(616b) Isolation and Identification of Subpopulations of Circulating Tumor Cells Using a Microchip with Bio-Inspired Patterns

Circulating tumor cells (CTCs) are highly heterogeneous and contain many cellular subpopulations. It is known that specific CTC subpopulations, rather than the whole, are responsible for cancer metastases. Recently, the phenotype of metastasis-initiating cells (MIC, EpCAM CD44 CD47 MET ) among CTCs have been experimentally demonstrated. Emerging microfluidic technologies have shown great promise for the complete capture of the CTCs population with high yield and enhanced purity. However, most existing devices simply isolate all CTCs in a blood sample without resolving them into distinct subpopulations, preventing researchers from acquiring true insights into the metastatic potential CTCs. Isolating, purifying, and profiling individual populations of heterogeneous CTCs remain significant challenges. In addition, cell capture is only the first step in a pipeline, where post-capture, immunofluorescence characterization is required for identification of CTCs from non-specifically adhered blood cells. Since fixation and permeabilization remain standards for CTC characterization, this destructive processing limits the potential for subsequent phenotypic assays and development of personalized disease models for cancer treatments. Quick non-invasive characterization of CTCs and recovery of CTCs with high viability are required for integrating CTC technology to clinical applications. Ideally, platforms for isolation and identification of CTCs need to be incorporated into clinical care settings.

The goal of this work is to develop a new platform for fractionation and profiling of CTC subpopulations and elucidate the metastatic potential of CTCs. To do so, I propose to isolate, in-situ identify, and selectively recover CTCs using a microchip with hyperuniform structure created by microposts (Figure 1). Hyperuniformity (HU) is an emerging concept of a packing pattern which contains local heterogeneity or randomness and global regularity or homogeneity. The HU concept will be integrated for the first time into affinity-based microfluidic devices for CTC isolation. I hypothesize that due to the controlled differences in local flow patterns induced by the hyperuniform structure, cell arrest in different locations on the microchip will require different adhesive strengths. Further, this adhesive strength is anticipated to be related to the types and densities of surface markers on the captured CTCs and, therefore, their metastatic character. Combined with methods to effectively reduce blood cell adhesion and non-invasive release of CTCs, this device can recover individual groups of CTCs on demand for downstream bio-analyses. The HU microchip offers a simple and unique resolution for fractionation of CTCs as its 1) global homogeneity provides equal possibility for all CTCs to adhere; and 2) local heterogeneity allows simultaneous differentiation of subpopulations by analyzing adhesive strength required for individual CTCs. As a result, subpopulations of CTCs can be identified using only their capture locations on the HU chip without requiring additional post-capture immunofluorescence characterization. This HU microchip is expected to profile CTC heterogeneity during cancer metastasis with respect to the numbers/fractions of CTCs with specific surface markers, quantitative expression of specific surface markers that relate to tumor progression, and dynamic change of biophysical properties of CTC subpopulations. This approach is expected to provide new insight into addressing the heterogeneity of CTCs, which will permit CTCs to be isolated and identified nondestructively to ensure that collected downstream genomic and proteomic information reflects the cells’ true characteristics, as current CTC characterization methods often created artifacts during sample processing.

Figure 1. Schematics for isolate, in-situ identify, and downstream analysis of CTCs using a microchip with hyperuniform structure created by microposts.