1152 - Radiotherapy-Induced Tumor Cell Senescence Reprograms Anti-Tumor Immunity in a p21 Dependent Manner

Z. Zhang¹, J. Yuan², D. Chen³, and J. Yu⁴; ¹Shandong Cancer Hospital and Institute, Jinan, Shandong, China, ²Department of Radiation Oncology and Shandong Provincial Key Laboratory of Radiation Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China, ³Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, ⁴Shandong Cancer Hospital and Institute, Jinan, China

Purpose/Objective(s): Radiotherapy is a classical way to induce cellular senescence. Senescent tumor cells exhibit unique surface proteome signature and secrete a range of senescence-associated secretory phenotypes (SASP), exerting immune activating or immunosuppressive functions. However, the role of radiotherapy-induced senescence and predictive value as a biomarker have not been fully understood. Therefore, this study aimed to identify the function of radiotherapy-induced tumor cell senescence, elucidate the underlying mechanism by which senescent cells reshape anti-tumor immunity and evaluate its clinical value as a potential biomarker to predict radiotherapy efficacy. Materials/

Methods: Multiplex immunofluorescence and luminex multiplex assays were performed on tissue and serum samples from patients receiving radiotherapy. To investigate the crosstalk between senescent tumor cells and immune cells, an abscopal mouse model with p21 knockdown was constructed and single-cell RNA-seq (scRNA-seq) and Flow Cytometry were used. CD4⁺ T cells were separated from OT-? transgenic mice and CD4⁺ T and NK cells from CD45.1 mice were adoptive transferred to CD45.1 mice to verify the interaction. Mass spectrum (MS), co-immunoprecipitation (co-IP), senescence-associated beta-galactosidase (SA-ß-gal) assay, qPCR, Western Blot and ELISA were used to explore the molecular mechanism.
Results: We found that radiotherapy induced tumor cell senescence and upregulated serum SASP level in samples of patients. The level of cellular senescence was significantly positively correlated with immune infiltration and patient prognosis. In addition, we demonstrated that 15Gy radiation led to senescence in tumor cell lines and tumor-bearing mice. p21 was essential in this process and p21 knockdown significantly reduced radiotherapy-induced senescence. In the abscopal mouse model, we found that P21 knockdown significantly attenuated the local control and abscopal effect of radiotherapy. Furthermore, RNA sequencing showed that the MHC-? antigen presentation pathway and SASP secretion were downregulated after p21 knockdown. We focused on the chemokine CCL5 through MS and verified that p21 knockdown downregulated radiotherapy induced secretion of CCL5 by qPCR, Western Blot and ELISA. CCL5 secreted by senescent tumor cells can act on CCR5 receptors on CD4⁺ T and NK cells, promote their infiltration and enhance the killing function. Further mechanism exploration revealed that p21 binding to JunB reduced its ubiquitination and inhibits its degradation post radiotherapy. JunB as a transcription factor can bind to CCL5 promoter, thereby regulating the expression level of CCL5.
Conclusion: Our study demonstrates that radiotherapy-induced tumor cell senescence reshapes surface proteome and secretome to enhance systemic anti-tumor immunity in a CD4⁺ T and NK cells dependent manner through p21/JunB/CCL5 axis.