Yoon Sun Yang

Discoidin Domain Receptor 1 Receptor Tyrosine Kinase Induces Cyclooxygenase-2 and Promotes Chemoresistance through Nuclear Factor-κB Pathway Activation

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase activated by various types of collagens and is known to play a role in cell attachment, migration, survival, and proliferation. However, little is known about the molecular mechanism(s) underlying the role of DDR1 in cancer. We report here that DDR1 induces cyclooxygenase-2 (Cox-2) expression resulting in enhanced chemoresistance. Depletion of DDR1-mediated Cox-2 induction using short hairpin RNA (shRNA) results in increased chemosensitivity. We also show that DDR1 activates the nuclear factor-kappaB (NF-kappaB) pathway and blocking this activation by an I kappaB superrepressor mutant results in the ablation of DDR1-induced Cox-2, leading to enhanced chemosensitivity, indicating that DDR1-mediated Cox-2 induction is NF-kappaB dependent. We identify the upstream activating kinases of the NF-kappaB pathway, IKK beta and IKK gamma, as essential for DDR1-mediated NF-kappaB activation, whereas IKK alpha seems to be dispensable. Finally, shRNA-mediated inhibition of DDR1 expression significantly enhanced chemosensitivity to genotoxic drugs in breast cancer cells. Thus, DDR1 signaling provides a novel target for therapeutic intervention with the prosurvival/antiapoptotic machinery of tumor cells.

A microfluidic chip for high resolution Raman imaging of biological cells

A microfluidic chip was designed, prepared and tested for integration with a confocal Raman imaging spectrometer with the specific purpose of enabling studies of individual biological cells. The design of the chip effectively overcomes the limitations arising from the high numerical aperture (NA) and short working distance objectives, which are necessary for high resolution imaging. The high confocal spatial resolution was achieved by a careful design of the geometry of the chip together with a thin, optically and Raman silent sealing window as the embedding medium for the channels. A leak-free microfluidic chip was obtained by surface plasma modification of polydimethylsiloxane (PDMS) and optimization of the liquid loading parameters. Raman images of biological cells, which were transported by flow into the microfluidic chip, are presented as an example of a Raman-microfluidics application. The broad band Raman spectra from −50 cm−1 to 3650 cm−1 were recorded in 1600 frequency intervals without any signal enhancement or sample labeling. Raman images were recorded in ∼400 seconds and they typically consisted of 64 × 64 pixels with a step size of 250 nm, thus containing ∼4 × 106 data points altogether.

Microfluidic devices for the interrogation of single circulating tumor cells

Introduction: Genetic and phenotypic characterization of Circulating Tumor Cells (CTC) offer the opportunity for a “real time liquid biopsy”. However, heterogeneity and rarity of CTC command the need for individual cell characterization. Following an enrichment procedure of CTC from blood, the identification, isolation and manipulation of single CTC for further analysis without cell loss remains challenging. Here, we present microfluidic devices for parallel single cell whole genome amplification (psc WGA) and parallel probing of drug response of single cancer cells (psc probing). Method: Microfluidic devices were designed using AUTOCAD software, and fabricated using PDMS multilayer soft-lithography. Cells from the SKBR-3 and MCF-7 breast cancer cell lines were used in the devices and identified using fluorescence microscopy after immunofluorescence staining. For pscWGA, the GE Single Cell GenomiPhi DNA Amplification kit was used under isothermal conditions. Results: In the 1st scWGA device, single cancer cells were addressed in 16 individual reaction chambers, subsequently lyzed, and their DNA amplified on a chip. We successfully amplified DNA of single cancer cells in a ca. 23 nanoliter reaction volume. 1,000-fold amplified DNAs were validated using qPCR targeting a set of genes on different chromosomes. For WGA of CTC present in a large number of other cells, we developed a 2nd scWGA platform by combining a self-sorting microwell cell sorter and a microfluidic device. After filtration of a cell suspension using a microwell plate, cancer cells were identified using fluorescence microscopy at the bottom of the plate. Cells of interest were subsequently punched into the open-well structures of the microfluidic device for further analysis. The lysis and WGA reaction buffers were loaded using peristaltic pumping of integrated micro-valves. After cell lysis, DNA was amplified in the open-well reaction chamber. For validation ∼ 100 ng of DNA was pipetted out of the well. We also developed microfluidic devices to study drug-dose response of single cancer cells. This device is capable of capturing single cells, dosing various concentrations of drugs and exposing the cells to the drugs. We optimized the capturing efficiency using different sizes of beads (3 μm, 6 μm, and 15 μm) as well as MCF-7 cells. We demonstrated that single cancer cells could be exposed to different drug candidates in the reagent chambers and their response measured. Conclusion: We successfully developed various microfluidic devices for individual cell characterization to be applied for CTC analysis. For genetic make-up, whole genome amplification of single cells either in suspension or in a self-sorting microwell plate was demonstrated. On-chip cell lysis and DNA amplification were performed and validated by qPCR targeting specific genes. In addition microfluidic devices were designed and tested to investigate single cell response to cancer drugs. Citation Format: Yoonsun Yang, Hoon Suk Rho, Joost F. Swennenhuis, Michiel Stevens, Arjan GJ Tibbe, Severine Le Gac, Han Gardeniers, Leon WMM Terstappen. Microfluidic devices for the interrogation of single circulating tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 367. doi:10.1158/1538-7445.AM2015-367

Chapter 9: Isolation and characterization of circulating tumor cells

Circulating tumor cells (CTCs) are tumor cells shed into the peripheral blood of cancer patients. The increasing number of treatment options for patients with metastatic carcinomas has created a concomitant need for new methods to establish which therapy will be effective and to monitor their use. Detection and characterization of CTCs is important not only to guide therapy, but also to increase our fundamental understanding of tumor progression and the formation of distant metastasis in which CTCs play a crucial role. However, identification of CTCs is quite challenging and different definitions lead to a large variation of CTC counts that will have different clinical implications. Here we will review the challenges in defining a CTC and data that have been obtained using CTCs in clinical studies emphasizing their importance as a prognostic and predictive biomarker. Furthermore, we summarize reported microfluidic platforms for CTC isolation, enumeration, and characterization developed to overcome technical challenges with current CTC detection platforms.