Candle soot-templated silica nanobiointerface chip for detecting circulating tumour cells from patients with urologic malignancies

Liquid biopsy, known as fluid biopsy or fluid phase biopsy, is of great clinical significance in cancer diagnosis and treatment monitoring. However, traditional techniques still meet restrictions when aiming for the detection of circulating tumour cells (CTCs) with high efficiency and low cost. Herein, we applied an easily prepared silica nanobiointerface chip for detecting CTCs in prostate cancer (PCa) and clear cell renal cell carcinoma (ccRCC) patients with high efficiency. The silica nanobiointerface chip was fabricated by depositing candle soot on a glass slide, followed by chemical vapour deposition, and then by modifying anti-epithelial cell adhesion molecule (EpCAM) antibody. The silica nanobiointerface chips exhibited excellent abilities to capture PC3 PCa cell lines, with average efficiency of 81.2 ± 1.4%. We demonstrate that the strong topographic interaction between targeted cells and nanostructured surface is critical to enhancing the capture efficiency of CTCs. We further tested peripheral blood samples from 10 preoperative PCa and 7 ccRCC patients. The results show that CTCs from 7 PCa cases and 4 ccRCC cases were successfully detected. We believe that the nanobiointerface chip will provide great potential for the clinical application of CTC.


Introduction
Circulating tumour cells (CTCs) have emerged as a promising biomarker for early diagnosis, monitoring and prognosis of multiple malignancies. 1,2 Prostate cancer (PCa) is the most common male cancer in western countries and has yielded considerable research on CTCs. [3][4][5] However, as another common urologic malignancy, there are limited studies on renal cell carcinoma CTCs. In general, CTC enrichment detection relies on cellular biological properties, such as micro beads, which select CTCs via surface marker or deplete unwanted leukocytes, 6,7 while some assays rely on the physical properties, such as size and density. 8,9 Despite boosted research interest in CTCs, its low concentrations in peripheral blood poses a serious challenge for any analytical system. 10 Previous studies on detecting CTCs in PCa patients with the "gold standard" CellSearch System showed controversial correlations between CTC and prognostic factors, resulting from the limited specicity and efficiency of the system. [11][12][13] Moreover, the high expense of some detecting assays hampered their clinical application. Developing more efficient methods with low cost is becoming increasingly necessary. In recent years, nanotechnology has shown great promises in CTC enrichment and detection. 14,15 In addition to the current promising nanosubstrates, we hope to nd out more cost-effective and easily-prepared substrates for CTC capturing.

Nanobiointerface chip preparation
Commercial glass slides were cut into 1 Â 2 cm pieces to t Lab-Tek Chamber Slides. The cut slides were held in a candle ame and moved back and forth uniformly for 12 seconds. Then, the slides were placed in a desiccator together with two open glass vessels containing 3 mL TEOS and the other containing ammonium hydroxide. Chemical vapour deposition was performed at 37 C for 24 hours in an enclosed desiccator. Silica shell was formed by hydrolysis and condensation of TEOS. The substrates were then calcinated at 600 C for 2 hours in air, and then cooled till room temperature. During calcination, the carbon cores thermally degraded, leaving behind hollow silica shell. Aer plasma treatment, the substrates were ready for the well-established modication procedures to link biotinylated capturing antibodies (Fig. 1a). 18 The at glass substrates were washed and then subjected to plasma treatment and the same modication. To embed the biotin-anti-EpCAM, the substrates were placed in 4well Lab Tek chamber slides, and then incubated with 25 mL biotin-anti-EpCAM solution (10 mg mL À1 in PBS) in each well for 30 min at room temperature. Aer washed once with PBS, the substrates were ready to use.

Scanning electron microscope (SEM) images for captured cells
We used diluted PC3 cell suspension (10 5 cells per mL) for cell capture on nanobiointerface and at glass chips. Aer capturing, the chips were washed for 3 times and xed in 2.5% glutaraldehyde solution at room temperature overnight. Dehydration was then performed using different concentrations of ethanol and puried water (30%, 50%, 70%, 85%, 95% and 100%). Then, the substrates were treated with 50% (in ethanol) and 100% hexamethyldisilazane for 10 min at room This journal is © The Royal Society of Chemistry 2018 temperature, in sequence. Aer gold sputtering, the chips were observed under SEM (HITACHI, SU8010).

Cell line capture
PC3 human prostate cancer cell line, Jurkat immortalized human T lymphocyte cell line and Daudi B lymphoblast cell line were cultured according to the general process from American Type Culture Collection (ATCC). For all cell lines, we applied our homemade device (a glass capillary connected to a winged needle set and an injector) to count approximately 50, 100, 200, 500 cells. The counted cells were added in 4-well Lab Tek chamber slides with a biotin-anti-EpCAM-embedded nanobiointerface chip and 1 mL DMEM in each well. PC3 cells were also added to biotin-anti-EpCAM embedded at glass chips. The cell suspensions in different concentration gradients were all incubated for 45 min (37 C, 5% CO 2 ). Then, the chips were washed with PBS three times, xed with 4% PFA, washed with PBS and treated by 0.2% Triton X-100. Aer incubating all the chips in 1% DAPI solution for 10 min, we counted captured cell number using a uorescence microscope (Leica, TCS SP8). All capturing assays were repeated 3 times for each cell line.

CTC capture from peripheral blood
All experiments were performed in accordance with the guidelines of "Ethics of Biomedical Research with Human Involved (National Health Commission of P. R. China)." The experiments were approved by the ethics committee of Peking University Third Hospital. Informed consents were obtained from human participants of this study. We randomly chose 10 preoperative prostate cancer patients. Peripheral blood was stored in vacuum tubes with EDTA as an anticoagulant and sent to test as quickly as possible. Then, 1 mL of blood from each patient was added to Lab Tek chamber slides with biotin-anti-EpCAM-embedded chips and incubated for 45 min (37 C, 5% CO 2 ). The chips were then washed gently in PBS 5 times (until there was little gross redness), xed in 4% PFA for 30 min, washed with PBS for three times and treated with 0.2% Triton X-100 for 30 min. Aer PBS washing for 3 times and BSA solution (2% BSA in PBS) blocking for 2 hours, the blocking solution was removed without washing. Then, the chips were incubated with 50 mL anti-CK and 50 mL anti-CD45 solution at 4 C in the dark overnight. Aer washing with PBS, the chips were incubated in 1% DAPI solution for 5 min and washed again 3 times with PBS. The cells were ready for uorescence microscopy observation (Leica, TCS SP8, a few by Nikon Ti-E, Fig. 1b). Procedures were the same using blood from 7 renal cancer patients. The chips were incubated with 50 mL mixed antibody solution comprising 25 mL biotin-anti-CA9 (10 mg mL À1 in PBS) and 25 mL biotin-anti-CD147 (10 mg mL À1 in PBS).

Results and discussion
PCa is the second leading cause of male death according to American Cancer Society, and it is a classic cancer type for CTC research. 3 First, we applied EpCAM positive PC3 prostate cancer cell suspension for cell capture. Nanobiointerface chips and at glass chips were put in chamber slides and modied with biotin-anti-EpCAM antibody. PC3 cell suspension was then added to the chamber slides and incubated for 45 min. Aer cell capturing, we observed the chips via SEM. SEM images displayed a nanostructure of irregular dendritic silica shells on the nanobiointerface chip surface (Fig. 2). Previous research showed that cells had limited interactions with the smooth surface of the chip and remained spherical. 17 In our experiment, however, SEM images revealed that the captured cell morphology changed remarkably with many lopodia protruded on the nanosurface. As a control, cells displayed more regular spherical shape on the at surface (Fig. 2). These lopodia enhanced the local topographic interactions between cell membrane and nanobiointerface chip surface, which amplied the antigen-antibody reaction and binding affinity.
We further examined the more precise capture efficiency of cell lines. We counted approximately 50, 100, 200 and 500 PC3 cells under the microscope, and also added the cells into 1 mL DMEM in Lab Tek chamber slides with a nanobiointerface or at glass chips. The cell suspensions in different concentration gradients were all incubated for 45 min (37 C, 5% CO 2 ). Aer gently washing with PBS for three times, xing with 4% PFA and staining with DAPI, we counted the captured cells under the uorescence microscope. On the nanobiointerface chips, the numbers of captured PC3 cells were 41 (41/50, 82%), 77 (77/100, 77%), 167 (167/200, 83.5%), and 403 (403/500, 80.6%), while on at glass chips, the numbers of captured cells were only 5 (5/50, 10%), 8 (8/100, 8%), 22 (22/200, 11%), and 43 (43/500, 8.6%). All capturing procedures were repeated another two times. Collectively, our nanobiointerface chips showed good capture efficiency with an average of 81.2 AE 1.4%, which is signicantly higher than the efficiency of at chips (Fig. 3). To determine the structure-derived non-specic binding, we also captured EpCAM negative Jurkat and Daudi cells. Under the same four concentration gradients, the average capture efficiency was only 9.2 AE 2.0% and 8.4 AE 2.1% for Jurkat and Daudi cells, respectively (Fig. 3). Compared with the signicant lower capture rate of at chips or EpCAM negative cells, the nanobiointerface chips could enhance the capture ability of targeted cells via dual structural and molecular recognition. Although manual errors could occur in the cell adding and counting process, the capture efficiency of the nanobiointerface chips for PC3 cells was comparable to that reported in previous research using such chips and higher than that of traditional SiNP substrates. 17,20 In addition, we chose the countable cell concentrations analogous to general CTC concentration in cancer patients' blood. The capture efficiency almost remained stable for cell concentration no more than 500/mL.
Due to the satisfying capture efficiency on cell lines, we further investigated the ability of our chips to catch CTCs under real clinical condition. Peripheral blood was taken from PCa patients preoperatively and transmitted using a vacuum tube for testing as quickly as possible. Then, 1 mL of the blood sample from each patient was added to chamber slides with biotin-anti-EpCAM-embedded chips and incubated for 45 min. The chips were then washed gently ve times in PBS to remove excess red blood cells and xed with PFA. Aer blocking and penetration, we marked cytokeratin (CK) with red uorescence, CD45 with green uorescence and nuclei with blue uorescence (DAPI) for uorescent microscope observation. PCa is epithelium originated, so its CTCs maintain the epithelial marker CK most of the time, while CK does not express in blood cells. We dened the cells meeting all the following criteria as CTCs: (1) positive CK and DAPI staining but negative CD45 staining (CK+/CD45À/DAPI+, white blood cells as CK-/CD45+/DAPI+), (2) cellular diameter between 13 mm to 50 mm, and (3) nuclear-cytoplasmic ratio over 50%. We took blood from 10 preoperative PCa patients. All patients underwent laparoscopic radical prostatectomy; the diagnosis of PCa was conrmed by pathology with stage ranging from T2aN0M0 to T3bN1M0, i.e., both localised and local advanced disease and low to high risk classication (Table 1). Of the 10 patients we examined, we could nd at least one CTC in 7 cases meeting all the criteria (Fig. 4). Moreover, we examined 5 patients with benign prostate hyperplasia (malignancy was excluded pathologically) and could not nd conrmed CTCs in all these ve patients. As we only detected CTCs with epithelial phenotype, CTCs undergoing epithelial mesenchymal transition (EMT) were yet to be identied with more markers. It should be noted that CTC formation is an early event in cancer progression, 21 and we detected CTCs not only in patients with local advanced disease, but also in early localized tumour without using much blood. Our chips may be sensitive for CTC detection with good specicity in PCa patients with relatively lower tumour burden and earlier stage diseases. Furthermore, the good sensitivity of the biointerface chips may also enable their application in diagnosis of suspicious cases and postoperative disease monitoring.
Motivated by the results of PCa, we decided to further detect CTCs in renal cancer patients. Renal cancer (including renal pelvis) is the 5 th most common cancer for males and 10 th for females. 3 Unlike prostate cancer, limited studies reported ideal results for detecting CTCs in renal cancer patients. 7,22 This may result from the insensitivity of both the methods and EpCAM antibody. Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cell carcinoma, but only 10-36.3% of ccRCC cases express EpCAM. [23][24][25] One study applied NanoVelcro substrates with anti-carbonic anhydrase 9 (CA9) and CD147 as dual capturing antibodies to test CTCs in 33 ccRCC patients, and 31 of them tested positive. According to this promising result, we further checked the validity of our chips. We chose 7 preoperative ccRCC patients (all conrmed by postoperative pathological assessment) with clinical stage of T3aN0M0 or above and took their peripheral blood ( Table 2). The chips were modied with biotin-anti-CA9/CD147 antibodies and blood samples were treated using the same procedures mentioned before. Of the 7 cases, we detected CTCs in 4 cases (57.1%) with the classic phenotype of CK+/CD45À/DAPI+ (Fig. 5). Although the positive rate is lower than the microuidic NanoVelcro assay, it is comparable or higher than that reported before. 7,22 Due to the underwater transparency of the chips, we could directly monitor the captured cells via bright eld imaging. In another case, we found some suspicious cells with the phenotype of CKÀ/CD45À/DAPI+ and irregular shape but larger size than blood cells (Fig. 6). As previous studies identied CTCs from ccRCC patients acquiring mesenchymal and stem cell phenotypes, 26,27 we assume that these suspicious cells may be CTCs without epithelial markers. Furthermore, it is possible to identify more phenotypes with our chips and apply other potential capturing markers to enhance positive rate in future. Since we chose the advanced ccRCC cases, the validity of the chips to detect CTCs in earlier stage diseases needs further studies.  1  T3aN0M0  High  2  2  T3aN0M0  High  2  3  T3bN1M0  High  3  4  T2cN0M0  High  0  5  T3bN0M0  High  0  6  T3aN1M0  High  1  7  T3aN0M0  High  1  8  T2aN0M0  Low  0  9  T2bN0M0  Intermediate  1  10 T2cN0M0    1  T3aN1M1  IV  3  2  T3aN1M1  IV  2  3  T3aN0M0  III  0  4  T3bN0M1  IV  1  5  T3bN1M0  III  3  6  T3aN0M0  III  0  7  T3aN0M0  III  0

Conclusions
In conclusion, the candle soot-moulded nanobiointerface chips were cost-effective and able to catch specic cancer cells. The irregular dendritic nanostructure enhanced the interaction between cell membrane and chip surface and amplied the binding affinity with targeted cells, contributing to an ideal efficiency of around 80% for PC3 cell line capture. Furthermore, our chips successfully captured CTCs from blood samples of PCa and ccRCC patients. It showed sensitivity to capture CTCs in early-stage PCa patients with low tumour burden and might be valid to distinguish more subtypes of CTCs in both PCa and ccRCC. These low-cost nanobiointerface chips can be used to further study their potential use for early diagnosis of clinically suspicious PCa and postoperative surveillance.

Conflicts of interest
There are no conicts to declare.  This journal is © The Royal Society of Chemistry 2018