Single Cell MALDI-MSI Analysis of Lipids and Proteins within a Replicative Senescence Fibroblast Model

Abstract

In this study, we evaluate lipids and select proteins in human lung fibroblasts (hLFs) to interrogate changes occurring due to aging and senescence. To study single cell populations, a comparison of cells adhered onto slides using poly-d-lysine versus centrifugal force deposition was first analyzed to determine whether specific alterations were observed between preparations. The poly-d-lysine approach was then utilized to interrogate the lipidome of the cell populations and further evaluate potential applications of the MALDI-immunohistochemistry (IHC) platform for single-cell-level analyses. Two protein markers of senescence, vimentin and p21, were both observed within the fibroblast populations and quantified. Lipidomic analysis of the fibroblasts found 12 lipids significantly altered because of replicative senescence, including fatty acids, such as stearic acid, and ceramide phosphoethanolamine species (CerPE). Similar to previous reports, alterations were detected in putative fatty acid building blocks, ceramides, among other lipid species. Altogether, our results reveal the ability to detect lipids implicated in senescence and show alterations to protein expression between normal and senescent fibroblast populations, including differences between young and aged cells. This report is the first time that the MALDI-IHC system has been utilized at a single-cell level to analyze both protein expression and lipid profiles in cultured cells, with a particular focus on changes associated with aging and senescence.

Figure 1.

Donor demographics and experimental design. A) Description of condition, age, sex, and passage number for the hLFs utilized within the studies. B) Experimental overview for preparation of hLFs for lipidomic and proteomic analyses. Workflow created using BioRender.com.

Figure 2.

Comparison of hLF cell architecture when prepared using a cytospin (A/C) or when grown onto slides coated with poly-d-lysine (B/D). Images A/B were taken at 10X objective on a brightfield microscope. MALDI-MSI images taken on timsTOF flex with MALDI-2 using DAN matrix at 5 μm resolution of each preparation method and normalized by root-mean-square. Displayed here are putative lipid species at 272.821 Da ± 10 ppm (orange) localizing primarily to the nucleus and 436.669 Da ± 10 ppm (purple) localizing to cytoplasm.

Figure 3.

Analyses of vimentin in hLF. A) Expression of vimentin as observed through the utilization of MALDI-IHC photocleavable mass tag at 1230 Da. B) Statistical analysis of spectral intensity from fibroblasts showed vimentin expression was nonsignificant between groups but a slight increase in average intensity was observed. C) Immunofluorescence staining of vimentin in hLF from young and old donors, as well as cells in replicative senescence (x10 magnification, 100 μm). Vimentin is shown in red, and nuclei are stained with DAPI in blue. D) Western blot analysis of vimentin protein levels in hLF homogenates from all groups. E) Quantitative densitometry of vimentin expression, with β-actin used as a loading control. F) Correlation between vimentin and p21 expression, with a Pearson correlation coefficient of r = 0.85. Data represent mean value ± SEM. Statistical significance was determined by one-way ANOVA followed by posthoc Tukey, p < 0.05.

Figure 4.

Analysis of p21 expression as a senescence marker in hLF by MALDI-IHC. A) MALDI MSI of AmberGen photocleavable mass tags to evaluate the expression of p21, indicated in red at m/z 1258.620 Da ± 0.5 ppm, and vimentin, indicated in green at m/z 1230.839 Da ± 0.5 ppm. MALDI MSI analyses were completed on a timsTOF fleX at 5 μm resolution with CHCA matrix, TIC normalized. B) Statistical analysis of the observed vimentin mass tag signal intensity for fibroblasts in early and late passages. C) Statistical analysis of the observed p21 mass tag signal intensity for fibroblasts in early and late passages.

Figure 5.

Analysis of p21 expression as a senescence marker in hLF by immunofluorescence and Western blot. A) Immunofluorescence analysis of p21 in hLF from young and old donors, and cells in replicative senescence. Images were captured at 20× magnification with a scale bar of 50 μm. p21 is indicated in red, and nuclei are labeled with DAPI in blue. B) Percentage of p21-positive cells in hLF from young and old donors, as well as in cells undergoing replicative senescence. C) Western blot analysis of p21 protein levels in hLF homogenates from all groups. D) Quantitative densitometry of p21 expression, with β-actin as the loading control. Data represent mean value ± SEM. Statistical significance was determined by one-way ANOVA followed by posthoc Tukey, p < 0.05.

Figure 6.

Observation of lipids previously reported within senescence. A) Comparison of average mass spectra from the segmented cells for each subtype. Spectra Colors: Orange -healthy young donor, purple - healthy old donor, and green -replicative senescence. B) Example of observed signal for putative stearic acid (283.265 Da ± 10 ppm) found to be statistically significant between healthy young donor and replicative senescence fibroblasts (C).