Biomechanical Microdevices to Study Circulating Cancer Cells In Hematogenous Metastasis

Ph.DDOCTOR OF PHILOSOPH

Fast and efficient microfluidic cell filter for isolation of circulating tumor cells from unprocessed whole blood of colorectal cancer patients

Liquid biopsy offers unique opportunities for low invasive diagnosis, real-time patient monitoring and treatment selection. The phenotypic and molecular profile of circulating tumor cells (CTCs) can provide key information about the biology of tumor cells, contributing to personalized therapy. CTC isolation is still challenging, mainly due to their heterogeneity and rarity. To overcome this limitation, a microfluidic chip for label-free isolation of CTCs from peripheral blood was developed. This device, the CROSS chip, captures CTCs based on their size and deformability with an efficiency of 70%. Using 2 chips, 7.5 ml of whole blood are processed in 47 minutes with high purity, as compared to similar technologies and assessed by in situ immunofluorescence. The CROSS chip performance was compared to the CellSearch system in a set of metastatic colorectal cancer patients, resulting in higher capture of DAPI+/CK+/CD45- CTCs in all individuals tested. Importantly, CTC enumeration by CROSS chip enabled stratification of patients with different prognosis. Lastly, cells isolated in the CROSS chip were lysed and further subjected to molecular characterization by droplet digital PCR, which revealed a mutation in the APC gene for most patient samples analyzed, confirming their colorectal origin and the versatility of the technology for downstream applications

Profiling Circulating Tumour Cells for Clinical Applications

Circulating tumour cells (CTCs) refer to cells found in the peripheral blood, which are derived from the primary or secondary tumour. They serve as an alternative to study the biology of the primary tumour especially when tissue biopsy is not available. However, major challenges in CTC analysis are the rarity of these cells and the purity of the isolated population. The advancement in technologies allows detection and enrichment of sufficiently pure CTCs at the single-cell level, facilitating downstream molecular characterisation. Single CTC analysis allows detection of key mutations that may be critical to disease management and helps to address the intercellular differences among tumour cells. In this chapter, we discuss the technologies for CTC isolation and the use of CTCs in achieving early detection and prognosis of cancer, real-time monitoring of cancer therapy and tailoring of personalised treatments

Microfluidics for antibody-independent separation of circulating tumor cells

Separation of circulating tumor cells (CTC) from the blood without using antibodies is a key challenge in the diagnosis of cancer. This article has reviewed few antibody-independent techniques governed by cell size on microfluidics platform. Microdevices employing micro-pore filters, dielectrophoresis, acoustophoresis and inertia that is popular in various research labs are studied in detail from the literature and discussed shortly. The inertia based technique in spiral microchannel seems to be more promising towards the development of future point-of-care (POC) microdevices for CTCs separation

Novel devices and protocols enabling isolation and enumeration of low abundant biological cells from complex matrices

The dimensions of microfluidic devices closely parallel those of biological cells; thusly, they are excellent platforms for the speciation, transport, manipulation, and analysis of cells. Electrokinetic transport of Escherichia coli and Saccharomyces cerevisiae was evaluated in microfluidic devices fabricated in pristine and UV-modified poly(methylmethacrylate) and polycarbonate. The magnitude and direction of transport of the cells was dictated by the buffer composition, conduit surface chemistry, and intrinsic cellular electrical properties. Baker’s yeast in all devices migrated toward the cathode, because of their smaller electrophoretic mobility compared to the electroosmotic flow of the polymer. E. coli cells suspended in 20 mM PBS migrated toward the anode, which indicated that the apparent mobility of the E. coli cells changed direction at higher ionic strengths. The observed differential migrations were exploited to sort cells, whereby judicious choice of the buffer concentration and the polymeric material in which the cell sorting was performed was controlled, allowed for cell enumeration via laser-based backscatter signals. A novel microfluidic device that selectively and specifically isolated the exceedingly small numbers of circulating tumor cells (CTCs) from whole blood through a monoclonal antibody (mAB) mediated process by sampling large input volumes (≥1 mL) of whole blood directly in short time periods (\u3c37 min) was designed, manufactured and implemented. Upon processing, the CTCs were concentrated into small volumes (190 nL) and the number of cells captured were read without the need for labeling by using an integrated conductivity sensor following an enzyme mediated release of the captured CTCs from the microchannel surface. The microchannel walls were covalently decorated with mABs directed toward breast cancer cells that over-express epithelial cell adhesion molecules. The released CTCs were then enumerated on-device using conductivity detection with 100% detection efficiency and exquisite specificity for CTCs. The CTC capture efficiency was made highly quantitative (\u3e97%) by designing capture channels with the appropriate widths and heights. Extension of the technique to environmental samples was performed using analogously patterned polyclonal anti-E. coli O157:H7 antibodies directed towards the virolent bacterial strain were used to isolate the enterohemorrhagic bacteria while E. coli K12 were not adsorbed to the antibody containing surface

A label free disposable device for rapid isolation of rare tumor cells from blood by ultrasounds

The use of blood samples as liquid biopsy is a label-free method for cancer diagnosis that offers benefits over traditional invasive biopsy techniques. Cell sorting by acoustic waves offers a means to separate rare cells from blood samples based on their physical properties in a label-free, contactless and biocompatible manner. Herein, we describe a flow-through separation approach that provides an efficient separation of tumor cells (TCs) from white blood cells (WBCs) in a microfluidic device, "THINUS-Chip" (Thin-Ultrasonic-Separator-Chip), actuated by ultrasounds. We introduce for the first time the concept of plate acoustic waves (PAW) applied to acoustophoresis as a new strategy. It lies in the geometrical chip design: different to other microseparators based on either bulk acoustic waves (BAW) or surface waves (SAW, SSAW and tSAW), it allows the use of polymeric materials without restrictions in the frequency of work. We demonstrate its ability to perform high-throughput isolation of TCs from WBCs, allowing a recovery rate of 84%±8% of TCs with a purity higher than 80% and combined viability of 85% at a flow rate of 80 µL/min (4.8 mL/h). The THINUS-Chip performs cell fractionation with low-cost manufacturing processes, opening the door to possible easy printing fabrication

On Chip Isolation and Enrichment of Tumor Initiating Cells

We report for the first time a microdevice that enables the selective enrichment and culture of breast cancer stem cells using the principles of mammosphere culture. For nearly a decade, researchers have identified breast cancer stem cells within heterogeneous populations of cells by utilizing low-attachment serum-free culture conditions, which lead to the formation of spheroidal colonies (mammospheres) that are enriched for cancer stem cells. While this assay has proven to be useful for identifying cancer stem cells from a bulk population, ultimately its utility is limited by difficulties in combining the mammosphere technique with other useful cellular and molecular analyses. However, integrating the mammosphere technique into a microsystem can enable it to be combined directly with a number of functions, including cell sorting and analysis, as well as popular molecular assays. In this work, we demonstrate mammosphere culture within a polydimethylsiloxane (PDMS) microsystem. We first prove that hydrophobic PDMS surfaces are as effective as commercial low-attachment plates at selectively promoting the formation of mammospheres. We then demonstrate the culture of mammospheres as large as 0.25 mm within a PDMS microsystem. Finally, we verify that reagents can be delivered to the cell culture wells exclusively by diffusion-based transport, which is necessary because the cells are unattached. This microsystem component can be integrated with other microfluidic functions, such as cell separation, sorting, and recovery, as well as molecular assays, to enable new discoveries in the biology of cancer stem cells that are not possible today

The potential for liquid biopsies in the precision medical treatment of breast cancer.

Currently the clinical management of breast cancer relies on relatively few prognostic/predictive clinical markers (estrogen receptor, progesterone receptor, HER2), based on primary tumor biology. Circulating biomarkers, such as circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs) may enhance our treatment options by focusing on the very cells that are the direct precursors of distant metastatic disease, and probably inherently different than the primary tumor's biology. To shift the current clinical paradigm, assessing tumor biology in real time by molecularly profiling CTCs or ctDNA may serve to discover therapeutic targets, detect minimal residual disease and predict response to treatment. This review serves to elucidate the detection, characterization, and clinical application of CTCs and ctDNA with the goal of precision treatment of breast cancer

Small Footprint High Flow Rate Microdevice for Rare Target Cell Capture

A novel high flow rate cell capture design was introduced to overcome the limitations of the current technologies or methods for rare target cell capture. Even though the rare target cell capture using BioMEMS technology has great potential for cancer diagnosis, previous rare cell capture research could not overcome the limitations of low flow rate or low recovery rate. Rare cell research requires precise sample preparation for accurate results. A new method of preparation for a single or a precise number of target cell was introduced. Current sample preparation methods which are not suitable for rare cell research, such as CTC capture or single cell analysis do not provide precise cell counts below 100. A cell collection chip was designed and used with a polyimide removed capillary tube to collect an exact number of target cells under a microscope. To optimize the dimensions of the high flow rate design, CFD (Fluent v6.3, ANSYS, Inc., Canonsburg, PA, USA) simulation was used. The design focused on a high flow rate at inlet and low axial and lateral velocities in the cell capture regions with a small footprint. Based on the simulation results, the dimensions for several prototypes were determined and fabricated in PMMA. The CTCs, MCF-7 cells, were captured flow rates up to 750 µL/min from 40% red blood cells with 80% recovery rate using the high flow rate device