Using Adhesion Molecules to Separate Circulating Tumor Cells

Circulating tumor cells (CTCs) have a characteristic identification in metastasis. In order to monitor, diagnose, and analyze early-stage non-hematologic cancers, these rare cells must be separated from the whole blood with high efficiency. Thus, a microfluidic chip coated with E-selectin and microscopic inclined three-dimensional groove and wave patterns, which increases the probability of cell contact and separates the leukocytes and CTCs even further, is created.

Circulating tumor cells (CTCs) have a characteristic identification in metastasis. In order to monitor, diagnose, and analyze early-stage non-hematologic cancers, these rare cells must be separated from the whole blood with high efficiency. The adhesion molecule, E-selectin, was discovered to form a ligand-receptor interaction with both leukocytes and CTCs. Based on preliminary trials, both cells have weak bonds with the E-selectin, resulting in cell rolling. However, CTCs have a weaker interaction. Thus, a microfluidic chip coated with E-selectin and microscopic inclined three-dimensional groove and wave patterns, which increases the probability of cell contact and separates the leukocytes and CTCs even further, is created. CTCs roll faster than other cells, separating them from the leukocytes, which are collected on one extreme side of the device, and normal blood, which flows in a straight streamline. In the wave pattern, 90% of all CTCs inserted into the device are separated and collected into one outlet. 97% of cells in that outlet are CTCs. Based on this high efficiency and purity, the microfluidic device offers an effective and inexpensive method in diagnosing the potential for metastasis (early-stage diagnosis of fatal epithelial cancers), individualizing patient treatment, and furthering the understanding of metastatic biology.

 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 Eselectin 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 an innovative, 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 110 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. 

CTCs initiate metastasis through cell rolling. Specific selectins mediate an interaction with CTCs for its recruitment to other areas of the body. Moreover, leukocytes, or white blood cells (WBCs), exhibit cell rolling during inflammation, where chemicals called chemokines attract WBCs to the site of injury. In literature, it was found that E-selectin, an adhesion molecule, is present on endothelial cells when chemotaxis of leukocytes (leukocyte migration) occurs. The ligand-receptor attraction between the cell and E-selectin allows for leukocyte rolling due to a relatively weak bond. This physiological process occurs mainly by the immunoaffinity between cells’ receptors and the vessels’ selectins (ligands). Specific selectins are expressed during inflammation: P-, L-, and E-selectin. Both P- and L-selectin ligands are found on leukocytes, but not on CTCs; therefore, CTCs have no interaction with those two surfaces. However, inside the blood vessel, CTCs travel throughout the circulation and through organs during metastasis due to its attraction to E-selectin. 

Through preliminary research and experimentation, a similar interaction of WBCs and E-selectin is noted with tumor cells. Through their binding to E-selectin, tumor cells are able to travel along blood vessels to different organs, thereby causing metastasis and spreading cancer. Inspired by a recent work [12], which shows different interaction performances of white blood cells (WBCs) and deformed cells (e.g. apoptotic cells) on an E-selectin coated surface, the design of a novel microfluidic chip coated with E-selectin was proposed to isolate and separate CTCs from the circulation. 

Both WBCs and CTCs are observed to form weak bonds on E-selectin coated surface, while red blood cells have no interaction with E-selectin. Furthermore, leukocytes exhibit a stronger interaction on E-selectin with stable, more numerous bonds. Conversely, CTCs maintain a weaker bond, as proved in the Preliminary Experimentation of this paper. With these two observations, a microfluidic chip consisting of inclined groove and wavy patterns coated with Eselectin could achieve the separation of CTCs. The rationale of the device is described: once the solution containing both WBCs and CTCs is injected into the device coated with groove patterns, both cells will flow through the helical flow pattern induced in the microfluidic chip. Due to the frequent cell-surface contact, the cells will form weak bonds, which allow them to roll along the surface. However, the difference of the bond forces for these two cells will gradually separate them into two different flow paths, followed by collections in two different outlets. Some cells may be trapped inside the groove’s trough and will thus follow the inclination until it reaches the end of the pattern. In this case, separation relies on the section after the grooves. Meanwhile, the red blood cells will collect into the extreme outer portion due to the focusing effect. The inclination of the 3D groove/wave pattern forces the red blood cells to travel in a straight streamline, unless non-specific binding occurs. A brief schematic figure of this device is shown in Figure 1.

Working Mechanism

The overall strategy to isolate tumor cells from normal blood cells in a wave design is described below. After designing an E-selectin coated microfluidic chip with inclined wavy patterns, blood samples spiked with tumor cells are injected through the chip. Different cells either flow or roll along different pathways. The waves create a more efficient vortexing action, allowing cell rolling to occur more easily. For the preliminary flow test, RBCs are lysed so that isolation of only WBCs and CTCs are tested. 

To achieve the cell-adhesive, rolling-based isolation mentioned above, the device performance was evaluated from two aspects. First, with the inclined wavy/groove pattern, the flow presents velocity components as shown in the cross-section in Fig. 2(a). This allows for more surface contact between E-selectin and cells. Due to the fluid mass conservation and the inclination direction, a large looped flow pattern in a clockwise direction is formulated as shown in Fig. 2(b). Regarding the forces exerted, the flowing cells experience the same gravitational force and buoyant force everywhere, while drag forces exhibit different directions in different locations. Since the densities of both CTCs and WBCs are larger than that of the fluid medium, cells undergo an unstable force balance and shift following the flow loop pattern. They finally focus at the interfaces where buoyant force and drag force balance with the gravity.

After the cells focus to one side of the channel, subsequent cell isolation is achieved based on different adhesive forces for CTCs and normal blood cells. It was found that RBCs have no interaction with the E-selectin coated surface, thus following the straight flow stream. Both CTCs and WBCs perform adhesive rolling on E-selectin while stronger adhesion force exists for WBCs. In combination with the inclined wavy pattern, which provides a guiding direction, it is expected that CTCs and WBCs will roll on the E-selectin coated surface following different pathways.

 Figure 2. (a) The velocity contour shows the fluid flow in the device; the red represents the area of highest flow rate, at approx. 2.5 m/s; the blue shows 0-0.5 m/s; the light blue is 0.5-1.5 m/s; the green-yellow is 1.5-1.75 m/s; and the orange to light red is 1.75-2.3 m/s. (b) A schematic image of the looped flow pattern and the forces acting on a flowing cell; The focusing section is on the left because gravity (G) balances the fluid flow (drag) and buoyancy (Fb), so the cells will not move horizontally, unlike the cell with unbalanced forces on the right. G > Fb because cell density (1.06 g/ml) > fluid density (1.007      g/ml).

 Focusing effect: Due to RBC’s non-interaction with the surface, it should flow mainstream with the blood. The inclination of the grooves allows the RBCs and plasma to be concentrated on one side of the device in a nearly straight line. If the RBC gets carried by the grooves to the other end of the device, it will rapidly return to its original path due to the net forces acting on the cell from the device’s design, as shown in the Cross Section image. In the front of the device, a focusing section is designed to set a definite path for the non-interacting fluid and cells.

Inclination: The inclination of the grooves is the primary method of making the cells roll at an angle to the horizontal line. This inclination, thus, is used for the wave designs as well. Therefore, the white blood cells will be at one end, the RBCs at the other, and the CTCs in the middle. 

Experimental Details (Materials and Methods)                  

Fabrication of silicon wafers (SI) covered with grooves

Silicon wafers covered with patterns with different parameters were fabricated by photolithography. In detail, the silicon surface was cleaned with deionized water, ammonia solution (25%) and hydrogen peroxide (30%), mixed in a ratio of 5: 1: 1. The solution was heated up to 80 and silicon chips were cleaned for 15 min. Afterwards, a layer of photoresist is covered over the SI wafer.  The photomask received by the company only allowed for the negative photoresist process, where the sections exposed to ultraviolet radiation became insoluble to the chemical substrate solution. To create a multi-layer pattern with the channel height included in the silicon wafer, another substrate needs to be added that will not be dissolved during the UV exposure step. The heights used for the design were 40-50 µm, and the depths of the grooves were 25-30 µm. Hence, the spinning rate would need to be adjusted to make the photoresist 65-80 µm thick. The SI wafer, after the photolithography process, was rinsed with acetone, ethanol and deionized water.   

Surface Functionalization of E-selectin

Prior to the surface functionalization, all the wafer substrates were washed using a three-step cleaning process (acetone, isopropyl alcohol and DI water), and dried under nitrogen.

Polydimethylsiloxane (PDMS) was then poured on top of the silicon wafer in a petri dish. PDMS is a viscous fluid that hardens at 4⁰C. Once hardened, the PDMS is peeled off the wafer, and the patterns from the wafer are transferred, backwards, to the PDMS. The PDMS is the main component of the device. It needs to be attached to a PDMS cover or a glass surface in order to cover the channel. To do this, the PDMS substrates were fixed in 35 x 10 mm Petri dishes and treated with oxygen plasma to introduce hydroxyl groups onto its surface. The cover was placed on top of this substrate, thereby creating the device. The inlets and outlets were made by carving out 2mm holes in their respective locations. 2mm tubing was then inserted into these holes. They were attached at the end to a microtube, which is used to collect the blood/fluid during flow testing. 

In order to chemically modify the substrates and coat E-selectin onto the surface, a three-step surface functionalization process was immediately applied. They were first pretreated and baked with 1% (3-Aminopropyl) triethoxysilane (APTES) in 200 proof Ethanol (EtOH) [3 µM of APTES if 300 µM of EtOH] for 30 minutes. This was followed by incubation of the recombinant protein G (5 µg/µL) in PBS for 60 minutes at room temperature (RT). E-selectin/F Chimera was then flowed through the device for 2 hours (5 or 10 µg/ml). One hour prior to running the cell test, the substrates were purged with 1% BSA or with 0.05% Tween20. After each reaction, PBS or ethanol, depending on the solvent used in the previous step, was used to remove unbound molecules. 

Wave Designing

Three methods were used to create the wave designs. All but one was post-fabrication. The first technique is preparing the initial pattern directly onto wax. The wax is poured over the silicon wafer and the pattern is ingrained. Wax is very sensitive to temperature changes; therefore, if it is heated to a certain temperature, the grooves will slightly melt, creating a smoother, wave-like surface. The second method is the thermal reflow process. This repeats the negative photoresist process but in an incubator. Repeated UV exposure damages the sharp edges of the groove, creating curved grooves, or waves. The final method, which proved to be the most effective and easiest, was producing a wave-pattern using a 3D printer. The wave pattern is programmed into the 3D printer and the PDMS is poured on the printed surface rather than a silicon wafer.  

Cell Culture and Preparation

PC3 (prostate cancer cells) were selected as appropriate platforms for optimization study of separation of CTCs, as a higher concentration of E-selectin ligands were found on the PC3 cells. PC3 cells and HL60 cells (leukocytes) were cultured at 37 ⁰C in 5% CO2 in F-12K growth medium containing 1.5 mM L-glutamine supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin with media change every 2–3 days. Cells were then released through incubation in 0.05% Trypsin–0.53 mM EDTA at 37 ⁰C for around 5 minutes. A hemocytometer was used to count cells and appropriate dilution was subsequently carried out to reach a cell concentration of around 105 mL-1. The cell concentration was selected also for ensuring the yield of a large result data pool to reach a reliable conclusion. Cell tracker, a fluorescent stain, was used for easier identification of the PC3 cells. During the culturing of the tumor cells, a fluorescent stain is added. Afterwards, it is either mixed with PBS or whole blood. When PBS is used, alginate is added to mimic the viscosity of the blood. Alginate also reduces the cell settling effect described earlier. During testing, a syringe pump pushes the blood inside the device. The syringe pump also controls the infusion rate. The velocities of all the cells are recorded by using a time interval of approximately 1 second, and measuring the distance the cell travelled in that time.

Analysis

The PC3 cells were stained with cell-tracker and then spiked into human whole blood or RBC lysed blood at a concentration of 106 cells/mL. The high concentration provides for lower error/variability and more accurate quantitative data. Cell solution was then injected into the Eselectin coated microfluidic chip using a syringe pump. The cells were flowed through the pump at different shear rates: 10, 20, 40, 80, 120, 200 /s. For the preliminary studies, the velocities and pathways of the cells were measured using ImageJ Analysis. Microscopic images were taken of the cells every second. In the groove/wave design, efficiency is measured by dividing the cells in the output tube by the total number of cells inserted. Purity is measured by calculating the percentage of cells that are CTCs in the middle, output tube.

Results and Findings

 

 

3D Design

Below are profilometer readings of the grooves and waves, which measure the specific heights and depths of channels and indentations, respectively.

In the groove design, the height of the channel, h, is approximately 41 µm. The depth of the grooves, d, is ≈ 32 µm. The height of the channel and depth of grooves also changed from 25 to 40/50 µm and 20 to 35 µm, respectively. This allowed for a more efficient focusing effect of the RBCs and plasma.       

Preliminary Trial Analysis

There is a visible weak adhesion force between E-selectin and WBCs and CTCs. This weak attraction allows for cell rolling and tethering. Cell tethering is the ability of a cell to wrap around an object, such as the groove, while rolling. E-Selectin ligands (receptors) are present on all leukocytes (granulocytes, monocytes, lymphocytes). There is a larger affinity of white blood cells to the surface, allowing for a stronger, more stable bond. CTCs have a weaker adhesion force compared to the leukocytes. Red blood cells have been shown to not consist of any surface receptors, thus, including the E-Selectin ligand. Therefore, it has no interaction with the surface. Different adhesion forces result in varying rolling velocities. A strong bond, such as WBCs, produced a slow velocity, whereas an extremely weak adherence or none at all, caused a fast or nearly normal flow rate. Different velocities lead to different pathways taken, ultimately separating the cells.

 Optimizing Geometric Parameters

Widths of channel: 1 mm and 5 mm. These were used because they are ideal widths for microfluidic devices (< 8 mm). Specifically, they were chosen to have two completely different widths for experimentation. The optimal width is determined by observing the isolation efficiency.

Inclination Angle: 30 and 45⁰. These are based on a computer simulation. The exact path of the cells was calculated by noting the velocities from the preliminary results. 

Groove Widths: 35 and 100 µm. The width has to allow the cells to roll for an extended period of time. The diameter of the CTC is 17-30 µm. Therefore, all widths have to be greater than 30.  

Outlets: Eight outlets were used to have a precise approximation of where each cell ended. This was used to evaluate the pathways taken by the majority of cells. For example, CTCs could culminate anywhere in the middle. Therefore, by having more than one outlet in the middle, there would be a higher purity with less tumor cells entering white blood cell outlets. 

Based on the results, a high shear rate (>200/s) is the most ideal flow for all designs of the microfluidic chip. It significantly reduces non-specific binding and the chances of CTCs or WBCs completely adhering to the surface. A small channel width (1 mm) minimized the occurrences of clotting within the chip, and it will eliminate non-specific binding due to higher pressures inside the device. A larger groove width allows for the most surface area contact and increased the probability of the PC3 cells weakly adhering to the surface and following the path of the groove. An interesting phenomenon was noted: if the flow of the fluid applied begins low and slowly increases, the HL60 and PC3 cells maintain a stronger bond than usual. New attachments must have formed in order to increase their bonding strength. Therefore, the flow rate must start high. 

Flow Results

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 travelled 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 1mm-100µm-45⁰ device, which was chosen based on previous flow tests. 

Wave Design: The waves increased the cell separation, on average, by .144, producing a mean efficiency of .892 or approx. 90%. The main reasons for this dramatic difference were: there was less damage to cells (sharp edges on grooves); there was easier separation of all three cells; and there was improved vortex action (circular movement), which in turn created enhanced cell rolling. The highest efficiency was reached, similar to the groove design, by the 1mm-100µm45⁰ device.

Purity: The purity of the device was similar in the groove and wave-design tests. Waves were slightly higher, at 0.97, whereas grooves were 0.95. This means 95-97% of the cells isolated in the CTC outlet were actual CTCs. The other 3-5% were red blood cells or leukocytes.

Discussion

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.   

Benefits and Significance

My project focuses on diagnosing early stages of non-hematologic and epithelial cancers, something that has never efficiently been achieved before. The diagnostic device is inexpensive and only requires 1 mL of blood; thus, it can be implemented in third-world countries, where normal blood testing is rare and expensive. This groundbreaking technique can be used not only in cancer treatment centers, but also in small blood testing centers around the world. The patient’s blood can also be processed in the device in less than one day. Separating rare cancer cells with a nearly perfect efficiency provides deeper insight into metastasis, its origin, and its biology. Precise treatment methods can be used depending on the stage of cancer, making this device not only patient-specific but also an indicator of early stage metastasis. Preparation of the device is fast and easy. Therefore, it is extremely reproducible, allowing even small blood testing centers to create the device. By further analyzing the cancer cells, the cause of metastasis can be found. For example, if chemokine receptors are abundant on the surface of the cell, it must have been attracted by concentration gradients in the bloodstream. Thus, the organ that the circulating tumor cells (CTCs) are targeting can be discovered, and proper treatment can be given.  CTCs act as biomarkers for noninvasively monitoring the development and evolution of tumors. With my device, an accurate measurement of disease progression is provided, and the effects of treatment on tumors can be examined precisely. Monitoring progression leads to testing the effectiveness of certain treatments in certain patients. By knowing this information, patients do not have to sustain all different types of treatments. This further expands the concept of personalized treatment.

In recent work, the advantages of combining E-selectin, which was proved to bind weakly to CTCs by this work, with liposomes, which are spherical, multilamellar carriers composed of lipids, was shown to prevent or slow the progression of metastasis. As previously stated, one of the significant benefits of the device is that it holds the key to completely understanding metastasis, and eventually, preventing it. This aspect has been supported with further research.

After examining CTCs that were separated using the device, it was noticed that the CTCs’ had a tendency to travel towards the sides of the blood vessel during laminar flow. It was also found that CTCs possessed structural abilities that allowed them to completely bind to phosphatidylcholine, a lipid, in combination with E-selectin. Using this information, liposomes composed of phosphatidylcholine and coated with E-selectin were shown to strongly bind to nearly all CTCs, even with its rarity. This means that if a death or apoptotic-inducing ligand is encapsulated within the liposome, most CTCs can be killed, slowing and, hopefully, preventing metastasis. This recent finding clearly shows the broad implications of the microfluidic device.

Acknowledgements

I worked in Lehigh University’s Bionanomechanics Laboratory under Dr. Yaling Liu’s supervision. Professor Dr. Yaling Liu allowed me to use his materials and lab space, and graduate student Shunqiang Wang supervised and approved my protocols and designs.

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Using Adhesion Molecules to Separate Circulating Tumor Cells

Circulating tumor cells (CTCs) have a characteristic identification in metastasis. In order to monitor, diagnose, and analyze early-stage non-hematologic cancers, these rare cells must be separated from the whole blood with high efficiency. Thus, a microfluidic chip coated with E-selectin and microscopic inclined three-dimensional groove and wave patterns, which increases the probability of cell contact and separates the leukocytes and CTCs even further, is created.

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