Isolation and Identification of Heterogeneous Circulating Tumor Cells Using a Microchip with Hyperuniform Patterns

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Cancer metastasis, the leading cause of cancer-related death, involves the spreading of malignant tumor cells from the primary tumor to form tumors in other organs. Counting circulating tumor cells (CTCs) in blood can measure cancer progression and evaluate the effectiveness of a cancer treatment. However, CTCs are highly diversified and only specific CTC subtypes are responsible for cancer metastasis, while current CTC-counting technologies do not distinguish between subpopulations. In this project, CTC subpopulations will be isolated and identified using a microchip containing novel structures (arrangements of posts with different spacings and diameters) that can control local flow patterns. The different flow patterns enable the capture of subtypes in different chip locations. This chip will permit CTCs to be analyzed where they are captured to ensure that the analyses reflect the true characteristics of these cells. Transformative knowledge obtained from this work will guide application of CTC technologies in assisting cancer diagnosis, predicting tumor progression, and monitoring therapeutic efficacy. If successful, the approach can be adapted to similarly separate subpopulations of immune cells or stem cells for curing cancers or other diseases. The proposed education and outreach plan will enhance the Biomedical Engineering curriculum and program at TTU and introduce undergraduate and graduate students to modern bioengineering techniques. Outreach activities will coordinate with existing TTU programs for junior-high and high school girls and are armed at sparking their interest in science and engineering. In addition, since TTU is a Hispanic Serving Institution, efforts will be made to include more underrepresented minorities into the project.The goal of this project is to understand the metastatic potential of circulating tumor cells (CTCs) through label-free fractionation and profiling of CTC subpopulations. A microchip with hyperuniform structure will be used to isolate, in-situ identify, and selectively separate CTCs. Hyperuniformity (HU) is an emerging concept of a packing pattern that contains local heterogeneity or randomness and global regularity or homogeneity. The concept of hyperuniformity will be integrated into affinity-based microfluidic devices for CTC isolation. Due to the controlled differences in local flow patterns induced by the hyperuniform structure, cell arrest in different locations on the microchip are expected to 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. To maximize capture efficiency, microchip coatings will be modified with tumor specific antibodies. Separation of individual CTC groups will be achieved by selective degradation of biodegradable nanofilm between the captured cells and the surface of the microposts. In order to correlate of CTC heterogeneity profile with metastatic status, downstream characterization and preliminary in vivo validation will be performed. The Research Plan is organized under three objectives. The FiRST Objective is to design and characterize HU structured microchip for CTC capture and analyze flow pattern and adhesion force in the device. The local fluidic pattern will be studied through experiments and modeling to determine the potential "pockets" where cancer cells accumulate and to correlate the presence of a cell within a given pocket with a characteristic surface adhesion. The SECOND Objective is to capture and identify subpopulations of single cell and mixed cancer cell lines with variable expression of surface markers using a HU microchip. A prototype microchip will be used for studying subpopulations in PC3 (human prostate cancer line) with cells with different sizes, culture conditions and cell cycles and for distinguishing subpopulations in mixed prostate cancer cell lines (PC3, and LNCaP (human prostate adenocarcinoma line)). The THIRD Objective is to develop selective release of subpopulations of cancer cells from a HU microchip and perform downstream characterization and preliminary in vivo validation. Selective release of a captured cancer cell mixture (PC3 and LNCaP) on a HU microchip will be conducted by degrading the nanoflm between cells and micropost surfaces and controlled local mechanical agitation. To study the progression of metastasis, a primary human prostrate cancel cell line (CP3TX), will be transplanted into the femurs of mice and blood samples will be collected at different time points and run through HU microchips to capture CTCs that can be used as metastasis indicators. Indicator profiles are expected to correlate with prostate cancer metastasis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.