Multi-modal liquid biopsy platform for cancer screening: screening both cancer-associated rare cells and cancer cell-derived vesicles on the fabric filters for a reliable liquid biopsy analysis

Circulating tumor cells (CTCs) are receiving a great amount of scientific interest as a diagnostic biomarker for various types of cancer. Despite the recent progress in the development of highly sensitive CTC isolation devices, post-capture analysis of CTCs is still hindered by technical challenges associated with their rarity. Herein, we present a multi-modal CTC screening platform which is capable to analyze CTCs and CTC-derived extracellular vesicles (EVs), simultaneously from a single sample. Cytochalasin B (CB) treatment promotes cells to release large number of EVs from their surface, as demonstrated by CB-treated cells (5 µg/mL for 3 h) secreting 3.5-fold more EVs, compared to the non-treated cells. CB further generates 1.7-fold more EVs from the cells captured on our CTC filtration device (the fabric filter), compared to those from the cell culture flasks, owing to its multiple pore structure design which reduces the non-specific binding of EVs. Both CB-treated cancer cells and CB-induced EVs are found to overexpress tumor-associated markers, demonstrating a potential for the development of CTC dual-screening platform. Collectively, the results presented in this study reveal that our multi-modal cancer screening platform can synergistically improve the reliability and efficacy of the current CTC analysis systems.


Cell Preparation
Human breast cancer cell line, MCF-7, was obtained from Korean Cell Line Bank (Seoul, South Korea). Cells were stored in liquid nitrogen in aliquots at a passage number below 10.
Every experiment was performed after 3-5 passages post thaw. Cells were grown continuously as a monolayer in T25 culture flasks in RPMI 1640 media supplemented with 10 % (v/v) fetal bovine serum (FBS; Gibco) and 1 % (v/v) penicillin-streptomycin (Invitrogen). The cells were kept in humidified condition at 37 °C with 5% CO2 and incubated for ~72 h before they reached at 80% confluency. Cells were gently harvested and resuspended to RPMI media, having cell density of 5 × 10 5 cells per/flask. Cells were incubated further 24 h before CB treatment.

Cytochalasin B Treatment
CB was diluted with 4 mL serum-free media at the concentration of 0, 5, 10, 15, and 20 μg mL -1 . Cells were treated with CB for 0.5, 3, or 24 h in humidified condition at 37 °C with 300 rpm agitation. CB-induced EVs were then collected by series of centrifugations. First centrifugation was conducted at 1,000 g for 10 min, in order to remove the cells and large debris.
EVs were then further purified by spinning the sample at 14,000 g for 1 h and removing the top 0.5 mL layer. The samples were stored at -70 °C, before it was used for the subsequent experiments.
In case of obtaining EVs from the cells on the fabric filters, we first assembled fabric filters with the filter holders, as described in our previous literatures [1 -3]. Previously, we confirmed that our fabric filters could isolate the cancer cells at the capture efficiency of ~70% [1][2][3]. In this study, 1 mL of cell suspension was gently pipetted through the fabric filter at a cell density of 7.5 × 10 5 cells/mL. Cells were incubated for two hours on the filter and processed at the flow rate of 5 mL/h for an hour. The number of cells remained on the filters was measured by subtracting the number of cells released from the filter. Approximately (5.0 ± 0.9) × 10 5 cells were captured on the filter.
Filters were gently removed from the filter holders and placed on Corning® Costar® Ultra-Low Attachment 6-well plate. The cells on the filters were further incubated with complete media for 24 h and subsequently treated with CB at the concentration of 5 µg/mL for 3 h, which is determined as the optimal CB concentration and treatment time.

Cell Viability Measurement after Cytochalasin B Treatment
Viability of the cancer cells was measured using two-colored live/dead viability assay

Field Emission Scanning Electron Microscope (FE-SEM) Imaging
The EVs collected by CB treatment (at the optimal condition) were examined with field emission scanning electron microscope (FE-SEM, SU5000, Hitachi) with an acceleration voltage 4 of 5 kV. EVs were gently laid above the glass slides and dried at 4°C for 6 h. The slide glass was coated with 3 nm-thick osmium, to minimize the charging effects of the EVs. The images of the EVs were acquired to confirm the size and morphology.

Nanoparticle Tracking Analysis (NTA) and BCA Assay
Nanoparticle tracking analysis (NTA) was conducted using Nanosight N5300 (Malvern Instruments), to obtain the size distribution and the concentration of CB-induced EVs. 200 μL of the sample was loaded on the center of the plate, which was located above the laser module. The module was gently mounted on the main instrument and the size distribution of EVs was visualized by tracking the movements of individual particles. Data processing was done by NanoSight N5300 Control Software.
Total amount of protein for each of different CB concentration and incubation time was measured by extracting from RIPA buffer and protease inhibitors mixture for 15 min. Micro BCA protein assay (Thermo scientific) was used to calculate total amount of proteins in EVs, following the manufacturer's instruction.

Immunocytochemistry Analysis
EGFR expressions were examined based on immunocytochemistry, to verify that cancerrelated markers were still capable to be observed after CB treatment. The cells on the fabric sheets were fixed and permeabilized as described in the previous section. Endogenous peroxidase was blocked by incubating the filters with 1 % H2O2 in methanol for 30 min at the room temperature. for 10 min at the room temperature, followed by another PBS washing. Modified Mayer's hematoxylin (Thermo scientific, MA, USA) was treated for 3 min and rinsed with deionized water (three times, 3 min respectively). The filters were kept in 6-well cell culture plate mounted above the inverted microscope (Carl Zeiss) and the EGFR expressions were measured by comparing the intensity of the cells, before and after CB treatment.

Immunofluorescence Analysis
We compared the signals of two cancer-related markers, EGFR and EpCAM, to verify the effect of CB treatment on surface protein expression. The cells on the fabric filters were fixed with 4 % paraformaldehyde (PFA, Santa Cruz Biotechnology) for 15 min and rinsed with PBS solution for 5 min after CB treatment. Cells were subsequently exposed to PBS solution with 3 % bovine serum albumin (BSA, Sigma chimicals) for 30 min to prevent the non-specific bindings.