Letter pubs.acs.org/NanoLett
Real-Time Visualization of Nanoparticles Interacting with Glioblastoma Stem Cells Elliot S. Pohlmann,†,‡ Kaya Patel,† Sujuan Guo,† Madeline J. Dukes,§ Zhi Sheng,†,‡,∥,# and Deborah F. Kelly*,†,‡,⊥,# †
Virginia Tech Carilion Research Institute, 2 Riverside Circle, Roanoke, Virginia 24016, United States Virginia Tech Carilion School of Medicine, 2 Riverside Circle, Roanoke, Virginia 24016, United States § Protochips, Inc., Applications Science, 616 Hutton Street, Raleigh, North Carolina 27606, United States ∥ Department of Biomedical Sciences and Pathology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia 24061, United States ⊥ Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061, United States # Faculty of Health Sciences, Virginia Tech, Blacksburg, Virginia 24061, United States ‡
S Supporting Information *
ABSTRACT: Nanoparticle-based therapy represents a novel and promising approach to treat glioblastoma, the most common and lethal malignant brain cancer. Although similar therapies have achieved significant cytotoxicity in cultured glioblastoma or glioblastoma stem cells (GSCs), the lack of an appropriate approach to monitor interactions between cells and nanoparticle-based therapies impedes their further clinical application in human patients. To address this critical issue, we first obtained NOTCH1 positive GSCs from patient-derived primary cultures. We then developed a new imaging approach to directly observe the dynamic nature of nanoparticles at the molecular level using in situ transmission electron microscopy (TEM). Utilizing these tools we were able to visualize real-time movements of nanoparticles interacting with GSCs for the first time. Overall, we show strong proof-of-concept results that real-time visualization of nanoparticles in single cells can be achieved at the nanoscale using TEM, thereby providing a powerful platform for the development of nanotherapeutics. KEYWORDS: Glioblastoma stem cells, in situ transmission electron microscopy, gold nanoparticles, nanorods, NOTCH1 receptor, microfluidics
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the level of tissues, however, information regarding the interaction between NPs and individual cells remains missing from these analyses. Developing new technologies to directly assess NP-based therapies at the molecular level may contribute a wealth of information toward evaluating therapeutic efficacy. To address this issue, we engineered a new toolkit that permits us to perform real-time analyses of NP interactions with single cell membranes. We elected to use glioblastoma stem cells (GSCs) because (1) primary glioblastoma remains a lethal threat to patients afflicted with these tumors;6 (2) malignant brain tumors remain difficult to treat, possibly due to the presence of GSCs;7,8 and (3) NP-based therapeutic agents can kill GSCs in vitro and in vivo, however, a molecular assessment of their therapeutic efficacy remains ill-defined.9−11
odern drug delivery vehicles commonly used in cancer research are comprised of chemical polymers, liposomal formulations, gold nanoparticles (NPs), or some combination of these reagents. Gold NPs are readily used within the body for both therapeutic and medical imaging purposes due to their small size (∼10−100 nm) and characteristically high contrast.1,2 The high biocompatibility and easily modified surface properties of gold NPs also contribute to their efficacy as drug carriers in the treatment of solid tumors.3,4 One example of such modifications is the use of small interfering RNAs (siRNAs) engineered onto the surface of gold NPs and used in recent preclinical studies. The si-RNA-conjugated gold NPs could effectively penetrate the blood brain barrier to transfect glioblastoma cells, while showing resistance to degradation. These combined effects worked to synergistically ameliorate tumor growth in mice.5 While NP-based therapies continue to gain popularity in clinical trials, the major limitations toward implementing their widespread use are safety and controlled dose evaluation. Current approaches can provide therapeutic measurements at © 2015 American Chemical Society
Received: November 21, 2014 Revised: February 25, 2015 Published: March 3, 2015 2329
DOI: 10.1021/nl504481k Nano Lett. 2015, 15, 2329−2335
Letter
Nano Letters Here, we demonstrate a significant advance to observe molecular displacements in a liquid environment at the nanoscale using in situ transmission electron microscopy (TEM). The use of an isolated microfluidic imaging chamber enabled us to visualize for the first time NPs interacting with GSCs in a time-resolved manner. Therefore, the technical advancements described here provide the basis for directly assessing the affects of NPs on cancer cells at the molecular level. To better understand how NP-based drug therapies interact with cancer cells, we first attempted to enrich for GSCs from a primary glioblastoma culture mixed with nondifferentiated and differentiated tumor cells (GS9-6 line)12 by using the NOTCH1 receptor as a protein marker. NOTCH1 is abundantly expressed on the surface of GSCs and is important for the maintenance and differentiation toward the endothelium lineage of these cells.13−17 As such, NOTCH1 is widely used as a therapeutic target for glioblastoma treatment, but its potential as a protein marker for GSCs has not been fully explored. Therefore, we tested whether monoclonal antibodies against the extracellular domain of the NOTCH1 receptor could be used to enrich for GSCs. To confirm that GS9-6 cells expressed sufficient quantities of NOTCH1 we performed immunoblotting assays and found that NOTCH1 was specifically detected in GS9-6 cells in comparison to negative control cells (MA104 line) that are not known to highly express NOTCH1 (Figure 1A). We then employed a magnetic-activated cell sorting procedure by decorating magnetic beads with the same monoclonal antibodies against the extracellular domain of NOTCH1. We passed
GS9-6 cells through the NOTCH1 antibody-conjugated magnetic beads and referred to the sorted cells as GS9-6/ NOTCH1+. We then assessed the stemlike characteristics of the GS9-6/ NOTCH1+ cells (i.e., the ability to self-renew and give rise to other cell types).12,18 To assay for differentiation, we tested the responses of GS9-6/NOTCH1+ cells to fetal bovine serum (FBS)-induced differentiation.12,19 The GS9-6/NOTCH1+ cells initially expressed high levels of a neural stem cell marker (nestin (NES)) but not the astrocyte marker GFAP (Figure 1B, FBS−). Notably, the addition of FBS substantially reduced the level of NOTCH1 and NES with a concurrent increase of GFAP (Figure 1B, FBS+) and induced the cells to change morphology from spherical suspension cells to adherent cells with filamentous processes. These results indicated that GS9-6/ NOTCH1+ cells were differentiated into astrocyte-like cells in the presence of FBS. Next, we monitored for the self-renewal capacity of the GS96/NOTCH1+ cells using the sphere formation assay.18 We plated from 0 to 20 cells (including parental GS9-6, GS9-6/ NOTCH1+, and GS9-6/CD133+) into individual wells of a 96well plate and quantified sphere formation throughout a twoweek incubation period. We then calculated the number of cells that were required to form at least 1 sphere per well. This analysis revealed that only 1.33 GS9-6/NOTCH1+ cells were required to generate 1 sphere per well, whereas the cell numbers for GS9-6 or GS9-6/CD133+ cells were 10.98 or 29.52, respectively (Figure 1C,D). Therefore, we found that parental GS9-6, GS9-6/NOTCH1+, and GS9-6/CD133+ cells exhibited different propensities for self-renewal (Figure 1C) with GS9-6/NOTCH1+ cells being more potent in their relative self-renewal properties. Intriguingly, GS9-6/CD133+ cells that were enriched using CD133+, the most commonly used stem cell marker for glioblastoma, showed a reduced ability to self-renew, suggesting that NOTCH1 might be a more suitable candidate marker for GSCs. Collectively, we demonstrated that NOTCH1-enriched GSCs possess more power to regenerate and can differentiate into astrocyte-like cells. Thus, GS9-6/NOTCH1+ cells provided us with an adequately pure population of GSCs, enabling the following single cell imaging studies. To perform real-time recordings of NPs interacting with the GS9-6/NOTCH1+ cells, we employed the Poseidon specimen holder (Protochips, Inc.) and tunable silicon nitride (SiN) microchips containing 50 nm thin imaging windows.20,21 In general, GSCs preferred to grow in suspension in serum-free stem cell culture media and did not easily adhere to the microchip surface. We therefore exploited the presence of the NOTCH1 receptor found on the surface of the GS9-6/ NOTCH1+ cells to tether them to the surface of the SiN microchips. To do this, the microchips were glow-discharged for 60 s using a PELCO easiglow instrument (Ted Pella, Inc.) then coated with 3-μL aliquots of protein A (0.01 mg/mL) followed by monoclonal antibodies (0.01 mg/mL) against the extracellular domain of the NOTCH1 receptor.22 In parallel, we also decorated the protein-A coated microchips with antibodies against the NOTCH1 intracellular domain (NICD), which served as a negative control as the NICD is not found on the external cell surface (Figure 2A). The microchips decorated with antibodies against the NOTCH1 extracellular domain retained GSCs that freely spread across the imaging window within a 2 min incubation period. Cells did not readily adhere to microchips decorated with antibodies against the NICD
Figure 1. Differentiation and self-renewal properties of GS9-6/ NOTCH1+ glioblastoma stem cells. (A) Expression of NOTCH1 in GS9-6 cells. MA104 is a kidney epithelial cell line and was used as a negative control. (B) For our differentiation assay, GS9-6/NOTCH1+ cells were treated with or without fetal bovine serum (FBS). Stem cell markers NOTCH1 or NES (nestin) and astrocyte marker GFAP were monitored using immunoblotting. ACTB (β actin) is used as the loading control. (C) Sphere formation assays indicate different GS9-6 lines were plated and assessed for their ability to form spheres. (D) Calculated number of wells necessary to achieve 100% of wells with a minimum of 1 sphere. 2330
DOI: 10.1021/nl504481k Nano Lett. 2015, 15, 2329−2335
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Nano Letters
Figure 2. Glioblastoma stem cells can be specifically tethered to tunable SiN microchips. (A) Stepwise protocol to tether GS9-6/NOTCH1+ glioblastoma stem cells onto SiN microchips. (B) Confocal microscopy images indicate GS9-6/NOTCH1+ glioblastoma stem cells do not readily adhere to microchips decorated with antibodies against the intracellular domain of the NOTCH1 receptor (Control). Glioblastoma stem cells readily adhere to microchips decorated with antibodies against the extracellular domain of the NOTCH1 receptor (Capture). (C) Microchips with integrated microwells (10 μm in diameter) can also be used to specifically capture GS9-6/NOTCH1+ glioblastoma stem cells (D) in comparison to the same negative control experiment. (E) Schematic to indicate a cross-sectional view of the microfluidic system containing GS9-6/NOTCH1+ glioma cells positioned in the TEM column.
within the same 2 min incubation period. These results suggested that the GS9-6/NOTCH1+ cells were specifically captured upon the microchips based on the presence of the external domains of the NOTCH1 receptor (Figure 2B). To ensure that the GSCs can be reproducibly tethered to different types of SiN microchips, we repeated the 2 min immunocapture experiments using SiN microchips containing integrated microwells (Figure 2C). These microchips have been previously used to image transcriptionally active viral complexes at the nanoscale using in situ TEM.20 Immunocapture experiments were performed using the microwell-integrated microchips coated with antibodies against the extracellular NOTCH1 receptor. We found that the microchips retained high specificity for the GS9-6/NOTCH1+ cells as a large number of bound cells were easily visualized using confocal microscopy (Figure 2D). A key advantage to using the microwell-integrated microchips is that the individual microwells (∼10 μm in diameter) are large enough to accommodate individual stem cells. Also, each well was sealed by the top microchip in the assembly, thus ensuring the NPs subsequently
added to the assembly were interacting with the enclosed cells, and not independently interacting above or below the imaging plane of the cell (Figure 2E). After successfully tethering the GSCs to the base microchip surface, we carefully added to the assembly polyvinylpyridine (PVP)-encapsulated gold nanorods (Nanopartz Inc.) diluted to 0.17 or 0.017 mg/mL in Milli-Q water. The chamber was then sealed with a metal face-plate that was held in place by 3 brass screws. Control cells lacking gold nanorods were also prepared for imaging purposes. After sealing the device, the tip of the specimen holder was pumped to 10−6 Torr using a Gatan 655 dry pumping station (Gatan Inc.) as previously described23 and the inlet and outlet lines were closed. The specimen holder was then inserted into a FEI Spirit Bio-Twin TEM equipped with a LaB6 filament and operating at 120 kV. We recorded serial images of regions of interest for up to 30 s under low-dose conditions (
