Identification Of The Neo-Genesis Of Glioblastoma Stem Cells Using Label-Free Quantitative Phase Imaging

Introduction: Glioblastoma (GBM) is the most aggressive neurological cancer with a very poor prognosis due to its propensity to regenerate after medical interventions. Our experimental model is U87-MG cells, which contain subsets of cancer stem cells (CSC) and have the unusual property of spontaneously forming neurospheres in culture. We are investigating the origins of these stem cells. Materials and Methods: We employ recently developed time label-free, time-lapse quantitative phase imaging (QPI) technology that allows precise quantification of the refractive index (RI) distributions on a sub-cellular level. These measurements are stored in 2D or 3D arrays which can be presented as images or videos at various resolutions. Cells were seeded into sealable culture Petrie dishes and imaged while maintaining appropriate environmental controls. Results: We first noted CSCs in late stage cultures in exploratory experiments, where they presented as small cells (10-15 um) compared to the 50-100 um range of mature U8-MR cells. Their refractive index (RI) is 1.42, compared to about 1.40 for the cytoplasm of mature cells. QPI technology sensitivity enables almost complete separation between the two cell types in RI histograms. We exemplify a grouping of cells in an 80 um2 field of view, tracked in multiple time segments ranging from 20 minutes to 12 hours over a period of about 18 hours, containing a mature U87 which serves as a reference marker. Around this cell, there are 3 distinct pro-stem (PS) cells 12 to 16 um in size: PS-1 with numerous 0.5 um particles in the size range of mitochondria, PS-2 with a nucleus with nucleoli but no nuclear membrane, and PS-3 with a distinct nucleus, nucleoli, and cytosol. The cells are all connected in a network of nanotubes and tunnels. PS-1 and PS-2 are not very active in the early analysis, but they eventually move together and appear to fuse while still maintaining their individual shapes. PS-3 is very actively interacting with another cell on the border of the analysis window. It is extending and retracting a tube and is apparently moving materials both to and away from the other cell in a very regular and linear fashion, suggesting a primitive form of transcription. Discussion: The processes we observed are consistent with tumor stem cell generation by autophagy, mitophagy, and catabolization followed by reprograming of the remnants and formation of neoplastic stem cells. The novelty of our approach is determined by technical capabilities of our QPI platforms, which is non-invasive label-free approach coupled with ultra-high-resolution analysis, previously unobtainable in traditional time-lapse imaging systems. Citation Format: Ed Luther, Livia P. Mendes, Vladimir P. Torchilin. Identification of the neo-genesis of glioblastoma stem cells using label-free quantitative phase imaging [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6019.