Investigation of Therapy-Induced Senescent Cancer Cell Phenotypes with Mass Spectrometry and Non-Linear Optical Microscopy

Ghislanzoni, Silvia (2024). Investigation of Therapy-Induced Senescent Cancer Cell Phenotypes with Mass Spectrometry and Non-Linear Optical Microscopy. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00101782

Abstract

In response to chemotherapy and radiotherapy, a subset of resistant cancer cells avoids cell death by entering a stable state known as senescence, characterized by prolonged cell cycle arrest, several macromolecular changes, and a hypersecretory phenotype. Senescent cells play a dual role in cancer development: on the one hand, the cytostatic state of senescent cells acts as a natural barrier against cancer growth; on the other hand, aggressive senescent cells can evade the senescent state and, along with the inflammatory factors secreted by persisting senescent cells, promote survival, proliferation, and stemness. To either exploit the anti-tumour potential of cellular senescence or target senescent cells with specific drugs to eliminate them, it is crucial to be able to accurately and reliably identify these cells. However, senescence is highly complex and dynamic, and it is characterized by heterogeneous phenotypes that vary greatly depending on the cell type and on the stressor that triggers senescence itself. This heterogeneity translates into a substantial lack of universal markers, and it is still challenging to visualize, select, and target senescent cells. An additional layer of complexity arises from the dyes employed to visualize these cells. Currently, the description, identification, and/or isolation of a biological specimen is commonly obtained by staining the molecular species that compose it or binding them to an easy-to-visualize label, generally a fluorescent dye. However, staining and labelling come with their own limitations: it is necessary to know all the potential, labellable targets within one’s sample and it is imperative for the target to adequately absorb and retain the group of selected markers. Furthermore, using dyes causes the perturbation of the model, often leading to artefacts and signals that are not proportionally and/or specifically related to the target. The aim of this PhD was the visualization and investigation of senescent cancer cells in a completely label-free manner; to this goal, we applied mass spectrometry, non-linear optical microscopy, and cellular tomography to different in vitro senescent models. To average the cell type-derived and stressor derived-variability, four different cancer cell lines (TPC-1, HeLa, MCF7, and HepG2) and three senescence-inducing stimuli (doxorubicin, deferoxamine, and γ-radiation) were employed. We described the remodelling of the lipid profile associated with the onset of senescence in each model using mass spectrometry. In parallel, we developed a method to describe and monitor the senescence-associated lipid accumulation and mitochondrial dysfunction at the cellular level through Raman and two-photon excited fluorescence microscopy. Finally, we investigated the senescence-associated engulfing phenotype and demonstrated the concrete feasibility of imaging and detailing complex cell-in-cell and vacuolar structures associated with senescence in live cells through confocal Raman microscopy and optical diffraction tomography