Identification of senescent cell subpopulations by CITE-seq analysis

Abstract

Cellular senescence, a state of persistent growth arrest, is closely associated with aging and age-related diseases. Deciphering the heterogeneity within senescent cell populations and identifying therapeutic targets are paramount for mitigating senescence-associated pathologies. In this study, proteins on the surface of cells rendered senescent by replicative exhaustion and by exposure to ionizing radiation (IR) were identified using mass spectrometry analysis, and a subset of them was further studied using single-cell CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) analysis. Based on the presence of proteins on the cell surface, we identified two distinct IR-induced senescent cell populations: one characterized by high levels of CD109 and CD112 (cluster 3), the other characterized by high levels of CD112, CD26, CD73, HLA-ABC, CD54, CD49A, and CD44 (cluster 0). We further found that cluster 0 represented proliferating and senescent cells in the G1 phase of the division cycle, and CITE-seq detection of cell surface proteins selectively discerned those in the senescence group. Our study highlights the heterogeneity of senescent cells and underscores the value of cell surface proteins as tools for distinguishing senescent cell programs and subclasses, paving the way for targeted therapeutic strategies in disorders exacerbated by senescence.

Keywords: CITE‐seq; cell cycle; proteome; senescence; single‐cell transcriptome; surface proteins; surfaceome.

FIGURE 1

Uncovering the senescence‐associated surface proteome. (a) SA‐β‐Gal activity was analyzed in WI‐38 fibroblasts at ~PDL24 (proliferating, P), after reaching replicative exhaustion at ~PDL54 (RS), or after exposure to 10 Gy of ionizing radiation and culture for an additional 10 days (IRIS). (b) Workflow for cell‐surface protein biotinylation and isolation for proteomic analysis; prepared using BioRender. (c) Overlap of the cell surface proteins elevated in RS vs P (green) and IRIS vs P (pink) after processing as in panels (a, b). (d) Top significantly (FDR <0.05) enriched GO Molecular Function terms after ShinyGO analysis performed on the shared cell surface proteins upregulated in senescent cells. (e) Heatmap illustrating the magnitude of induction in a subset of surface proteins, comparing RS to P cells and IRIS to P cells that were prepared as explained in (a). In the heatmap, 5NTD, ICAM1, DPP4, NECT2, and ITA1 are CD73, CD54, CD26, CD112, and CD49A, respectively. (f) WI‐38 cells were processed as in (a), and the levels of CD49A, CD73, CD109, CD54, CD112, CD44, HLA‐ABC, CD26, and loading control β‐Actin (ACTB) were assessed in Input (whole‐cell proteins) and Surface (cell‐surface proteins) by western blot analysis. “Input”, 10 μg of the protein lysate before pulldown; “Surface”, 5% of the eluted protein volume after pulldown. As the lysis buffer did not fully solubilize membrane proteins, only “Surface” proteins are quantified (ImageJ). Data are representative of three independent replicates.

FIGURE 2

UMAP representation and clustering analysis of CITE‐seq. (a) Workflow illustrating the use of a cocktail of CITE‐seq antibodies to incubate with cells, followed by single‐cell sequencing analysis on the 10× Genomics platform. Illustration was created using BioRender. (b) ADT levels of cell‐surface proteins in cell populations that were either proliferating (P) or rendered senescent by exposure to IR and additional culture for 10 days (IRIS). Color indicates scaled average expression and dot size represents the percentage of cells expressing ADT. (c) UMAP of integrated samples, color‐labeled for P and IRIS cell populations. (d) UMAP illustrating the cell clusters (top), and percent composition of each cluster (bottom) in P and IRIS populations. (e) Genes (x‐axis) corresponding to the ten most highly expressed marker RNAs of each cluster. Color indicates average expression scaled across all clusters and dot size represents the percentage of cells expressing specific RNAs.

FIGURE 3

Surface proteins in cell clusters. (a) Levels of the studied cell‐surface proteins in each cluster of P and IRIS populations. Color indicates average expression scaled across all clusters and dot size represents the percentage of cells expressing specific ADTs. (b) Expression of surface proteins from (a) distributed in UMAP space for proliferating (P) and senescent (IRIS) cells.

FIGURE 4

Functional assessment of cell clusters. (a) Cell cycle states (top) and percentage of cells in each phase (bottom) of P and IRIS cells assigned by the Seurat CellCycleScoring analysis. (b) Expression of mRNAs encoding specific cell cycle regulatory proteins in each cluster. (c) Cell‐surface protein levels in cell populations expressing transcriptomes consistent with G1, S, and G2/M phases. Color indicates scaled average expression and dot size represents the percentage of cells expressing specific ADTs. (d) Average expression of selected mRNAs encoding proteins representing the indicated hallmarks of senescence in each cluster of P and IRIS populations.

FIGURE 5

Subclustering of cluster 0 cells. (a) UMAP representation of the integrated cells of cluster 0 from Figure 2d, color‐labeled for P and IRIS cell populations. (b) UMAP illustrating the cell subclusters (top) and percent composition of each subcluster (bottom) in P and IRIS populations. (c) Expression levels of the cell‐surface proteins in each subcluster of P and IRIS populations. (d) Top ten highly expressed RNA markers of each subcluster. Color indicates average expression scaled across all subclusters, and dot size represents the percentage of cells expressing specific RNAs. (e) Cell cycle phase of P and IRIS cells assigned by the Seurat CellCycleScoring analysis. (f) Average expression of mRNAs encoding SASP factors in each subcluster of P and IRIS populations, using the SenMayo dataset as reference.