Senescence at the Heart of Cancer and Neurodegeneration DOJINDO LABORATORIES

Cellular senescence is recognized as an important contributor to cancer development through its effects on the tumor microenvironment, and also to age-related dysfunction in both the peripheral and central nervous systems. This ScienceNote introduces recent findings that highlight the tumor-promoting role of senescent fibroblasts in breast cancer, the contribution of senescent neurons to age-associated pain, and the accumulation of senescent neurons through abnormal cell cycle activity in neurodegenerative conditions.

Senescent CAFs mediate immunosuppression and drive breast cancer progression (Cancer Discov., 2025)
Summary: This study identifies a subtype of senescent cancer-associated fibroblasts (senCAFs) in breast tumors that suppresses natural killer (NK) cell activity and promotes tumor growth. Eliminating these senCAFs enhances NK cell–mediated tumor killing and may offer a new therapeutic strategy to prevent breast cancer progression.

Highlighted technique: To examine how senescent CAF-derived ECM affects NK cell function, doxorubicin-treated mouse mammary fibroblasts were cultured for six days to deposit ECM in vitro. After cell removal, the ECM was used in NK cytotoxicity assays, with NK cells pre-incubated on the matrix before co-culture with labeled target cells.

Related technique SA-βGal Detection(Cell)

Aging and injury drive neuronal senescence in the dorsal root ganglia (Nature Neuroscience, 2025)
Summary: This study shows that peripheral sensory neurons (DRG neurons) undergo senescence with aging and nerve injury, leading to increased pain sensitivity via pro-inflammatory signaling and excitability. Eliminating these senescent neurons alleviated pain, suggesting a potential therapeutic strategy for age-related sensory dysfunction.

Highlighted technique: This study assessed DRG neuron senescence using multiple markers. Aging-related senescence was confirmed by increased SA-β-gal activity, and injury-induced senescence was evaluated by examining the upregulation of p16, p21, and SASP factors including IL-6.

Related technique SA-βGal Detection(Tissue)

Neuronal cell cycle reentry events in the aging brain are more prevalent in neurodegeneration and lead to cellular senescence (PLoS Biol., 2024)
Summary: This study reveals that terminally differentiated neurons in the human brain, which are normally post-mitotic, can aberrantly re-enter the cell cycle under aging and neurodegenerative conditions. These neurons ultimately undergo senescence, with disease contexts like Alzheimer’s and Parkinson’s showing accumulation of proinflammatory and metabolically dysregulated senescent neurons.

Highlighted technique: To identify rare neurons that start dividing again, the researchers analyzed RNA data from individual cell nuclei collected from multiple human brain samples. They used a list of 350 genes related to the cell cycle and a scoring method to find excitatory neurons that are normally non-dividing but showed signs of abnormal cell cycle activity.

Related technique Cell Cycle Assay

Previous Science Note

Related Techniques (click to open/close)

Target	Kit & Probes
Cellular senescence detection	SPiDER-βGal for live-cell imaging or flow cytometry / microplate reader / tissue samples. Blue cellular senescence detection dye for fixed cells, SPiDER Blue
Cell Cycle Measurement	Cell Cycle Assay Solution Blue / Deep Red
Mitochondrial membrane potential detection	JC-1 MitoMP Detection Kit, MT-1 MitoMP Detection Kit
Total ROS detection	Highly sensitive DCFH-DA or Photo-oxidation Resistant DCFH-DA
Oxygen Consumption Rate(OCR) Detection	Extracellular OCR Plate Assay Kit
Apoptosis detection in multiple samples	Annexin V Apoptosis Plate Assay Kit
Cell proliferation/ cytotoxicity assay	Cell Counting Kit-8 and Cytotoxicity LDH Assay Kit-WST

Application Note I (click to open/close)
> Senescent Cells Lose Mitochondrial Activity

The senescent cell detection dye SPiDER Blue (SG07) and the mitochondrial membrane potential (MMP) dye MT-1 (MT13) were used to stain human microglial cells. Microscopy revealed that, compared to control cells, senescence-induced cells showed reduced MMP and increased SPiDER Blue fluorescence, reflecting elevated SA-β-Gal activity.

*This data was kindly provided by Dr. Supriya D. Mahajan, Department of Medicine, Jacobs School of Medicine & Biomedical Sciences.

1.　Seed human microglia cells into a dish and incubate in an incubator set at 37 ℃ and equilibrated with 95% air and 5% CO₂.
2.　Dilute the MT-1 Dye (1:1000) in the cell culture medium.
3.　Add MT-1 working solution to cells.
4.　Incubate the cells for 30 minutes in an incubator set at 37 ℃ and equilibrated with 95% air and 5% CO₂.
5.　Discard the supernatant and wash the cells with HBSS twice.
6.　Add 4% PFA/ PBS solution to the cells and incubate at room temperature for 30 minutes.
7.　Discard the 4% PFA / PBS solution and wash the cells with PBS.
8.　Add 15 µmol/l Spider Blue working solution and incubate at 37°C for 30 minutes.
9.　Remove the working solution, and wash the cells with PBS.
10. Add Imaging Buffer solution and observe the cells under a fluorescence microscope.

Application Note II (click to open/close)
> Increased Senescence in Aged Adipose Tissue

Frozen liver adipose tissue sections from 8-week-old and 35-week-old mice were stained with senescence detection probes SPiDER Blue (SG07) and SPiDER-βGal (SG02). Confocal microscopy revealed a marked increase in fluorescence intensity only in the 35-week-old samples, indicating an age-associated accumulation of senescent cells in older tissue.

1. 8-week-old and 35-week-old mouse liver adipose tissue (frozen sections) samples were prepared on glass slides.
2. After washing once with PBS, 200 µl of 4% paraformaldehyde (PFA)/PBS solution was added and fixed at room temperature for 30 minutes.
3. The supernatant was removed and washed once with PBS.
4. Add 200 µl of 15 µM SPiDER Blue and 15 µM SPiDER-βGal prepared in Assay buffer and incubate at 37°C for 2 hours.
5. The supernatant was removed and washed once with PBS.
6. Add 1 drop of encapsulant (ProLong Glass Antifade Mountant, Thermo) and encapsulate with cover glass.
7. Observed under a confocal laser microscope (40x magnification).

[Detection conditions]
SPiDER Blue: 405 nm (Ex), 400–500 nm (Em), 2.0%, 700V
SPiDER-βGal: 488 nm (Ex), 500–600 nm (Em), 1.0%, 600V