Xu, F. X. | Search bioRxiv

bioRxiv2024

Assessing drug uptake and response differences in 2D and 3D cellular environments using stimulated Raman scattering microscopy

The architecture of cell culture--two-dimensional (2D) versus three-dimensional (3D)--significantly impacts various cellular factors, including cell-cell interactions, nutrient and oxygen gradients, metabolic activity, and gene expression profiles. This can result in different cellular responses during cancer drug treatment, with 3D-cultured cells often exhibiting higher resistance to chemotherapeutic drugs. While various genetic and proteomic analyses have been employed to investigate the underlying mechanisms of this increased resistance, complementary techniques that provide experimental evidence of spatial molecular profiling data are limited. Stimulated Raman scattering (SRS) microscopy has demonstrated its capability to measure both intracellular drug uptake and growth inhibition. In this work, we applied three-band SRS imaging to 2D and 3D cell cultures and provided a comparative analysis of drug uptake and response with the goal of understanding whether the difference in drug uptake explains the drug resistance in 3D culture compared to 2D. Our investigations revealed that despite similar intracellular drug levels in 2D and 3D A549 cells during lapatinib treatment, the growth of 3D spheroids is less impacted, supporting an enhanced drug tolerance in the 3D microenvironment. We further elucidated drug penetration patterns and the resulting heterogeneous cellular responses across different spheroid layers. Additionally, we investigated the role of the extracellular matrix in modulating drug delivery and cell response, and we discovered that limited drug penetration in 3D could also contribute to lower drug response. Our study provides valuable insights into the intricate mechanisms of increased drug resistance in 3D tumor models during cancer drug treatments.

cell biology↗

bioRxiv2021

Quantitative imaging of intracellular density with ratiometric stimulated Raman scattering microscopy

Cell size and density impact a wide range of physiological functions, including tissue homeostasis, growth regulation, and osmoregulation. Both are tightly regulated in mammalian cells. In comparison, density variation of a given cell type is much smaller than cell size, indicating that maintenance of cell type-specific density is important for cell function. Despite this importance, little is known about how cell density affects cell function and how it is controlled. Current tools for intracellular cell density measurements are limited either to suspended cells or cells growing on 2D substrates, neither of which recapitulate the physiology of single cells in intact tissue. While optical measurements have the potential to measure cell density in situ and noninvasively, light scattering in multicellular systems prevents direct quantification. Here, we introduce an intracellular density imaging technique based on ratiometric stimulated Raman scattering microscopy (rSRS). It quantifies intracellular drymass density through vibrational imaging of macromolecules. Moreover, water is used as an internal standard to correct for aberration and light scattering. We demonstrate real-time measurement of intracellular density quantification and show that density is tightly regulated across different cell types and can be used to differentiate cell types as well as cell states. We further demonstrate dynamic imaging of density change in response to osmotic challenge as well as intracellular density imaging of a 3D tumor spheroid. Our technique has the potential for imaging intracellular density in intact tissue and understanding density regulation and its role in tissue homeostasis.

biophysics↗