A New Label-Free Approach to Glioblastoma Cancer Stem Cell Sorting and Detection

Abstract

Cancer stem cells (CSCs) play critical roles in cancer, making them important targets for new diagnostic and therapeutic approaches. Since CSCs are heterogeneous and not abundant in tumors, and few specific markers for these cells currently exist, new methods to isolate and characterize them are required. To address this issue, we developed a new label-free methodology to isolate, enrich, and identify CSCs from an heterogeneous tumor cell subpopulation using a cell sorting method (sedimentation field flow fractionation, SdFFF) and a biosensor as a detector. Enrichment was optimized using an original protocol and U87-MG glioblastoma cells cultured in a normal (N) or defined (D) medium (± fetal bovine serum, FBS) under normoxic (N, p O 2 = 20%) or hypoxic (H, p O 2 < 2%) conditions to obtain four cell populations: NN, NH, DN, and DH. After elution of CSCs via SdFFF using the hyperlayer mode (inertial elution mode for micrometer-sized species), we isolated eight subpopulations with distinct CSC contents based on phenotypical and functional properties, ranging from NN F1 with a lower CSC content to DH F3 with a higher CSC content. Reflecting biological differences, the intrinsic intracellular dielectric permittivity increased from NN to DH conditions. The largest difference in electromagnetic signature was observed between NN F1 and DH F3, in which the CSC content was lowest and highest, respectively. The results demonstrate that microwave dielectric spectroscopy can be used to reliably and efficiently distinguish stem cell characteristics. This new instrumental and methodological approach is an important innovation that allows both enrichment and detection of CSCs, opening the door to novel diagnostic and therapeutic approaches.

Autophagy inhibition reinforces stemness together with exit from dormancy of polydisperse glioblastoma stem cells

“…Thus, larger and less dense cells generating more hydrodynamic lift forces within the mobile phase and not very sensitive to centrifugal force (blue cells on Figure 5A ), will be eluted before smaller and denser cells (red cells, Figure 5A ) generating less hydrodynamic forces and more sensitive to the multigravitational field. As for a number of other cell models [ 27 , 29 , 31 ], we found that under the elution conditions, U87 cells were eluted under the hyperlayer elution mode [ 28 , 30 ] schematized in Figure 5A .…”

“…In each case, we observed the classical two-peak profile ( Figure 5B ); the first peak corresponds to the void peak (VP, t 0 /t 0 = R obs ≈ 1) containing unretained species (cell debris, proteins, cell aggregates…), and the second corresponds to the cell peak (CP, t 0 /t r = R obs < 1). These broad cell peaks ( Figure 5B ) correspond to very polydisperse populations, reflecting the heterogeneity of these GB cell lines containing a cell continuum, from fully differentiated cells to cancer stem cells [ 28 , 30 , 35 ].…”

“…Over the past decade, applications of SdFFF cell sorting have been developed in many fields, including oncology. A pioneering study of CSCs sorting from within a GB cell line [ 28 ] made it possible to sort other cancer cells [ 29 ] and to improve cell culture conditions ahead of cell sorting and CSCs detection [ 30 ]. Moreover, these studies allowed an opening towards the field of stem cell biology [ 31 ].…”

Benefits of functional assays in personalized cancer medicine: more than just a proof-of-concept

“…However, rarer, more aggressive cells still end up lost within the dynamic range. Either label-free cell sorting technologies 120 or co-staining protocols coupled to image analysis and detection algorithms may improve the specific detection of rare cells at any relevant step of the CSRA workflow. Circulating tumour cells remain easily accessible and represent a target of choice in that regard.…”

Characterization of Glioblastoma Cancer Stem Cells Sorted by Sedimentation Field-Flow Fractionation Using an Ultrahigh-Frequency Range Dielectrophoresis Biosensor

“…One of the most interesting label-free cell sorting methods is the now well-known sedimentation field-flow fractionation (SdFFF) technique. [22][23][24] . The SdFFF is a gentle and noninvasive technique.…”