Identifying cancer origin using circulating tumor cells

Abstract

Circulating tumor cells (CTCs) have become an established clinical evaluation biomarker. CTC count provides a good correlation with the prognosis of cancer patients, but has only been used with known cancer patients, and has been unable to predict the origin of the CTCs. This study demonstrates the analysis of CTCs for the identification of their primary cancer source. Twelve mL blood samples were equally dispensed on 6 CMx chips, microfluidic chips coated with an anti-EpCAM-conjugated supported lipid bilayer, for CTC capture and isolation. Captured CTCs were eluted to an immunofluorescence (IF) staining panel consisting of 6 groups of antibodies: anti-panCK, anti-CK18, anti-CK7, anti-TTF-1, anti-CK20/anti-CDX2, and anti-PSA/anti-PSMA. Cancer cell lines of lung (H1975), colorectal (DLD-1, HCT-116), and prostate (PC3, DU145, LNCaP) were selected to establish the sensitivity and specificity for distinguishing CTCs from lung, colorectal, and prostate cancer. Spiking experiments performed in 2mL of culture medium or whole blood proved the CMx platform can enumerate cancer cells of lung, colorectal, and prostate. The IF panel was tested on blood samples from lung cancer patients (n = 3), colorectal cancer patients (n = 5), prostate cancer patients (n = 5), and healthy individuals (n = 12). Peripheral blood samples found panCK(+) and CK18(+) CTCs in lung, colorectal, and prostate cancers. CTCs expressing CK7(+) or TTF-1(+), (CK20/ CDX2)(+), or (PSA/ PSMA)(+) corresponded to lung, colorectal, or prostate cancer, respectively. In conclusion, we have designed an immunofluorescence staining panel to identify CTCs in peripheral blood to correctly identify cancer cell origin.

Keywords: CMx chip; Cancer cell origin; IF panel; circulating tumor cells; immunofluorescence staining; microfluidic chip; supported lipid bilayer.

Figure 1.

The CMx system for binding, releasing, and analyzing circulating tumor cells (CTCs). (A) Schematic of CTC capture on a CMx chip. A blood sample is drawn from a 5 mL volume syringe connected at the inlet of CMx chip and processed through the microfluidic channel on the CMx chip by syringe pump withdraw. CTCs can be captured by the anti-EpCAM antibody layer that is linked to the functional supported lipid bilayer (SLB) coated on the glass substrate. (B) Cell release mechanism of the CMx platform. Once the blood sample has flowed through the microfluidic channel, the channel is rinsed with PBS to remove excess blood cells. Captured CTCs are then released by injecting air foam into the channel, which disrupts the SLB coating without damaging the cells. (C) CMx platform workflow for the IF staining panel. Blood samples are drawn in EDTA tubes. 12mL of blood is mixed with 3mL of Streck Cell Preservative and processed within 24 hours. Six CMx chips are used for blood processing resulting in a sample volume of 2.5 mL/chip. An additional chip with a baseline sample is used for quality control (QC). After sample processing and PBS rinse, captured cells are released into individual Eppendorf tubes and are transferred to separate collection membrane chips for staining with immunofluorescence (IF) markers. Each collection membrane chip was applied to its corresponding primary antibody condition. The QC chip processed a control sample 200 cells of CMFDA-labeled cancer cells, in or culture medium. The efficiency of detection in the control sample must be >70% to ensure the CMx chip quality.

Figure 2.

Immunofluorescence (IF) staining of cancer markers on cell lines from lung, colorectal, and prostate cancer origin. The array of images were representative of NSCLC (H1975), CRC (DLD-1), prostate carcinoma (LNCaP), and lymphoma (HL-60) cell lines stained with anti-panCK, anti-CK18, anti-CK7, anti-TTF-1, anti-CK20, anti-CDX2, anti-CK20/anti-CDX2 mixture, anti-PSA, ant-PSMA, and anti-PSA/anti-PSMA mixture. 200 cells were dripped and fixed on the collection membrane chip, underwent IF staining with rabbit hosted primary antibody for each marker, and labeled with Alexa Flour 647 conjugated goat-anti-rabbit IgG secondary antibody (red). Cell nuclei were labeled with DAPI (blue). The scale bar represents 50 µm.

Figure 3.

Decision tree for predicting CTC tissue origin. CTCs from lung cancer origin could be identified in chip1/chip2/chip3/chip4. CTCs from colorectal cancer could be identified in chip1/chip2/chip5. CTCs from prostate cancer could be identified in chip1/chip2/chip6. Additionally, CTCs of tissue origins other than lung, colorectal, and prostate could be identified in chip1/chip2.

Figure 4.

Cell line overall efficiency of CMx chip and staining efficiency test. 200 cells, stained with CellTracker CMFDA, were spiked in 2 ml of culture medium or whole blood. After CMx chip capturing and releasing to an Eppendorf tube, the cells were dripped and fixed on the collection membrane chip. The number of pre-stained cells were counted for the calculation of the system overall efficiency (triplicate individual tests of each cell line). The overall efficiency (%) was defined as: number of CellTracker CMFDA stained cells on the collection membrane chip / number of CellTracker CMFDA stained cells spiked into the CMx chip x 100%

Figure 5.

CTC count and analysis of blood samples using the CMx platform and staining panel. Blood from 12 healthy individuals, 3 NSCLC patients, 5 CRC patients, and 5 prostate cancer patients were processed through the CMx platform, and the enumerated CTCs were applied in the IF staining panel. The distribution of CTCs in the panel is shown for each group of patients.

Figure 6.

CTCs of cancer patients identified in the IF staining panel. Representative images of CTCs and white blood cells (WBC) from patients of (A) NSCLC, (B) CRC, and (C) prostate cancer. The cancer related markers were labeled with Alexa Flour 647, and WBC marker CD45 was labeled with FITC. Cell nuclei were labeled with DAPI. (D). Identified circulating tumor microemboli (CTM) morphology. All images were taken with a Nikon Ti-Eclipse microscope at 100X magnification. Scale bar = 10 µm.