The methods for isolating rare cells such as circulating tumor cells (CTCs) can be generally classified into two categories: those based on physical properties (e.g., size) and methods based on biological properties (e.g., immunoaffinity). CellSearch, the only FDA-approved method for the CTC-based cancer prognosis, relies on immunoaffinity interactions between CTCs and antibodies immobilized on magnetic particles. Immunoaffinity-based CTC isolation has also been employed in microfluidic devices, which show higher capture efficiency than CellSearch. We report here our investigation of combining size-based microfiltration into a microfluidic device with immunoaffinity for enhanced capture efficiency of CTCs. The device consists of four serpentine main channels, and each channel contains an array of lateral filters that create a two-dimensional flow. The main flow is through the serpentine channel, allowing the majority of the sample to pass by while the secondary flow goes through the lateral filters. The device design is optimized to make all fluid particles interact with filters. The filter sizes range from 24 to 12 µm, being slightly larger than or having similar dimension of CTCs. These filters are immobilized with antibodies specific to CTCs and thus they function as gates, allowing normal blood cells to pass by while forcing the interactions between CTCs and antibodies on the filter surfaces. The hydrodynamic force experienced by a CTC was also studied for optimal experimental conditions to ensure immunoaffinity-enabled cell capture. The device was evaluated by capturing two types of tumor cells spiked in healthy blood or a buffer, and we found that their capture efficiency was between 87.2 and 93.5%. The platform was further validated by isolating CTCs from blood samples of patients with metastatic pancreatic cancer. Circulating tumor cells (CTCs) have been considered an important biomarker for early detection of cancer metastasis, therapy monitoring, and disease prognosis 1-3. However, they are exceptionally rare, with only a few CTCs in billions of normal blood cells in each milliliter of peripheral blood 4. As a result, their isolation is technically challenging. CellSearch is the only CTC isolation method approved by the U.S. Food and Drug Administration (FDA) for cancer prognosis and it is based on the immunological interaction between epithelial cell adhesion molecules (EpCAM) on CTCs and anti-EpCAM immobilized on magnetic particles. Similar immunoaffinity-based isolation has also been implemented in microfluidic devices to differentiate CTCs from normal blood cells 5-10. Over the past decade, many efforts have been made to increase CTC capture efficiency in microfluidicsenabled, immunoaffinity-based methods; some representative works are listed in Table S1 in Supplementary Information 5-7,11,12. A prevalent and effective approach is to enhance the interaction between CTCs and the antibodies immobilized in devices. For instance, microstructures such as microposts of different sizes 13 , in various shap...

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The characterization of single cells within heterogeneous populations has great impact on both biomedical sciences and cancer research. By investigating cellular compositions on a broad scale, pertinent outliers may be lost in the sample set. Alternatively, an investigation focused on the behavior of specific cells, such as circulating tumor cells (CTCs), will reveal genetic biomarkers or phenotypic characteristics associated with cancer and metastasis. On average, CTC concentration in peripheral blood is extremely low, as few as one to two per billion of healthy blood cells. Consequently, the critical element lacking in many methods of CTC detection is accurate cell capture efficiency at low concentrations. To simulate CTC isolation, researchers usually spike small amounts of tumor cells to healthy blood for separation. However, spiking tumor cells at extremely low concentrations is challenging in a standard laboratory setting. We report our study on an innovative apparatus and method designed for low-cost, precise, and replicable single-cell spiking (SCS). Our SCS method operates solely from capillary aspiration without the reliance on external laboratory equipment. To ensure that our method does not affect the viability of each cell, we investigated the effects of surface membrane tensions induced by aspiration. Finally, we performed affinity-based CTC isolation using human acute lymphoblastic leukemia cells (CCRF-CEM) spiked into healthy whole blood with the SCS technique. The results of the isolation experiments demonstrate the reliability of our method in generating low-concentration cell samples.

We report two microfluidic devices that combine filtration and immunoaffinity for the detection of circulating tumor cells (CTCs) from peripheral blood samples of pancreatic and colorectal cancer patients. Our approach contrasts with most CTC isolation methods that are based either on cells’ physical property (e.g., size-based filtration) or on biologic property (e.g., immunoaffinity). The combined approach addresses the challenges related to CTCs’ heterogeneity in their properties. For instance, immunoaffinity-based methods such as CellSearch® that employ antibodies against epithelial cell adhesion molecule (EpCAM) cannot isolate those CTCs expressing little or no EpCAM. Filtration-based methods cannot detect CTCs of a size smaller than the predefined filter pore diameter while many normal blood cells of a size larger than the pore are retained by the filter. Our microfluidic devices contain a serpentine main channel and an array of lateral microfilters. The key difference of our devices from the conventional filtration platforms is that our filters are not in the direction of the main flow. The unique design of the device layout leads to a two-dimensional flow that allows the majority of a sample to pass by while all cells have opportunities to interact with filters, resulting in a larger throughput, reduced cell clogging, and increased purity of cells isolated. Another advantage of our devices is that a series of filter sizes can be created in one device to accommodate different CTC diameters, which is difficult to achieve in traditional filtration platforms. Our devices are further functionalized by immobilizing antibodies on the surfaces of microfilters to combine filtration with immunoaffinity for CTC isolation. We have designed one device with filter sizes of 6-10 μm and another device with filter sizes of 12-24 μm, and we tested them for the isolation of L3.6pl cells (pancreatic cancer cells) spiked in a buffer or blood. We compared an antibody-immobilized device with the same device containing filters only. Without antibody, the device has a capture efficiency from 69.8% to 83.9%, depending on the flow rate used. With antibody, the device with combined CTC isolation mechanisms can accomplish a capture efficiency of (98.7 ± 1.2)% at a flow rate of 1.8 mL/h. Other tumor cells with different sizes have also been evaluated in the devices. Further, we employed the devices for enumerating CTCs from blood samples of pancreatic and colorectal cancer patients. We compared our integration devices with a previously reported device containing herringbone-based micromixers. We found that the integration devices generally detected a higher number of CTCs. In summary, microfluidic devices have been developed to integrate filtration with immunoaffinity for CTC isolation. The devices offer better performance than those based on one isolation mechanism only, with a potential to address CTC heterogeneity and detect CTCs with different sizes and various expression levels. Citation Format: Hugh Fan, Kangfu Chen, Pablo Dopico, Jacob Amontree, Thomas George. Integration of filtration with immunoaffinity for isolating circulating tumor cells from pancreatic and colorectal cancer patients [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B24.