Ching-Te Kuo

Modeling of cancer metastasis and drug resistance via biomimetic nano-cilia and microfluidics

Three-dimensional (3D) tissue culture platforms that are capable of mimicking in vivo microenvironments to replicate physiological conditions are vital tools in a wide range of cellular and clinical studies. Here, learning from the nature of cilia in lungs - clearing mucus and pathogens from the airway - we develop a 3D culture approach via flexible and kinetic copolymer-based chains (nano-cilia) for diminishing cell-to-substrate adhesion. Multicellular spheroids or colonies were tested for 3-7 days in a microenvironment consisting of generated cells with properties of putative cancer stem cells (CSCs). The dynamic and reversible regulation of epithelial-mesenchymal transition (EMT) was examined in spheroids passaged and cultured in copolymer-coated dishes. The expression of CSC markers, including CD44, CD133, and ABCG2, and hypoxia signature, HIF-1α, was significantly upregulated compared to that without the nano-cilia. In addition, these spheroids exhibited chemotherapeutic resistance in vitro and acquired enhanced metastatic propensity, as verified from microfluidic chemotaxis assay designed to replicate in vivo-like metastasis. The biomimetic nano-cilia approach and microfluidic device may offer new opportunities to establish a rapid and cost-effective platform for the study of anti-cancer therapeutics and CSCs.

A novel microfluidic driver via AC electrokinetics

A novel ac electrokinetic microfluidic driver based on alternating current electro-osmosis flow induced by asymmetrically capacitance/chemistry-modulated microelectrode arrays has been successfully developed and demonstrated. Asymmetric capacitance modulation (ACM) is made of comb electrode arrays and parts of individual electrode surfaces are modulated/deposited with a SiO(2) dielectric layer. This proposed design can be utilized to shift the optimal operation frequency of maximum velocity to a higher frequency to minimize electrolytic bubble generation and enhance micropumping performance. The pumping velocity, described in this paper, is measured via the tracing of microbeads and is a function of applied potential, signal frequency, buffer concentration, and dielectric layer thickness. A maximum pumping velocity up to 290 microm s(-1) in 5 mM buffer solution with the applied potential of 10 Vpp is observed in our prototype device, and the estimated maximum flow rate is up to 26.1 microl h(-1). This is the first successful demonstration regarding bubble-free ac electrokinetic micropumping via such asymmetrically capacitance-modulated electrode arrays. Design, simulation, microfabrication, experimental result, and theoretical model are described in this paper to characterize and exhibit the performance of the proposed novel bubble-free ac electrokinetic microfluidic driver.

TTBK2: a tau protein kinase beyond tau phosphorylation.

Tau tubulin kinase 2 (TTBK2) is a kinase known to phosphorylate tau and tubulin. It has recently drawn much attention due to its involvement in multiple important cellular processes. Here, we review the current understanding of TTBK2, including its sequence, structure, binding sites, phosphorylation substrates, and cellular processes involved. TTBK2 possesses a casein kinase 1 (CK1) kinase domain followed by a ~900 amino acid segment, potentially responsible for its localization and substrate recruitment. It is known to bind to CEP164, a centriolar protein, and EB1, a microtubule plus-end tracking protein. In addition to autophosphorylation, known phosphorylation substrates of TTBK2 include tau, tubulin, CEP164, CEP97, and TDP-43, a neurodegeneration-associated protein. Mutations of TTBK2 are associated with spinocerebellar ataxia type 11. In addition, TTBK2 is essential for regulating the growth of axonemal microtubules in ciliogenesis. It also plays roles in resistance of cancer target therapies and in regulating glucose and GABA transport. Reported sites of TTBK2 localization include the centriole/basal body, the midbody, and possibly the mitotic spindles. Together, TTBK2 is a multifunctional kinase involved in important cellular processes and demands augmented efforts in investigating its functions.

A Bubble-Free AC Electrokinetic Micropump Using the Asymmetric Capacitance-Modulated Microelectrode Array for Microfluidic Flow Control

A novel ac electrokinetic micropumping device based on ac electro-osmotic flow induced by asymmetrically capacitance/chemistry-modulated microelectrode arrays has been successfully developed and demonstrated. Asymmetric capacitance modulation is made of comb electrode arrays and parts of individual electrode surfaces are modulated/deposited with a SiO2 dielectric layer. This proposed design can be utilized to shift the optimal operation frequency of maximum velocity from tens of kilohertz to megahertz to minimize electrolytic bubble generation and enhance micropumping performance. The pumping velocity, described in this paper, is measured via the tracing of microbeads and is a function of applied potential, signal frequency, buffer concentration, and dielectric layer thickness. A maximum pumping velocity up to 290 ?m ·s-1 in 5-mM buffer solution with the applied potential of 10 Vpp is observed in our prototype device, and the estimated maximum flow rate is up to 26.1 ?l ·h-1. This is the first successful demonstration regarding bubble-free ac electrokinetic micropumping via such an asymmetrically capacitance-modulated electrode arrays. Design, simulation, microfabrication, experimental result, and theoretical model are described in this paper to characterize and exhibit the performance of proposed novel bubble-free ac electrokinetic micropump.

Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array

Biomaterial-based tissue culture platforms have emerged as useful tools to mimic in vivo physiological microenvironments in experimental cell biology and clinical studies. We describe herein a three-dimensional (3D) tissue culture platform using a polydimethylsiloxane (PDMS)-based hanging drop array (PDMS-HDA) methodology. Multicellular spheroids can be achieved within 24 h and further boosted by incorporating collagen fibrils in PDMS-HDA. In addition, the spheroids generated from different human tumor cells exhibited distinct sensitivities toward drug chemotherapeutic agents and radiation as compared with two-dimensional (2D) cultures that often lack in vivo-like biological insights. We also demonstrated that multicellular spheroids may enable key hallmarks of tissue-based bioassays, including drug screening, tumor dissemination, cell co-culture, and tumor invasion. Taken together, these results offer new opportunities not only to achieve the active control of 3D multicellular spheroids on demand, but also to establish a rapid and cost-effective platform to study anti-cancer therapeutics and tumor microenvironments.

ParaStamp and Its Applications to Cell Patterning, Drug Synergy Screening, and Rewritable Devices for Droplet Storage

Engineered materials have been employed as versatile tools to explore fundamental processes in cell biology/drug development as well as an approach towards intelligent devices, thereby becoming key components in modern technology. Herein, a ParaStamp technique is revealed to possess applications for cell patterning, drug screening, and rewritable functional patterning. The ParaStamp includes a micropatterned polydimethylsiloxane master and a liquid‐phased paraffin oil generated at high temperature, which can transfer the patterned paramembrane onto varied material surfaces such as glass, polystyrene, and flexible foil. This technique is simple and cost‐effective to meet the high‐throughput requirement for industries. Taken together, the findings herein should provide general insights in cell biology, biodetection, and the development of smart hydrophobic surfaces.

Experimental demonstration of bindingless signal delivery in human cells via microfluidics

The cellular signal transduction is commonly believed to rely on the direct “contact” or “binding” of the participating molecule reaction that depends positively on the corresponding molecule concentrations. In living systems, however, it is somewhat difficult to precisely match the corresponding rapid “binding,” depending on the probability of molecular collision, existing in the cellular receptor-ligand interactions. Thus, a question arises that if there is another mechanism (i.e., bindingless) that could promote this signal communication. According to this hypothesis, we report a cellular model based on the examination of intracellular calcium concentration to explore whether the unidentified signal delivery in cells exists, via a microfluidic device. This device was designed to isolate the cells from directly contacting with the corresponding ligands/molecules by the particular polydimethylsiloxane (PDMS) membranes with different thicknesses. Results show a significant increment of calcium mobilization in human prostate cancer PC-3 cells by the stimulation of endothelin-1, even up to a separated distance of 95 μm. In addition, these stimulated signals exhibited a bump-shaped characteristics depending on the membrane thickness. When the PDMS membrane is capped by SiO2, a particular trait that resembles the ballistic signal conduction was observed. A theoretical model was developed to describe the signal transport process across the PDMS membrane. Taken together, these results indicate that the unidentified signal (ligand structural information) delivery could occur in cells and be examined by the proposed approach, exhibiting a bindingless communication manner. Moreover, this approach and our finding may offer new opportunities to establish a robust and cost-effective platform for the study of cellular biology and new drug development.

PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway

Abstract The DNA-dependent protein kinase (DNA-PK), consisting of the DNA binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, has been well characterized in the non-homologous end-joining mechanism for DNA double strand break (DSB) repair and radiation resistance. Besides playing a role in DSB repair, DNA-PKcs is required for the cellular response to replication stress and participates in the ATR-Chk1 signaling pathway. However, the mechanism through which DNA-PKcs is recruited to stalled replication forks is still unclear. Here, we report that the apoptosis mediator p53-induced protein with a death domain (PIDD) is required to promote DNA-PKcs activity in response to replication stress. PIDD is known to interact with PCNA upon UV-induced replication stress. Our results demonstrate that PIDD is required to recruit DNA-PKcs to stalled replication forks through direct binding to DNA-PKcs at the N’ terminal region. Disruption of the interaction between DNA-PKcs and PIDD not only compromises the ATR association and regulation of DNA-PKcs, but also the ATR signaling pathway, intra-S-phase checkpoint and cellular resistance to replication stress. Taken together, our results indicate that PIDD, but not the Ku heterodimer, mediates the DNA-PKcs activity at stalled replication forks and facilitates the ATR signaling pathway in the cellular response to replication stress.

A novel round bottom μ-well array chip with biomimetic nano-cilia promotes 3D tumor cultures and metastatic bioassays

We present a microarray chip integrated with novel round bottom μ-well arrays and biomimetic nano-cilia, which can, for the first time, not only recapitulate in vivo-like tumor microenvironment but also be achieved in situ for hallmarks of tumor-based bioassays.

Observation of "wired" cell communication over 10-μm and 20-μm poly(dimethylsiloxane) barriers in tetracycline inducible expression systems

Communication between cells and extracellular environments is of interest because of its critical roles in cell development and differentiation. Particularly, this signal transduction is commonly believed to rely on the contact and binding of the participating molecules/proteins, suggesting that the binding distance needed is less than a few nanometers. However, it is difficult to precisely match the rapidly binding interaction which depends on the probability of molecular collision in living systems, raising a hypothesis that another mechanism exists, could promote this signal communication, and remains unknown. Here we report that a long-range signal delivery over 10-μm and 20-μm polydimethylsiloxane(PDMS) barriers can be observed in microfluidically tetracycline (Tet) inducible expression systems. Results show that a significant increment of the long-range induced green fluorescent protein in human embryonic kidney 293T (HEK 293T) cells by the stimulation of Tet is demonstrated, and that such a signal induction is not dominated by Tet diffusion and displays a specific bindingless property. In addition, our experimental results, combined with theoretical modeling, suggest that this communication exhibits a bump-shaped characteristic depending on barrier thickness, materially structural property, surface roughness, and agonist concentration. It strongly relies on the PDMS barrier to delivery signal; therefore, we call such a mechanism as “wired” cell communication instead of wireless. These results could ignite interests in the novel and “wired” cell communication, which we call it X-signal, and in the use of such systems for the study of cellular biology and development of new drug.