Optical Diffraction Tomography and Raman Confocal Microscopy for the Investigation of Vacuoles Associated with Cancer Senescent Engulfing Cells

Abstract

Wild-type p53 cancer therapy-induced senescent cells frequently engulf and degrade neighboring ones inside a massive vacuole in their cytoplasm. After clearance of the internalized cell, the vacuole persists, seemingly empty, for several hours. Despite large vacuoles being associated with cell death, this process is known to confer a survival advantage to cancer engulfing cells, leading to therapy resistance and tumor relapse. Previous attempts to resolve the vacuolar structure and visualize their content using dyes were unsatisfying for lack of known targets and ineffective dye penetration and/or retention. Here, we overcame this problem by applying optical diffraction tomography and Raman spectroscopy to MCF7 doxorubicin-induced engulfing cells. We demonstrated a real ability of cell tomography and Raman to phenotype complex microstructures, such as cell-in-cells and vacuoles, and detect chemical species in extremely low concentrations within live cells in a completely label-free fashion. We show that vacuoles had a density indistinguishable to the medium, but were not empty, instead contained diluted cell-derived macromolecules, and we could discern vacuoles from medium and cells using their Raman fingerprint. Our approach is useful for the noninvasive investigation of senescent engulfing (and other peculiar) cells in unperturbed conditions, crucial for a better understanding of complex biological processes.

Bio‐Engineering Yeast Cells Biolenses by Reshaping Intracellular Vacuoles

“…For example, TPI has been recently employed to study the association between vacuoles and cancer senescent engulfing cells. [27] In fact, vacuoles are often correlated to diseases, inflammations, and cell malfunctioning. [28,29] In particular, intracellular vacuolar structures have been described in the peripheral blood cells of patients affected by several genetic disorders belonging to the lysosomal storage diseases family (LSDs), [30] cancer, [31][32][33][34][35] and viral infections, [36][37][38] including the more recent SARS-CoV-2.…”

Label-free morpho-molecular phenotyping of living cancer cells by combined Raman spectroscopy and phase tomography

“…A shared aspect of these label-free multimodal approaches and similar reports 21 , 25 is the use of 2D QPI maps, capable of delivering only an integrated projection of the RI along sample thickness, to extract the morphological counterpart of the cell profile. On the other hand, TPM achieves 3D morphological imaging 20 , 26 , reconstructing a tomogram from multiple 2D phase images acquired at different illumination angles. 3D QPI unlocks additional quantitative data about cell morphology (e.g., cell volume, surface area, cell dry mass and density), and we consider that its combination with biomolecular fingerprints holds the potential of describing cell types in greater detail, boosting the accuracy of phenotype inference, even with limited population sampling.…”