Algorithmic assessment of cellular senescence in experimental and clinical specimens

Abstract

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies-pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells-that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies.

Figures

Extended Data Fig. 1∣. Sensitive detection of senescence in a variety of cell types using GL13 in cellular systems.

Positive GL13 staining is depicted in normal (a and b) and cancerous (f and l) cells of epithelial origin, normal (c, d, j, o), premalignant (e) and malignant mesenchymal (g, h, m, n) cells as well as in differentiating megakaryocytes (k) and peripheral blood mononuclear cells (PBMCs) from patients suffering from a chronic aged-related disease (p). Chromogenic assays: Diaminobenzidine (DAB)-brown cytoplasmic signal (a, b, ci-ii, a, d-gi, h, l, mi, n-p) and Alkaline phosphatase (BCIP/NBT)-blue purple cytoplasmic signal (white arrowheads in ciib, gii, k); Fluorescent assay: granular red cytoplasmic signal (j, mii -white arrowheads). Double staining experiments showing nuclear p16INK4A or p21WAF1/Cip1 expression (yellow arrowheads) in senescent cells that are concurrently positive with GL13 (white arrowheads) (ciib, gii and miii). Black arrowheads depict double negative cells. giii: Mutually exclusive staining pattern between nuclear Ki67 positivity (yellow arrowhead) and GL13 staining (white arrowhead). Images adopted from: a, c, g, h, m and n; b; j. Counterstain: Hematoxylin (chromogenic assay) and DAPI (fluorescent assay). Scale bars: 5 μm (l), 10μm (a-c, f-k-m-p,), 15μm (e) and 20 μm (d).

Extended Data Fig. 2 ∣. Sensitive detection of senescence using GL13 in a variety of tissues obtained from different animal models.

Identification of senescent cells in the following animal models with established senescence: a. K-rasV12 induced lung adenoma mouse model (images ai and aii adopted from; for material depicted in panel b see), c. H-Ras G12V knock-in mouse model (,), d. Aged Rat (age: 22 months), e. Bleomycin induced lung fibrosis mouse model (images adopted from), f. Palbociclib induced tumor (melanoma) mouse model (image adopted from), g. Floxed Ercc1 allele in Lys2-Ercc1F/– mouse model (image adopted from) h. Hela tumor xenografts subjected to G-quadruplex ligand 20A treatment (image adopted from). Red arrows denote GL13 positive cells. Double staining assays revealing nuclear p16INK4A expression (yellow arrowheads) in concurrently positive with GL13 senescent cells (white arrowheads) (aii, adopted from), as well as inverse staining between Ki67 (yellow arrowhead) and GL13 (white arrowhead) eii, adopted from). Chromogenic assays: DAB-brown cytoplasmic signal (ai, c, d, ei, f-h) and Alkaline phosphatase (BCIP/NBT)-blue purple cytoplasmic signal (aii, b and eii). Counterstain (when applied): Hematoxylin for DAB and Nuclear Fast Red for BCIP/NBT. Scale bars: 25 μm (g), 50μm (aii, b-d, eii), 100μm (ai, ei, f,) and 200 μm (h).

Extended Data Fig. 3 ∣. Sensitive detection of senescence using GL13 in a variety of tissues from human clinical samples.

Senescent cells of different cell origin (mesenchymal: a-d, p, r, s; megakaryocytes: e; lymphoid/histiocytic: f-h; epithelial: j-n; neuronal:o, nevoid:q) identified via GL13 staining in various clinical settings (red arrows and double black arrowheads in image h). Chromogenic assays: DAB-brown cytoplasmic signal (a, bi, c-g, j, k, l, n-s) and Alkaline phosphatase (BCIP/NBT)-blue purple cytoplasmic signal (bii, fii-iv, h, m); Double staining experiments showing nuclear p16INK4A or p21WAF1/Cip1 expression (yellow arrowheads) in senescent cells that are simultaneously positive with GL13 (red arrows) (bi, fii-iii, mii). Mutually exclusive staining pattern between nuclear Ki67 positivity (yellow arrowhead) and GL13 reactivity (red arrows) is evident (fiv). Green arrowhead depicts a double negative cell (fiv). Images adopted from: b, q; f, g, j; h; k; m; o; s. Counterstain: Hematoxylin for DAB and Nuclear Fast Red for BCIP/NBT. Scale bars: 25 μm (e, p), 50μm (bii, d, f, h, m, n, q, r), 100μm (a, bi, g, j, k, o) and 200 μm (c, l, s).

Fig. 1 ∣. Schematic representation of the protocol.

Phase 1 aims to validate the presence of senescence, phase 2 to specify the senescence subtype.

Fig. 2 ∣. Step-by-step protocol.

A summary of the procedures described in the two-phase protocol, highlighting the distinction between in vitro and in vivo samples. Phase 1 focuses on analyzing the expression of general markers of senescence. Phase 2 measures markers that are specific for senescence subtypes. MS, mass spectrometry. Procedure step numbers are shown in red.

Fig. 3 ∣. Double staining SA-β-Gal or LF and proliferation markers.

a, Human BJ fibroblasts were treated with 4 μM vorinostat (SAHA) for 8 d. Drug was refreshed daily. Eight days post-treatment, mutually exclusive staining between SA-β-Gal and EdU was observed. The purple arrow denotes an EdU+/SA-β-Gal− cell and the white arrow depicts an EdU−/SA-β-Gal+ (senescent) cell. Scale bar, 150 μm. b, Double immunohistochemical/hybrid histo-immunohistochemical staining in human primary classical Hodgkin lymphomas (cHLS). A mutually exclusive staining pattern between GL13 and Ki-67 in neoplastic Hodgkin and Reed-Sternberg cells was observed. The purple arrow denotes a Ki-67+/GL13− cell and the white arrow depicts a Ki-67−/GL13+ (senescent) cell. Alkaline phosphatase (AP) chromogenic reaction: dark blue cytoplasmic product; diaminobenzidine (DAB) reaction: nuclear brown signal; counterstain: nuclear fast red. Scale bar, 50 μm.