Targeted Reduction of Senescent Cell Burden Alleviates Focal Radiotherapy-Related Bone Loss

Abstract

Clinical radiotherapy treats life-threatening cancers, but the radiation often affects neighboring normal tissues including bone. Acute effects of ionizing radiation include oxidative stress, DNA damage, and cellular apoptosis. We show in this study that a large proportion of bone marrow cells, osteoblasts, and matrix-embedded osteocytes recover from these insults only to attain a senescent profile. Bone analyses of senescence-associated genes, senescence-associated beta-galactosidase (SA-β-gal) activity, and presence of telomere dysfunction-induced foci (TIF) at 1, 7, 14, 21, and 42 days post-focal radiation treatment (FRT) in C57BL/6 male mice confirmed the development of senescent cells and the senescence-associated secretory phenotype (SASP). Accumulation of senescent cells and SASP markers were correlated with a significant reduction in bone architecture at 42 days post-FRT. To test if senolytic drugs, which clear senescent cells, alleviate FRT-related bone damage, we administered the senolytic agents, dasatinib (D), quercetin (Q), fisetin (F), and a cocktail of D and Q (D+Q). We found moderate alleviation of radiation-induced bone damage with D and Q as stand-alone compounds, but no such improvement was seen with F. However, the senolytic cocktail of D+Q reduced senescent cell burden as assessed by TIF⁺ osteoblasts and osteocytes, markers of senescence (p16 ^Ink4a and p21), and key SASP factors, resulting in significant recovery in the bone architecture of radiated femurs. In summary, this study provides proof of concept that senescent cells play a role in radiotherapy-associated bone damage, and that reduction in senescent cell burden by senolytic agents is a potential therapeutic option for alleviating radiotherapy-related bone deterioration. © 2020 American Society for Bone and Mineral Research.

Keywords: OSTEOPOROSIS; RADIOTHERAPY; SENESCENCE; SENOLYTICS; TELOMERE DYSFUNCTION.

Fig. 1.

Identification of senescent osteoblasts and osteocytes in radiated bones. (A,B) The right legs of C57BL/6 mice were radiated (24 Gy) in a 5-mm region near the femoral metaphysis, while the left leg served as control. R and NR femurs at 28 days post-FRT were harvested, μCT scanned (n = 4 animals), fixed, decalcified, and processed for frozen sectioning. Representative 3D-reconstructed images are shown in A; BV/TV is shown in B. (C) SA-β-gal staining shows the presence of senescent osteoblasts (red arrows) in frozen femur sections (n = 3 animals) at 28 days post-FRT. (D) Quantification of SA-β-gal⁺ cells lining the bone surface. (E) R and NR femurs (n = 4 animals) at 42 days post-FRT were harvested and processed for MMA embedding. Five-micrometer (5-μm) deplasticized sections were processed for TIF staining and TIFs were detected. Telomeres (red) and γH2AX (green) are shown in Ot of nondecalcified NR and R femurs, 42 days post-FRT. The co-localization detects yellow-orange TIF foci. Insets “a” and “b” show images representative of TIF⁺ (R) osteocyte nuclei. (F) Quantification of TIF⁺ osteocytes from NR and R femurs. (G) Detection of TIF⁺ bone lining cells including osteoblasts in nondecalcified NR and R femurs, 42 days post-FRT. Inset “a” shows a representative image of a TIF from an osteoblast. (H) Quantification of TIF⁺ bone lining cells on the trabecular bone surface in NR and R femurs. Statistical analysis was done using GraphPad Prism and p value was calculated using a two-tailed paired t test to compare the R and NR bones from the same animals. BV/TV = bone volume fraction of the total volume; Ot = osteocytes.

Fig. 2.

Focal radiation induces markers of senescence in bone. NR and R femurs were collected on days 1, 7, 14, 21, and 42, and qRT-PCR was performed to detect p21 (A) and p16^Ink4a (B). (C,D) Heat maps of CDKi genes were determined by RT-qPCR from NR and R femurs collected 14 days (C, n = 3 mice) and 21 days (D, n = 5mice) post-FRT. Statistical analysis was done using GraphPad Prism and p value was calculated using a two-tailed paired t test to compare the R and NR bones from the same animals.

Fig. 3.

Radiation of bone induces a SASP similar to that of aged bone cells. (A) Heat map of SASP genes from NR and R femurs (n = 3 mice) at 14 days post-FRT. (B) Venn diagram representing SASP genes that were commonly expressed between R femurs from 4-month-old male mice and old bone cells from 24-month old male mice (table). (C) Comparison of expression levels of SASP genes Mmp12, Icam1, and Igfbp4, between R bones and NR bones at 14 days post-FRT, with enriched cell populations from bones of young (Y) versus old (O) animals. y axis: fold change normalized against NR (for comparison between NR versus R), and normalized against Y (for comparison between Y versus O). Expression of SASP genes that are classified as interleukins and chemokines are shown (D and E, respectively). y axis: fold change normalized against NR (for comparison between NR versus R). Statistical analysis was done using GraphPad Prism and p value was calculated using a two-tailed paired t test to compare the R and NR bones from the same animals, and using a two-sampled t test for comparison of young and old bone cells.

Fig. 4.

Targeted clearance of senescent cells alleviates radiation-induced bone damage. Four-month-old C57BL/6 male mice received radiation (24 Gy) in a 5-mm region of the right femoral metaphysis, accompanied with intermittent treatments on days 0 and 14, with either vehicle (n = 5 mice) or senolytic drug treatment (F: fisetin, 50 mg/kg, n = 5 mice; D: dasatinib, 5 mg/kg, n = 4 mice; Q: quercetin, 50 mg/kg, n = 5 mice) or a cocktail of D+Q (5 mg/kg D and 50 mg/kg Q, n = 5 mice); bones were collected on day 42 post-FRT. (A) Images represent 3D representations of the bone architecture generated by ex vivo μCT scans using Viva40. (B) Percentage of bone volume over total volume. (C) Experiment was performed as described in A. 3D representation of μCT data of vehicle (n = 5) and D+Q (n = 5). (D) Bone architecture parameters for vehicle-treated and D+Q-treated NR and R femurs. Statistical analyses were done using GraphPad Prism and p values were calculated using a two-way ANOVA with a Tukey post hoc analysis.

Fig. 5.

D+Q preserves functional osteoblasts and restores bone formation in radiated femurs. Four-month-old C57BL/6 male mice received radiation (24 Gy) in a 5-mm region of the right femoral metaphysis, accompanied with intermittent treatments on days 0 and 14, with either vehicle (n = 4 mice) or D+Q (5 mg/kg D and 50 mg/kg Q, n = 4 mice); bones were collected on day 42 post-FRT and processed for MMA embedding. (A) A representative Goldner’s Trichrome–stained bone section showing the region of interest (1–4 mm below the growth plate) used for static histomorphometry. (B) Representative images from vehicle-treated and D+Q-treated NR and R femurs (arrows indicate osteoblasts). (C) Quantification for N.Ob per mm of bone surface N. Ad per mm² of total area, and total N.Ot per mm² of total area. Results are expressed as medians with interquartile range. Statistical analyses were done using GraphPad Prism and p values were calculated using a two-way ANOVA (α = 0.05) with a Tukey post hoc analysis. N.Ad = number of adipocytes; N.Ob = number of osteoblasts; N.Ot = number of osteocytes.

Fig. 6.

D+Q preserves bone formation in radiated femurs. Four-month-old C57BL/6 male mice received radiation (24 Gy) in a 5-mm region of the right femoral metaphysis, accompanied by intermittent treatments on days 0 and 14, with either vehicle (n = 4 mice) or D+Q (5 mg/kg D and 50 mg/kg Q, n = 4 mice); bones were collected on day 42 post-FRT and processed for MMA embedding. (A) Animals were injected with Alizarin and Calcein on days 9 and 2, respectively, before bones were harvested for MMA embedding. Unstained 8-μm MMA sections were used to visualize Alizarin (red) and Calcein (green) fluorescence. (B) MS/BS and BFR/BS were quantified. (C) Serum protein measurement of P1NP from vehicle-treated and D+Q-treated animals (n = 5/group) by ELISA. Results are expressed as medians with interquartile range. Statistical analyses were done using GraphPad Prism and p values were calculated using a two-way ANOVA (α = 0.05) with a Tukey post hoc analysis. BFR/BS = bone formation rate per bone surface; MS/BS = mineralizing surface per bone surface.

Fig. 7.

The senolytic cocktail, D+Q, reduces the senescent cell burden in radiated bones. Four-month-old C57BL/6 male mice received radiation (24 Gy) in a 5-mm region of the right femoral metaphysis, accompanied with intermittent treatments on days 0 and 14, with either vehicle (n = 8 mice) or D+Q (5 mg/kg D and 50 mg/kg Q, n = 8 mice); bones were collected on day 42 post-FRT and processed for mRNA (n = 4/group) or histology (n = 4/group). Gene expression levels of p21 (A) and p16^Ink4a (B) were detected by qRT-PCR in NR-femurs and R-femurs 42 days post-FRT. (C) Representative images showing the co-localization of telomere foci with γH2AX in osteocytes (inset in Veh-R panel is a TIF+ osteocyte). (D) TIF+ osteocytes (from n = 4 animals/group) were quantified by visualizing the co-localization of telomere foci (red) and γH2AX (green). TIFs were scored by overlapping staining (yellow). (E) TIF⁺ bone lining cells including osteoblasts were scored as cells on the trabecular bone surface with co-localization of telomere foci and γH2AX (inset in Veh-R panel is a TIF⁺ osteoblast). (F) Quantification of TIF⁺ bone lining cells including osteoblasts (from n = 4 animals/group) on the bone surface. (G) Quantification of TIF⁺ bone marrow cells (from n = 4 animals/ group). Results are expressed as medians with interquartile range. Statistical analyses were done using GraphPad Prism and p values were calculated using a two-way ANOVA (α = 0.05) with a Tukey post hoc analysis.

Fig. 8.

Clearance of senescent cells by D+Q reduces the SASP in radiated bones. Four-month old C57BL/6 male mice received radiation (24 Gy) in a 5-mm region of the right femoral metaphysis, accompanied with intermittent treatments on day 0 and 14, with either vehicle (n = 4 mice) or D+Q (5 mg/kg D and 50 mg/kg Q, n = 4 mice). Bones were collected on day 42 post-FRT and processed for mRNA analysis. Gene expression levels for 12 SASP factors at 42 days post-FRT from vehicle-treated and D+Q–treated NR femurs and R femurs are presented. Results are expressed as medians with interquartile range. Statistical analyses were done using GraphPad Prism and p values were calculated using a two-way ANOVA (α = 0.05) with a Tukey post hoc analysis.