Natural Fish Trap‐Like Nanocage for Label‐Free Capture of Circulating Tumor Cells

Abstract Nanomaterials have achieved several breakthroughs in the capture of circulating tumor cells (CTCs) over the past decades. However, artificial fabrication of label‐free nanomaterials used for high‐efficiency CTC capture is still a challenge. Through billions of years of evolution and natural selection, various complicated and precise hierarchical structures are developed. Here, a novel fish trap‐like “nanocage” structure derived from the natural Chrysanthemum pollen is reported and a nanocage‐featured film for the label‐free capture of CTCs and CTC clusters is constructed. The nanocage‐featured film effectively captures 92% rare cancer cells with a broad spectrum of cancer types, due to the synergistic effect of nanocage‐CTC filopodia matching, high contact area, and strong adhesion force between the cancer cells and the nanocage. Furthermore, the nanocage‐featured film successfully detects CTCs and CTC clusters in 2 or 4 mL blood taken from 21 cancer patients (stages I–IV) suffering from various types of cancers. This novel, abundant, and economical fish trap‐like “nanocage” may provide new perspectives for the application of natural nanomaterials in clinical CTC capture and analysis.

sputter-coated with gold, observed and photographed with a NOVA NanoSEM 450 (FEI, U.S.A.). SEM images of the EChry pollens and nanocage were taken and the images were used for the quantitative and morphological analysis of the pollens and nanocage using the software of Nanomeasurer.

Surface Spray-coating of EChry films:
PVB film was fabricated on a flat glass substrate by applying a PVB ethanol solution (10%, v/v) via spin-coating at a rotation rate of 4000 rev. min −1 for 30 s in air, or via spraying the same PVB solution on an irregular or curved substrate, such as silicone tube, and metal coin. EChry pollens-suspended ethanol solution (25 mg mL -1 ) was then sprayed over the PVB film using an airbrush (U-STAR, A-119) at a nozzle height of 10 cm, a carrier gas pressure of 30 psi and a spraying time of 10 s. The obtained EChry PVB film was purged with N 2 to remove the excess pollens and then blown-dried. After complete drying, the film was sputter-coated with gold, observed and photographed with a NOVA NanoSEM 450 (FEI, U.S.A.). Fresh 0.25% trypsin-EDTA in PBS was used to resuspend cells.
Cell capture experiments performed in culture medium: EChry films (1 × 1 cm 2 ) were incubated with 1% BSA for 1 h at room temperature and then placed in a 24-well plate (1 piece per well) loaded with cell suspension (1 mL per well, containing 10 5 cells). After incubation at 37 °C and 5% CO 2 for a certain time, the cell suspension was removed and the films were gently rinsed with PBS for three times. After fixed with 4% PFA and gently rinsed with PBS, the captured cells were incubated with PBS containing 10 μg mL -1 DAPI for 15 min, and then photographed and the number of captured cells was counted from the image (10 × objective) using Image-pro-plus. Cell capture yield was calculated using the number of cells per unit area as the following formula: is the practical number of capture cells per image, and is the theoretical cell number per image, calculated as 1000 in this experiment.
To give an average captured cell number of , twenty-five fluorescent images (10 × objective) were uniformly taken of the whole area of cell-capture films (1 × 1 cm 2 ) without any subjective intention. Five parallel experiments (films were different batches) were carried out to give an error bar for each cell-capture yield.

SEM analysis of Captured cells:
Cells captured on the films were rinsed with PBS and fixed with 2.5% glutaraldehyde for 2 h at room temperature. The cells were then dehydrated through a series of ethanol concentrations (30%, 50%, 70%, 80% and 90%), each lasting for 5 min, followed by lasting for 15 min in absolute ethanol for two times. After that, the cells were dried in liquid CO 2 using a supercritical point dryer to maintain the morphology of the captured cells. After complete drying, the samples were sputter-coated with gold, observed and photographed with a NOVA NanoSEM 450 (FEI, U.S.A.). The FE-SEM images were used to directly determine the number of filopodia and their diameters using the software of Nanomeasurer.

TEM Observation:
Cells captured on the EChry pollens were fixed with 2.5% glutaraldehyde at room temperature for 30 min followed by fixation at 4 °C overnight. The sample was centrifuged and rinsed with PBS for three times, and then dehydrated in a graded ethanol series. Subsequently, absolute ethanol (Gold Shield) was exchanged for acetonitrile. This was followed by infiltration with a graded Embed 812 resin series.
The resin-block sample was incubated at room temperature for 1.5 h and then baked at 65 °C for 24 h. The resin block was then trimmed and sectioned on a Leica Ultracut ultramicrotome into 70 nm-thickness sections and imaged on a Tecnai F30 (FEI, U.S.A.).

Immunofluorescence Staining of Captured Cells:
The captured cells were treated with 4% PFA for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. Then the captured cells were incubated with 1% BSA in PBS for 1 h at room temperature and then incubated with 10 μg mL -1 rabbit anti-vinculin primary antibody at 4 o C for 12 h and 10 μg mL -1 Alexa Fluor 488-conjugated goat antirabbit IgG secondary antibody at 37 o C for 1 h. Afterward, the cells were subsequently incubated with 10 μg mL -1 rhodamine-conjugated phalloidin (in PBS) for 30 min, and then incubated with 10 μg mL -1 DAPI (in PBS) for 5 min. Finally, the cells were examined under a confocal laser scanning microscope and images of the cells were recorded and used to determine the heights of nucleus using the software of Image-Pro Plus.

Anti-EpCAM modified assay:
The PVB, PS, EChry-bl and EChry films were separately loaded into a 24-well cell culture plate, each well of which was immersed in 1 mL of biotin-BSA solution (0.5 mg mL -1 in PBS) at 37 °C for 2 h and then rinsed with PBS for three times. Afterward, the films were treated with 10 μg mL -1 of streptavidin (SA) at 37 °C for 30 min. After rinsing with PBS for three times, 100 μL of biotin-anti-EpCAM (10 μg mL -1 in PBS) was added onto the films at 37 °C for 30 min and then rinsed with PBS for three times.

Collecting and Processing Human Blood Specimen:
Blood specimens were drawn from healthy donors. Sodium citrate was added to the blood samples as an anticoagulant. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood using a human mononuclear cell separation kit (Beijing Solarbio Co. Ltd, China) according to the manufacturer's specifications.

Rare Cell Capture Experiments Performed in Isolated PBMCs:
Prior to cell-capture assays, the targeted MCF-7 cancer cells were pre-labeled with DiO (10 μg mL -1 in PBS) for 30 min. The target-number cells (10-4000) were added into the 1-mL PBMC suspension (10 6 mL -1 ). The mixtures were loaded onto the EChry films (2 cm 2 in round), and incubated at 37 °C and 5% CO 2 for 60 min. After image with 4 × objective) were taken from one EChry film without missing or overlapping any area. The total number of the captured rare cancer cells was the sum of captured cells number on each image. Cell capture yield was calculated using the ratio of the total number of captured cells to loaded cancer cell numbers. Additionally, the accuracy of the rare cell capture yields was double-checked through counting the number of uncaptured cells to further calculate the captured yields with a subtraction method. The difference in capture yields between these two methods should be less than 1%.

Cell Adhesion Strength Measurements:
The abilities of MCF-7, A431, Hela, HL-60 cells and PBMCs to adhere to the EChry films were measured according to the method reported by Reyes' work. [1] Cell suspensions (1 mL per well, containing 10 5 MCF-7, A431 and Hela cells or 10 6 HL-60 cells and PBMCs) were loaded on the EChry films (1 × 1 cm 2 ) in a 24-well plate (1 piece per well). Following the cell-capture assay, the cells adhered to the films were incubated with 2 μg mL -1 FDA in PBS solution for 5 min, and the images of the cells were then taken under an inverted fluorescence microscope (10 × objective).
Subsequently, 10-mL tapered plastic centrifuge tubes were filled with PBS, individual films containing the cells were then carefully laid on the bottom of the tubes with the side containing the cells facing downward. The tubes were gradually centrifuged in an Eppendorf centrifuge (5840 R) at 6, 34, 88, 166, 309, 550, 859, 1238, and 3834  g, each for 5 min at room temperature. After each round of centrifugation, the cells that remained on the film were examined and counted using the same microscope and under the same magnification (10 × objective). The percentage of cells adhered to the film was calculated using the following formula:

Percentage of remained adhesion cells =
where x is the number of cells attached to the EChry films before centrifugation, and is the number of cells remained on the film after centrifugation. A total of 25 images were taken to present an average value for x and y. Five experiments were carried out in parallel using different batches of films.
The relative centrifugal force imposed on the cells varies according to the following equation: where F c is the relative centrifugal force, is the specific density of the cell (1.07 g cm -3 ), is the specific density of the medium (1 g cm -3 ), is the cell volume (4000 µm 3 for MCF-7 cell, 500 µm 3 for PBMC), 0 is the radius of rotation (determined by the dimensions of the rotor), and x is the lateral distance from the bottom of the tube to the center of centrifuge. The force value at which 50% of the cells were detached was determined as the population adhesion strength.
The measurement was carried out in PBS. The EChry pollen-modified cantilever was then positioned on the center of a cell, and force-distance (F-z) curves were acquired by approaching the cell with the cantilever at 1 µm s -1 , and the contact was maintained for 15 s before the cantilever was retracted at the same speed. The maximum indentation force applied was 100 pN. To collect the adhesion force data, force-curves were obtained for at least nine cells from a minimum of three independent experiments, and an average value of the collected force data was obtained with the help of JPK Data Processing software.

Quantification of Capture Purity of Cancer Cells in the Mixture with PBMCs:
Prior to cell capture assays, the targeted MCF-7 cancer cells were labeled with DiO (10 μg mL -1 in PBS) for 30 min. 4000 MCF-7 cells and 10 6 PBMCs (1:250) were mixed in 1 mL of cell culture medium, and then loaded onto the EChry film (2 cm 2 in round tailoring the size and quantity of EChry films. A 2-mL (or 4-mL) blood sample was drawn from each patient with advanced (or early-stage cancer). First, the blood samples were treated with red blood cell lysis buffer to remove the red blood cells.
The remaining cells in each 2-mL blood were resuspended with 10 mL DMEM, and loaded 1-mL onto 2 cm 2 -EChry film, which has been coated with 1% BSA for 1 h at room temperature before used. After 60 min of incubation (37 °C, 5% CO 2 ), the films were washed with PBS for three times. After that, the cells captured on the films were rinsed three times with PBS, fixed with 4% PFA for 15 min, and 1% BSA for 1 h at room temperature. Subsequently, the cells were incubated with a rabbit CD45-polyclonal antibody (10 μg mL -1 in PBS) for 30 min at 37 °C. This was followed by another three washes in 1% BSA and further incubation with donkey anti-rabbit antibody (IgG -Alexa Fluor 546 at 2 μg mL -1 in PBS) for 30 min at 37 °C.
After that, the captured cells were permeabilized with 0.1% Triton X-100 for 10 min, blood was the sum of captured cells on each EChry film.

Statistical Analysis:
Data are reported as mean ± SD and the statistical significance is determined using one-way ANOVA analysis, which were considered at p < 0.05, p < 0.01 and p < 0.001 levels.

Preparation of H 2 SO 4 -etched Pine and Rape Pollens:
The H 2 SO 4 -etched pine (EPine) and H 2 SO 4 -etched rape (ERape) pollens were prepared with the same process as for EChry pollens, except that the EChry pollens were replaced with EPine and ERape pollens.

Stability Analysis of pollens on EChry film:
The peeling test was carried out according to previously reported methods. In brief, a piece of 3M Scotch tape (KST1046, adhesive strength higher than 1.23 N cm -1 ) was pressed down firmly on the EChry film and then quickly removed. [2] The area of pollens remained attached to the EChry film was determined under SEM and reflected light microscope (10 × objective). The images were saved as uncompressed color images (8-bit). The areas covered by the EChry pollen before and after the peeling test were counted using Image-Pro Plus based on the differences in color. The abscission rate of EChry film was determined by the following formula: where, 1 and 2 are the areas covered by the EChry pollen before and after the peeling test, respectively. is the area of the image taken by the microscope (10 × objective).

Preparation of EChry films with different covering-ratios:
EChry pollens were suspended in absolute ethanol to a concentration of 25 mg mL -1 and a piece of PVB film was then sprayed with this pollen suspension at a nozzle height of 10 cm and a carrier gas pressure of 30 psi for 1 ~ 10 s. The EChry film was then characterized using a reflected light microscope (10 × objective), and the images taken were saved as uncompressed color images (JPEG format). The area covered by the EChry pollen (Α EChry ) was counted using Image-Pro Plus based on the color differences. Then, the ratio of EChry pollen coverage to PVB film was determined by the following formula: Covering-ratio = ΑEChry Αimage where Α EChry is the area covered by the EChry pollen, and Α image is the area of the image (10 × objective) taken by the microscope.

Surface Spray-coating of PS and EChry-bl films:
PS film was prepared under the same condition as for the EChry film, except that the EChry pollens were replaced with PS particles. For the construction of the EChry-bl film, EChry pollens were suspended in 1.5% PVB ethanol solution (v/v), and then sprayed onto the PVB film.

Surface Spray-coating of EPine and ERape films:
EPine and ERape films were prepared under the same condition as for the EChry film, except that the EChry pollens were replaced with EPine and ERape pollens. Characterization: X-ray photoelectron spectroscopy (XPS) experiments were performed with an ESCALAB 250Xi X-ray photon-electron spectrometer (Thermo Scientific, U.S.A.) using Mg Kα radiation under a vacuum of 2 × 10 −8 Pa. The binding energy (BE) scale was calibrated by comparing with the neutral adventitious C 1s peak at 284.6 eV.

Fourier transform infrared (FTIR) spectrum was measured with a BRUKER-MPA
FTIR spectrometer from 4000 to 400 cm -1 at room temperature (Bruker, Germany).
Fluorescence spectra were recorded on a Fluorescence Spectrophotometer-FP-650 at an excitation wavelength of 405 nm (Jasco, Japan). Roughness (Ra) was measured using a LASER-OLS4000 confocal microscope (Olympus, Japan). The nitrogen adsorption and desorption isotherms were measured at 77 K on a Quantachrome instrument Quadrasor SI. The specific surface area (SSA) was calculated by multi-point BET.

Cell capture experiments performed with EChry-bl, PS, EPine, ERape films and TCPS in culture medium:
EChry-bl, PS, EPine, ERape films and TCPS were coated with 1% BSA before used, and the experimental process was the same as that described for EChry films.

The effective contact area of per filopodia with the nanocage and the bed-of-nails nanostructures:
In the process of filopodia inserting and adhering to the nanocage, the contact area A filo between filopodia and nanocage was estimated as one-second of the area of the flank area of the cylinder, [3] using the following formula: where d is the diameter of filopodia was measured as 131 nm and is the length of the nanocage-inserted filopodia which was measured in TEM images. To ensure that the filopodia evaluation was statically significant, more than 40 filopodia were used for the evaluation of inserted filopodia length.
For the reported bed-of-nails nanostructures, the contact area ( ) between the filopodia tip and the nanostructure was considered as the cross section of filopodia tip, [4] calculated using the formula： where d is the diameter of filopodia which was statistics from the previous reports as 100 ~ 150 nm. [5]                 Figure S17. ai-bi) SEM images of EChry and ERape films. aii-bii) Amplifying SEM images of the surficial aperture of EChry and ERape pollen. The average diameter of the entrance is labeled. aiii-biii) Schematic of the effective filopodia-contact area of EChry and Erape pollen. Specific surface area (SSA) is labeled below. ci-di) Presentative DAPI-stained fluorescence images of MCF-7 cells captured on the indicated films. cii-dii) The captured cell was costained for the nucleus (DAPI, blue) and actin cytoskeleton (rhodamine, red). EChry and Erape pollens were pointed with white arrows. ciii-diii) The captured cells on the indicated films. e) Capture yields of MCF-7 cells on EChry and ERape films (mean ± SD, n = 5). **: p < 0.01. f) Percentage of the remaining MCF-7 cells on EChry and ERape films under indicated dethatched centrifugal forces (mean ± SD, n = 5) Figure S18. a) Quantitative evaluation of the capture performance of EChry films for 10 5 mL -1 MSC and HL-60 cells in the culture medium (left, mean ± SD, n = 5). ***: p < 0.001. Presentative DAPI-stained fluorescence images of MSC and HL-60 cells captured on the EChry films (right) (Scale bar = 200 µm). b, c) The captured MSCs and HL-60 cells were costained for the nucleus (DAPI, blue) and actin cytoskeleton (rhodamine, red). Scale bar =50 µm. Figure S19. Capture yields and purities with MCF-7 cells. a,b) Capture yields of a) MCF-7 cells and b) PBMCs before and after centrifugation with 1238 × g (mean ± SD, n = 5). c) Capture purities of MCF-7 cells on the EChry films before and after centrifugation with 1238 × g (mean ± SD, n = 5). d) Captured MCF-7 cells and PBMCs on EChry films before and after centrifugation with 1238 × g (mean ± SD, n = 5). The images below are partially enlargements (scale bar = 200 µm).  Figure S21. The total number of 50, 100, 200, and 500 MCF-7 cells were loaded into 1 mL of PBMC suspension (10 6 mL -1 ) and lysed blood sample (diluted by 5 times). Capture yields of rare cancer cells showed no significant difference between the PBMC suspension and diluted lysed blood sample (mean ± SD, n = 5). (N. S.) denotes not significant at p > 0.05.