AFM Indentation Measurements and Viability Tests on Drug Treated Leukemia Cells
Creators: name: Fortier, Hélène
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2016
Subjects: atomic force microscopy
cancer cell mechanics
leukemia
automated data processing

description: A significant body of literature has reported strategies and techniques to assess the mechanical properties of biological samples such as proteins, cellular and tissue systems. Atomic force microscopy has been used to detect elasticity changes of cancer cells. However, only a few studies have provided a detailed and complete protocol of the experimental procedures and data analysis methods for non-adherent blood cancer cells. In this work, the elasticity of NB4 cells derived from acute promyelocytic leukemia (APL) was probed by AFM indentation measurements to investigate the effects of the disease on cellular biomechanics. Understanding how leukemia influences the nanomechanical properties of cells is expected to provide a better understanding of the cellular mechanisms associated to cancer, and promises to become a valuable new tool for cancer detection and staging. In this context, the quantification of the mechanical properties of APL cells requires a systematic and optimized approach for data collection and analysis, in order to generate reproducible and comparative data. This Thesis elucidates the automated data analysis process that integrates programming, force curve collection and analysis optimization to assess variations of cell elasticity in response to processing criteria. A processing algorithm was developed by using the IGOR Pro software to automatically analyze large numbers of AFM data sets in an efficient and accurate manner. In fact, since the analysis involves multiple steps that must be repeated for many individual cells, an automated and un-biased processing approach is essential to precisely determine cell elasticity. Different fitting models for extracting the Young’s modulus have been systematically applied to validate the process, and the best fitting criteria, such as the contact point location and indentation length, have been determined in order to obtain consistent results. The designed automated processing code described in this Thesis was used to correlate alterations in cellular biomechanics of cancer cells as they undergo drug treatments. In order to fully assess drug effects on NB4 cells, viability assays were first performed using Trypan Blue staining for primary insights before initiating thorough microplate fluorescence intensity readings using a LIVE/DEAD viability kit involving ethidium and calcein AM labelling components. From 0 to 24 h after treatment using 30 µM arsenic trioxide, relative live cell populations increased until 36 h. From 0 to 12 h post-treatment, relative populations of dead cells increased until 24 h post-treatment. Furthermore, a drastic drop in dead cell count has been observed between 12 and 24 h. Additionally, arsenic trioxide drug induced alterations in elasticity of NB4 cells can be correlated to the cell viability tests. With respect to cell mechanics, trapping of the non-adherent NB4 cells within fabricated SU8-10 microwell arrays, allowed consistent AFM indentation measurements up to 48 h after treatment. Results revealed an increase in cell elasticity up to 12 h post-treatment and a drastic decrease between 12 and 24 h. Furthermore, arsenic trioxide drug induced alterations in elasticity of NB4 cells can be correlated to the cell viability tests. In addition to these indentation and viability testing approaches, morphological appearances were monitored, in order to track the apoptosis process of the affected cells. Relationships found between viability and elasticity assays in conjunction with morphology alterations revealed distinguish stages of apoptosis throughout treatment. 24 h after initial treatment, most cells were observed to have burst or displayed obvious blebbing. These relations between different measurement methods may reveal a potential drug screening approach, for understanding specific physical and biological of drug effects on the cancer cells.