We followed two conceptionally different approaches in parallel. In these, we aim to improve optical properties of cells (published), and tissue spheroids (ongoing work).
By directed evolution (published in Subramanian et al, J BioPhot 2021), we managed to make living cells considerably more transparent while preserving their physiological state. Specifically, we generated numerous cell lines with improved optical properties (i.e. with reduced side scattering, SSC-A = Integrated side scattering signal per cell) using FACS technology (compare figure 1). Sequencing and computational transcriptome enabled us to find multiple pathways frequently associated with changes in optical properties.
After successful selection of cells with improved optical properties, we performed competitive growth assays to select by fitness such that an heterogenous population of evolved cells was exposed to repeated cell stress, i.e. to freeze–thaw and expansion cycles. This process counter-selected many, but not all, low-scattering cells. A plurality of single-cell origin cell lines from these experiments were sequenced individually. This data revealed that evolved cells fall into transcriptomically distinct groups, that are significantly different not only from the WT cells but also form the mutated, but not optically selected cells. Further, our selection by fitness was indeed successful as also transcriptome reflected a physiological state. That is the mRNA content did not show elevated levels of increased cell stress or apoptosis. Specifically, the GO term ‘repones to stress’ was not found significantly upregulated and there was no strong difference in the expression of important housekeeping genes.
We hypothesized, that improvements in the optical properties of a cell were associated with nuclear architecture or altered chromatin compaction states. Using high resolution microscopy, we indeed observed that nuclear granularity showed a distinct correlation with side scattering. Cell lines showing least light scattering also possessed the lowest granularity.
These results established that a modulation of nuclear architecture of cells in culture, and potentially also in cell spheroids, as a highly promising way to improve their optical properties. Specifically, chromatin compaction states seem a relevant parameter in this regard.
The above results successfully demonstrated a significant optical plasticity of mammalian cells.
This further motivated us to i) get better understanding of the pathways involved, ii) find ways to leverage modern genetics to improve optical properties iii) transition from random mutation to gain of function phenotypes. The finding motivates more systematic screens for genes and pathways affecting these (currently ongoing work).
