On-Chip Clonal Analysis of Glioma-Stem-Cell Motility and Therapy Resistance

Abstract

Enhanced glioma-stem-cell (GSC) motility and therapy resistance are considered to play key roles in tumor cell dissemination and recurrence. As such, a better understanding of the mechanisms by which these cells disseminate and withstand therapy could lead to more efficacious treatments. Here, we introduce a novel micro-/nanotechnology-enabled chip platform for performing live-cell interrogation of patient-derived GSCs with single-clone resolution. On-chip analysis revealed marked intertumoral differences (>10-fold) in single-clone motility profiles between two populations of GSCs, which correlated well with results from tumor-xenograft experiments and gene-expression analyses. Further chip-based examination of the more-aggressive GSC population revealed pronounced interclonal variations in motility capabilities (up to ∼4-fold) as well as gene-expression profiles at the single-cell level. Chip-supported therapy resistance studies with a chemotherapeutic agent (i.e., temozolomide) and an oligo RNA (anti-miR363) revealed a subpopulation of CD44-high GSCs with strong antiapoptotic behavior as well as enhanced motility capabilities. The living-cell-interrogation chip platform described herein enables thorough and large-scale live monitoring of heterogeneous cancer-cell populations with single-cell resolution, which is not achievable by any other existing technology and thus has the potential to provide new insights into the cellular and molecular mechanisms modulating glioma-stem-cell dissemination and therapy resistance.

Keywords: Living single-cell analysis; anti-microRNA; cancer stem cell; cell motility; glioblastoma; nanochannel electroporation.

Figure 1

Patient-derived GSC exhibition of significant inter- and intrapopulation differences in disseminative capabilities. (a) Schematic diagram illustrating the concept of single-cell dissemination in invasive gliomas. GSCs migrate along highly oriented fiberlike structures (e.g., white-matter tracts) and invade the surrounding brain parenchyma. (b) Micro-/nanofabricated chip for GSC motility biointerrogation at the single-cell level. Top images show an atomic-force-microscopy micrograph (left) of the topography mimicking the fiberlike structures within the tissue. GSCs exhibit guided alignment and motility in response to the topography (right). (c) Motility patterns followed by single GSCs. Patient-derived GSC populations exhibit different motility capabilities. GBM528 remain immobile on the biomimetic surface, while GBM157 show enhanced and guided single-cell motility along the textured surface. (d) Single-cell quantification of motility for GBM528 and GBM157. Although GBM157 show overall improved motility capabilities compared to GBM528, there are significant interclonal variations in motility within the GBM157 population. (e,f) On-chip nanochannel-based electroporation (NEP) was then used to identify interclonal differences in the expression patterns of migration-related genes by in situ hybridization. (g) NEP-delivered molecular beacons for vimentin mRNA showed significant interclonal differences in the expression pattern of its gene, which correlated with the motility capabilities of the cells. Asterisks indicate p < 0.05 (Dunn’s method).

Figure 2

Large-scale screening of GSCs revealing of significant variations across and within (single-cell resolution) populations. (a) Microarray data showing that GBM157 tend to overexpress migration-related genes compared to GBM528. (b–d) Large-scale single-cell screening based on 3D NEP. (b) GSCs are first loaded on the chip surface. (c) Molecular beacons are then delivered into each cell by applying a focused, pulsed electric field through arrayed nanochannels. (d) Following in situ hybridization, the cells show varying levels of fluorescence depending on the expression level of the target gene. (e) Single-cell fluorescence is then quantified via fluorescence microscopy. The results show that both the GBM157 and GBM528 populations reveal similar levels of expression for CD133, which is a proneural GSC marker. CD44 expression was more pronounced for the GBM157 population, with a number of clones showing markedly higher expression (inset) compared to the rest of the population. Such clones are presumed to exhibit more aggressive behavior.

Figure 3

Therapy efficacy studies on a 3D NEP platform revealing the existence of a subset of clones exhibiting high antiapoptotic capabilities. Schematic diagram of the experimental setup in which GBM157 were exposed to (a) chemotherapy via direct-contact delivery of temozolomide (TMZ) or (b) oligo-RNA therapy via NEP-based delivery of anti-miR 363. (c) NEP-based delivery of anti-miR 363 shows a dose-dependent (two vs five pulses) decrease in cell viability over time, compared to the control. (d) Direct and concomitant exposure to temozolomide increased the proportion of cell death, with approximately 10% of the population still showing a high degree of resistance to drug- and oligo-RNA-induced apoptosis.

Figure 4

Therapy-resistant GSC exhibition of enhanced motility capabilities. (a) Single-cell motility assays showing that although both TMZ and anti-miR363 therapy had a significant negative effect on cell viability, only the anti-miR363 caused a significant and concomitant decrease in cell motility. (b) Continuous monitoring of single-cell motility following anti-miR363 delivery revealed that only the cells that were prone to undergoing apoptosis within the first 48 h showed a significant decrease in single-clone motility. Cells that had not undergone apoptosis past 48 h still showed marked motility, comparable to untreated and control cells. (c) In situ hybridization experiments coupled with drug-resistance studies by 3D NEP allowed us to identify the surviving and highly migratory cell population as being high in CD44 expression. Asterisks indicate p < 0.05 (Dunn’s method).