Polyploid giant cancer cells (PGCCs) contribute to the genetic heterogeneity and evolutionary dynamics of tumors. Their size, however, complicates their isolation from mainstream tumor cell populations. Standard techniques like fluorescence-activated cell sorting (FACS) rely on fluorescent labeling, introducing potential challenges in subsequent PGCC analyses. In response, we developed the Isosceles Trapezoidal Spiral Microchannel (ITS mu C), a microfluidic device optimizing the Dean drag force (FD) and exploiting uniform vortices for enhanced separation. Numerical simulations highlighted ITS mu C's advantage in producing robust FD compared to rectangular and standard trapezoidal channels. Empirical results confirmed its ability to segregate larger polystyrene (PS) particles (avg. diameter: 50 mu m) toward the inner wall, while directing smaller ones (avg. diameter: 23 mu m) outward. Utilizing ITS mu C, we efficiently isolated PGCCs from doxorubicin-resistant triple-negative breast cancer (DOXR-TNBC) and patient-derived cancer (PDC) cells, achieving outstanding purity, yield, and viability rates (all greater than 90%). This precision was accomplished without fluorescent markers, and the versatility of ITS mu C suggests its potential in differentiating a wide range of heterogeneous cell populations. Polyploid giant cancer cells (PGCCs) contribute to the genetic heterogeneity and evolutionary dynamics of tumors.

Background: Most high-throughput screening (HIS) systems studying the cytotoxic effect of chimeric antigen receptor (CAR) T cells on tumor cells rely on two-dimensional cell culture that does not recapitulate the tumor micro-environment (TME). Tumor spheroids, however, can recapitulate the TME and have been used for cytotoxicity assays of CART cells. But a major obstacle to the use of tumor spheroids for cytotoxicity assays is the difficulty in separating unbound CART and dead tumor cells from spheroids. Here, we present a three-dimensional hanging spheroid plate (3DHSP), which facilitates the formation of spheroids and the separation of unbound and dead cells from spheroids during cytotoxicity assays.

Results: The 3DHSP is a 24-well plate, with each well composed of a hanging dripper, spheroid wells, and waste wells. In the dripper, a tumor spheroid was formed and mixed with CART cells. In the 3DHSP, droplets containing the spheroids were deposited into the spheroid separation well, where unbound and dead T and tumor cells were separated from the spheroid through a gap into the waste well by tilting the 3DHSP by more than 20 degrees. Human epidermal growth factor receptor 2 (HER2)-positive tumor cells (BT474 and SKOV3) formed spheroids of approximately 300-350 pm in diameter after 2 days in the 3DHSP. The cytotoxic effects ofT cells engineered to express CAR recognizing HER2 (HER2-CAR T cells) on these spheroids were directly measured by optical imaging, without the use of live/dead fluorescent staining of the cells. Our results suggest that the 3DHSP could be incorporated into a HTS system to screen for CARs that enable T cells to kill spheroids formed from a specific tumor type with high efficacy or for spheroids consisting of tumor types that can be killed efficiently by T cells bearing a specific CAR.

Conclusions: The results suggest that the 3DHSP could be incorporated into a HTS system for the cytotoxic effects of CART cells on tumor spheroids.

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We have developed a microfluidic-based culture chip to simulate cancer cell migration and invasion across the basement membrane. In this microfluidic chip, a 3D microenvironment is engineered to culture metastatic breast cancer cells (MX1) in a 3D tumor model. A chemo-attractant was incorporated to stimulate motility across the membrane. We validated the usefulness of the chip by tracking the motilities of the cancer cells in the system, showing them to be migrating or invading (akin to metastasis). It is shown that our system can monitor cell migration in real time, as compare to Boyden chambers, for example. Thus, the chip will be of interest to the drug-screening community as it can potentially be used to monitor the behavior of cancer cell motility, and, therefore, metastasis, in the presence of anti-cancer drugs.

Affinity biosensing is one of many analytical techniques that have high potential for the detection of drug leads. In this thesis the use of ellipsometry, surface plasmon resonance (SPR) as well as quartz crystal microbalance (QCM) as screening methods in drug discovery are presented. In particularly, attention was given to the sensing surfaces and their incorporation in multi-sensor arrays, or biochips, for monitoring the specific affinity binding of low molecular weight molecules.

Silicon dioxide and gold surfaces were rendered porous and used as the biosensing surface. Porous surfaces enhanced the performance of the biosensor considerably, due to the increase in the capacity of binding ligands. In the case of porous silicon dioxide the increase in the response is 10-fold, while porous gold showed a 6-fold increase, when employing ellipsometric measurements. It was also shown that porous gold can successfully be incorporated in SPR systems when used as a biosensor, resulting in a small increase in sensitivity when employing rough gold surface and an 20-fold increase in case of thick porous gold layers, compared to planar surfaces. The surface area of the gold electrode on the crystals was increased by rendering the electrodes porous. This increased the response up to a factor 3. Thus, porous surfaces together with ellipsometry, SPR or QCM have the advantage of not requiring labelling, such as fluorescence, isotope or antigen tags, for achieving the necessary sensitivity.

Further, ellipsometric and SPR imaging systems were introduced employing a 1 cm² surface, containing 900 targets. These biochips are a step towards a faster highthroughput screening process. Two methods of fabrication of these biochips are shown: one based on wet etching of a silicon surface and the other on the preparation of so called tension wells on the silicon surface.

Affinity models were used throughout this work.Streptavidin was immobilised to the porous silicon to bind biotin and an oligopeptide, synthesised by means of combinatorial chemistry monitored with the ellipsometer. A protein model system consisting of anti-human myoglobin as analyte and sheep skeletal myoglobin as ligand was used in different concentrations. Measurements on the biochips were performed with carbohydrate model substances selected for six common lectins.