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We demonstrated an efficient antibody-enabled CTC capture device based on micromixer structures which increased cell-surface collisions. We achieved ~92% capture efficiency and ~85% purity for the L3.6pl cell line, and ~90% capture efficiency and 80% purity for the BxPC-3 cell line. Capture efficiency was defined as the number of target cells captured over the number of target cells introduced while purity is the number of target cells captured over the number of all cells captured (both target and non-target cells). As expected, the control cell line, MIA PaCa-2, was captured with extremely low efficiency anc purity. The flow cytometry results, shown in Figure 3, display the relative EpCAM expression levels of the tested cell lines. Data shows that anti-EpCAM binds well with L3.6pl and BxPC-3 cells, but not with MIAPaCa-2 cells, indicating the expression or lack of expression of surface epithelial cell-adhesior molecules.

Figure 3 We demonstrated an efficient antibody-enabled CTC capture device based on micromixer structures which increased cell-surface collisions. We achieved ~92% capture efficiency and ~85% purity for the L3.6pl cell line, and ~90% capture efficiency and 80% purity for the BxPC-3 cell line. Capture efficiency was defined as the number of target cells captured over the number of target cells introduced while purity is the number of target cells captured over the number of all cells captured (both target and non-target cells). As expected, the control cell line, MIA PaCa-2, was captured with extremely low efficiency anc purity. The flow cytometry results, shown in Figure 3, display the relative EpCAM expression levels of the tested cell lines. Data shows that anti-EpCAM binds well with L3.6pl and BxPC-3 cells, but not with MIAPaCa-2 cells, indicating the expression or lack of expression of surface epithelial cell-adhesior molecules.

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Figure 1: (a) The layout of microfluidic devices used for this work. (b) Schematic of the micromixer geometry in microchannels and surface modification schemes for CTC capture. Bovine serum albumin (BSA) is used to passivate the uncoated channel surfaces to decrease non-specific cell capture. While leukocytes (white blood cells) and erythrocytes (red blood cells) flow through the channel, CTCs are captured by the antibodies immobilized on the channel surfaces. Drawing is not to scale. ~ Figure la shows the layout of the device. The surface modification scheme is depicted in Figure 1b, in which avidin is physically adsorbed onto microchannel surfaces, followed by conjugation with biotinylated epithelial cell-adhesion molecules (EpCAM) that are present on cells of epithelial origin.
Figure 1: (a) The layout of microfluidic devices used for this work. (b) Schematic of the micromixer geometry in microchannels and surface modification schemes for CTC capture. Bovine serum albumin (BSA) is used to passivate the uncoated channel surfaces to decrease non-specific cell capture. While leukocytes (white blood cells) and erythrocytes (red blood cells) flow through the channel, CTCs are captured by the antibodies immobilized on the channel surfaces. Drawing is not to scale. ~ Figure la shows the layout of the device. The surface modification scheme is depicted in Figure 1b, in which avidin is physically adsorbed onto microchannel surfaces, followed by conjugation with biotinylated epithelial cell-adhesion molecules (EpCAM) that are present on cells of epithelial origin.
Figure 2: CTC enumeration antibody markers panel. After CTCs are isolated from a pancreatic cancer patient using anti-EpCAM, they are verified using fluorescent images, including DAPI-positive (for nucleus staining), anti-cytokeratin-positive (for epithelial origin), and anti-CD45-negative (for non- leukocytes). Also shown is the merged image. To assess the capabilities of our device, we conducted capture experiments with two cell lines know1 to express EpCAM, L3.6pl and BxPC-3, as well as a cell line, MIAPaCa-2, that does not expres EpCAM. Flow cytometry tests were carried out to determine the relative expression levels of EpCAM fo each cell line. After using cell lines to assess the capture parameters of the device, we applied it t capture and enumerate tumor cells in the blood of patients with advanced pancreatic cancer. To verif captured cells as CTCs, we utilized the FDA-approved, triple-color immunocytochemistry technice definition, illustrated in Figure 2. In order to enumerate a captured cell as a CTC, it must be a nucleate cell, verified with a staining dye (DAPI), and be cytokeratin-positive as well as CD45-negative Additionally, cell morphology and size are taken into consideration for the enumeration process.
Figure 2: CTC enumeration antibody markers panel. After CTCs are isolated from a pancreatic cancer patient using anti-EpCAM, they are verified using fluorescent images, including DAPI-positive (for nucleus staining), anti-cytokeratin-positive (for epithelial origin), and anti-CD45-negative (for non- leukocytes). Also shown is the merged image. To assess the capabilities of our device, we conducted capture experiments with two cell lines know1 to express EpCAM, L3.6pl and BxPC-3, as well as a cell line, MIAPaCa-2, that does not expres EpCAM. Flow cytometry tests were carried out to determine the relative expression levels of EpCAM fo each cell line. After using cell lines to assess the capture parameters of the device, we applied it t capture and enumerate tumor cells in the blood of patients with advanced pancreatic cancer. To verif captured cells as CTCs, we utilized the FDA-approved, triple-color immunocytochemistry technice definition, illustrated in Figure 2. In order to enumerate a captured cell as a CTC, it must be a nucleate cell, verified with a staining dye (DAPI), and be cytokeratin-positive as well as CD45-negative Additionally, cell morphology and size are taken into consideration for the enumeration process.
Figure 4: (a) The number of CTCs per mL of blood from one pancreatic cancer patient at different stages of chemotherapy treatment. Each cycle represents 2 weeks. (b) The patient’s CT scan at the anti-cancer treatment cycle 1. (c) The patient’s CT scan after treatment cycle 11. There is a significant reduction in tumor size from the beginning of the treatment to cycle 11. adenocarcinoma patients who went through clinical trial using a drug cocktail, referred to as FOLFOX -D, consisting of 5-fluorouracil, leucovorin, oxaliplatin, and dasatinib. Figure 4 shows a representative example of one patient whose CTC numbers, per mL of blood, reduced over the initial 10 cycles (2 weeks per cycle), which is in agreement with the decrease in sizes of both primary tumor and metastasized tumor during the treatment period, according to CT scans.
Figure 4: (a) The number of CTCs per mL of blood from one pancreatic cancer patient at different stages of chemotherapy treatment. Each cycle represents 2 weeks. (b) The patient’s CT scan at the anti-cancer treatment cycle 1. (c) The patient’s CT scan after treatment cycle 11. There is a significant reduction in tumor size from the beginning of the treatment to cycle 11. adenocarcinoma patients who went through clinical trial using a drug cocktail, referred to as FOLFOX -D, consisting of 5-fluorouracil, leucovorin, oxaliplatin, and dasatinib. Figure 4 shows a representative example of one patient whose CTC numbers, per mL of blood, reduced over the initial 10 cycles (2 weeks per cycle), which is in agreement with the decrease in sizes of both primary tumor and metastasized tumor during the treatment period, according to CT scans.
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