POLY-Senolytic nanoplatform for tumor-specific eradication of senescent tumor cells and mitigation of radiotherapy-induced immune resistance of cancer

Yi Lai; Shunan Zhang; Jiaxing Pan; Wenyue Lan; Min Li; Zifan Zhu; Leiming Xu; Bruno G De Geest; Twan Lammers; Wen Zhang; Haijun Yu; Zhiai Xu

doi:10.1038/s41467-026-73109-w

. 2026 Jun 18;17:7720. doi: 10.1038/s41467-026-73109-w

POLY-Senolytic nanoplatform for tumor-specific eradication of senescent tumor cells and mitigation of radiotherapy-induced immune resistance of cancer

Yi Lai ^1,^2,^#, Shunan Zhang ^1,^#, Jiaxing Pan ^3,^#, Wenyue Lan ^2,⁴, Min Li ², Zifan Zhu ¹, Leiming Xu ⁵, Bruno G De Geest ⁶, Twan Lammers ⁷, Wen Zhang ¹, Haijun Yu ^2,^4,^✉, Zhiai Xu ^1,^✉

PMCID: PMC13434853 PMID: 42315837

Abstract

Radiotherapy (RT) efficacy is limited by RT-induced immune resistance. Here we show that RT upregulates programmed death ligand 1 (PD-L1) on senescent tumor cells (STCs) via bromodomain-containing protein 4 (BRD4) signaling, thereby promoting immune evasion. To counter this, we develop POLY-Senolytic, a polymeric senolytic nanoparticle formed by conjugating an acid-responsive polymer to a peptide-based BRD4 PROteolysis-TArgeting Chimera via a reduction-cleavable disulfide bond. The POLY-Senolytic is activated in the acidic and reductive intracellular environment of tumor cells, leading to BRD4 degradation, suppression of RT-induced PD-L1 expression and enhanced immune clearance of STCs. Combined with RT, the POLY-Senolytic suppresses tumor growth and metastasis in orthotopic mouse models of pancreatic and breast tumors. We further engineer a β-galactosidase-responsive POLY-Tracker for real-time monitoring of senolytic therapy. Together, this study identifies an RT-driven BRD4-PD-L1 axis in STCs that promotes immune resistance and provides a practical strategy to eliminate and track them.

Subject terms: Radiotherapy, Drug delivery

Radiotherapy-triggered BRD4 signaling drives PD-L1 upregulation and immunosuppression in senescent tumor cells. This study introduces BRD4-degrading polymeric nanoparticles to enhance antitumor immunity and real-time tracking of senescence.

Introduction

Radiotherapy (RT) is one of the most prevalent modalities for clinical treatment of solid tumors, with over half of all cancer patients receiving RT during the course of their disease^1,2. RT is administered either alone or in combination with surgery or chemotherapy in 40–60% of cancer cases^3,4. The antitumor effects of RT have traditionally been identified as ionizing radiation-induced DNA damage and cell death. In the past decade, RT has been exploited to elicit local and abscopal antitumor immune responses by promoting the release of tumor-associated antigens and activating antigen-presenting cells^5–7. Despite the clinical advances in RT-based cancer immunotherapy, the immunostimulatory effects of RT are severely impeded by the intrinsic and adaptive immune resistance of solid tumors^8–10. It remains a formidable challenge to optimize radioimmunotherapy by overcoming RT-induced immune resistance.

Recently, accumulated evidence suggested that RT-induced senescent tumor cells (RISTCs) play ambivalent roles in eliciting antitumor immunity and adaptive immune evasion. The senescent tumor cells induce a proinflammatory tumor microenvironment with upregulated expression of major histocompatibility complex class I (MHC-I) and secretion of senescence-associated secretory phenotypes (SASPs), which recruit tumor-infiltrating cytotoxic T lymphocytes (CTLs) for tumor eradication^11,12. On the other hand, a recent study by Ruscetti et al. reported that RISTCs in pancreas tumors inactivate natural killer cells and suppress immune surveillance via enhancer of zeste homolog 2 (EZH2)-mediated epigenetic repression of proinflammatory SASP genes¹³. Furthermore, chronic secretion of immunosuppressive SASPs by senescent tumor cells recruits immunosuppressive cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), which drives immune evasion and therapy resistance¹⁴. These findings highlight the urgent challenge to delineate the immunosuppressive mechanisms of RISTCs.

In this work, we demonstrated that RISTCs upregulate programmed death-ligand 1 (PD-L1) via the bromodomain-containing protein 4 (BRD4) signaling pathway, thereby promoting immune resistance. We further engineered a polymeric senolytic nanoparticle (POLY-Senolytic) to degrade BRD4 in RISTCs and restore immune clearance, potentiating RT-driven antitumor immunity. This POLY-Senolytic was synthesized by integrating a BRD4-targeted peptide PROteolysis Targeting Chimeras (PROTACs) onto the backbone of an acid-sensitive amphiphilic copolymer via a glutathione (GSH)-responsive disulfide linker (Fig. 1a). The POLY-Senolytic self-assembled into nanoparticles and passively accumulated at the tumor site. Upon cellular uptake by tumor cells, the POLY-Senolytics degraded BRD4 protein and suppressed PD-L1 expression in RISTCs. Consequently, tumor-infiltrating CTLs were activated for the eradication of RISTCs (Fig. 1b). In combination with RT, the POLY-Senolytic nanoparticles dramatically boosted antitumor immunity, delayed tumor growth, and metastasis in mouse models of orthotopic pancreatic and breast tumors. Furthermore, we developed a POLY-Tracker nanoparticle (namely PDPC@Cygal) for fluorescence imaging of senescence-associated β-galactosidase (SA-β-Gal), which therefore enables in vivo monitoring of RISTC and clearance (Fig. 1a, b). Taken together, our work unveiled the mechanism of RISTC-induced immune resistance, and the POLY-Senolytic might represent a practical strategy for potentiating RT of cancer.

Fig. 1 — a Chemical design of tumor-specific POLY-Senolytics and RISTC-activatable POLY-Trackers based on an acid-responsive copolymer functionalized with either a reduction-responsive PROTAC or a β-gal-responsive fluorogenic probe. b Schematic showing that RISTCs induce immune resistance through BRD4–PD-L1 upregulation and SASP secretion, whereas POLY-Trackers enable RISTC monitoring and POLY-Senolytics promote BRD4 degradation, PD-L1/SASP reduction, and CTL-mediated immune clearance of RISTCs.

Results

RISTCs drive an immunosuppressive tumor microenvironment via PD-L1 upregulation and SASP secretion

To elucidate the role of RISTCs in suppressing antitumor immunity, we first performed bioinformatic analysis of The Cancer Genome Atlas (TCGA) database. High expression of senescence signature genes was significantly associated with poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC), breast invasive carcinoma (BRCA), skin cutaneous melanoma (SKCM), and head and neck squamous carcinoma (HNSC) (Fig. 2a). Correlation analysis further revealed a positive correlation between senescent signature genes (CDKN1A and GLB1) and immunosuppressive genes, particularly PD-L1-encoding gene CD274 across these cancer types (Fig. 2b and Supplementary Fig. 1).

Fig. 2 — a Prognostic impact of senescence signature genes (*CDKN1A* and *GLB1*) expression in PDAC (pancreatic ductal adenocarcinoma), BRCA (breast invasive carcinoma), HNSC (head and neck squamous cell carcinoma), and SKCM (skin cutaneous melanoma) patients. b The correlation between senescent signature genes and immunosuppressive genes expression across four human cancer types. c Uniform Manifold Approximation and Projection (UMAP) showing two subclusters, including senescent tumor cells and non-senescent tumor cells, and proportion of senescent tumor cells and non-senescent tumor cells (n = 3 biologically independent mice per group). d The expression of immunosuppressive genes in senescent tumor cells and non-senescent tumor cells (n = 3 biologically independent mice per group). e Time-dependent proportion of PD-L1⁺ senescent tumor cells in total PD-L1⁺ tumor cells. f, g FCM analysis of p21 and PD-L1 expression in Panc02 (f) and 4T1 (g) tumor cells treated with various doses of RT (0, 4, 8, 12 Gy) (n = 3 independent experiments performed using independently cultured cells). h, i CLSM images of SA-β-Gal and PD-L1 staining in Panc02 (h) and 4T1 (i) cells with or without radiation treatment (scale bars = 25 μm). Experiments were independently repeated three times using independently cultured cells. j FCM analysis of RT-induced PD-L1 expression in p21⁺ tumor cells, and intratumor infiltration of CD8⁺ T cells in Panc02 tumor model in vivo (n = 6 biologically independent mice per group). k Correlation analysis between p21⁺ tumor cells vs. PD-L1⁺ tumor cells, and p21⁺ tumor cells vs. tumor-infiltrating CD8⁺ T cells, as well as PD-L1⁺ tumor cells vs. tumor-infiltrating CD8⁺ T cells. l Immunofluorescence staining of p21 (green), PD-L1 (red) expression, and CD8⁺ T cells (white) in tumor sections with or without RT treatments (scale bars = 100 μm). Images are representative of tumors collected from n = 3 biologically independent mice per group. m The positive area of p21⁺ tumor cells, PD-L1⁺ tumor cells, and tumor-infiltrating CD8⁺ T cells with or without RT treatments (n = 3 biologically independent mice per group). n Schematic illustration of RT-induced immune resistance by PD-L1 upregulation in RISTCs. Data are presented as the mean ± SD. Statistical analysis was calculated using the log-rank (Mantel–Cox) test (a), two-sided Pearson correlation analysis (b, k), two-sided paired Student’s t-test (f, g), and one-way ANOVA with Tukey’s test (j). Exact P-values are indicated in the figure or Source Data file. Source data are provided as a Source Data file.

Single-cell RNA-sequencing (scRNA-seq) analysis was next performed to validate the immunosuppressive role of RISTCs in an orthotopic mouse tumor model of PDAC (Supplementary Fig. 2a). Clustering analysis based on uniform manifold approximation and projection (UMAP) identified five major clusters (Supplementary Fig. 2b, c). The key populations were defined by established marker genes of tumor cells (Sox9, Krt18, Krt19) and ductal cells (Cftr, Muc1), respectively. Tumor cells were further categorized into two distinct subclusters of senescent and non-senescent tumor cells based on a set of senescence-associated gene signatures (Fig. 2c and Supplementary Fig. 2d, e). Quantification analysis revealed that RT significantly increased the proportion of RISTCs, which reached a peak of approximately 27.7% at one day post-RT (Fig. 2c). Gene expression analysis of immune checkpoints revealed that PD-L1-encoding gene Cd274 was the most significantly upregulated marker in RISTCs (Fig. 2d). Moreover, PD-L1⁺ senescent tumor cells further accumulated after RT (Fig. 2e).

To verify the upregulation of PD-L1 in RISTCs, we selected four tumor cell lines, including Panc02 (PDAC tumor cell), 4T1 (triple-negative breast cancer tumor cell), B16-F10 (SKCM tumor cell), SCC7 (HNSCC tumor cell), and treated them with RT at the indicated doses in vitro. Flow cytometric (FCM) analysis demonstrated that RT robustly increased PD-L1 expression in a radiation dose-dependent manner, with the upregulation occurring predominantly in p21⁺ senescent Panc02, 4T1, SCC7, and B16-F10 tumor cells (Fig. 2f, g and Supplementary Figs. 3, 4a, b). Of note, RT-induced PD-L1 and p21 expression in senescent tumor cells showed a positive correlation.

RT-induced senescence of tumor cells was visualized by chemical staining of intracellular senescence-associated β-galactosidase (SA-β-gal)¹⁵, and the concomitant upregulation of membrane PD-L1 on RISTCs was examined by immunofluorescence (IF) staining (Fig. 2h, i and Supplementary Fig. 4c). RT-induced PD-L1 expression in Panc02 and 4T1 RISTCs was further validated by fractionated RT (2 × 2 Gy or 4 × 2 Gy, respectively) mimicking the clinical fractionation scheme (Supplementary Fig. 5). These data consistently demonstrated that RT effectively induced PD-L1 expression on the surface of a broad spectrum of RISTCs.

To validate the above observations in vivo, subcutaneous Panc02 and 4T1 tumor models were established in immunocompetent mice and subjected to RT at the indicated doses. FCM analysis of PDAC tumor tissues revealed 7.5- and 2.6-fold increases in p21⁺ and PD-L1⁺ tumor cells, respectively, and a 3-fold decrease in tumor-infiltrating CD8⁺ T lymphocytes at 3 days post-RT compared to the RT-free group (Fig. 2j and Supplementary Fig. 6). Pairwise correlation analysis revealed that the fractions of p21⁺ or PD-L1⁺ tumor cells were negatively associated with the frequency of tumor-infiltrating CD8⁺ T cells in vivo (Fig. 2k). IF staining of the Panc02 tumor sections further displayed that compared to the control group, RT increased p21 and PD-L1 expression by 26.6-fold and 8.8-fold, respectively, while decreasing tumor-infiltrating CD8⁺ T cells by 4.3-fold (Fig. 2l, m). Similar patterns of immune microenvironment remodeling were observed in the 4T1 tumor model (Supplementary Fig. 7), further supporting a pivotal role of RISTCs in mediating immunosuppression across distinct tumor types. Collectively, these results suggested that PD-L1⁺ RISTCs play a pivotal role in RT-induced immune evasion by inactivating CD8⁺ T cells in vivo (Fig. 2n).

BRD4 degradation with a peptide PROTAC abolished RT-induced PD-L1 upregulation in RISTCs and potentiated radioimmunotherapy in vivo

Previous studies, including ours, have linked BRD4 to therapy-associated PD-L1 upregulation¹⁶ and treatment resistance in solid tumors^17,18. For example, it has been reported that BRD4 can cooperate with stress-responsive transcription factors (e.g., via a BRD4-IRF1 axis) to drive chemoradiotherapy-induced PD-L1 expression and immune escape¹⁹. Building on this framework, we asked whether RT-induced senescence activates a BRD4-dependent program that drives PD-L1 expression in RISTCs. Bioinformatic analysis of TCGA dataset revealed positive correlations of the expression between BET (e.g., BRD4) and CD274 or senescent signature genes (e.g., CDKN1A) in PDAC, BRCA, HNSC and SKCM patients (Fig. 3a and Supplementary Fig. 8). Further analysis of PDAC and BRCA patients confirmed noticeable BRD4 upregulation in CDKN1A-higher (a senescent biomarker) groups compared to the lower ones (Fig. 3b). Western blot assay of Panc02, 4T1, B16-F10 and SCC7 tumor cells in vitro further showed RT simultaneously upregulated BRD4 and PD-L1 expression in a dose-dependent manner (Fig. 3c and Supplementary Fig. 9a, b). Subsequent BRD4 knockdown by short hairpin RNA (shRNA) significantly attenuated RT-induced PD-L1 upregulation in RISTCs (Supplementary Fig. 9c), indicating that BRD4 acts as an upstream driver of RT-induced PD-L1 expression. In addition, we further found that chemotherapeutic doxorubicin-induced senescence elicited PD-L1 expression while marginally affected endogenous BRD4 level (Supplementary Fig. 10), indicating broad PD-L1 upregulation occurs in senescence tumor cells, whereas BRD4 upregulation is more pronounced in RT-induced senescence.

Fig. 3 — a TCGA correlation analysis of *BRD2*, *BRD3*, and *BRD4* expression vs *CDKN1A* and *CD274* expression in PDAC, BRCA, HNSC, and SKCM patients. b TCGA analysis of BRD4 expression in p21 high-expressed groups compared to the low-expressed groups. c Western blot assay determined a positive correlation of BRD4 and PD-L1 expression in Panc02, 4T1, B16-F10 and SCC7 tumor cells in vitro (n = 3 independent experiments performed using independently cultured cells). d Schematic diagram of the chemical structure and the BRD4 degradation mechanism of the BRD4d. e Western blot analysis of BRD4 and PD-L1 expression in Panc02 and 4T1 tumor cells after 8 Gy irradiation, followed by BRD4d treatment at 24 h post-RT, and incubated for an additional 24 h (n = 3 independent experiments performed using independently cultured cells). f Western blot assay of protein expression upon treatment by RT (8 Gy) or RT + BRD4d (n = 3 independent experiments performed using independently cultured cells). g Multiplex cytometric bead array analysis-determined SASP (senescence-associated secretory phenotype)-associated factor expression (n = 3 independent experiments performed using independently cultured cells). h ELISA-determined extracellular secretion of IL-1β with indicated treatments (n = 3 independent experiments performed using independently cultured cells). i Flow cytometric data of PD-L1 expression upon treatment by IL-1β or IL-1β + JAKi (n = 3 independent experiments performed using independently cultured cells). j FCM plots and quantification of PD-L1 expression upon treatment with wild-type tumor cell-conditioned medium (CM), RISTC-conditioned medium (Sen-CM), or Sen-CM + BRD4d (n = 3 independent experiments performed using independently cultured cells). k Schematic illustration of the therapeutic schedule. l, m Averaged Panc02 tumor growth curves (l) and survival curves (m) of Panc02 tumor-bearing mice receiving the indicated treatments (n = 6 biologically independent mice per group). n IHC staining and o semiquantitative analysis of BRD4 expression in the tumor sections examined at the end of the antitumor study (scale bars = 100 μm). Images are representative of tumors from n = 3 biologically independent mice per group. p CLSM examination of p21⁺ (green), PD-L1⁺ (red), and CD8⁺ (white) expression in the tumor sections with indicated treatments (scale bars = 100 μm). Images are representative of tumors from n = 3 biologically independent mice per group. q Schematic illustration for the mechanism of BRD4d-mediated PD-L1 downregulation via reducing SASP-JAK/STAT pathways. CTL, cytotoxic T lymphocyte. Data are presented as the mean ± SD. Statistical analysis was calculated using two-sided Pearson correlation analysis (a, c), two-sided unpaired Student’s t-test (b), one-way ANOVA with Tukey’s test (h–j, o), two-way ANOVA with Tukey’s test (l), and log-rank (Mantel–Cox) test (m). Exact P-values are indicated in the figure or Source Data file. Source data are provided as a Source Data file.

To achieve effective BRD4 intervention by protein degradation technology, we next synthesized a peptide-based PROTAC degrader of BRD4 (namely BRD4d) by coupling a Von Hippel-Lindau (VHL)-binding peptide with a BRD4 inhibitor JQ1 (Fig. 3d and Supplementary Fig. 11, 12). BRD4 degradation with the BRD4d dramatically reversed RT-induced PD-L1 upregulation in both Panc02 and 4T1 tumor cells (Fig. 3e and Supplementary Fig. 13a, b). Notably, BRD4d preferentially degraded BRD4 over other BET family members (BRD2 and BRD3) (Supplementary Fig. 13c, e), supporting its functional selectivity toward BRD4. In addition, BRD4d displayed 5-fold lower DC₅₀ in RISTCs than normal tumor cells (0.18 ± 0.06 μM vs 0.53 ± 0.06 μM) (Supplementary Fig. 13d, e), indicating increased susceptibility of RISTCs to BRD4 degradation. BRD4d-mediated BRD4 degradation and PD-L1 downregulation were further validated in RT-treated pancreatic tumor specimens from patients (Supplementary Fig. 14).

RNA-sequencing (RNA-seq) analysis was next performed to investigate the mechanism underlying BRD4d-mediated PD-L1 downregulation in RISTCs. Differential gene expression analysis confirmed RT-induced tumor cell senescence via upregulation of senescent signature genes (e.g., Trp53 and H2ax), SASP-related genes (e.g., Il1a and Il6), and downregulation of proliferative genes (e.g., Ki67) and anti-apoptotic genes (e.g., Bcl2 and Bcl6) in RISTCs compared to normal tumor cells (Supplementary Fig. 15). Cd274 and Brd4 expression were markedly increased in RT-treated tumor cells. In contrast, BRD4d treatment significantly reversed the transcription of apoptotic genes. GO enrichment analysis revealed that RT markedly activated the JAK-STAT and SASP secretion-associated pathways, which were dramatically suppressed by BRD4d (Supplementary Fig. 16).

Western blot analysis further validated that RT significantly activated the JAK-STAT-IRF signaling axis and increased PD-L1 expression in Panc02 tumor cells in vitro, which were reversed to basal levels by BRD4d-mediated BRD4 degradation (Fig. 3f and Supplementary Fig. 17). Multiplex cytometric bead array assay of Panc02 tumor cells displayed that RT markedly upregulated various SASPs, particularly IL-1α and IL-1β, which are key mediators known to promote tumor progression and immune evasion (Fig. 3g). The secretion of these cytokines was significantly reduced upon BRD4d treatment. Enzyme-linked immunosorbent assay (ELISA) further validated that RT induced a 7.2-fold increase in IL-1β secretion compared to the RT-free group, while BRD4d treatment reduced IL-1β level by 3.3-fold (Fig. 3h).

FCM analysis showed that the frequency of the PD-L1⁺ Panc02 tumor cells was significantly increased from 16.5 ± 2.3% to 27.6 ± 4.5% after IL-1β treatment, but reduced after the addition of a JAK1 inhibitor (JAKi) (Fig. 3i, Supplementary Fig. 18). RT-induced PD-L1 upregulation was partially reduced in IL-1 receptor (IL-1R) knockdown cells, supporting the contribution of IL-1/IL-1R signaling to RT-induced PD-L1 expression (Supplementary Fig. 19). Co-culture of non-senescent tumor cells with the conditioned medium derived from RISTCs (sen-CM) elicited a 10.4-fold increase of PD-L1 expression, but it was completely reversed by BRD4d treatment (Fig. 3j). It was reported that BRD4 directly binds to the promoter and enhancer regions of CD274, thereby promoting PD-L1 transcription^20,21. Our data further showed that RT upregulated PD-L1 via BRD4-induced SASP secretion and activation of the JAK/STAT pathway, suggesting the rationale of BRD4 degradation to downregulate PD-L1.

Given the efficient BRD4d-mediated PD-L1 downregulation in RISTCs, we next evaluated the BRD4d for boosting radioimmunotherapy in subcutaneous Panc02 tumor models. The tumor-bearing mice were treated with fractionated RT (4 × 2 Gy) followed by intratumoral (i.t.) injection of free BRD4d (1.0 mg/kg) three days post-RT (Fig. 3k). The combination of RT and BRD4d (RT + BRD4d) inhibited 72.1% of tumor growth and doubled the median survival compared to the RT groups (Fig. 3l, m and Supplementary Fig. 20a, b). Immunohistochemistry (IHC) staining of the tumor sections showed that RT alone increased BRD4 expression by 1.5-fold compared to the PBS-injected control group, which was restored by BRD4d treatment (Fig. 3n, o).

Western blot assay of the tumor lysates displayed that PD-L1 and p21 expression in the RT + BRD4d group decreased to 61% and 31% of the RT + PBS group, respectively (Supplementary Fig. 20c). Multiplex immunofluorescence (mIF) staining of the tumor sections further demonstrated obvious BRD4 degradation and PD-L1 downregulation by BRD4d, which dramatically increased the tumor-infiltrating CD8⁺ T cells and eradicated RISTCs in vivo (Fig. 3p). Taken together, these data validated that the BRD4d blocked RT-induced PD-L1 expression and SASP secretion via the BRD4-JAK-STAT3 pathway, and BRD4-targeted senolytic therapy is promising to sensitize RT of cancer (Fig. 3q).

The POLY-Senolytic nanoparticles suppressed PD-L1 expression in RISTCs in vitro

Given the satisfactory BRD4 degradation efficacy of the peptide BRD4d in vitro and in vivo, we next developed the POLY-Senolytic for systemic delivery of the BRD4d by leveraging the acidic and reductive microenvironment of tumor cells. The POLY-Senolytic was synthesized by conjugating BRD4d onto the backbone of the amphiphilic PEG-b-PDPA diblock copolymer via a thiol-maleimide free-radical addition reaction (termed PDPS@BRD4d). To elucidate the contribution of polymer acid responsiveness and the GSH-cleavable disulfide linkage in the POLY-Senolytic design, we synthesized two control nanoparticles. PDPC@BRD4d remaind acid-responsive but became GSH-insensitive by substituting the disulfide bond with a stable C-C bond, whereas PBPC@BRD4d was both acid-insensitive and GSH-insensitive due to replacing 2-(diisopropylamino)ethyl methacrylate (DPA) with butyl methacrylate (BMA) (Fig. 4a and Supplementary Fig. 21–24). In the PDPS@BRD4d POLY-Senolytic design, the hydrophilic PEG segment was employed for elongating blood circulation of the BRD4d, while the acid-sensitive segment and GSH-sensitive disulfide bond enable selective nanoparticle dissociation and drug release inside the tumor cells.

Fig. 4 — a Chemical structures of the POLY-Senolytic library, and their acid/GSH-responsive mechanism. b DLS-determined hydrodynamic diameter of the PDPC@BRD4d and PBPC@BRD4d nanoparticles (n = 3 independently prepared nanoparticle samples). c TEM images of the PDPC@BRD4d and PBPC@BRD4d nanoparticles examined at pH 7.4 and 6.0 (scale bar = 100 nm). Images are representative of three independently prepared nanoparticle samples. d Acid-activatable fluorescence profile of the ICG-labeled PDPC@ICG and PBPC@ICG nanoparticles (fluorescence intensity normalized to pH 7.4, n = 3 independently prepared nanoparticle samples). The inset displayed the fluorescence image of the PDPC@ICG and PBPC@ICG suspensions across varied pH values. e CLSM examination of intracellular distribution of the PDPC@ICG and PBPC@ICG nanoparticles following 6 h of incubation. Nuclei were stained with DAPI (blue), ICG was shown in red, and lysosomes were stained with LysoTracker (green). Scale bars, 25 μm. Experiments were independently repeated three times using independently cultured cells, with similar results. f GSH-triggered BRD4d release from the PDPS@BRD4d nanoparticles at pH 6.0 with 10 mM GSH addition (n = 3 independently prepared nanoparticle samples). g Western blot analysis of BRD4 levels in senescent Panc02 cells treated with free BRD4d, PDPC@BRD4d, or PDPS@BRD4d, and in RAW 264.7 and 3T3 cells treated with PDPS@BRD4d for 24 h (n = 3 independent experiments performed using independently cultured cells). h CLSM images of BRD4 (green) and PD-L1 (red) expression following indicated treatments (BRD4d dose of 1 μM, RT at 8 Gy, scale bar = 25 μm). Experiments were independently repeated three times using independently cultured cells, with similar results. i FCM plots showing reversal of RT-induced PD-L1 upregulation by PDPS@BRD4d (n = 3 independent experiments performed using independently cultured cells). j FCM analysis-determined IFN-γ⁺ CD8⁺ T cells upon indicated treatments (n = 3 independent co-culture experiments using primary CD8⁺ T cell preparations isolated from 3 biologically independent mice). k Schematic of the PDPS@BRD4d-mediated PD-L1 downregulation and CD8⁺ T cell activation. l Fluorescence images of PBPC@ICG and PDPC@ICG distribution in Panc02 tumor-bearing nude mice and the major organs and tumors harvested 48 h post-injection (n = 5 biologically independent mice per group). He heart, Li liver, Sp spleen, Lu lung, Ki kidney, Tu tumor. m Normalized fluorescence intensities of the tumor tissue in vivo and major organs and tumors ex vivo (n = 5 biologically independent mice per group). n CLSM examination of the tumor sections at 48 h post nanoparticle injection (scale bar = 25 μm). CD31 is shown in green, ICG in red, and nuclei in blue. Images are representative of tumors from n = 3 biologically independent mice per group. o Pharmacokinetics of i.v. injected BRD4d and PDPS@BRD4d nanoparticles (n = 3 biologically independent mice per group). p Intratumoral distribution of free BRD4d and PDPS@BRD4d nanoparticles examined at the indicated time points post-injection (n = 3 biologically independent mice per group). Data are presented as the mean ± SD. Statistical analysis was calculated using one-way ANOVA with Tukey’s test (j). Exact P-values are indicated in the figure. Source data are provided as a Source Data file.

The resultant POLY-Senolytics and their counterparts all self-assembled into micellar nanoparticles in aqueous solution. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) examination displayed that PDPS@BRD4d nanoparticles prepared via nanoprecipitation possessed an average hydrodynamic diameter of 77.8 ± 6.0 nm and a narrow PDI of 0.19 ± 0.02 (Supplementary Fig. 25). The PDPS@BRD4d nanoparticles maintained good stability in phosphate buffer solution (pH 7.4), fetal bovine serum and cathepsin B solution over 48 h (Supplementary Fig. 26). PDPC@BRD4d and PBPC@BRD4d nanoparticles exhibited spherical morphology of the PDPS@BRD4d, with average hydrodynamic diameters of ~75 nm and narrow size distributions at neutral pH (Fig. 4b, c). PDPC@BRD4d nanoparticles dissociated at pH 6.0 due to acid-induced protonation of the DPA groups. In contrast, the PBPC@BRD4d nanoparticles remained intact due to the lack of an acid-sensitive segment.

The acid-sensitive and acid-insensitive POLY-Senolytic nanoparticles were then covalently labeled with a fluorescence dye ICG (namely PDPC@ICG and PBPC@ICG, respectively) for investigating the distribution of the POLY-Senolytic nanoparticles in vitro and in vivo (Supplementary Figs. 27 and 28). The PDPC@ICG nanoparticles displayed a sharp fluorescence transition (ΔpH <0.2, pH 6.0–6.2) due to their acid-responsive property (Fig. 4d). CLSM examination in Panc02 tumor cells showed obvious activation of the fluorescence signal in the PDPC@ICG group compared to the PBPC@ICG one (Fig. 4e).

GSH-triggered BRD4d release from the POLY-Senolytic nanoparticles was examined by high-performance liquid chromatography (HPLC) examination. Fig. 4f showed that 60% of BRD4d was restored from the PDPS@BRD4d nanoparticles after 24 h incubation with 10 mM GSH. However, no BRD4d was released from the GSH-insensitive PDPC@BRD4d nanoparticles. Western blot assay displayed an excellent BRD4 degradation profile of the PDPS@BRD4d nanoparticles in Panc02 tumor cells in vitro. In contrast, the PDPC@BRD4d nanoparticles marginally affected BRD4 expression (Fig. 4g), validating the crucial role of the disulfide bond to release the peptide PROTAC for BRD4 degradation. The PDPS@BRD4d nanoparticles displayed a DC₅₀ of 0.35 μM in senescent Panc02 tumor cells in vitro. Of note, the POLY-Senolytics marginally affected the BRD4 expression profiles of fibroblasts (e.g., 3T3 cells) and macrophages, demonstrating tumor cell-specific BRD4 degradation property of the POLY-Senolytics (Fig. 4g and Supplementary Fig. 29a). The BRD4 degradation property of the PDPS@BRD4d nanoparticles was abolished by a proteasome inhibitor MG132 (Supplementary Fig. 29b), verifying ubiquitin-proteasome-dependent BRD4 degradation profile of the POLY-Senolytic nanoparticles. FCM and CLSM examination showed that the PDPS@BRD4d nanoparticles markedly reduced 96.3% of PD-L1 expression in RT-induced Panc02 senescent tumor cells in vitro, much more efficiently than free BRD4d (Fig. 4h, i).

To investigate whether BRD4 degradation and PD-L1 downregulation mitigate the immune resistance of RISTCs in vitro, tumor-infiltrating CD8⁺ T cells were isolated from Panc02 tumor suspensions and incubated with PDPS@BRD4d-treated RISTCs. FCM examination displayed that the fraction of IFN-γ⁺CD8⁺ T cells increased from 2.3 ± 0.3% to 5.6 ± 0.4% compared to the RT group (Fig. 4j and Supplementary Fig. 30a), implying PD-L1 downregulation with the PDPS@BRD4d nanoparticles efficiently relieved the immune suppression of RISTCs. Apoptosis assays further showed that BRD4 degradation induced by PDPS@BRD4d nanoparticles significantly enhanced RT-induced apoptosis of Panc02 tumor cells, whereas PD-L1 blockade with anti-PD-L1 antibody (αPD-L1) negligibly affected the apoptotic effect of RT (Supplementary Fig. 30b, c). This phenomenon represents a unique advantage of BRD4d-based senolytic therapy over αPD-L1 by promoting apoptosis of RISTCs. The above data demonstrated that the BRD4d-integrated POLY-Senolytic degraded BRD4, downregulated PD-L1 in RISTCs, and thereby effectively alleviated RISTC-induced immunosuppression of CTLs (Fig. 4k).

The POLY-Senolytic nanoparticles specifically delivered the BRD4d into the tumor in vivo

We next investigated the tumor-targeted delivery capability of POLY-Senolytic nanoparticles in vivo using the Panc02 tumor-bearing BALB/c nude mouse model. The acid-responsive nanoparticles (PDPC@ICG) and their pH-insensitive analogs (PBPC@ICG) were administered via intravenous (i.v.) injection at an identical ICG dose of 2.5 mg/kg when the tumor volume reached 100 mm³. Fluorescence imaging displayed significantly higher fluorescence intensity of the PDPC@ICG than the PBPC@ICG control at the selected time points. The fluorescence intensity of PDPC@ICG nanoparticles at the tumor site increased gradually over time, peaking at 36 h post-injection with a 2.4-fold enhancement relative to the control group (Fig. 4l, m).

Ex vivo fluorescence imaging at 48 h showed 2.6-fold higher tumor signal with PDPC@ICG than with the non-responsive PBPC@ICG (Fig. 4l, m). IF staining of Panc02 tumor sections at 48 h post-injection showed a markedly stronger and more widespread ICG signal in the PDPC@ICG group than in the PBPC@ICG group (Fig. 4n). Together with the distribution pattern observed in the highly vascularized CT26 colorectal tumor model (Supplementary Fig. 31), these data support passive accumulation of the POLY-Senolytic nanoparticles mediated by the enhanced permeability and retention effect.

We next investigated the pharmacokinetics and intratumoral distribution of the POLY-Senolytic nanoparticles in a Panc02 tumor-bearing mouse model. HPLC examination displayed that compared to free BRD4d, the PDPS@BRD4d nanoparticles exhibited ~4-fold-higher bioavailability than free BRD4d (area under curves, AUC), indicating reduced blood clearance of the BRD4d payload (Fig. 4o). Moreover, the POLY-Senolytic nanoparticles displayed a 6-fold higher tumor accumulation (AUC) compared to the BRD4d group over 36 h post-injection, validating their tumor-targeted BRD4d delivery profile (Fig. 4p). These results highlight that the pH and GSH dual-activatable POLY-Senolytic nanoparticles are capable of efficiently delivering and precisely releasing the BRD4 PROTAC in tumor tissues.

The POLY-Senolytic nanoparticles suppressed tumor growth and mitigated RT-induced immune evasion by promoting immune eradication of RISTCs in vivo

Inspired by the tumor-specific BRD4 degradation property of the POLY-Senolytic nanoparticles in vivo, we next sought to evaluate their antitumor performance in a mouse model of subcutaneous Panc02 tumor. Tumor-bearing C57BL/6 mice were randomly divided into six groups (n = 6) and exposed to the indicated treatments, including PBS (i.v.), PDPS@BRD4d (i.v.), RT + PBS (i.v.), RT + BRD4d (i.v.), RT + PDPS@BRD4d (i.v.), or RT + αPD-L1 at an X-ray dose of 4 × 2 Gy and an identical BRD4d dose of 10 mg/kg. αPD-L1 was intraperitoneally injected for 3 times at 3 days post-RT, with a dosage of 5 mg/kg (Fig. 5a). The tumor growth curves showed that RT + PBS or RT + BRD4d moderately inhibited Panc02 tumor growth by 27.3% and 35.4%, respectively. The combination of RT + PDPS@BRD4d and RT + αPD-L1 comparably regressed ~76.2% of tumor growth (Fig. 5b, c and Supplementary Fig. 32a). Of note, compared to the RT + PBS or RT + BRD4d group, RT + PDPS@BRD4d much more efficiently prolonged the median survival of the tumor-bearing mice, with 4 of 6 mice surviving beyond 81 days (Fig. 5d).

Fig. 5 — a Experimental schedule for evaluating the therapeutic performance of the POLY-Senolytic nanoparticles in a subcutaneous Panc02 tumor model. b, c Averaged (b) and individual (c) tumor growth curves recorded during the antitumor study (n = 6 biologically independent mice per group). d Survival curves of the tumor-bearing mice during the experimental period (n = 6 biologically independent mice per group). e IHC analysis of BRD4 expression in the tumor sections (scale bars = 40 μm). Images are representative of tumors from n = 3 biologically independent mice per group. f CLSM examination of p21 (green) and PD-L1 (red) expression in the tumor sections (scale bars = 100 μm). Images are representative of tumors from n = 3 biologically independent mice per group. g, h FCM plots of p21⁺ tumor cells (g) and PD-L1⁺ tumor cells (h) after indicated treatments at 7-day post-treatment in vivo (n = 6 biologically independent mice per group). i The frequency of the tumor-infiltrating CD8⁺ T cells (n = 6 biologically independent mice per group). j The frequency of tumor-infiltrating IFN-γ⁺CD8⁺ T cells (n = 6 biologically independent mice per group). k The CD8⁺ T to Tregs ratio was determined at 7-day post-treatment in vivo (n = 6 biologically independent mice per group). l CLSM examination of tumor-infiltrating CD8⁺ T cells (red) and Foxp3⁺ Tregs (green) examined at 7 days post-treatment (scale bars = 100 μm). Images are representative of tumors from n = 3 biologically independent mice per group. m FCM determined tumor-infiltrating T_EM (CD62L⁻CD44⁺CD8⁺CD3⁺) (n = 6 biologically independent mice per group). n Schematic of the POLY-Senolytic nanoparticle-mediated BRD4 degradation and elimination of RISTCs by activating T cell immune response. CTL, cytotoxic T lymphocyte; Treg, regulatory T cell; T_EM, effector memory T cell. For b-m, treatment groups are defined as follows: G1, PBS; G2, PDPS@BRD4d; G3, RT + PBS; G4, RT + BRD4d; G5, RT + αPD-L1; G6, RT + PDPS@BRD4d. Data are presented as the mean ± SD. Statistical analysis was calculated using two-way ANOVA with Tukey’s test (b), log-rank (Mantel–Cox) test (d), and one-way ANOVA with Tukey’s test (g–k, m). Exact P-values are indicated in the figure. Source data are provided as a Source Data file.

To elucidate the mechanism underlying POLY-Senolytic-potentiated RT, IHC analysis of the tumor sections was performed to unveil remarkable BRD4 degradation in the RT + PDPS@BRD4d group. In contrast, the RT + PBS and RT + αPD-L1 groups displayed highly upregulated BRD4 expression (Fig. 5e). IF staining showed markedly downregulated PD-L1 and p21 expression in the RT + PDPS@BRD4d group (Fig. 5f). FCM analysis further revealed that RT treatment upregulated 4.4- and 3.5-fold of p21 and PD-L1 expression in tumor cells, respectively, compared to PBS due to RT-induced senescence of tumor cells in vivo. In contrast, the combo of RT + PDPS@BRD4d dramatically reduced PD-L1⁺ Panc02 tumor cells from 15.4% to 5.4%, and p21⁺ RISTCs from 32.2% to 9.0%, respectively (Fig. 5g, h and Supplementary Fig. 33a), indicating that BRD4 degradation with the PDPS@BRD4d nanoparticles promoted immune eradication of PD-L1⁺ RISTCs in vivo.

Subsequently, we investigated whether the POLY-Senolytic nanoparticles reprogrammed the RISTC-mediated immunosuppressive tumor microenvironment. FCM analysis revealed that RT + PDPS@BRD4d significantly recruited 3.2- and 2.3-fold higher tumor-infiltrating CD8⁺ T lymphocytes than RT + PBS and RT + αPD-L1, respectively (Fig. 5i, Supplementary Fig. 33b). Moreover, the RT + PDPS@BRD4d group displayed 3.2- and 2.1-fold higher tumor-infiltrating IFN-γ⁺CD8⁺ T cells than the RT + PBS and RT + αPD-L1 groups, respectively (Fig. 5j, Supplementary Fig. 33c). Of note, the RT + PDPS@BRD4d group displayed 7.2-fold higher CD8⁺ T cell/Treg ratio than the RT + αPD-L1 group (Fig. 5k, l and Supplementary Fig. 34a), implying activation of a protective immune response by RT + PDPS@BRD4d treatment. Meanwhile, RT + PDPS@BRD4d 2.0-fold more efficiently expanded effector memory T cells (T_EM) compared to RT + αPD-L1 (Fig. 5m, Supplementary Fig. 34b), implying tumor-specific BRD4 degradation elicited immune memory for long-term tumor regression. These data collectively validated that the PDPS@BRD4d POLY-Senolytic nanoparticles more efficiently elicited antitumor immune response than αPD-L1 for overcoming RISTC-induced immune resistance of RT (Fig. 5n).

To further investigate whether the combinatory therapy by PDPS@BRD4d nanoparticles + αPD-L1 cumulatively boosts RT, we performed an antitumor study in a subcutaneous mouse model of PDAC tumor in vivo. Compared to RT alone or single-agent treatments, combinatory therapy with RT + PDPS@BRD4d + αPD-L1 most efficiently inhibited Panc02 tumor growth compared to the combinations of RT + BRD4 inhibitor JQ1 + αPD-L1, RT + PDPS@BRD4d, or RT + αPD-L1 (Supplementary Fig. 35). These results suggested improved antitumor effects by BRD4 degradation with PDPS@BRD4d and PD-L1 blockade with αPD-L1.

We next evaluated the biosafety of the PDPS@BRD4d nanoparticles. All animals showed slightly increased body weights during the experimental period, indicating good biosafety of the PDPS@BRD4d nanoparticles (Supplementary Fig. 32b). Systemic toxicity assessment in healthy mice treated with PDPS@BRD4d or αPD-L1 revealed negligible histopathological abnormalities in major organs (e.g., heart, liver, spleen, lung, and kidney) (Supplementary Fig. 36a). Hematology and serum biochemistry analyses revealed negligible abnormalities in all the treatment groups (Supplementary Fig. 36b). These results indicated good biosafety and biocompatibility of PDPS@BRD4d POLY-Senolytic nanoparticles in vivo.

The POLY-Senolytic nanoparticles elicited robust antitumor immunity in mouse models of orthotopic pancreatic and breast tumors in vivo

Given the impressive antitumor performance of the tumor-specific POLY-Senolytic nanoparticles in the subcutaneous tumor model of PDAC, we further evaluated their effectiveness in an orthotopic model of PDAC tumor by implanting luciferase-expressing Panc02 (Panc02-Luc) tumor cells into the pancreas. The ICG-labeled acid-responsive nanoparticles (PDPC@ICG) were i.v. injected when the orthotopic tumors were established (Fig. 6a). Ex vivo bioluminescence and fluorescence imaging of the pancreas and spleen displayed a significant colocalization of PDPC@ICG accumulating sites with the primary tumor foci and splenic metastatic lesion (Fig. 6b). Notably, fluorescence imaging of the major organs (heart, liver, spleen, lung, kidney, pancreas) and tumors ex vivo at 24 h post-injection validated tumor-specific accumulation of the nanoparticles, with 1.8-fold higher fluorescence intensity in the tumor lesions than the peritumoral pancreatic tissue (Supplementary Fig. 37a, b). CLSM examination of the tumor sections further validated colocalization between ICG fluorescence and the Ki67-positive tumor region (Supplementary Fig. 37c). All these data demonstrated the excellent tumor specificity of the PDPS@BRD4d nanoparticles in the orthotopic tumor model.

Antitumor study in the orthotopic Panc02 tumor model demonstrated the combo of RT + PDPS@BRD4d significantly inhibited Panc02-Luc tumor growth, as evidenced by 20.0- and 5.2-fold lower bioluminescence than the PBS and RT + PBS groups, respectively (Fig. 6c, Supplementary Fig. 37d). Furthermore, RT + PDPS@BRD4d markedly prolonged the median survival of the tumor-bearing mice up to 78 days, in striking contrast to 28 days in the PBS group and 40 days in the RT + PBS group (Fig. 6d). Subsequent IHC and IF analyses revealed effective BRD4 degradation and significant elimination of PD-L1⁺ RISTCs in the RT + PDPS@BRD4d group (Fig. 6e, f). Moreover, RT + PDPS@BRD4d treatment significantly increased tumor-infiltrating CD8⁺ T cells but decreased Tregs compared to RT alone, signifying POLY-Senolytic-mediated activation of antitumor immunity in the orthotopic tumor model (Fig. 6g).

We next extended these findings in an orthotopic 4T1-Luc tumor model of triple-negative breast cancer (Fig. 6h). Bioluminescent imaging of the tumor-bearing mice displayed a significant tumor size reduction in the RT + PDPS@BRD4d group compared to RT alone (Fig. 6i). Moreover, RT + PDPS@BRD4d significantly prolonged the median survival of the tumor-bearing mice from 27 days (PBS) to 68 days (Fig. 6j). IHC analysis showed the POLY-Senolytic nanoparticles successfully degraded BRD4 in 4T1 tumor cells in vivo (Fig. 6k). mIF imaging showed that RT markedly increased intratumoral p21 and PD-L1 expression, which were dramatically abolished by RT + PDPS@BRD4d treatment (Supplementary Fig. 37e). FCM quantification indicated that RT raised the proportion of PD-L1⁺ tumor cells from 30.1% to 72.6%, while PDPS@BRD4d treatment reversed the proportion to 37.4% (Fig. 6l). PDPS@BRD4d reduced RISTCs by 2.1-fold and increased tumor-infiltrating CD8⁺ T cells by 2.5-fold compared to RT alone (Fig. 6m, n). The CD8⁺ T/Treg ratio increased by 6.0-fold and 30.6-fold compared to the PBS and RT + PBS groups, respectively (Fig. 6o).

Moreover, RT led to a substantial increase in tumor-infiltrating myeloid-derived suppressor cells (MDSCs) to approximately 18.0% of total immune cells, which was 2.3-fold higher than the PBS group (~8%). In contrast, RT + POLY-Senolytic nanoparticles treatment dramatically reduced MDSC levels to ~6% (Supplementary Fig. 38a, b), implying relieved immunosuppression of the tumor microenvironment. RT + PDPS@BRD4d treatment also reduced the number of lung metastatic lesions of 4T1 tumor cells (Fig. 6p). Immune assay determined increased maturation of dendritic cells (DCs) from 2.1% (PBS) to 4.2% (RT) and 5.3% (RT + PDPS@BRD4d). CD8⁺ T_EM cells rose from 8.1% (PBS) to 14.2% (RT) and 18.5% (RT + PDPS@BRD4d), respectively (Supplementary Fig. 38c, d). These data indicated that the combination of RT + PDPS@BRD4d induced systemic immunity to prevent distant metastases of breast tumor cells (Fig. 6q).

RISTC-activatable POLY-Trackers performed real-time and in-situ evaluation of POLY-Senolytic-potentiated RT of cancer

It remains a formidable challenge for real-time and in vivo detection of senescent tumor cells, which is crucial for the development of senolytic therapy^22–24. To facilitate in vivo monitoring of RISTCs and evaluation of the POLY-Senolytics, we rationally engineered a senescence-activatable POLY-Tracker (termed PDPC@Cygal) by integrating a d-galactose-caged hemicyanine dye (CyOH) with the same polymer backbone of PDPS@BRD4d (Supplementary Figs. 39–44). The d-galactose cage quenches the intrinsic near-infrared fluorescence of CyOH and is specifically cleaved by β-Gal, a well-established biomarker of senescent cells²⁵, thereby restoring the fluorescence signal for senescence imaging (Fig. 7a). Upon cellular internalization, endosomal acidification triggers PDPC@Cygal nanoparticle dissociation and exposes the Cygal probe, allowing β-Gal in RT-induced senescent tumor cells to remove the galactose cage and switch on strong fluorescence (Fig. 7b).

Fig. 7 — a Chemical structure and β-gal-activated fluorescence mechanism of PDPC@Cygal, in which RT-induced senescent tumor cells trigger fluorescence switching from “off” to “on”. b Schematic illustrating that PDPC@Cygal dissociates in acidic endosomes and β-gal in senescent cells activates fluorescence, whereas non-senescent cells remain non-fluorescent. c Time-dependent activation of the PDPC@Cygal nanoparticles with 1.0 U/mL β-gal (λ_ex = 680 nm, F. I., fluorescence intensity). Representative spectra from three independently prepared samples. d The fluorescence spectral changes of PDPC@Cygal in the presence of β-gal (0-1.0 U/mL). Representative spectra from n = 3 independently prepared samples are shown. e Absorption spectra of PDPC@Cygal in the absence or presence of β-gal. Inset: Color change before and after β-gal treatment. Representative spectra from n = 3 independently prepared samples are shown. f The linear relationship of PDPC@Cygal activation vs β-gal concentration (0-1.0 U/mL). Experiments were independently repeated three times with similar results (n = 3). g The fluorescence emission of PDPC@Cygal (10 μM, λ_em = 700 nm) in the presence of various interferents (for example, Cys, cysteine; HCy, homocysteine; GSH, glutathione; ClO⁻, H₂O₂, Na⁺, Mg²⁺, Ca²⁺, β-gal). Experiments were independently repeated three times with similar results (n = 3). h The fluorescence intensity of the PDPC@Cygal nanoprobe (Cygal of 10 μM) in the presence of β-gal and various interferents (λ_em = 700 nm). Experiments were independently repeated three times with similar results (n = 3). i FCM analysis-determined fluorescent intensity of the PDPC@Cygal (5 μM) in Panc02 tumor cells following RT at the indicated dosages (n = 3 independent experiments performed using independently cultured cells). j FCM-determined linear correlation between p21⁺ expression and β-gal staining in RISTCs in vitro (n = 3 independent experiments performed using independently cultured cells). k Experimental schedule for in-situ detection of RISTCs in vivo. l, m Fluorescence images (l) and normalized fluorescence intensities (m) of PDPC@Cygal activated by RT-upregulated β-gal in tumor mass (n = 3 biologically independent mice per group). n Ex vivo fluorescence imaging of the tumor sections harvested 36 h post-injection (n = 3 biologically independent mice per group). o, p IF images (o) and normalized fluorescence intensities (p) of the tumor sections examined at 36 h post-injection (scale bar = 25 μm). Images are representative of tumors from n = 3 biologically independent mice per group. q Experimental schedule for monitoring senolytic therapy with the PDPC@Cygal nanoprobe. r, s Fluorescence imaging in vivo (r), and normalized fluorescence intensities (s) of the PDPC@Cygal nanoprobe recorded during RT + PDPS@BRD4d therapy (n = 3 biologically independent mice per group). Data are presented as the mean ± SD. Statistical analysis was calculated using one-way ANOVA with Tukey’s test (m), two-sided Pearson correlation analysis (j), and two-way ANOVA with Sidak’s test (s). Exact P-values are indicated in the figure. Source data are provided as a Source Data file.

DLS measurement exhibited a hydrodynamic diameter of ~66 nm and a uniform size distribution of the PDPC@Cygal nanoparticles (Supplementary Fig. 45). The PDPC@Cygal nanoparticles were readily activated by β-Gal as a function of incubation time and β-gal concentration (Fig. 7c, d). Upon activation, the maximum absorption of the hemicyanine dye shifted from 600 to 690 nm (Fig. 7e). The limit of detection (LOD) of β-gal with the PDPC@Cygal nanoparticles was 6.8 × 10^‒3U/mL (Fig. 7f). Importantly, the fluorescence signal was unaffected by common biological interferents including biothiols (e.g., Cys, HCy, GSH), reactive oxygen species (e.g., H₂O₂, ClO^‒), and common metal ions (e.g., Na⁺, Mg²⁺, Ca²⁺), underscoring the high β-gal sensitivity and selectivity of the PDPC@Cygal nanotracer (Fig. 7g, h).

To evaluate the ability of the PDPC@Cygal nanoparticles for detecting senescent tumor cells, Panc02 tumor cells pretreated with RT were incubated with PDPC@Cygal (5 μM of Cygal) for 30 min. FCM analysis revealed an RT dose-dependent increase of intracellular fluorescence (Fig. 7i). CLSM examination revealed intracellular activation of the POLY-Tracker in lysosomes of senescent tumor cells (Supplementary Fig. 46). A linear correlation was found between p21 expression and β-gal level, further validating the potential of the β-gal-activatable POLY-Tracker for detecting RISTCs (Fig. 7j).

Achieving real-time monitoring of RISTCs in vivo, the PDPC@Cygal nanoparticles were i.v. administered to Panc02 tumor-bearing mice (Fig. 7k). Fluorescence images in vivo demonstrated specific activation of the PDPC@Cygal nanoparticles at the tumor site, with gradually increased fluorescence intensity post-RT (Fig. 7l–n), suggesting RT-induced senescence of tumor cells in vivo. This speculation was validated by CLSM examination of the tumor section ex vivo, by revealing colocalization of the fluorescence signal in p21⁺ senescent tumor cells (Fig. 7o, p).

To further explore the potential of the PDPC@Cygal nanoparticles for monitoring the therapeutic effect in vivo, the POLY-Tracker was i.v. injected into the Panc02 tumor-bearing mice at 5-days post fractionated RT, and 7 days post triplicate PDPS@BRD4d treatments (Fig. 7q). Fluorescence imaging indicated gradual accumulation of RISTCs in the tumor mass over time in the RT group (Fig. 7r). In contrast, the RT + PDPS@BRD4d group showed notable reduction of fluorescence intensity at 19-day post the first RT treatment, verifying effective elimination of RISTCs by the POLY-Senolytic nanoparticles (Fig. 7s). These results validated the capacity of the POLY-Tracker for real-time monitoring of senolytic therapy-potentiated RT.

Discussion

Previous studies have reported that senescent tumor cells elicit antitumor immunogenicity in the acute or early phase of senescence. Senescent tumor cells display upregulated immunogenic molecules (MHC-I, NKG2D ligands, and ICAM-1) and increased SASP secretion to induce a proinflammatory tumor microenvironment. The tumor-infiltrating CTLs were therefore recruited for tumor eradication^11,12. However, a recent study by Ruscetti et al. reported that therapy-induced senescence suppresses NK cell and T cell surveillance in the pancreatic tumor through EZH-mediated epigenetic repression of SASP genes¹³. Furthermore, chronic secretion of SASPs by senescent tumor cells recruits immunosuppressive cells such as regulatory T cells and myeloid-derived suppressor cells, thereby driving immune evasion and therapy resistance¹⁴. In this study, we demonstrated that RISTCs displayed elevated PD-L1 expression, thereby shifting the proinflammatory tumor microenvironment toward immunosuppression. Mechanistic studies identified that RT induced tumor cell senescence and elicited PD-L1 expression via the BRD4-JAK-STAT axis. We further observed that BRD4 activates SASP-associated signaling, particularly the IL-1β-JAK/STAT cascade, which collectively amplifies PD-L1 induction in RISTCs (Fig. 3q). Thus, BRD4 functions as an upstream hub to link senescence signaling to PD-L1 upregulation.

Recent advances in protein degradation technology, particularly PROTACs, have shown their potential as senolytic agents, surpassing traditional small-molecule inhibitors^26–28. However, existing PROTACs designed for senescent cell elimination exhibit poor pharmacokinetics and lack tumor specificity, raising concerns regarding systemic toxicity^29,30. While some studies have utilized nanoplatforms to deliver ABT-263 for senescent fibroblast clearance, these approaches often rely on physical encapsulation methods, which limit drug loading and increase the risk of drug leakage³¹.

Achieving tumor-specific eradication of RISTCs, we herein proposed a PROTAC-based senolytic strategy for the elimination of the senescent tumor cells. The POLY-Senolytic nanoplatform enabled precise delivery of the senolytic agents to the tumor lesion via the enhanced permeability and retention effect, and release BRD4 PROTAC via GSH-mediated cleavage of disulfide bonds. The POLY-Senolytic nanoparticles intervened the upstream of this signaling cascade by preventing both SASP-driven JAK/STAT activation and PD-L1 upregulation. Apart from inhibiting PD-L1 expression, BRD4 degradation with the POLY-Senolytic nanoparticles exerts broader immunomodulatory effects, including selectively induced apoptosis of RISTCs (Supplementary Fig. 30b, c), suppressed intratumoral secretion of immunosuppressive SASP factors such as IL-1α, IL-1β, and IL-6, and reduced intratumoral infiltration of Tregs and MDSCs (Fig. 3g, Supplementary Fig. 34a, 38a, b). A combination of fractionated RT and the POLY-Senolytic disrupted the immunosuppressive signaling axis and boosted the antitumor performance in multiple mouse models of orthotopic breast and pancreatic tumors.

The emerging advances of senolytic therapies have escalated the importance of in-situ monitoring of senescent cells in vivo^23,25. Several small-molecule fluorescent probes have been exploited to visualize senescent tumor cells by detecting β-Gal activity in vitro; however, the application of these probes in vivo was hindered by rapid metabolic clearance and insufficient tumor specificity^15,32. To this end, we further engineered a POLY-Senolytic nanotracer to monitor RT-induced senescence of tumor cells in vivo and provide feedback on the therapeutic efficacy. Together, this study revealed mechanistic insight into RT-induced tumor cell senescence and immune resistance, and provided a practical strategy for overcoming the immune resistance of solid tumors.

Despite the promising performance of POLY-Senolytic nanoparticles and the β-gal nanoprobe, their clinical translation is restricted by several concerns. First, despite the POLY-Senolytic nanoparticles more efficiently degrading BRD4 in RISTCs than in non-senescent tumor cells, RISTC-targeted PROTAC delivery remains to be exploited for the selective eradication of senescent tumor cells. Second, although we demonstrated a senescent tumor cell-activatable fluorescent POLY-Tracker for monitoring senolytic therapy in this study, the clinic-relevant imaging modalities, including magnetic resonance imaging and ultrasound imaging, would be preferred to enable imaging-guided precision senolytic therapy. Finally, the sequential combination treatment via RT + POLY-Senolytics has shown enhanced antitumor efficacy in mouse models; however, its translation potential remains to be investigated in larger animal models. Future endeavors could explore the development of β-gal-responsive bonds to boost senescent tumor cell-specific immune responses, as well as imaging-guided senolytic therapy.

In summary, our study demonstrates that RISTCs contribute to RT-induced immune resistance via the BRD4-associated signaling pathway. POLY-Senolytic nanoparticles-mediated BRD4 degradation in RISTCs effectively downregulated PD-L1 expression, and therefore restored CTLs' function and suppressed tumor progression. Furthermore, the RISTC-activatable POLY-Tracker enabled in-situ monitoring of senescent cell clearance. Collectively, this work highlights the potential of PROTAC-based senolytic strategy to overcome RT-induced immune resistance by targeted eradication of senescent tumor cells.

Methods

Ethics statements

This research complies with all relevant ethical regulations. All animal procedures were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (No. 2024-09-YHJ-10). Human pancreatic cancer tissue collection was approved by the Medical Ethics Committee of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine (No. XHEC-D-2025-026).

Reagents

Comprehensive details regarding commercial reagents can be found in the “Materials availability” section of the Supplementary Information. Supplementary Table 1 enumerates the antibody library employed in this study. Furthermore, extensive data on chemical synthesis and compound characterization are available in the Supplementary Information.

Cell lines and animals

Panc02 murine PDAC, 4T1 murine breast, B16-F10 murine melanoma, and SCC7 murine squamous carcinoma cells were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). Panc02-Luc and 4T1-Luc cells were purchased from Shanghai Model Organisms Center. Panc02, Panc02-Luc, 4T1, 4T1-Luc, B16-F10, and SCC7 cells were cultured with complete DMEM medium consisting 10% FBS and 1% Penicillin-Streptomycin in an atmosphere containing 5% CO₂ at 37 °C. All experiments were performed in the logarithmic phase of cell growth.

C57BL/6 mice (female, 4–5 weeks, 18 ~ 20 g) and Balb/c nude mice (female, 4–5 weeks, 18 ~ 20 g) were obtained from Shanghai Experimental Animal Center (Shanghai, China). Animals were housed under SPF conditions in groups of 5 mice per cage and maintained at a temperature of ~25 °C in a humidity-controlled environment with a 12 h light/dark cycle, with free access to standard food and water. All animal procedures were carried out under the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Fabrication and characterization of the PDPS@BRD4d, PDPC@BRD4d, PBPC@BRD4d, and PDPC@Cygal nanoparticles

mPEG₁₁₃-b-P(DPA₅₀-r-HESSMA₄-BRD4d₄) (2.0 mg) or mPEG₁₁₃-b-P(DPA₅₀-r-HEMA₅-BRD4d₄) (2.0 mg) or mPEG₁₁₃-b-P(BMA₅₀-r-HEMA₅-BRD4d₄) (2.0 mg) or mPEG₁₁₃-b-P(DPA₅₀-r-HEMA-Cygal₃) (2.0 mg) were dissolved in 100 μL of DM, and added dropwise into 900 μL of DI water under ultrasonication to obtain the PROTAC nanoparticles (termed PDPS@BRD4d, PDPC@BRD4d, and PBPC@ BRD4d nanoparticles, respectively). The organic solvent was removed by dialyzing against DI water. The size distribution and morphology of the nanoparticles were examined by dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern Instrument, UK) and transmission electron microscopy (TEM) measurements (Talos L120C, USA, 120 kV), respectively.

Acid responsiveness of PDPC@BRD4d and PBPC@BRD4d nanoparticles

To examine the acid-sensitivity of the PDPC@BRD4d and PBPC@BRD4d nanoparticles, they were incubated in buffers at pH 6.0 and 7.4. Subsequently, the hydrodynamic particle size and morphology were assessed using DLS and TEM examination, respectively. Additionally, to further explore the pKa of PDPC@BRD4d and PBPC@BRD4d nanoparticles, ICG was covalently attached to the polymer chain instead of BRD4d. Fluorescence spectrum analysis was conducted by incubating the nanoparticles in a series of buffer solutions with varying pHs (specifically pH 5.2, 5.8, 6.0, 6.2, 6.8, and 7.4). The fluorescence spectra of nanoparticles were recorded using a Cary Eclipse Spectrophotometer (USA).

Peptide PROTAC release from the PDPS@BRD4d nanoparticles in vitro

To evaluate the BRD4d release profile of the PDPS@BRD4d nanoparticle, the suspension of PDPS@BRD4d nanoparticles or PDPC@BRD4d nanoparticles was dialyzed against 1wt% Tween 80-containing buffer solution at 37 °C (MWCO 3500 Da; e.g., pH 6.0 without GSH, pH 6.0 + 10 mM GSH). BRD4d concentration in the buffer solution was examined by HPLC measurement at the predetermined time points (e.g., 0.5, 1.0, 2.0, 4.0, 8.0, 12, and 24 h).

Cellular uptake

To investigate the cellular uptake profile of the nanoparticles in vitro, Panc02 tumor cells were seeded in 6-well plates at a density of 3 × 10⁴ cells per well and incubated for 24 h. Subsequently, the cells were treated with PDPC@ICG and PBPC@ICG nanoparticles for 6 h. Following the treatment, the cells were stained with DAPI and Lysotracker Green and analyzed using confocal laser scanning microscopy (CLSM, Leica TCS-SP8 STED, Germany).

Western blot assay

To investigate PROTAC-induced protein degradation in vitro, Panc02 cells were seeded in 6-well plates at a density of 4 × 10⁵ cells per well and then incubated with free BRD4d or BRD4d-based nanoparticles at the predetermined conditions for 24 h. Following incubation, the cells were lysed using RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 nM NaCl, 1% NP-40, 0.1% SDS) supplemented with 1 mM of phenylmethanesulfonyl fluoride (PMSF). The protein lysate was centrifuged at 12000 g for 20 min at 4 °C. Then, the lysates were denatured at 100 °C and resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto PVDF membranes (Merck Millipore), which were then blocked using a 5% BSA buffer for 2 h at room temperature. After blocking, the membranes were incubated overnight at 4 °C with specific primary antibodies (anti-BRD4 antibody, 1:1000; anti-β-tubulin antibody, 1:1000; anti-GAPDH antibody, 1:1000; anti-PD-L1 antibody, 1:1000; anti-P21 antibody, 1:1000). Subsequently, the membranes were washed thrice with TBST buffer and incubated with corresponding secondary antibody (Peroxidase-Conjugated Goat Anti-Rabbit IgG (H + L), 1:5000 and Peroxidase-Conjugated Goat Anti-Mouse IgG (H + L), 1:5000) in 5% BSA solution for 2 h at room temperature. After several washes with TBST buffer, the membranes were imaged using the Bio-Rad Chemi Doc XRS imaging system.

SA–β-galactosidase staining

SA–β-gal staining was performed using a SA-β-gal staining kit (catalog no. 40754ES60, YEASEN, Shanghai, China) according to the manufacturer’s instructions. Briefly, Panc02, 4T1, SCC7, and B16-F10 cells were incubated in 6-well plates at a density of 2 × 10⁴ cells per well for 24 h, and then the irradiation groups were exposed to X-ray irradiation (8 Gy). 48 h later, the cells were washed with PBS and fixed with β-galactosidase staining fixative for 15 min. Following three washes with PBS, the cells were incubated in a specific staining solution at 37 °C overnight. The stained cells were then viewed and counted using a standard light microscope (Nikon ECLIPSE Ts2).

Extraction and culture of primary splenic CD8⁺ T lymphocytes

CD8⁺ T lymphocytes were positively isolated from mouse splenocytes using magnetic-activated cell sorting (MACS). Briefly, the single-cell suspension was incubated with anti-CD8 microbeads directly conjugated to monoclonal antibodies against the CD8 antigen. After incubation and washing to remove unbound beads, the cell suspension was loaded onto a MACS column placed within a magnetic field. The magnetically labeled CD8⁺ T cells were retained within the column. The column was then removed from the magnetic field, and the positively selected CD8⁺ T cells were eluted as the target fraction. The isolated cells were immediately ready for downstream applications.

RNA-sequencing analysis

Panc02 cells received RT or RT + BRD4d treatments, and the control counterparts were collected for RNA-sequencing analysis. Total RNA was extracted using TRIzol® Reagent, followed by genomic DNA removal with DNase I. RNA integrity and contamination were assessed by 1% agarose gel electrophoresis. RNA quality was verified using the 2100 Bioanalyzer (Agilent Technologies) and quantified with the ND-2000 (NanoDrop Technologies). High-quality RNA samples (OD260/280 = 1.8–2.2, OD260/230 ≥ 2.0, RIN ≥ 8.0, 28S:18S ≥ 1.0, and total RNA ≥ 1 μg) were used for library preparation.

RNA purification, reverse transcription, library construction, and sequencing were performed by Shanghai Major Bio-Pharm Biotechnology Co., Ltd. (Shanghai, China) following the manufacturer’s instructions (Illumina, San Diego, CA). Briefly, mRNA was enriched using oligo (dT) beads and fragmented. Double-stranded cDNA was synthesized with random hexamer primers, followed by end repair, adenylation, and adapter ligation. cDNA libraries of ~300 bp were selected and amplified by PCR. The resulting libraries were sequenced on an Illumina NovaSeq Xplus platform (paired-end mode). Gene expression levels were quantified using TPM (transcripts per million) and RSEM. Differential expression analysis was performed with DESeq2 or DEGseq, identifying significant DEGs under the thresholds of |log2FC| ≥ 1 and FDR ≤ 0.05 (DESeq2) or FDR ≤ 0.001 (DEGseq). Functional enrichment analysis for GO terms and KEGG pathways was conducted using Goatools and KOBAS, respectively, with a significance cutoff of Bonferroni-corrected P-value ≤ 0.05.

Establishment of BRD4 or IL-1R-knockdown Panc02 tumor cell line

For BRD4 knockdown, independent shRNA sequences targeting mouse Brd4 (shBRD4-1 and shBRD4-2) and a scrambled control (shNC) were cloned into the pLKO.1 vector. For IL-1R knockdown, independent shRNA sequences targeting mouse Il1r1 (shRNA-1, shRNA-2, and shRNA-3) and a scrambled control (shRNA-NC) were cloned into the pLKO.1 vector. Lentiviral particles were produced in HEK293T cells using psPAX2 and pMD2.G packaging plasmids (Addgene #12260 and #12259). Panc02 cells were infected at an MOI of 5 with 8 μg/mL polybrene, followed by puromycin selection (4 μg/mL, 7 days).

Ex vivo cultures of the PDAC patient-derived tumor tissues

Fresh tissue was sectioned into 300-500 μm slices using a Vibratome VT1200 (Leica). Tissue slices were cultured on 0.4 μm pore Teflon membrane inserts (Millipore; PICM0RG50) at 37 °C and 5% CO₂ in Ham’s F-12 medium supplemented with 20% FBS, antibiotics, and antifungals. At each time point, tissue slices were harvested for IF analysis.

Single-cell RNA-sequencing ex vivo

Tumors were dissociated into single-cell suspensions by enzymatic digestion (2% collagenase II, 0.1% Dispase, 0.1% Pancreatic Trypsin Inhibitor, and DNase I) at 37 °C. The suspensions were sequentially filtered through 70 μm and 30 μm strainers, followed by red blood cell lysis. Single-cell libraries were prepared using the 10× Chromium Platform (v2 chemistry) and sequenced on the DNBSEQ-T7. Raw sequencing data were processed with Fastp for quality control. CellRanger was used for alignment, barcode assignment, and count matrix generation. Downstream analysis was performed in Seurat: cells were filtered (≥200 genes, mitochondrial ratio <20%), normalized, and integrated with Harmony to remove batch effects. Cell clusters were identified via graph-based clustering and visualized with UMAP. Major cell types were annotated using established marker genes. Differential expression analysis was conducted with FindMarkers. Pathway enrichment was assessed by GSVA using MSigDB gene sets, and cell-cell communication was inferred with CellChat based on ligand-receptor interactions.

Monitoring of RISTCs with the POLY-Tracker (PDPC@Cygal) in vitro

To investigate the efficacy of the POLY-Tracker for detecting RISTCs in vitro, Panc02 cells were seeded in 6-well plates at a density of 1.2 × 10⁵ cells/well and cultured for 24 h. Subsequently, the cells were exposed to varying doses of radiation (0 Gy, 4 Gy, 8 Gy, 12 Gy). After 48 h, the cells were treated with the PDPC@Cygal nanoparticles at a concentration of 5 μM for 30 min. Following treatment, the cells were collected for FCM analysis (BD FACS Calibur, BD, USA).

To validate the ability of the BRD4 PROTAC nanoparticles (PDPS@BRD4d) to eliminate RISTCs, Panc02 cells were seeded in 6-well plates at a density of 1 × 10⁴ cells per well and received RT (8 Gy). The following day, the cells were treated with PDPS@BRD4d nanoparticles at a concentration of 1 μM. After 24 h, the cells were treated with PDPC@Cygal nanofluorescent probes for 30 min and collected for CLSM examination.

Monitoring RT-induced tumor senescence with the POLY-Senolytic nanotracer in vivo

To evaluate tumor senescence monitoring efficacy of the POLY-Senolytic nanotracer in vivo, we established a Panc02 mouse tumor model. Once the tumor volume reached approximately 100 mm³, the mice in the Day 1, Day 3, and Day 5 groups were subjected to RT at a dose of 8 Gy at the specified time points and all mice were intravenously (i.v.) injected with PDPC@Cygal probes at an identical Cygal dose of 2.5 mg/kg on the same day for fluorescence imaging by IVIS imaging system (Xenogen, Alameda, CA).

To assess the RISTCs elimination efficacy of the BRD4 PROTAC nanoparticles (PDPS@BRD4d) in vivo, mice were randomly divided into three groups (n = 3): PBS, RT, RT + PDPS@BRD4d. Three days after RT (2 Gy × 4), i.v. injections of PDPC@Cygal nanoparticles were administered to enable fluorescence imaging for the detection of RT-induced tumor senescence. Subsequently, PDPS@BRD4d nanoparticles were i.v. injected for three consecutive days. Following this, PDPC@Cygal nanoparticles were i.v. injected again at the designated time to monitor the clearance of RISTCs.

Biodistribution of the POLY-Senolytic nanoparticles in vivo

To investigate the biodistribution of POLY-Senolytic nanoparticles, a Panc02 tumor model of PDAC was established. The tumor-bearing mice were randomly grouped when the tumor volume reached ∼100 mm³ and intravenously (i.v.) injected with ICG-labeled nanoparticles at an identical ICG dose of 2.5 mg/kg. Then, fluorescence imaging was conducted using the IVIS imaging system at predetermined time points (Xenogen, Alameda, CA). The mice were euthanized at 48 h post-injection. The major organs and tumor tissue were harvested for fluorescence imaging ex vivo. Afterwards, the tumor tissues were fixed, sectioned, and stained with indicated antibodies and DAPI. The fluorescence signal of the tumor sections was measured by CLSM examination.

Pharmacokinetics and tumor distribution of the PDPS@BRD4d nanoparticles in vivo

Free BRD4d or PDPS@BRD4d nanoparticles were i.v. injected into Panc02 tumor-bearing mice at an identical BRD4d dose of 10 mg/kg (n = 3). Blood samples and tumor tissues were collected at the indicated time points post-injection. Blood samples and tumor homogenates were incubated with GSH to release BRD4d, followed by methanol extraction. The BRD4d concentration was examined by HPLC measurement.

Antitumor performance of the POLY-Senolytic nanoparticles in vivo

To investigate the antitumor performance of the POLY-Senolytic nanoparticles (PDPS@BRD4d), a Panc02 tumor model of PDAC was established. When the tumor volume reached around 100 mm³, the tumor-bearing mice were randomly divided into six groups (n = 6): PBS, PDPS@BRD4d, RT + PBS, RT + BRD4d, RT + PDPS@BRD4d, or RT + αPD-L1, at an identical BRD4d and αPD-L1 dose of 10 mg/kg and 5 mg/kg, respectively. The tumors in the RT + PBS, RT + BRD4d, RT + PDPS@BRD4d, or RT + αPD-L1 groups were locally irradiated at a dose of 2 × 4 Gy and i.v. injected with BRD4d or PDPS@BRD4d or intraperitoneally injected with αPD-L1 three times following RT. Body weight and tumor volume were monitored every three days during the observation period. The tumor volume was calculated as half the product of the longest dimension and the square of the shortest dimension.

The mice were considered dead when their tumor volume exceeded 1200 mm³ according to the animal ethics of our institute. The tumor tissues were harvested at the end of the antitumor study, fixed in 4% formalin solution, dehydrated, and subjected to H&E staining. BRD4 degradation in the tumor tissue was validated by western blot assay and IHC. The expression of PD-L1 and P21 was validated by Western blot, FCM, and IF analysis.

Antitumor immunity evaluation

To evaluate the effects of PDPS@BRD4d on antitumor immunity, tdLNs and spleens of the tumor-bearing mice were harvested from the tumor-bearing mice. Tumor tissues were cut into small pieces after weighing, then suspended in RPMI 1640 with indicated digestive enzymes and dissociated by the gentle MASC^TM dissociator (Miltenyi, Germany). The single-cell suspension was obtained by filtering through 70 µm filters. The tumor-infiltrating CD3⁺ T cells, CD8⁺ T cells, CD4⁺ T cells, Tregs, and IFN-γ⁺CD8⁺ T cells were analyzed by FCM. To evaluate the central memory T lymphocytes (CD45⁺CD3⁺CD8⁺CD44⁺CD62L⁺) and the effector memory T lymphocytes (CD45⁺CD3⁺CD8⁺CD44⁺CD62L^‒) in vivo, the spleens of the Panc02 tumor-bearing mice were harvested 7 days post the last treatment and processed into single-cell suspensions, and then stained with anti-CD45-PE/FITC, anti-CD3-PerCP-Cy5.5, anti-CD4-APC-Cy7, anti-CD8-PE, anti-CD44-APC, anti-CD62L-BV-421, anti-CD25-APC, and anti-FOXP3-PE for FCM examination (n = 6 mice).

Biosafety assay in vivo

To investigate the biosafety of PDPS@BRD4d nanoparticles, the mice were randomly divided into two groups (n = 3) and then i.v. injected with PBS or PDPS@BRD4d ([BRD4d] = 10 mg/kg) for three times. After treatments, serum biochemistry analysis and HE staining of the major organs (heart, liver, spleen, lung, and kidney) were performed to examine biosafety.

Statistics and reproducibility

All data are presented as mean ± SD. Statistical analyses were performed using GraphPad Prism 10.0. A two-tailed Student’s t-test was used for comparisons between two groups. One-way analysis of variance (ANOVA) with Tukey’s post hoc test was used for comparisons among multiple groups with one variable. Two-way ANOVA with Tukey’s multiple-comparison test or Sidak’s multiple-comparison test was used, as appropriate, for comparisons among groups with two variables. Survival was analyzed using the two-sided log-rank (Mantel–Cox) test. Cell-based experiments were independently repeated three times with similar results, and representative images or blots are shown. For in vivo histology and immunofluorescence analyses, representative images from biologically independent mice are shown as indicated in the corresponding figure legends. No statistical method was used to predetermine sample size. No data were excluded from the analyses. Animals were randomly assigned to treatment groups where indicated. Investigators were not blinded to allocation during experiments or outcome assessment.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information^{(13.9MB, pdf)}

Reporting Summary^{(4.9MB, pdf)}

Transparent Peer Review file^{(9.2MB, pdf)}

Source data

Source Data^{(3MB, xlsx)}

Acknowledgements

This work was supported by the New Organizational Model Initiative (2025ZD0552300 to H.Y.), National Natural Science Foundation of China (32571707 to Y.L., 22474042 to Z.X., U22A20328 and W2412035 to H.Y.), Science and Technology Commission of Shanghai Municipality (23490712700 to Z.X., 23ZR1475000 and 20430711800 to H.Y.), Shanghai Oriental Talents Program (BJKJ2024046 to Z.X.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB1060000 to H.Y.), and State Key Laboratory of Chemical Biology. The Mass Spectrometry System and the cell sorter BD Influx of the National Facility for Protein Science in Shanghai (NFPS), Shanghai Advanced Research Institute, CAS, are gratefully acknowledged.

Author contributions

Y.L., S.Z., H.Y. and Z.X. conceived the project. H.Y. and Z.X. supervised the study. Y.L., S.Z. and J.P. designed the study, analyzed the data, and wrote the manuscript. W.L., M.L. and Z.Z. assisted with the experiments. L.X. provided pancreatic cancer patient samples. B.G.D.G., T.L., W.Z., H.Y. and Z.X. revised the manuscript.

Peer review

Peer review information

Nature Communications thanks David Gewirtz, REYAZ RASOOL, and the other anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

Data availability

The data supporting the findings of this study are available within the Article, the accompanying Source Data file, and the Supplementary Information. The single-cell RNA-sequencing data generated in this study have been deposited in the Genome Sequence Archive of the China National Center for Bioinformation under accession code PRJCA036335. The RNA-seq data generated in this study have been deposited in the NCBI Sequence Read Archive under accession code PRJNA1414145 [https://www.ncbi.nlm.nih.gov/bioproject/1414145]. Publicly available TCGA datasets analyzed in this study were obtained from the Genomic Data Commons (GDC) Data Portal. The specific projects used were TCGA-PAAD (Pancreatic Adenocarcinoma; https://portal.gdc.cancer.gov/projects/TCGA-PAAD), TCGA-BRCA (Breast Invasive Carcinoma; https://portal.gdc.cancer.gov/projects/TCGA-BRCA), TCGA-HNSC (Head and Neck Squamous Cell Carcinoma; https://portal.gdc.cancer.gov/projects/TCGA-HNSC), and TCGA-SKCM (Skin Cutaneous Melanoma; https://portal.gdc.cancer.gov/projects/TCGA-SKCM). Source data are provided with this paper.

Materials availability

Unique reagents generated in this study are available from the corresponding authors upon reasonable request.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Yi Lai, Shunan Zhang, Jiaxing Pan.

Contributor Information

Haijun Yu, Email: hjyu@simm.ac.cn.

Zhiai Xu, Email: zaxu@chem.ecnu.edu.cn.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-026-73109-w.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information^{(13.9MB, pdf)}

Reporting Summary^{(4.9MB, pdf)}

Transparent Peer Review file^{(9.2MB, pdf)}

Source Data^{(3MB, xlsx)}

Data Availability Statement

Unique reagents generated in this study are available from the corresponding authors upon reasonable request.

[CR1] 1.Buckley, A. M., Lynam-Lennon, N., O’Neill, H. & O’Sullivan, J. Targeting hallmarks of cancer to enhance radiosensitivity in gastrointestinal cancers. Nat. Rev. Gastroenterol. Hepatol.17, 298–313 (2020). [DOI] [PubMed] [Google Scholar]

[CR2] 2.Nishiga, Y. et al. Radiotherapy in combination with CD47 blockade elicits a macrophage-mediated abscopal effect. Nat. Cancer3, 1351–1366 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR3] 3.Khor, R. C. et al. Tolerability and outcomes of curative radiotherapy in patients aged 85 or more years. Med. J. Aust.202, 153–155 (2015). [DOI] [PubMed] [Google Scholar]

[CR4] 4.Tabasso, A. F. S., Jones, D. J. L., Jones, G. D. D. & Macip, S. Radiotherapy-induced senescence and its effects on responses to treatment. Clin. Oncol.31, 283–289 (2019). [DOI] [PubMed] [Google Scholar]

[CR5] 5.Groelly, F. J., Fawkes, M., Dagg, R. A., Blackford, A. N. & Tarsounas, M. Targeting DNA damage response pathways in cancer. Nat. Rev. Cancer23, 78–94 (2023). [DOI] [PubMed] [Google Scholar]

[CR6] 6.Lynch, C., Pitroda, S. P. & Weichselbaum, R. R. Radiotherapy, immunity, and immune checkpoint inhibitors. Lancet Oncol.25, e352–e362 (2024). [DOI] [PubMed] [Google Scholar]

[CR7] 7.Herrera, F. G., Bourhis, J. & Coukos, G. Radiotherapy combination opportunities leveraging immunity for the next oncology practice. CA Cancer J. Clin.67, 65–85 (2017). [DOI] [PubMed] [Google Scholar]

[CR8] 8.Cytlak, U. M. et al. Immunomodulation by radiotherapy in tumour control and normal tissue toxicity. Nat. Rev. Immunol.22, 124–138 (2022). [DOI] [PubMed] [Google Scholar]

[CR9] 9.Zhai, D., An, D., Wan, C. & Yang, K. Radiotherapy: brightness and darkness in the era of immunotherapy. Transl. Oncol.19, 101366 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR10] 10.Brooks, E. D. & Chang, J. Y. Time to abandon single-site irradiation for inducing abscopal effects. Nat. Rev. Clin. Oncol.16, 123–135 (2019). [DOI] [PubMed] [Google Scholar]

[CR11] 11.Prata, L., Ovsyannikova, I. G., Tchkonia, T. & Kirkland, J. L. Senescent cell clearance by the immune system: emerging therapeutic opportunities. Semin. Immunol.40, 101275 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR12] 12.Marin, I. et al. Cellular senescence is immunogenic and promotes antitumor immunity. Cancer Discov.13, 410–431 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.Chibaya, L. et al. EZH2 inhibition remodels the inflammatory senescence-associated secretory phenotype to potentiate pancreatic cancer immune surveillance. Nat. Cancer4, 872–892 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Wang, H. et al. Immunoregulatory mechanisms in the aging microenvironment: targeting the senescence-associated secretory phenotype for cancer immunotherapy. Acta Pharm. Sin. B.15, 4476–4496 (2025). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR15] 15.Lozano-Torres, B. et al. Real-time in vivo detection of cellular senescence through the controlled release of the NIR fluorescent dye Nile blue. Angew. Chem. Int. Ed.59, 15152–15156 (2020). [DOI] [PubMed] [Google Scholar]

[CR16] 16.Li, F. et al. Dual-targeting prodrug nanotheranostics for NIR-Ⅱ fluorescence imaging-guided photo-immunotherapy of glioblastoma. Acta Pharm. Sin. B.12, 3486–3497 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Nicolas, A. M. et al. Inflammatory fibroblasts mediate resistance to neoadjuvant therapy in rectal cancer. Cancer Cell40, 168–184.e13 (2022). [DOI] [PubMed] [Google Scholar]

[CR18] 18.Zhang, S. et al. PROTAC prodrug-integrated nanosensitizer for potentiating radiation therapy of cancer. Adv. Mater.36, e2314132 (2024). [DOI] [PubMed] [Google Scholar]

[CR19] 19.Wang, J. et al. BRD4-IRF1 axis regulates chemoradiotherapy-induced PD-L1 expression and immune evasion in non-small cell lung cancer. Clin. Transl. Med.12, e718 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR20] 20.Zhu, H. et al. BET bromodomain inhibition promotes anti-tumor immunity by suppressing PD-L1 expression. Cell Rep.16, 2829–2837 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.Yamaguchi, H., Hsu, J. M., Yang, W. H. & Hung, M. C. Mechanisms regulating PD-L1 expression in cancers and associated opportunities for novel small-molecule therapeutics. Nat. Rev. Clin. Oncol.19, 287–305 (2022). [DOI] [PubMed] [Google Scholar]

[CR22] 22.Wang, T. et al. Emerging prodrug and nano-drug delivery strategies for the detection and elimination of senescent tumor cells. Biomaterials318, 123129 (2025). [DOI] [PubMed] [Google Scholar]

[CR23] 23.Garcia-Fleitas, J. et al. Chemical strategies for the detection and elimination of senescent cells. Acc. Chem. Res.57, 1238–1253 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Lee, S. & Schmitt, C. A. The dynamic nature of senescence in cancer. Nat. Cell Biol.21, 94–101 (2019). [DOI] [PubMed] [Google Scholar]

[CR25] 25.Gao, Y. et al. Two-dimensional design strategy to construct smart fluorescent probes for the precise tracking of senescence. Angew. Chem. Int. Ed.60, 10756–10765 (2021). [DOI] [PubMed] [Google Scholar]

[CR26] 26.Chang, J. et al. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Nat. Med.22, 78–83 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR27] 27.Wakita, M. et al. A BET family protein degrader provokes senolysis by targeting NHEJ and autophagy in senescent cells. Nat. Commun.11, 1935 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR28] 28.He, Y. et al. Using proteolysis-targeting chimera technology to reduce navitoclax platelet toxicity and improve its senolytic activity. Nat. Commun.11, 1996 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR29] 29.Gao, J. et al. Stimuli-activatable PROTACs for precise protein degradation and cancer therapy. Sci. Bull.68, 1069–1085 (2023). [DOI] [PubMed] [Google Scholar]

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POLY-Senolytic nanoplatform for tumor-specific eradication of senescent tumor cells and mitigation of radiotherapy-induced immune resistance of cancer

Yi Lai

Shunan Zhang

Jiaxing Pan

Wenyue Lan

Min Li

Zifan Zhu

Leiming Xu

Bruno G De Geest

Twan Lammers

Wen Zhang

Haijun Yu

Zhiai Xu

Abstract

Introduction

Fig. 1. Schematic illustration of the POLY-Senolytic and POLY-Tracker nanoparticles for eliminating and monitoring RT-induced senescent tumor cells (RISTCs).

Results

RISTCs drive an immunosuppressive tumor microenvironment via PD-L1 upregulation and SASP secretion

Fig. 2. PD-L1-positive RISTCs account for RT-induced immune resistance.

BRD4 degradation with a peptide PROTAC abolished RT-induced PD-L1 upregulation in RISTCs and potentiated radioimmunotherapy in vivo

Fig. 3. BRD4 degradation abolished RT-induced PD-L1 expression on RISTCs and boosted RT efficacy of PDAC tumors in vivo.

The POLY-Senolytic nanoparticles suppressed PD-L1 expression in RISTCs in vitro

Fig. 4. The acid and GSH dual-responsive POLY-Senolytic nanoparticles suppressed PD-L1 expression in RISTCs in vitro and achieved tumor-specific delivery of BRD4d in vivo.

The POLY-Senolytic nanoparticles specifically delivered the BRD4d into the tumor in vivo

The POLY-Senolytic nanoparticles suppressed tumor growth and mitigated RT-induced immune evasion by promoting immune eradication of RISTCs in vivo

Fig. 5. Antitumor performance of the PDPS@BRD4d POLY-Senolytic nanoparticles in a subcutaneous PDAC tumor model.

The POLY-Senolytic nanoparticles elicited robust antitumor immunity in mouse models of orthotopic pancreatic and breast tumors in vivo

Fig. 6. Antitumor effects of the POLY-Senolytic nanoparticles in mouse models of orthotopic pancreatic and breast tumors.

RISTC-activatable POLY-Trackers performed real-time and in-situ evaluation of POLY-Senolytic-potentiated RT of cancer

Fig. 7. Engineering of acid-responsive POLY-Trackers for real-time and in-situ monitoring of senolytic therapy.

Discussion

Methods

Ethics statements

Reagents

Cell lines and animals

Fabrication and characterization of the PDPS@BRD4d, PDPC@BRD4d, PBPC@BRD4d, and PDPC@Cygal nanoparticles

Acid responsiveness of PDPC@BRD4d and PBPC@BRD4d nanoparticles

Peptide PROTAC release from the PDPS@BRD4d nanoparticles in vitro

Cellular uptake

Western blot assay

SA–β-galactosidase staining

Extraction and culture of primary splenic CD8+ T lymphocytes

RNA-sequencing analysis

Establishment of BRD4 or IL-1R-knockdown Panc02 tumor cell line

Ex vivo cultures of the PDAC patient-derived tumor tissues

Single-cell RNA-sequencing ex vivo

Monitoring of RISTCs with the POLY-Tracker (PDPC@Cygal) in vitro

Monitoring RT-induced tumor senescence with the POLY-Senolytic nanotracer in vivo

Biodistribution of the POLY-Senolytic nanoparticles in vivo

Pharmacokinetics and tumor distribution of the PDPS@BRD4d nanoparticles in vivo

Antitumor performance of the POLY-Senolytic nanoparticles in vivo

Antitumor immunity evaluation

Biosafety assay in vivo

Statistics and reproducibility

Reporting summary

Supplementary information

Source data

Acknowledgements

Author contributions

Peer review

Peer review information

Data availability

Materials availability

Competing interests

Footnotes

Contributor Information

Supplementary information

References

Associated Data

Supplementary Materials

Data Availability Statement

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Extraction and culture of primary splenic CD8⁺ T lymphocytes