Separating CTCs for Prostate Cancer Diagnosis
Developing a microfluidic device to separate circulating tumor cells, the primary cancer cell in metastasis, from whole blood.
Research performed at Lehigh University’s Bionanomechanics Lab.
Summary
Tumor-shed cells from an epithelial lining lesion, or circulating tumor cells (CTCs), have been of significant concern with its highly characteristic identification and origin in metastatic diseases. CTCs can provide an insightful guide on detection, monitoring, diagnosis, and analysis of epitheliomas. However, its rarity is a pressing issue. Current methods of isolating CTCs for further purification are low-yielding and rely on physical properties (e.g. size, density). None show clinical potential. Characterizing and collecting CTCs can confirm the stage of cancer and can intricately unscramble its DNA and polymorphisms. This project creates a new and more effective approach towards the isolation of circulating tumor cells. Based on previous studies, both cancer cells and leukocytes possess adhesive bonds with E-selectin, although the strength of these bonds are unknown.
Therefore, in this study, microfluidic devices with immobilized E-selectin antibodies on the surface were used to test this cell adhesion. Hypothetically, the surface would induce different adhesion forces depending on the cell. Each type of blood cell was put into the plain device coated with E-selectin. Preliminary trials showed that the white blood cells (WBCs) and cancer cells revealed cell rolling on the E-selectin surface; however, the specific strength of the bond varied between the two cells. Red blood cells (RBCs) exhibited no adhesion to the surface. Two concentrations of E-selectin were used: 5 and 10 μg/ml. In 10 μg/ml, at a shear rate of 80/s, the WBC velocity was 6.12 μm/s, compared to the faster CTC’s velocity at 14.53 μm/s. In 5 μg/ml, at the same 80/s shear rate, the WBC’s velocity was 11.73 μm/s and the CTC’s was 15.84 μm/s. The results indicated different rolling velocities.
Thus, taking advantage of this property, a E-selectin coated microfluidic device with geometric patterns (inclined three dimensional micro-grooves and waves) incorporated into the surface was created to completely separate CTCs from the whole blood. The inclination allowed each individual cell type to flow in different pathways. With the groove pattern, approximately 75% of CTCs were separated into a single tube. The purity was 95%, meaning that of the cells inside that tube, 95% were cancer cells. In the wave design, the efficiency increased to 90%. These results demonstrate aninnovative, inexpensive technique for CTC separation.
Introduction
Metastasis accounts for more than 90% of the deaths in cancer-related mortalities. Circulating tumor cells (CTCs) are cells that shed from a tumor mass and circulate in peripheral blood. This has been recognized as the precursor to metastasis in many cancers. Detection and analysis of these cells can provide a guide on diagnosis, prognosis and treatment of non-hematologic and epithelial cancers, along with furthering the research on how to prevent metastasis [1–3]. It can predict the potential for metastasis before the cancer spreads. Isolation of CTCs from a patient’s blood acts as the first step and has attracted significant attention. The ultimate goal in this field is to isolate these cancer cells from normal blood cells with a high efficiency, purity, and viability. Various technologies have been developed in previous studies, which fall into two main categories: physical property (e.g., size, density and deformability) [4–6] and immunoaffinity based approaches (e.g. biomarkers) [7–11].
Due to the complexity of cell biology, there is currently no definite method, yet, to achieve the above-mentioned goal. Only 1-10 CTCs exist in 1 mL of peripheral blood, surrounded by millions of normal body cells. Consequently, these cells’ rarity makes most methods fail to show clinical potential due to limited isolation efficiency and purity. The current techniques that aim to separate CTCs from whole blood also require complex blood processing and complicated approaches to detach captured cells, thus yielding a low viability. As a result, there is an urgent need to develop a technique to improve CTC isolation efficiency, and thus diagnose early-stage metastatic cancers, in a convenient, reliable, and inexpensive manner.
Results
WBCs have been found to exhibit a strong bonding based on the results. This concludes that CTCs also express E-selectin ligand receptors, and have different adhesion forces compared to the HL60 cells. As proved again from the literature, RBCs had no interaction with the surface. These differences in the adhesive properties of the cells open a new method of separation.
Focusing Effect: The focusing effect was successful in keeping the RBCs and non-interactive cells in a single streamline, as shown in Figure 3(a). Surprisingly, red blood cells were focused to the side immediately after entering the device, demonstrating that the focusing effect eliminated RBC contact with tumor cells and leukocytes. This ensured that complete adhesion could occur.
Groove Design: The average efficiency of 100 tests was .748, or approximately 75%, with a standard deviation of .012. These ¾ of PC3 cells traveled at an incline parallel to the grooves before they formed the weak bond, and they eventually entered the CTC outlet, separating them from the regular blood. The attraction proved to be weak, as discovered in the preliminary studies, making these cells travel faster than the HL60. The 100 tests were performed in the 1 mm-100μm-45⁰ device, which was chosen based on previous flow tests.
Conclusions & Findings
Preliminary results demonstrate the cell focusing effect and adhesive force difference in the geometrically optimized microfluidic chip coated with E-selectin. Rare CTC capture proved to be nearly 90% efficient using a 3D wave pattern. This approach provides a convenient strategy to isolate tumor cells from whole blood without any blood processing and cell detachment operation, thus providing a convenient platform which is essential for tumor cell profiling and genomic analysis. Future work will be performed to optimize the full functionality of this device.
Advantages
This method of separating tumor cells achieves a high efficiency and high purity. Unlike most other tests, this is very inexpensive and requires only a few droplets of blood. High efficiency allows for accuracy of diagnostics. Additionally, this does not require post-labeling. Most current tests, even ones unrelated to cancer diagnosing, require pre- and post-labeling. These tests capture the cell; therefore, there needs to be an additional process in order to release it for evaluation and further analysis. This extra process could be expensive, requires more materials, and takes a much longer time. Separating CTCs using the technique mentioned in this paper is fast, efficient, resourceful, and inexpensive.
