Tony Dickherber, National Cancer Institute
Rong Fan, Yale University
Sharon Gerecht, Johns Hopkins University
Sebastian Maerkl, Ecole Polytechnique Federale de Lausanne
Miqin Zhang, University of Washington
K2: Advancing Liquid Biopsies in the Clinic
Monday PM, November 30, 2015
Hynes, Level 3, Room 301
2:30 AM - *K2.01
Magnetic Ranking Cytometry for Ultrasensitive Analysis of Heterogeneous CTCs
Reza M. Mohamadi 1 Shana O. Kelley 1
1University of Toronto Toronto CanadaShow Abstract
The analysis of circulating tumor cells (CTCs) is an important capability that may lead to new approaches for cancer management. Here, we present a new device for CTC analysis that captures these cells and spatially arranges the cells according to their surface phenotype. Antibody-functionalized magnetic nanoparticles facilitate CTC sorting, and permit deconvolution of phenotypic subpopulations. Working with patient blood samples and animal models of cancer, we obtain profiles that elucidate the heterogeneity of CTCs and dynamically track how these cells change as a function of tumor growth.
3:00 AM - *K2.02
Affinity Selection of Rare Circulating Tumor Cells (CTCs) For Cancer Diagnostics Using Polymer Microfluidics: Is the Role of the Polymer Material Important?
Steven Allan Soper 1
1University of North Carolina Chapel Hill United StatesShow Abstract
While metastic disease causes 90% of all cancer deaths, determining the role of various cancer cells in the metastatic process has been difficult due to the rare nature of these cells and the continuum of phenotypes they possess. To assist in studies based on the use of rare CTCs that may be invovled in metastasis, a microfluidic fabricated via micro-replication from a polymer material was used to affinity select different CTC types. The microfluidic contained a series of 190 nL fluidic channels (50-500) configured in a serpentine geometry. The walls of the channels were decorated with antibodies used to recognize and select various subpopulations of CTCs. The covalent attachment to a polymeric surface could be affected by activating the polymer surface using UV/O3 light, which creates a surface scaffold of carboxylic acids that could subsequently be reacted with antibodies via EDC/NHS chemistry. The microfluidic could process 7.5 mL of whole blood in ~20 min with a recovery of 97% and purity >80%. These performance metrics were highly dependent on the type of polymer used as the substrate material for the microfluidic. Following selection from whole blood, the CTCs could be released from the surface-immobilized antibodies; this was affected by using single-stranded DNA linkers that contained a uracil residue that could be enzymatically cleaved releasing the CTCs. The CTCs were then enumerated by measuring impedance signatures of single cells that traversed through a pair of electrodes. The use of the microfluidic for determining the molecular characteristics of CTC subpopulations involved in an epithelial-to-mesenchymal transition were evaluated using pancreatic cancer as the example disease. In this presentation, we will discuss the microfluidic used for the CTC selection and the role of the selection of polymeric material on the performance of the device.
3:30 AM - *K2.03
Rare Circulating Tumor Cell (CTC) Capture Using Micro-Patterned Silicon Nanowire Platform and Its Drug Response of Captured CTCs within Cancer Patient Bloods
Sang-Kwon Lee 1 Dong-Joo Kim 3 Mun-Ki Choi 1 Jin-Tak Jeong 1 Wonshik Han 2
1Chung-Ang University Seoul Korea (the Republic of)2Seoul National University College of Medicine Seoul Korea (the Republic of)3UNIST Ulsan Korea (the Republic of)Show Abstract
Specific cell separation, further enumeration and further characterization (e.g. molecule analysis and drug response within patient whole bloods) of tumor cells are necessary in a variety of immunology, neuroscience, stem cell, and cancer research. Here we present on the rapid and direct quantification of specific cell captures using a micro-patterned streptavidin (STR)-functionalized silicon nanowire (SiNW) platform, which was prepared by Ag-assisted wet chemical etching and a photo-lithography process. This platform operates by high-affinity cell capture rendered by the combination of antibody-epithelial cell surface-binding, biotin-STR binding, and the topologically enhanced cell-substrate interaction on a 3-dimensional SiNWs array.
4:30 AM - *K2.04
Cancer Cell Migration in 3D
Denis Wirtz 1 Pei-Hsun Wu 1
1Johns Hopkins University Baltimore United StatesShow Abstract
Cell migration is a tightly regulated function critical to the normal development of organs and tissues. Cell migration is abnormally activated in a wide range of human diseases, including cancer metastasis, immunological responses, and wound healing. Most of what we know about eukaryotic cell migration at a mechanistic level has stemmed from well-controlled studies of cell migration on flat dishes. However, the onset of migration in disease and development often forces cells to negotiate, exert pulling forces on, and more through a 3D matrix.
Recent work has highlighted how 3D migration is fundamentally different from its 2D counterpart, in particular the biophysical forces driving 3D migration. Migration on 2D dishes, which induces an artificial baso-apical polarization of the cell, is driven by actomyosin contraction of stress fibers between focal adhesions and the formation of a wide lamellipodium terminated by thin filopodial protrusions at the leading edge. The same cells in a more physio-pathologically relevant collagen-rich 3D matrix do not display a lamellipodium or filopodia. Instead, cells display highly dendritic protusions controlled by distinct proteins that do not rely nearly as much on acto-myosin contractility, but rather microtubule assembly/disassembly dynamics. Recent work has also shown how cells in 3D can alternate between a mesenchymal and an amoeboid phenotype depending on the physical properties of the matrix (density, pore size, fiber alignment). Several biophysical assays have recently been developed and validated to monitor and measure cell motility in 3D microenvironments for applications in basic life science, biomedical research, and drug screening and testing.
5:00 AM - K2.05
Microfluidics Technology for Cancer Diagnosis & Personalized Treatment
Chwee Teck Lim 1
1National University of Singapore Singapore SingaporeShow Abstract
The presence of Circulating Tumor Cells (CTCs) in bloodstream of patients with epithelial cancers is an important intermediate step in cancer metastasis and can provide valuable insights into disease detection, staging and personalized treatment. As compared to obtaining a tissue biopsy which is invasive and painful, ‘‘liquid biopsy&’&’ for CTCs detection can be easily performed via a routine blood draw. The presence and number of CTCs in peripheral blood has been associated with the severity of the disease and have potential use for early detection, diagnosis, prognosis and treatment monitoring purposes. The isolation of CTCs using microfluidics is attractive as the flow conditions can be accurately manipulated to achieve an efficient separation. Here, we demonstrate several effective separation methods by utilizing the unique differences in size and deformability of cancer cells from that of blood cells. By exploiting the fluid dynamics in specially designed microfluidic channels, CTCs which are generally stiffer and larger can be physically separated from the more deformable blood constituents. Using this label-free approach, we are able to retrieve viable CTCs that are not only suitable for downstream molecular analysis such as genetic or RNA sequencing, but also for expansion and culture. With blood specimens from cancer patients, we not only confirmed successful detection, isolation and retrieval of CTCs, but also identification of actionable mutation which will enable precision medicine and personalized treatment of cancer patients.
5:15 AM - K2.06
In Situ Electrochemical ELISA for Specific Identification of Captured Cancer Cells
Tina Saberi Safaei 1 Reza M. Mohamadi 2 Edward H. Sargent 1 Shana O. Kelley 2 3 4
1University of Toronto Toronto Canada2University of Toronto Toronto Canada3University of Toronto Toronto Canada4University of Toronto Toronto CanadaShow Abstract
Circulating tumor cells (CTCs) are the cancer cells disseminated from a tumor into the blood stream. Presence of these cells in blood is known to be associated with metastasis of the disease. Therefore isolation and detection of CTCs have diagnostic and prognostic importance. However, due to their low abundance in blood only highly sensitive detection methods can be potentially utilized for clinical applications. Despite the many successful examples of clinical trials on importance of detection of rare tumor cells, they are yet to be widely considered as a routine clinical test or for prognosis and screening purposes. Most of the current methods for detection of cancer cells are based on immunostaining and require experienced technician. Here we report an integrated chip design that enables isolation and detection of rare cancer cells, using a simple yet sensitive electrochemical ELISA method. With this design, detection of very low number of isolated cancer cells is achieved in whole blood, which demonstrates the clinically relevant specificity and sensitivity of the device.
5:30 AM - K2.07
Identification of Various Stages of Induced Apoptosis and Treatment Sensitivity in Non-Small Cell Lung Carcinoma Using Dielectrophoresis
Rajeshwari Taruvai Kalyana Kumar 1 Shanshan Liu 2 John D. Minna 2 Shalini Prasad 1
1Univ of Texas-Dallas Richardson United States2University of Texas Southwestern Medical College Dallas United StatesShow Abstract
It is important to understand various biomolecular events that contribute towards classification of programmed cell death (apoptosis) and how tumors evade apoptotic death. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) are two major categories of lung cancer that differ in their sensitivity to undergo apoptosis. With new drugs being formulated to control abnormal cell proliferation (cancer) caused by mutations, effective drug-induced apoptosis will yield the relationship between cancer genetics and treatment sensitivity. The objective of this study was to build non-invasive techniques (without altering inherent cell properties) to isolate cells that are undergoing varying stages of apoptosis for a high-throughput screening application. We have used dielectrophoresis to determine and isolate various stages of apoptotic cells (early, mid and late) for two NSCLCs adenocarcinoma cell lines (HCC1833 and H1755). Our studies have shown significant differences in apoptotic cells by chromatin condensation, formation of apoptotic bodies and exposure of phosphatidylserine (PS) on the extracellular surface when the cells where exposed to a potent Bcl-2 family inhibitor drug (ABT-263). Time lapse dielectrophoretic studies were performed over a period of 48 hours upon exposure of varying concentrations of ABT-263 ranging between 50 nM to 500 nM. As a result of physical and biochemical changes, inherent dielectric properties of cells undergoing varying stages of apoptosis showed amplified changes in their cytoplasmic and membrane capacitance, measured using dielectrophoretic technique. In addition, zeta potential (potential at the electrical double layer of cell-buffer interface) of these fixed isolated cells were measured to obtain direct correlation to biomoelcular events. Western blot was used to examine the changes in the expression levels of Bax, Bcl-2, caspase-3, Hsp-90. As a control, the obtained results were compared against standard apoptotic assays for respective stages; JC-1, a mitochondrial membrane potential dye for marking early stage apoptosis, Annexin-V for the detection of PS as a mid-stage marker and defragmentation of DNA, verified by immunofluorescence staining using fluorescein-deoxyuridnie triphosphate (FITC-dUTP) and flow cytometry. The use of non-invasive dielectrophoresis is to provide a sophisticated method to characterize and isolate cells for potential downstream analysis. Effective separation of carcinoma cells at different apoptotic stages should enable a more rational approach to anticancer drug design and therapy.
5:45 AM - K2.08
Microfluidic Device Based on Tunable Thermo-Responsive Polymer-Graphene Oxide (GO) Composite Film for Efficient CTC Isolation and Enumeration
Apoorv Shanker 1 Hyeun Joong Yoon 2 Yang Wang 2 Molly Kozminsky 2 Qu Jin 2 Nallasivam Palanisamy 8 Monika Burness 3 Ebrahim Azizi 3 Diane Simeone 3 Max Wicha 3 Jinsang Kim 4 1 5 Sunitha Nagrath 2 6 7
1University of Michigan Ann Arbor United States2University of Michigan Ann Arbor United States3University of Michigan Ann Arbor United States4University of Michigan Ann Arbor United States5University of Michigan Ann Arbor United States6University of Michigan Ann Arbor United States7University of Michigan Ann Arbor United States8Henry Ford Health System Detroit United StatesShow Abstract
Compared to tissue biopsy, the standard method for cancer diagnosis, circulating tumor cells (CTCs) provide easy and non-invasive access to tumor cells and have proven to be valuable biomarkers for disease diagnosis and progression. Furthermore, CTCs represent the heterogeneous nature of tumor and hence potential surrogates of tumor cells for functional and genomic analysis. However, CTCs occur at very low frequency (1-10 cells/ml) in blood and are obscured by millions of blood cells, which makes their isolation difficult and, hence, has impeded their clinical application. Over the last decade, different technologies and devices have been developed to facilitate isolation and enumeration of these rare cells. Most technologies are based on micro/nanostructures coated with antibodies against the cell surface markers to aid cell capture. However, this makes device fabrication costly and time consuming. Most such devices also lack the mechanism for releasing the captured cells thereby making downstream analysis difficult.
We developed a planar microfluidic device based on tunable polymer-graphene oxide (GO) nanocomposite film made through simple drop-casting on a patterned and surface modified glass substrate. The platform architecture is composed of functionalized GO as the carrier of anti-EpCAM, a tumor cell recognition unit, and embedded in matrix of a thermo-responsive polymer. The polymer is designed to provide selective dissolution only below 13°C (lower critical solution temperature (LCST)) which allows the device to be used for cell capture at room temperature and cell release at temperature lower than the LCST. The non-fouling PEG monolayer-modified substrate further aids the release of the cells upon film dissolution. Since the captured cells are tethered to graphene sheets and not the polymer chains, cell isolation is easier and loss of cell viability is minimized. To test the performance of these devices, fluorescently labeled human breast cancer cell line MCF-7 cells (1000 cells/mL) were spiked into buffer and flowed at different flow rates (1-10mL/h). An average capture efficiency of >82% was achieved in the range of 1-3mL/h flow rate. Cell release experiments showed that on average >90% of the captured cells were released upon flowing cold PBS. Over 90% of the cells remained viable after release. We also tested clinical samples from ten metastatic breast cancer and three pancreatic cancer patients. CTCs were isolated from eight breast and two pancreatic cancer patient samples in the range of 2-20 CTCs/mL. Additionally, to demonstrate the potential for single cell genomic analysis of released CTCs, Fluorescence in situ hybridization (FISH) was conducted using probes for HER2 and chromosome 17 control probe, revealing HER2 amplification in one breast cancer patient. Effective downstream analysis of rare CTC populations facilitated by easy operation of the fabricated device highlights its potential for clinical application.
K3: Poster Session I: Advancing Basic Cancer Research Capabilities
Monday PM, November 30, 2015
Hynes, Level 1, Hall B
9:00 AM - K3.01
Enhancement of the Photoactivity RuO2 Doped Nanoparticles TiO2 Supported on Disordered Mesoporous Silica as Photocatalyts for Methylene Blue Photodegradation
Yuliana de Jesus Acosta-Silva 1 Arturo Mendez-Lopez 1
1CINVESTAV Distrito Federal MexicoShow Abstract
RuO2 doped nanoparticles TiO2 supported on disordered mesoporous silica (DMS-1) was synthesized by the sol-gel method, its ex-situ modification with TiO2 nanoparticles. The photoactivity was examined for methylene blue photodegradation in aqueous medium. To assist in interpreting the photocatalytic behavior of 0.76%RuO2-40%TiO2/DMS-1 and 0.79%RuO2-30TiO2/DMS-1, reference systems consisting of x%TiO2/DMS-1 photocatalysts undoped RuO2 and TiO2 pure (Degussa P-25). The load RuO2 <1.0 wt% was prepared by impregnation method and vary amount of TiO2 (30 and 40 wt.%). The photocatalysts were characterized by N2 adsorption-desorption isotherms, X-ray diffraction (XRD), UV-vis diffuse reflectance spectroscopy (UV-vis DRS) and transmission electron microscopy (TEM). The results showed that RuO2 on TiO2 present greater activity photocatalytic due to the presence of RuO2 that promoting efficiently charge separation, causes inhibition of recombination of electron-hole pairs.
9:00 AM - K3.02
Mesoporous Fe2O3/CdS Heterostructures for Real-time Photoelectrochemical Dynamic Probing of Cu2+
Jun Li 1 Jing Tang 1 Yongcheng Wang 1 Gengfeng Zheng 1
1Fudan University Shanghai ChinaShow Abstract
Three dimensional (3D) aligned mesoporous α-Fe2O3/CdS heterostructures are prepared for solar-driven, real-time, and selective photoelectrochemical sensing of Cu2+ in the living cells. Fabrication of aligned mesoporous α-Fe2O3 substrates is realized by an interfacial aligned growth and self-assembly process based on the van der drift model and subsequent selective in situ growth of CdS nanocrystals. The as-prepared mesoporous iron oxide/CdS heterostructures achieve significant enhancement (~2-fold) in the photocurrent intensity compared with mesoporous iron oxide. The electrochemical impedance spectroscopy measurement and the density functional theory calculation exhibit that the considerably improved performance of 3D aligned mesoporous α-Fe2O3/CdS heterostructures is attributed to the enhancement of charge transfer and the increase of charge carrier density. This photocurrent increase is ascribed to unique 3D Fe2O3 and CdS with multiple features including their excellent flexibility, high surface area (~ 87 m2/g) and large pore size (~ 20 nm), which give rise to enhancement of the PEC performance by facilitating ion transport and providing more active electrochemical reaction sites. In addition, the introduction of Cu2+ enables the activation of quenching the charge transfer efficiency, thus leading to sensitive photoelectrochemical recording of Cu2+ level in buffer and cellular environments. Furthermore, real-time monitoring (~ 0.5 nM) of Cu2+ released from apoptotic HeLa cell is performed using the as-prepared 3D aligned mesoporous iron oxide/CdS biosensor, suggesting the capability of studying the nanomaterial-cell interfaces and illuminating the role of Cu2+ as trace element.
9:00 AM - K3.03
Nanoporous GaN Substrates for Directing Stem Cell Function and Differentiation
Lin Han 1 Jing Zhou 1 Yubing Sun 2 Yu Zhang 3 Jung Han 3 Jianping Fu 2 Rong Fan 1
1Yale University New Haven United States2University of Michigan Ann Arbor United States3Yale University New Haven United StatesShow Abstract
Recently, nanostructured surfaces are finding applications in rare cell analysis and stem cell engineering via nanoscale cell-surface interactions. Studies have shown that nanoscale cellular structures, such as, focal adhesion and integrin, interact with the nanotopography of the substrate, which in turn affects cell behavior such as cell morphology, adhesion, spreading, motility, gene expression, and differentiation. Here, we report the use of a novel nanotopographic substrate - single-crystalline nanoporous GaN substrate fabricated over a large area via electrochemical reaction and showing precisely tunable pore size for the study of human mesenchymal stem cell function and differentiation. This material is not only an optically active semiconductor but also relatively biocompatible and non-toxic. Single-crystalline nanoporous gallium nitride (GaN) thin films were fabricated with the pore size readily tunable in 20-100 nm. We found that the ability for human mesenchymal stem cells (hMSCs) to adhere uniformly on these substrates shows a maximum with the substrate pore size of 30 nm. Substantial cell elongation was observed on the films with pore size of 80 nm. Interestingly, the osteogenic differentiation of hMSCs also occurs preferentially on the substrate with 30 nm sized nanopores, which is correlated with the optimized conditions for uniform cell spreading, which suggests that adhesion, spreading, and stem cell differentiation are inter-linked and can be co-regulated by nanotopography.
9:00 AM - K3.04
Microchip Platforms for Multiplex Measurement of microRNA Biomarkers in Single Cells
Nayi Wang 1 Yu Wu 1 Yao Lu 1 Steve Chapin 2 Patrick S. Doyle 2 Jun Lu 1 Rong Fan 1
1Yale University New Haven United States2MIT Cambridge United StatesShow Abstract
MicroRNAs (miRNAs) are a group of small RNAs that are important regulators of normal development, immune defense and a range of human diseases including cancer. The level, variety and kinetics of miRNAs in a cell population could contribute to the formation of substantial non-genetic cell heterogeneity and alters the collective response of, i.e., tumor cells, to treatment. Measurement of an array of oncogenic and tumor-suppressive miRNAs from a population of cancer cells at single-cell resolution will enable the possibility to quantify cellular heterogeneity and corresponding miRNA profiles in cancer. Herein we developed a microchip platform for amplification-free, multiplexed detection of miRNAs at the single cell level. This microchip consists of a nanoliter microchamber array to isolate single cells and a high-density DNA oligomer array to perform multiplexed detection of miRNA. We tested two different approaches on this microchip platform. It employed a microfluidic double-T unit to conduct on-chip cell lysis and release of cytoplasmic miRNAs, which are detected directly by the DNA oligomers immobilized in the microchambers via hybridization and ligation. Our preliminary results demonstrated up to 12-plexed measurements of miRNA targets from single human monocytes (THP-1) and identified the high expression of miR-16, miR-150, miR-223 and miR221, and an appreciable level of cellular heterogeneity. This microchip platform has the potential to be utilized to invetigate the heterogeneity of cancer cells and the associated microRNA signatures from clinical specimens.
9:00 AM - K3.05
Cancer Detection from Breath: Aldehyde Sensing by Microsensor Arrays
Andreas Guentner 1 Samuel Regli 1 Kiran Chikkadi 1 Vitaly Koren 1 Marco Righettoni 1 Sotiris E Pratsinis 1
1ETH Zurich Zurich SwitzerlandShow Abstract
Cancer is one of the major health problems of modern society with predicted 12.7 million newly diagnosed cases per year (2008).1 Its early detection could significantly improve medical therapy and thus reduce morbidity and mortality rates. Promising breath markers for lung2 and breast cancer3 are aldehydes as their increased levels were detected in affected patients. Portable and simple#8209;in#8209;use aldehyde analyzers could contribute significantly to the implementation and widespread application of breath analysis for medical self-diagnostics and simple body parameter monitoring. Chemo-resistive gas sensors are ideal as they offer sufficient analyte sensitivity, fast response and recovery times for real-time monitoring, simple operation and they have been tested already in a portable design.4 Well-known chemo-resistive materials, however, suffer from insufficient aldehyde selectivity in the complex5 analyte matrix of human breath. To overcome this challenge, arrays of multiple non-specific sensors are a viable option where selectivity is obtained by statistical analysis of the sensor responses.6
Here, we present a microsensor array consisting of four differently doped SnO2 films for selective formaldehyde (FA) detection. The microsensor substrates are silicon wafer-based and fabricated by state-of-the-art micro-processing technology. Rapid flame aerosol synthesis is applied to produce the sensing nanoparticles (~10 nm) that are accurately deposited onto the substrates in designated areas (d = 500 mu;m). The resulting nanostructured and highly porous film morphology offers high sensitivity that is quite attractive for breath analysis. These sensors are combined in arrays featuring a compact size and low power consumption (~2 W) at operational conditions. The derived arrays are tested on simulated breath mixtures at 90% rh. Breath-relevant FA concentrations (30 - 200 ppb) are clearly identified that could allow detection of cancer. Such microsensor arrays have high potential for further development towards a portable breath analyzer. The compact size and low power consumption make them attractive for integration into portable electronic devices (e.g. smart phone).
(1) Bray, F.; Jemal, A.; Grey, N.; Ferlay, J.; Forman, D. The Lancet Oncology2012, 13, 790-801.
(2) Fuchs, P.; Loeseken, C.; Schubert, J.K.; Miekisch, W. Int. J. Cancer2010, 126, 2663-2670.
(3) Ebeler, S.E.; Clifford, A.J.; Shibamoto, T. J. Chromatogr. B. Biomed. Appl.1997, 702, 211-215.
(4) Righettoni, M.; Tricoli, A.; Gass, S.; Schmid, A.; Amann, A.; Pratsinis, S.E. Anal. Chim. Acta2012, 738, 69-75.
(5) De Lacy Costello, B.; Amann, A.; Al-Kateb, H.; Flynn, C.; Filipiak, W.; Khalid, T.; Osborne, D.; Ratcliffe, N.M. J. Breath Res.2014, 8, 014001 (29 pp.)
(6) Di Natale, C.; Paolesse, R.; Martinelli, E.; Capuano, R. Anal. Chim. Acta2014, 824, 1-17.
9:00 AM - K3.06
A Microchip Platform to Monitor Proliferation and Protein Fluctuation of Individual Cancer Cells
Iva Xhangolli 1 Franziska Michor 2 3 Rong Fan 1
1Yale University New Haven United States2Dana-Farber Cancer Institute Boston United States3Harvard T.H. Chan School of Public Health Boston United StatesShow Abstract
Tumor suppressor genes inhibit cancer development and oppose oncogene function providing a physiological balance for the regulation of cell proliferation. Due to random stochastic events, the levels of tumor suppressor proteins (TSP) may fluctuate to very low levels even when one of the alleles remains functional giving the cells an evolutionary advantage to lock them in the low-TSP-expression states over multiple generations. As a result, the cells constantly experience a condition similar to complete deletion/mutation of both TSG alleles. To test this hypothesis, we need to follow the same individual cancer cells over multiple divisions and simultaneously monitor the fluctuation of tumor suppressor protein expression, which is challenging at the population level. We have developed a novel high-throughput single-cell capture, culture, and perfusion microfluidic system to correlate cell proliferation and the level of a given tumor suppressor protein for thousands of single cells. The capture chambers (length 60 um x width 20 um x depth 20 um) provide sufficient space for cell division in the x-plane while communication channels between each chamber and the inlet/outlet will provide continuous nutrient and gas perfusion. Passive perfusion will allow the transportation of cytokines and other factors promoting an environment mimicking growth on culture flasks preferred by adherent cell types. Gravity driven fluid transport in-between the inlet/outlet of the microfluidic network will be modified using height adjustment of the media reservoir since shear force exerted by fluid flow has shown to influence cell attachment and growth. By combining this cell segregation ability with other powerful analytics such as live cell protein level tracking and in situ hybridization, we will be able to correlate the fitness of individual cells and their progenies, and correlate with the level of TSP in real time. Using this platform, we can answer whether or not the initial neoplastic (uncontrolled growth) transition can indeed occur even when the TSG are not completely deleted generating new insight for cancer prevention and control.
9:00 AM - K3.07
Functional Nanoparticles for SERS Imaging and Detection of Biomolecular Activities
Zakiya Skeete 1 Jing Li 1 Christian Salazar 1 Alice Wu 1 Xavier Hamilton 1 Christopher Manahan 1 Jin Luo 1 Chuan-Jian Zhong 1
1SUNY-Binghamton Binghamton United StatesShow Abstract
As interests in the application of noble metal nanoparticles, especially gold nanoparticles, in medical theranostics rapidly expand in many cutting-edge areas, there is an increasing need for the development of effective strategies for the fabrication of nanomaterials and the detection of the biomolecules. One important strategy is to establish an effective plasmonic coupling of gold-based nanoparticles for an enhanced detection of proteins or biomarkers associated with cancer. This report describes recent findings of an investigation of gold-based nanoparticles to construct a microfluidic platform for the detection of proteins or DNA in the biomolecular recognition process. In addition to building a well-defined gold-based nanoparticle array on a substrate, one important emphasis is the understanding of molecular and biomolecular interactions at the interfaces of the array, which creates “hot-spots” for surface enhanced Raman scattering of reporters labeled on the nanoparticles through controlled small aggregates via specific protein binding activities. Examples of gold-based nanoprobes conjugated with biomolecules such as proteins and DNA sequences linked to the regulatory region of a cell cycle gene will be discussed.
K1: Novel Materialsmdash;Enabled Treatment Strategies I
Monday AM, November 30, 2015
Hynes, Level 3, Room 301
10:00 AM - *K1.01
Nanopore-Based Detection of Biomarker toward Cancer Diagnostics
Rashid Bashir 1
1University of Illinois at Urbana - Champaign Urbana United StatesShow Abstract
Epigenetic alterations involving DNA methylation are early and frequently observed events in carcinogenesis. Hypermethylation is reported to be associated with cancers of the prostate, colon, lung, liver, breast head and neck, and further correlated with metastatic potential in many other tumor types. Thus, methylation analysis in DNA can play a critical role in the diagnosis of cancer, especially at an early, pre-cancerous stage. A simple, rapid, and reliable method to detect epigenetic modification of DNA, which uses small samples and eliminates bisulfite treatment and PCR amplification, has potential to impact cancer diagnostics. We drilled single nanopores in free standing 10nm thick SiN membranes supported on Silicon substrate. The nanopore measurements were performed in 1M KCl at pH 7.6 or in 0.2 M NaCl at pH 7.6 containing 10mM Tris and 1mM EDTA. The methylated-DNA/MBP complexes were prepared and incubated for 15 minutes at room temperature (25 ± 2 °C) immediately before nanopore experiment. Nanopore current traces were recorded using Axopatch 200B and Digidata 1440A at 10 kHz built-in low pass Bessel filter and 10 µs sampling rates. Instrumental control and data analysis was performed using Clampex 10.2 and Clampfit 10.2. We demonstrate the detection of methylation in 30, 60, and 90 bp double stranded oligos. Hypermethylated DNA (hyMethDNA) were selectively labeled using methylation specific proteins (MBP), and can be detected without the need for any further processes, such as bisulfite conversion, tagging with fluorescent agent, or sequencing. Discrimination of hyMethDNA fully bound with MBP mixed with unmethylated DNA (unMethDNA) revealed through nanopore ionic signatures of current blockage and duration. Specifically at 300 mV, 90 bp hyMethDNA/MBP was discriminated from 90 bp unMethDNA by a 6.5-fold difference in current blocking and a 23-fold difference in transport duration. This approach could be compatible with small amounts of genomic extracts and direct methylation detection without fluorescence-labeling and bisulfite-conversion. However, nanopore-based methylation assay should improve efficiency for low sample volume obtained from body fluids.
10:30 AM - *K1.02
Measuring Kinase Activity in Living Cells with Chemical Biology
Laurie Parker 1
1University of Minnesota Minneapolis United StatesShow Abstract
Our research program is broadly directed at assay development for post-translational modifications (PTMs), with a focus on protein phosphorylation by tyrosine kinases. Protein tyrosine kinases play key roles in disease and are particularly important in cancer: mutations in several protein tyrosine kinase genes have been identified as drivers of many tumor types and drugs targeted at inhibiting these enzymes represent ~20% (>$9 billion) of the current oncology market. We use a “decoy” substrate biosensor approach in which an artificial, optimized substrate peptide is designed to report the activity of a specific enzyme in living cells. Delivery is achieved using cell-penetrating peptides, and enzymatic modification is measured using a range of readout strategies—some that require extraction of the cell contents and some that leave the cell intact. Targeting the function of the enzyme in its intracellular environment preserves protein-protein interactions, localization, and scaffolding-dependent activation, and decoy substrates provide a snapshot of enzymatic activity that circumvents the need for pre-knowledge of every endogenous substrate site. We also develop multiplex-compatible readouts, so we can use a suite of biosensors for different enzymes in order to profile pathways. We have established our approach and laid the groundwork of a substrate development workflow to expand our repertoire of biosensors for kinases and other enzymes. Long term, our lab will maintain a pipeline of biosensor and read-out technology development while also taking an active role in studying signaling biology with our tools.
11:30 AM - K1.03
Electrical Monitoring of 3D Cell Models Using Organic Electronics
Vincenzo Curto 1 Miriam Huerta 1 Jonathan Rivnay 1 Adel Hama 1 Roisin Owens 1
1Ecole des Mines-St. Etienne Gardanne FranceShow Abstract
The development of electrical techniques for monitoring of biological phenomena is a field that is fast gathering pace. Advantages of electrical techniques are manifold, including the fact that they are label-free, and have the potential to be very efficient transducers, since the signal measured is already in an electrical readout format. Electronic methods for live-cell sensing can be applied to applications involving extracellular recording of electrical activity from electrically active cells (neurons/myocytes), but also for monitoring of non-electrically active cells and tissue assemblies. Electrical impedance sensing (EIS) has emerged as a dynamic method, with demonstrated potential for use in monitoring barrier function, cellular adhesion, proliferation, micro motion, and wound healing many of which are processes that are fundamentally altered in cancer. Although significant progress has been made in developing systems for electrical monitoring of cells in 2D, the transition to 3D cell models, which has been shown repeatedly to be necessary for representative cell models of cancer, presents many challenges. The advent of organic bioelectronics has the potential to meet these challenges. Organic bioelectronics refers to the coupling of conducting polymer based devices with biological systems, proven repeatedly in the last decade to provide numerous advantages to a wide variety of biomedical applications in terms of sensitivity, specificity and most importantly, bridging of the biotic/abiotic interface. Flexibility in design, patterning and processing means that the materials are amenable for integration into novel device formats, compatible with complex 3D cell culture models. Two key examples will be used to show the literal flexibility of organic electronic materials and devices: 1. The adaptation of an organic electronic device for monitoring of 3D cysts and spheroids, and 2. The integration of an ‘organ on chip&’ cell model with microfluidics and embedded electronics. In both cases the device format is adapted to suit the 3D format of the cell model in question. Continuous electronic monitoring provides a very useful dynamic parameter to assess cell growth and health, while the optical transparency of the polymeric materials allows continuous optical monitoring.
11:45 AM - K1.04
Synthesis Strategies to Achieve Optimal Pd@Au Core-Shell Radioactive Nanoparticles for Medical Physics (Brachytherapy) Applications
Myriam Laprise-Pelletier 1 2 3 Marc-Andre Fortin 1 2 Jean Lagueux 2 Marie-France Cote 2 Pascale Chevallier 2 3
1Universite Laval Quebec Canada2CR-CHU de Quebec Quebec Canada3Centre Quebecois des Mateacute;riaux Fonctionnels (CQMF) Quebec CanadaShow Abstract
The radioisotope palladium (103Pd; 20kV photons; 17-days half-life), encapsulated in millimetre-size seed implants, is widely used in prostate cancer brachytherapy (internal radiotherapy). A new strategy under development to replace millimetre-size implants, consist in injecting radioactive nanoparticles (NPs) in the affected tissues. In particular, the interaction of low-energy 103Pd photons (20 keV) with gold (Au), produce a large number of photoelectric events, that strongly enhance the dose to tumors (the "radiosensitization effect"). Therefore, injections of 103Pd-containingAu NPs could represent an alternative to millimetre-sized implants, which are associated to discomfort and dose heterogenities. However, the production of 103Pd-containing Au NPs requires the development of NP synthesis procedures that are a) highly efficient (to minimise radioactive waste), b) rapid (to minimise the exposure of manipulators), c) performed in water (to avoid tedious ligand exchange), d) precise (Pd/Au ratios, size distribution and core-shell characteristics), and e) performed with biocompatible molecules. In this study, an original NP synthesis procedure allowing the production of 103Pd@Au NPs, was explored for optimal efficiencies, production rates and volumes, core-shell characteristics, and overall 103Pd radioisotope encapsulation efficiency.1,2 First, ultra-small cores of Pd were synthesized by using an original and direct reduction route in water (NP measured by high-resolution TEM and dynamic light scattering - DLS), using dimercaptosuccinic acid (DMSA) as a surface ligand and ascorbic acid as a reductor. Comparisons were made between non-radioactive and radioactive Pd chloride solutions. The synthesis of Pd cores (sim;10 nm core size) was found to be highly susceptible to pH, with synthesis efficiencies as high as 87% in optimised conditions. Then, Pd NPs were purified from the non-reacted synthesis products, and used as seeds for the growth of gold (Au) coatings, followed by steric stabilisation by polyethylene-glycol (thiol reaction). Thus-generated Pd@Au and 103Pd@Au core-shell nanoparticles (sim; 30 nm) were analysed in high-resolution TEM (particle size distribution, core-shell characteristics by EDX mapping), XPS, FTIR, UV-Visible and DLS. The imaging properties of radioactive nanoparticles were assessed in computed tomography (CT; attenuation by high-Z elements) and single-photon emission computed tomography (SPECT; radioactive emissions from 103Pd; for in vivo tracking of the 103Pd@Au NPs). Overall, this study confirms the conditions allowing optimal production of 103Pd@Au radioactive nanoparticles for more precise, uniform, and less invasive brachytherapy procedures.
1. Djoumessi, Laprise-Pelletier, Fortin et al, RSC J. Mater. Chem. B, (2015), 3, 2192-2205.
2. Laprise-Pelletier, Fortin et al; IUPESM World Congress (WC) on Medical Physics (MP) and Biomedical Engineering (BME); Toronto June 10, 2015.
12:00 PM - K1.05
Engineering Gold -Silver Based Theragnostic Nanocages for Combinatorial Therapy of Breast Cancer
Sreejith Raveendran 1 Anindito Sen 2 Toru Maekawa 1 Sakthi Kumar 1
1Toyo University, Bionano Electronics Research Center Saitama Japan2JEOL Limited Tokyo JapanShow Abstract
Gold (Au) nanostructures as a multifaceted nanoplatform have shown their versatility in the fields of biomedical, bionanoscience and material science applications. Based on their unique optical properties they are well explored for several applications including live cellular- imaging, therapy and optoelectronics. Au nanoparticles of various shapes are capable of displaying Localized Surface Plasmon Resonance (LSPR) in the near- infrared region spanning to a range of 600- 1200 nm . Au based nanoparticles are profoundly used for cancer therapeutic applications as they are less cytotoxic and highly biocompatible in nature. Here, we demonstrate the synthesis of a gold- silver nanocage based theragnostic nanoparticles passivated with extremophilic polysaccharide, functionalized with antibodies for combinatorial therapy of breast cancer. Silver (Ag) nanocubes were made as sacrificial templates for the preparation of Au nanocages by employing Galvanic Replacement (GR) reaction; characterization was performed using JEM-ARM200F Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM). On replacement of Ag by Au, several random holes were appeared on the edges and faces of the nanocubes. Statistical analysis revealed a dominance of 220-240 nm sized Au nanocages with a small population of cuboid shaped particles. 3D tomographic analysis of nanoparticles revealed the overall structure of nanoparticle with encapsulation of the drug and the functionalizing moieties; using computational analysis we have analyzed the plausible locations of nanoparticle adsorption and accumulation in the cells. The formulation would be a pivotal step in the combinatorial therapeutic models for breast cancer treatment.
 S. Jain, D. G. Hirst, J. M. O&’Sullivan, Br. J. Radiol.2012, 85, 101.
 X. Xia, Y. Xia, Front. Phys.2014, 9, 378.
 J. P. M. Almeida, E. R. Figueroa, R. A. Drezek, Nanomedicine Nanotechnology, Biol. Med.2014, 10, 503.
12:15 PM - K1.06
Cancer Cell Membrane-Coated Gold Nanorods as Theranostic Agents for Cancer Therapy
Valeria Marangoni 1 Juliana Cancino 1 Valtencir Zucolotto 1
1Univ de Sao Paulo Sao Carlos BrazilShow Abstract
With the recent advances in nanomedicine, there is an increasing expectation for the development of multifunctional nanostructures combining specificity with diagnostic and therapeutic properties. These so-called theranostic materials represent the state-of-the-art in the development of nanoscale-based materials for fighting cancer. Gold nanorods (AuNRs) have found promising applications in medicine, mainly because of the two surface plasmon resonance (SPR) bands resulting from the coherent motion of the conduction band electrons along the short (visible region) and long axes (near infrared region) of the particle. The absorption in the near infrared region makes them appropriate for in vivo photothermal applications due to the maximum radiation penetration through tissue. However, a major obstacle related to the use of AuNRs is related to their low blood circulation time and, consequently, their low accumulation in the tumor. To overcome these challenges, we developed a nanosystem comprising cell membrane-coated AuNRS, which have been synthetized by colloidal seed-mediated, surfactant-assisted approach, followed by coating with human lung adenocarcinoma epithelial cell (A549) membrane. The nanoconjugates presented higher toxicity to cancer cells compared to healthy fibroblasts. The incorporation of gold nanorods into real membrane monolayers was also studied using Langmuir techniques via kinetics absorption and surface pressure measurements and revealed significant differences on how the AuNRs interact with the cell membranes depending on the size of the gold nanorods, indicating that the lipids present in the covering membrane exerted high influence on the uptake process. These results revealed the potential of cell membrane-coated nanomaterials and open opportunities for the development of more efficient nanosystems for cancer applications.
12:30 PM - K1.07
Biosynthesis of Magnetite Nanoparticles for Specific Targeting of Breast Cancer Cells
John David Obayemi 1 2 Winston O Soboyejo 1 2 Olushola S Odusanya 2 Karen Malatesta 1
1Princeton University Princeton United States2African University of Science and Technology, AUST Abuja NigeriaShow Abstract
This paper presents the results of an experimental study of uniquely biosynthesized magnetite nanoparticles (BMNPs) using Magnetospirillum magnetotacticum bacteria. The BMNPs were functionalized by conjugation to luteinizing hormone releasing hormone (LHRH), a molecular recognition unit (MRU) with chemically synthesized nanoparticles (CMNPs) as control. The resulting nanoparticle structure, morphology and characteristics properties were examined using X-ray diffraction (XRD), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), Raman Spectroscopy, Fourier Transform Infrared (FTIR) Spectroscopy as well as quantitative image analysis. Insights on the adhesive forces of the functionalized magnetite nanoparticles needed to overcome the hydrodynamic and shear forces to target the breast cancer cells were explored. The adhesion forces between BMNPs and human breast cancer cells (MDA-MB-231 cell line) for improved selectivity and specificity were demonstrated using the Atomic Force Microscope (AFM). The BMNPs constituents had adhesion forces to breast cancer cells that were greater than those of CMNPs. The implications of the results are very useful for the development of nano-targets and magnetite nanoparticles for the detection and treatment of breast cancer.
12:45 PM - K1.08
Synthesis and Characterization of Poly(1,2-glycerol carbonate)-Graft-Succinic Acid-Paclitaxel Conjugate Nanoparticles
Iriny Ekladious 1 Heng Zhang 1 Mark W Grinstaff 1
1Boston Univ Boston United StatesShow Abstract
Polymeric nanoparticle (NP) drug delivery systems possess several advantages over conventional small molecule chemotherapeutics. Among these is the ability to provide controlled and sustained drug release, specific drug targeting and delivery, and high loading of insoluble agents such as paclitaxel (Pax). Pax is one of the most widely used chemotherapeutic agents for a variety of solid organ malignancies (lung, ovarian, breast, head and neck cancers, and advanced forms of Kaposi&’s sarcoma). However, despite its widespread use, Pax suffers from poor solubility, rapid systemic clearance, limited tumor exposure, and low target tissue concentrations (~0.4% of the systemically administered dose). Due to its poor aqueous solubility, Pax is often delivered in a Cremophor EL (C/E) adjuvant; and C/E itself is known to cause adverse side-effects and hypersensitivity reactions. We have engineered a novel poly(1,2-glycerol carbonate)-graft-succinic-acid-paclitaxel (PGC-Pax) NP system in which Pax can be incorporated at high loadings (>60 wt%). Additionally, the polymer backbone is readily degradable and biocompatible, with glycerol, succinic acid, and carbon dioxide as the degradation byproducts. We herein demonstrate the synthesis and characterization of PGC-Pax NPs with tunable release kinetics, in vitro cytotoxic activity, and in vivo efficacy in a small animal cancer model. We also show the cellular internalization of rhodamine labeled PGC-Pax (PGC-Pax-Rho) NPs via flow cytometric analysis and confocal microscopy.