Discovery of surface biomarkers for cell mechanophenotype via an intracellular protein-based enrichment strategy

Abstract

Cellular mechanophenotype is often a defining characteristic of conditions like cancer malignancy/metastasis, cardiovascular disease, lung and liver fibrosis, and stem cell differentiation. However, acquiring living cells based on mechanophenotype is challenging for conventional cell sorters due to a lack of biomarkers. In this study, we demonstrate a workflow for surface protein discovery associated with cellular mechanophenotype. We sorted heterogeneous adipose-derived stem/stromal cells (ASCs) into groups with low vs. high lamin A/C, an intracellular protein linked to whole-cell mechanophenotype. Proteomic data of enriched groups identified surface protein candidates as potential biochemical proxies for ASC mechanophenotype. Select surface biomarkers were used for live-cell enrichment, with subsequent single-cell mechanical testing and lineage-specific differentiation. Ultimately, we identified CD44 to have a strong inverse correlation with whole-cell elastic modulus, with CD44^lo cells exhibiting moduli three times greater than that of CD44^hi cells. Functionally, these stiff and soft ASCs showed enhanced osteogenic and adipogenic differentiation potential, respectively. The described workflow can be replicated for any phenotype with a known correlated intracellular protein, allowing for the acquisition of live cells for further characterization, diagnostics, or therapeutics.

Keywords: Atomic force microscopy; CD44; Elastic modulus; Lamin; Mechanobiology; Proteomics.

Fig. 1

Enrichment of fixed ASCs by whole-cell mechanophenotype using intracellular, lamin A/C protein levels. a ASCs labeled with AlexaFluor 488-conjugated anti-lamin A/C antibody were gated to collect the top and bottom 20% of cells based on fluorescence. b Relative lamin A/C levels were confirmed by western blot with bands at 74 and 67 kDa, with β-tubulin as a loading control at 55 kDa. c The lamin A and lamin C bands were quantified via densitometry, as well as combined for a total lamin A/C amount, and normalized to the loading control, which confirmed expected expression levels in the two sorted groups

Fig. 2

Proteomic heat map of identified surface proteins for low and high lamin A/C ASC samples. The top half of listed proteins showed a positive correlation with lamin A/C levels while the bottom half were negatively correlated. Surface proteins were pulled from the whole proteome dataset (1300 proteins) based on UniProt subcellular location classification. Results show n = 4 technical replicates from singular low and high lamin A/C samples. Figure produced with ClustVis online tool

Fig. 3

Single-cell mechanical testing was performed by AFM on two different human donor ASC samples sorted by CD90 or CD44 surface protein levels. a Expected trends and statistically significant differences were observed between “stiff” CD90^hi and CD44^lo cells and “soft” CD90^lo and CD44^hi cells (n = 20–30). Statistical significance between low and high protein levels denoted by asterisks above bars (mean ± standard error). b, c The amount of lamin A/C in the CD44-sorted samples was quantified via western blot to confirm an inverse correlation existed between the two proteins

Fig. 4

Lineage-specific differentiation potential differed for ASCs based on CD44 levels. a CD44^lo/hi ASCs were chemically differentiated down the osteogenic lineage and compared to cells kept in control medium. b, c Osteogenesis samples were assessed for ALP activity after 7 days and stained with alizarin red S after 21 days to visualize calcium deposits. Induced CD44^lo cells, the stiffer phenotype, showed more robust osteogenic differentiation based on these two assays (*p < 0.05). d CD44^lo/hi were also chemically differentiated down the adipogenic lineage and lipids stained with oil red O. Insets are 2.5 × the original magnification. Scale bars are 100 µm in all images (a, d). e, f Adipogenesis samples were stained with oil red O to visualize intracellular lipids, and the number of large lipids/image (diameter > 2 µm) was quantified along with the mean large-lipid diameter (*p < 0.05). Error bars signify standard error