stage, middle stage, terminal stage) (Figure 10). Early stage sample number is #1-1, #1-2, #1-3, middle stage sample number is #2-1, #2-2, #2-3, terminal stage sample number is #3-1, #3-2, #3-3, using anti-EpCAM to obtain the CTC numbers from these different stage patients ranged from 5-18/0.5ml, and the average number of captured-cells from these different stage patients are 7, 11 and 16, respectively. Meanwhile, in order to prove our CTC-capture platforms have highly capture efficiency in patient samples, we adopt magnetic beads to isolate the CTC from the same patient’ blood sample, the average number is 9, 11



and 18, respectively. The bright field microscopy images of the CTCs (Figure 11) which isolated from blood samples displayed that some WBCs adsorption in CTC capture. In order to further observe the cell morphology, SEM was used to characterize (figure S8). More interesting things happened in our experiment, we found that with the change of cancer stage, the number of CTC is increasing, and meanwhile, the morphology of cells is also changing greatly. At the early stage of cancer, the size of the cell was about 35um, but at the terminal stage, the size of the cell was just 18um. It is indicated that our capture platform showed fairly high capture efficiency in the peripheral blood samples. Basing on FITC-labeled anti-CD45, PE-labeled anti-Cytokeratin, and DAPI nuclear staining, many reports used the three-colour immunefluorosence method to authenticate and enumerate CTCs from non-specifically trapped WBCs29. Between CTCs and WBCs, the expression levels of CK and CD45 have a large difference. Our experiment (Figure 12) displayed that CTCs present a high CK expression while WBCs show a high CD45 expression. Moreover, we can obtain that the size of CTCs is larger than WBCs. The above experiment results can lead to a conclusion: CTCs (DAPI+, CK+), WBCs ( DAPI+,CD45+).

Conclusions: We have designed a novel two-dimensional combined with a three-dimensional platform to capture and release circulating tumor cells. The light transmittance of these substrates are ranged from 50%-80%. MnO2/TiO2/FTO substrate can afford much antibody modification sites for target cell contact, coupled with strong combination,cell-TiO2 or MnO2/TiO2 nanorod arrays affinity and interaction, the optimal capture efficiency is as high as 92.9% and the release efficiency is up to 90%, the released cells could be further cultured. Our platform is applied to the artificial


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blood samples, the capture yield above 80% in DMEM medium and about 60% in whole blood samples, respectively. Compared to adopting magnetic beads to isolate CTCs from the different stage breast cancer patients, our platform showed fairly high capture efficiency, we also found that with the change of cancer stage, the number of CTCs is increasing, and meanwhile, the morphology of cells is also changing greatly. Our device has a great potential in capture and release circulating tumor cells, which facilitate further cancer diagnosis and treatment.

ASSOCIATED CONTENT Supporting Information Information of fabrication of the capture platform, materials and methods, surface modification with streptavidin, cells, blood samples and Figure S1-S8.

Author information Corresponding author *E-mail: [email protected] *E-mail: [email protected] Author Contributions R. Li and F.F. Chen contributed equally to this work. Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by the National Key R&D Program of China (No. 2017YFF0108600) and the National Natural Science Foundation of China (No. 81572860, 81672668).



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Figures

Figure 1: A scheme showing the modification of the TiO2 nanorod arrays coated with soluble MnO2 nanoparticles and added an antibody for tumor cell capture.



Figure 2: Cell capture efficiency on different substrates (FTO, MnO2/FTO, TiO2/FTO, MnO2/TiO2/FTO).


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Figure 3: Capture efficiency for cancer cells at different incubation times.



Figure 4: SEM of captured breast cancer cells on the different substrates (a)FTO, (b)MnO2/FTO, (c)TiO2/FTO, (d) MnO2/TiO2/FTO. Scale bar: 5 μm.


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Figure 5: The recovery efficiencies of breast cancer cells at different cell numbers from artificial CTCs DMEM medium and blood samples on MnO2/TiO2/FTO substrate. (a) The number of captured cells and against the number of spiked cells. (b) The capture efficiency against the number of spiked cells.



Figure 6: Zeta potential of white cells, SW480 cells and MCF-7 cells.


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Figure 7: (a) Release efficiency of the SW480 and MCF-7 cell lines on the different substrates (FTO, MnO2/FTO, TiO2/FTO, MnO2/TiO2/FTO), (b) Release number of the SW480 and MCF-7 cell lines on MnO2/TiO2/FTO substrate (c) Release efficiency of the SW480 and MCF-7 cell lines on MnO2/TiO2/FTO substrate.


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Figure 8: The activity of released MCF-7 cells by 0.2mM concentration of oxalic acid and cultured at different times. (a) 6h, (b) 12h, (c) 24h, (d) 48h. Scale bar: 50 μm.



Figure 9: The viable cells were captured at different times. (a) 6h, (b) 12h, (c) 24h, (d) 48h. Scale bar: 50 μm.


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Figure 10: CTC enumeration results obtained from 0.5 mL blood samples of breast cancer patients.



Figure 11: Using anti-EpCAM and immune-magnetic beads to capture the CTCs. Using anti-EpCAM to obtain the circulating tumor cells from patient blood samples (a) Terminal stage; (b) Middle stage; (c) Early stage. Using immune-magnetic beads to capture the CTCs from patient blood samples (d) Terminal stage; (e) Middle stage; (f) Early stage. Scale bar: 50 μm.


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Figure 12: Fluorescent micrographs of CTCs captured from blood samples from a breast cancer patient. Scale bar: 50 μm.



TOC


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Efficient Capture and High Activity Release of Circulating Tumor Cells

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