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. 2026 Sep 22;6(10):5734–5743. doi: 10.1021/jacsau.6c01028

A Dual-Locked β‑Galactosidase/pH Near-Infrared Sensor Enables High-Fidelity Monitoring of Therapy-Induced Senescence

Lele Ding 1, Yuxuan Zhang 1, Na Wang 1, Zijian Li 1, Lijun Jia 1,*
PMCID: PMC13714839  PMID: 42953165

Abstract

Therapy-induced senescence is increasingly recognized as a critical determinant of chemotherapy efficacy, tissue toxicity, and senolytic intervention. However, conventional senescence-associated β-galactosidase (SA-β-Gal) assays and single β-Gal-responsive probes often suffer from limited specificity because elevated β-Gal activity is not exclusive to senescent cells. Here, we report ECD-Gal, a dual-locked near-infrared fluorescent sensor that integrates β-Gal enzymatic activity with lysosomal pH variation for high-fidelity sensing therapy-induced senescence. ECD-Gal shows weak emission in β-Gal positive cells with an acidic lysosomal microenvironment but is strongly activated in senescent cells where β-Gal accumulation coincides with lysosomal alkalinization. Mechanistic studies using agents that induced lysosomal acidification or lysosomal alkalinization supported that both β-Gal activity and elevated lysosomal pH are required for fluorescence activation. ECD-Gal enabled confocal imaging, flow-cytometric quantification, in vivo visualization of doxorubicin or bleomycin-induced senescence, and its fluorescence readout correlated with SA-β-Gal staining, senescence marker expression, and fibrosis-associated pathological remodeling. This dual-locked sensing strategy provides a robust molecular tool for monitoring therapy-induced senescence and evaluating senolytic interventions in living systems.

Keywords: therapy-induced senescence, dual-locked, senescence-associated β-galactosidase, lysosomal pH, fluorescent sensor

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Introduction

Cellular senescence is a stable cell-cycle arrest state induced by diverse stressors, including DNA damage, oxidative stress, telomere shortening, and therapeutic intervention. , Although transient senescence plays important roles in tissue remodeling and injury repair, persistent accumulation of senescent cells can promote a pathological senescence-associated microenvironment through continuous secretion of the senescence-associated secretory phenotype (SASP). SASP comprises a broad spectrum of cytokines, chemokines, and proteases, which drive inflammatory responses, tissue dysfunction, and the onset and progression of multiple age-related diseases. , Chemotherapeutic agents are widely used in cancer treatment, which can cause therapy-induced senescence (TIS) in both tumor and normal cells while eliminating malignant tumor cells. Senescent tumor cells can promote therapeutic resistance and relapse, whereas senescent normal cells contribute to treatment-related toxicities. Therefore, senolytics, which can selectively eliminate senescent cells, represents a promising strategy to enhance chemotherapy efficacy and reduce adverse effects. − To support the development and evaluation of such interventions, high-fidelity molecular tools that enable real-time, precise visualization of TIS in live organisms are indispensable for deciphering senescence-related biological processes and assessing the efficacy of senolytics.

Senescence-associated β-galactosidase (SA-β-Gal) is one of the most widely used biomarkers for identifying senescent cells. , Conventional SA-β-Gal staining has been extensively applied to cultured cells and tissue sections, but such a method is labor-intensive, time-consuming, end point-based, and poorly compatible with living cells or in vivo real-time monitoring. − Fluorescent sensors responsive to β-Gal activity have been developed for detecting senescent cells in vitro and in vivo. Nevertheless, these probes that rely solely on β-Gal activity can suffer from limited specificity. , The primary challenge in developing fluorescent probes for senescent cells detection is not simply detecting β-Gal activity but distinguishing senescence-associated β-Gal from nonsenescent β-Gal activity in complex biological systems. Elevated β-Gal activity is not unique to senescent cells and can also be observed in certain tumor cells, such as SKOV-3 cells, potentially resulting in false-positive fluorescence signals. − Therefore, a more reliable senescence-sensing strategy should integrate β-Gal activity with another senescence-associated feature to minimize false-positive fluorescence signals.

Cellular senescence is accompanied not only by the accumulation of β-Gal but also by extensive lysosomal remodeling and altered lysosomal acidity. In normal cells, lysosomal pH is maintained within an acidic range (pH 4.5–5.0) by vacuolar-type H+-ATPases (V-ATPases), which pump protons into the lysosomal lumen through ATP hydrolysis. During senescence, lysosomal remodeling accompanied by reduced V-ATPases activity and lysosomal pH shift to a mildly acidic state (pH 6.0). In contrast, β-Gal-positive nonsenescent tumor cells may retain a relatively acidic lysosomal environment. − This difference provides a rational basis for integrating lysosomal pH as a second microenvironmental input to improve the accuracy of β-Gal-based senescence sensing. In this dual-locked strategy, β-Gal-mediated cleavage served as the enzymatic lock, whereas lysosomal pH elevation functions as a microenvironmental lock. Only cells containing both elevated β-Gal activity and senescence-associated lysosomal alkalinization can generate a strong fluorescence output. ,

pH-sensitive sensors are widely applied to enable direct visualization and quantification of the dynamic pH changes in varying biological systems with spatiotemporal precision. Coumarin-based fluorophores have attracted broad interest in fluorescence sensing and bioimaging because of their favorable biocompatibility, bright and stable emission, and high structural tunability. Notably, the photophysical properties of coumarin derivatives can be modulated by protonation–deprotonation equilibria and intramolecular charge transfer, making them ideal scaffolds for pH-sensitive fluorescent sensors. − By coupling coumarin units with hemicyanine-type electron-accepting structures, it could extend absorption and emission wavelengths into the near-infrared region, affording long wavelength pH-responsive fluorophores for biological imaging.

Herein, we reported (E)-3-ethyl-1,1-dimethyl-2-(2-(2-oxo-7-((2,3,4,5-tetrahydroxycyclohexyl) oxy)-2H-chromen-3-yl) vinyl)-1H-benzo­[e]­indol-3-ium iodide (ECD-Gal), a β-Gal/pH dual-locked near-infrared fluorescent sensor for high-fidelity sensing of TIS in vitro and in vivo. ECD-Gal was designed not as a conventional β-Gal probe but as a lysosomal microenvironment-gated β-Gal sensor that requires two concurrent senescence-associated inputs for fluorescence activation (Scheme ). We first synthesized a series of coumarin-based fluorophores and evaluated their pH-responsive photophysical properties. Among them, (E)-3-ethyl-2-(2-(7-hydroxy-2-oxo-2H-chromen-3-yl) vinyl)-1,1-dimethyl-1H-benzo­[e]­indol-3-ium iodide (ECD) exhibited a pH-response window within pH 4.0–6.0, which closely matches the lysosomal pH transition associated with cellular senescence. Then, ECD-Gal was constructed by introducing a β-galactoside trigger onto the phenolic hydroxyl group of ECD. Upon β-Gal-mediated cleavage, the pH-responsive fluorophore ECD is released, and its fluorescence is further activated under the mildly acidic lysosomal microenvironment of senescent cells. With this dual-locked design, ECD-Gal enables discrimination of senescent cells from normal cells and β-Gal-positive nonsenescent tumor cells and supports live-cell imaging, flow-cytometric detection, and in vivo visualization of therapy-induced senescence and senolytic response.

1. Schematic Illustration of the Precise Differentiation between Senescent Cells and Normal Cells by ECD-Gal.

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Results and Discussion

Screening of ECD as a Senescence-Matched pH-Responsive Sensor

To obtain a suitable sensor for lysosomal microenvironment-gated senescence sensing, several coumarin-based sensors were synthesized using 3-formyl-7-hydroxycoumarin as the starting material through Knoevenagel condensation (Schemes S1 and S2). Their pH-responsive photophysical properties were systematically evaluated by recording UV–vis absorption and fluorescence emission spectra under different pH conditions (pH 3.0 and 6.0). The spectroscopic results showed that fluorophores containing a coumarin-hemicyanine hybrid structure exhibited pronounced pH-dependent spectral changes. Compared with fluorophores lacking the hemicyanine motif, the coumarin-hemicyanine derivatives displayed an evident red shift in absorption and stronger fluorescence enhancement within the tested pH range. In contrast, fluorophores without the hemicyanine structure showed significant fluorescence enhancement at pH values above 6.0, which is less suitable for monitoring the lysosomal pH transition associated with cellular senescence (Figures , S1 and S2).

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The response capability of ECD toward pH. (a) pH-dependent naked-eye visible changes of ECD. (b) Absorption spectra of ECD (10 μM) at different pH values. (c) Fluorescence spectra changes of ECD (10 μM) at different pH values. (d) Normalized pH-dependent fluorescence intensity changes of ECD at 640 nm. (e) The ability of ECD to resist interference from various ions (λex/em = 560/640 nm).

The above results suggested that the coumarin-hemicyanine hybrid system provides a pH response window that closely matches the lysosomal pH transition from acidic (pH 4.5–5.0) to mildly acidic conditions (pH 6.0) during cellular senescence, making them an ideal scaffold for constructing β-Gal/pH dual-locked sensors. Among these candidates, the fluorophore ECD displayed a characteristic pH-dependent response, its maximum absorption wavelength red-shifted from 470 nm at pH 3.0 to 586 nm at pH 6.0 (Figure b). Meanwhile, its fluorescence intensity at 640 nm gradually increased within the pH range of 4.0–6.0 and reached a plateau at pH values above 6.0 (Figure c,d). Notably, this fluorescence signal remained stable in the presence of various biologically relevant ions, indicating an excellent anti-interference capability (Figure e). Based on these results, ECD was selected as the fluorophore core for subsequent sensor development.

Design and In Vitro β-Gal Activation of ECD-Gal

After identifying ECD as the fluorophore core, ECD-Gal was synthesized by appending a β-galactoside moiety to the phenolic hydroxyl group of ECD. This glycosylation strategy was applied to mask the pH-responsive ability of ECD until the enzymatic cleavage of β-galactoside moiety by β-Gal (Scheme ). The enzymatic responsiveness of ECD-Gal was first evaluated using commercially available E. coli β-galactosidase as a model enzyme. Upon incubation with β-Gal under physiological conditions (pH 7.4, 37 °C), the fluorescence intensity of ECD-Gal at 640 nm gradually increased over time (Figure a,b), indicating β-Gal-triggered activation of the sensor. The fluorescence response also increased with β-Gal concentration and showed a robust linear relationship (R 2 > 0.95) within the tested concentration range (Figure c,d). This result suggests that ECD-Gal provides a concentration-dependent near-infrared fluorescence readout for β-Gal activity. Moreover, the enzymatic kinetics assay was conducted and the kinetics parameters including K m and V max were calculated to be 203.4 μmol/L and 0.6432 nmol/min/U β-Gal, respectively (Figure S3). Then, the selectivity of ECD-Gal was further evaluated against various biologically relevant ions, analytes, and enzymes. Incubation of ECD-Gal with potential interfering species produced negligible fluorescence enhancement, and with acid phosphatase producing weak fluorescence enhancement, whereas a pronounced increase in the fluorescence intensity at 640 nm was observed in the presence of β-Gal (Figures and S4).

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The response capability of ECD-Gal toward β-Gal. (a) Time-dependent and (c) concentration-dependent fluorescence spectral changes of ECD-Gal (10 μM) upon incubation with β-Gal and the corresponding calibration curves were shown in (b) and (d), respectively. (e) Fluorescence spectral responses of ECD-Gal (10 μM) in the presence of various ions and (f) the corresponding fluorescence intensity changes. (g) Fluorescence spectra of ECD-Gal (10 μM) incubated with β-Gal in PBS buffers at different pH values at 37 °C for 30 min. (h) Heatmap analysis of fluorescence intensities at 640 nm for β-Gal-treated ECD-Gal, ECD-Gal alone, and ECD across different pH values (λex/em = 560/640 nm).

Moreover, we further evaluated the cooperative effect of pH and β-Gal activity on the fluorescence response of ECD-Gal. In the absence of β-Gal, ECD-Gal showed a negligible fluorescence variation in PBS buffers (pH 4.0–7.4) after incubation at 37 °C for 30 min, confirming that the caged sensor is minimally affected by pH alone. In contrast, the fluorescence of the liberated fluorophore ECD increased with rising pH and reached a plateau at approximately pH 6.0 (Figure S5). Upon β-Gal treatment, ECD-Gal exhibited a distinct pH-dependent fluorescence profile, with the signal increasing from pH 4.0 to 6.0 and decreasing at pH values above 6.0 (Figure g,h). The above results suggest that the fluorescence of ECD-Gal is governed by both β-Gal-mediated cleavage and the pH variation in a microenvironment. Importantly, the enhanced fluorescence response in the weakly acidic range matches the lysosomal pH shift during cellular senescence. Collectively, these results highlight the potential of ECD-Gal for accurate senescence imaging.

Confocal Imaging of Therapy-Induced Senescent Cells

Prior to live-cell imaging, the cytotoxicity of ECD-Gal was evaluated using a CCK-8 assay. As shown in Figure S6, cell viability exceeded 90% after 24 h incubation with ECD-Gal at concentrations up to 40 μM, indicating low cytotoxicity and suitability for live-cell imaging.

Therapy-induced senescence can be elicited by diverse pharmacological stressors and is intimately linked to cancer treatment outcomes and chemotherapy-induced side effects. To evaluate whether ECD-Gal can detect senescence induced by different therapeutic agents, A549 cells were pretreated with etoposide, alisertib, MLN4924, bleomycin (BLM), or doxorubicin (DOX) to establish a cell senescence model. − In all tested models, ECD-Gal elicited a robust fluorescence turn-on response in senescent cells (Figure S7). Collectively, these results suggested that ECD-Gal can image senescent cells induced by multiple pharmacological stimuli.

Lysosomal pH Gating Distinguishes SA-β-Gal from Tumor-Associated β-Gal

To confirm whether ECD-Gal functions as a β-Gal/pH dual-locked sensor in living cells, we next examined the contribution of lysosomal pH to fluorescence activation. A DOX-induced senescence model was first established. DOX-induced senescent cells showed strong fluorescence after incubation with ECD-Gal, consistent with the concurrent presence of elevated SA-β-Gal activity and senescence-associated lysosomal pH elevation. Concomitantly, these cells exhibited typical senescence-associated morphological changes, including enlarged cell size, flattened or rounded morphology, and multinucleation. Subsequently, EN6, which has been reported to covalently target the ATP6 V1A subunit of lysosomal V-ATPase and enhance lysosomal acidification, was employed to modulate lysosomal pH in senescent cells. Upon cotreatment of DOX-induced senescent cells with EN6 (50 μM) for 4 h, the fluorescence signal of ECD-Gal was significantly attenuated (Figure a,c). This finding indicates that lysosomal pH is a critical determinant of ECD-Gal fluorescence activation in senescent cells.

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Response of ECD-Gal toward lysosomal β-Gal and pH in living cells. (a) ECD-Gal was activated in DOX-induced senescent A549 cells and treatment with EN6 (a lysosomal acidification agent, 50 μM, 4 h) could significantly weaken the cellular fluorescence signal, and corresponding to statistical data (n = 6) of fluorescence intensities (c). (b) ECD-Gal is activated in SKOV-3 cells but exhibited weak fluorescence, while enhanced fluorescence could be observed in DOX-induced senescent cells or chloroquine (a lysosomal alkalinizing agent, 5 μM, 4 h) pretreated cells, and corresponding to statistical data (n = 6) of fluorescence intensities (d). Statistical significance was determined by the Student’s t-test (two-tailed): *P < 0.05, ***P < 0.001, (λex = 560 nm, emission window: 610–660 nm).

To further elucidate the role of lysosomal pH in fluorescence activation of ECD-Gal, SKOV-3 cells were employed as a β-Gal-positive but nonsenescent control because these cells exhibit high β-Gal expression while maintaining an acidic lysosomal microenvironment (Figure S8). After incubation with ECD-Gal, only a weak fluorescence signal was observed in SKOV-3 cells while a bright fluorescence signal was observed in DOX-induced senescent cells. This finding suggests that β-Gal activity alone is insufficient to generate a strong fluorescence signal when the lysosomal microenvironment remains acidic. Chloroquine, which was accumulated in the lysosomal compartment and induces an increase in lysosomal pH, was then used to increase lysosomal pH in SKOV-3 cells. Notably, SKOV-3 cells pretreated with chloroquine (5 μM, 4 h) exhibited a bright fluorescence signal after incubation with ECD-Gal (Figure b,d). Coupled with the EN6 experiment, these results demonstrate that lysosomal alkalinization is required as a second lock for robust ECD-Gal activation. Therefore, ECD-Gal does not merely report β-Gal abundance but functions as a lysosomal microenvironment-gated β-Gal sensor capable of discriminating SA-β-Gal from β-Gal activity in nonsenescent tumor cells such as SKOV-3 cells.

Monitoring the Progression of Therapy-Induced Senescence in Living Cells

During senescence progression, β-Gal activity and lysosomal pH undergo dynamic changes, which are well aligned with the dual-locked mechanism of ECD-Gal. Therefore, we next investigated whether ECD-Gal could distinguish different degrees of cellular senescence. A549 and MRC-5 cells were treated with DOX or BLM for different durations to establish cellular models with progressive senescence. Based on the emission profile of ECD-Gal, fluorescence imaging was performed using excitation at 560 nm and emission window in the 610–660 nm range. In normal cells, ECD-Gal produced only weak fluorescence. In contrast, the fluorescence intensity progressively increased with the duration of DOX or BLM treatment (Figures and S9). Western blotting and qPCR assays were applied to analyze the expression level of senescence-associated markers, including p21, p53, and β-Gal in therapy-induced senescence cells. Western blotting analysis exhibited that these senescence-associated markers were accumulated in both BLM- and DOX-induced senescent cells (Figure d), but only p21 was robustly increased in the mRNA level while p53 and β-Gal did not increase, this result was similar to the reported result (NCBI: GSE292003) (Figure S10). These findings suggested that the accumulation of β-Gal and p53 may be associated with the lysosomal dysfunction and was consistent with the impaired protein turnover during cellular senescence. Collectively, these findings confirmed that ECD-Gal can report the progression of therapy-induced cellular senescence in living cells.

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The ability of ECD-Gal to detect cells at varying degrees of senescence in A549 cells. (a) Confocal imaging of A549 cells, which were pretreated with BLM or DOX for 24 , 48, and 72 h to induced senescence at different stages followed by treated with ECD-Gal (10 μM) for 15 min, (b,c) statistical data (n = 5) of fluorescence intensities. (d) Western blotting analysis of senescent associated marker. Statistical significance was determined by the Student’s t-test (two-tailed): *P < 0.05, ***P < 0.001 (λex = 560 nm, emission window: 610–660 nm).

Flow-Cytometric Quantification of Therapy-Induced Senescence

Compared with confocal microscopy, flow cytometry provides a higher throughput and more accurate quantitative analysis of fluorescence signals at the single-cell population level. Therefore, we investigated whether ECD-Gal could be used for the flow-cytometric detection of senescent cells. Relative to normal A549 cells, both DOX- and BLM-induced senescent A549 cells showed increased fluorescence intensity following pretreatment with ECD-Gal, and the fluorescence signal increased in a concentration- and time-dependent manner (Figure ). Similar results were observed in MRC-5 cells (Figure S11). These findings exhibited that ECD-Gal is compatible with flow-cytometric analysis of therapy-induced senescence. This capability extends ECD-Gal from fluorescence imaging to the high-throughput quantitative analysis of senescent cells populations, further strengthening its value as a practical senescence sensor.

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The ability of probe ECD-Gal was used to detect senescent cells by flow cytometry. (a) A549 cells pretreated with BLM for 48 h, followed by treatment with ECD-Gal for different times before flow-cytometry analysis and (b) corresponding statistical data (n = 3). (c) A549 cells pretreated with BLM for 48 h, followed by treatment with ECD-Gal for different concentrations before flow-cytometry analysis and (d) corresponding statistical data (n = 3). (e) A549 cells pretreated with DOX for 48 h, followed by treatment with ECD-Gal for different times before flow-cytometry analysis and (f) corresponding statistical data (n = 3). (g) A549 cells pretreated with DOX for 48 h, followed by treated with ECD-Gal for different concentrations before flow-cytometry analysis and (h) corresponding statistical data (n = 3). Statistical significance was determined by the Student’s t-test (two-tailed): ***P < 0.001.

In Vivo Imaging of Therapy-Induced Senescence and Senolytic Efficacy

Cellular senescence is widely present in vivo and plays a critical role in the onset and progression of numerous diseases. In particular, chemotherapy-induced senescence has been closely associated with tumor resistance and the adverse effects of anticancer treatments. , Senolytics, a class of drugs that selectively eliminate senescent cells, have been demonstrated to improve physical function, mitigate tumor resistance, and alleviate chemotherapy-induced side effects. − Therefore, noninvasive imaging of senescent cells in vivo is of great importance for assessing the efficacy of senolytics.

To investigate whether ECD-Gal can monitor therapy-induced senescence in vivo, a multiorgan senescence model was established by intraperitoneal administration of DOX (5 mg/kg) twice on days 1 and 7. Following the first DOX adminstration on day 1, mice were treated with the classical senolytic combination 5 mg/kg Dasatinib plus 50 mg/kg quercetin (D + Q), followed by administration of ECD-Gal (IP, 10 μM, 100 μL) and fluorescence imaging (Figure a). In vivo imaging revealed a robust fluorescence signal in DOX-treated mice, whereas the fluorescence signal was markedly attenuated after D + Q treatment. Ex vivo imaging of major organs, including the heart, liver, spleen, lung, and kidney, showed that the fluorescence signal was predominantly localized in the liver and kidney (Figure ).

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Evaluation of ECD-Gal for in vivo imaging of senescent cells in therapy-induced senescent mice. (a) Schematic illustration of the DOX-induced mouse senescence model. (b) Representative in vivo imaging of senescent cells in mice with ECD-Gal were obtained by Small Animal Imaging System (λex/em = 560/640 nm), the mice were treated with DOX to induce senescence, followed by the administration of classical senolytic combination D + Q, intraperitoneal injection of ECD-Gal (8 h) was performed to detect senescent cells in mice and (d) corresponding quantification of relative fluorescence intensity in different groups (n = 3). (c) Representative fluorescence images and (e) corresponding quantitative analysis of fluorescence signals in major organs (n = 3). (f) Representative β-Gal staining of liver and kidney and the (g) corresponding quantitative analysis results of beta-gal positive area (n = 12). (h) Representative Masson staining of liver and kidney and (i) the corresponding quantitative analysis results of Masson positive area (n = 12). Statistical significance was determined by the Student’s t-test (two-tailed): **P < 0.01, ***P < 0.001.

To evaluate whether the ECD-Gal fluorescence signal was correlated with tissue senescence burden, SA-β-Gal staining was performed on liver and kidney sections. As shown in Figure f, the SA-β-Gal-positive area was significantly increased in the liver and kidney tissues from DOX-treated mice, and the positive area was substantially reduced after D + Q treatment. The change in SA-β-Gal staining was consistent with the fluorescence imaging results, which underscores the potential of ECD-Gal to image therapy-induced senescence cells in vivo.

Senescent cells are closely associated with fibrotic remodeling, partly through the secretion of SASP factors that promote inflammation, extracellular matrix deposition, and tissue fibrosis. Accordingly, elimination of senescent cells can effectively alleviate tissue fibrosis. , Masson’s trichrome staining was therefore performed to evaluate fibrosis-associated pathological changes. DOX treatment resulted in increased collagen deposition and fibrosis in liver and kidney tissues, whereas D + Q treatment alleviated these pathological changes (Figure h). In addition, the mRNA expression levels of several representative SASP-associated factors, including IL-6, IL-1β, TGF-β, and TNF-α, were analyzed in the liver and kidney tissues by RT-qPCR. The expression levels of these factors were elevated in the DOX-treated group and decreased following D + Q treatment (Figure S12). The concordance among ECD-Gal fluorescence imaging, SA-β-Gal staining, mRNA expression levels of SASP factors, and Masson’s trichrome staining indicates that ECD-Gal provides a fluorescence readout associated with senescence burden and senescence-related tissue remodeling. To further evaluate the applicability of ECD-Gal in another therapy-induced senescence model, a BLM-induced pulmonary fibrosis model was established. In this model, strong ECD-Gal fluorescence was observed in the lung tissue, accompanied by increased SA-β-Gal-positive area and Masson-positive fibrotic area (Figure S13). These results further support the potential of ECD-Gal for imaging therapy-induced senescence burden and senescence-associated pathological remodeling in vivo.

Taken together, the in vivo results demonstrate that ECD-Gal enables fluorescence imaging of therapy-induced senescence cells in living animals and provides a readout associated with the reduction of the senescence burden after senolytic intervention. The consistency between fluorescence imaging and histopathological analyses supports the utility of ECD-Gal as a practical molecular sensor for monitoring senescence-associated biological processes in vivo.

Conclusion

In summary, we developed ECD-Gal as a β-Gal/pH dual-locked near-infrared fluorescent sensor for high-fidelity monitoring of therapy-induced senescence. ECD-Gal was constructed by caging a newly developed pH-responsive coumarin–hemicyanine fluorophore with a β-galactoside trigger. This design integrates two senescence-associated features, SA-β-Gal activity and lysosomal pH alkalization, into a single fluorescence output. Unlike conventional SA-β-Gal-responsive probes that mainly report enzyme activity, ECD-Gal requires both β-Gal-mediated cleavage and a senescence-associated lysosomal pH microenvironment for strong fluorescence activation.

ECD-Gal exhibited favorable pH responsiveness, selective β-Gal-triggered activation, low cytotoxicity, and robust fluorescence turn-on behavior in senescence cells induced by multiple therapeutic agents. Cellular mechanistic studies using EN6 and chloroquine demonstrated that lysosomal pH acts as a critical gating input, enabling ECD-Gal to distinguish senescence-associated β-Gal from β-Gal activity in nonsenescent tumor cells such as SKOV-3 cells. ECD-Gal further enabled fluorescence monitoring of senescence progression, flow-cytometric detection of senescent cells, and in vivo visualization of DOX- and BLM-induced senescence. Importantly, ECD-Gal fluorescence was reduced after D + Q senolytic treatment and the imaging results were consistent with β-Gal staining and fibrosis-associated histopathological changes. These findings demonstrate that combining enzymatic activity with lysosomal microenvironmental information is an effective strategy for improving fidelity in senescence cell sensing. The β-Gal/pH dual-locked sensing strategy described here provides a useful molecular platform for monitoring therapy-induced senescence cells and evaluating senolytic interventions in living systems.

Supplementary Material

au6c01028_si_001.pdf (873.2KB, pdf)

Acknowledgments

This work was financially supported by the Postdoctoral Fellowship Program (Grade C) of China Postdoctoral Science Foundation (GZC20252563), Jiangsu Fund Program for Excellent Postdoctoral Talent, the National Natural Science Foundation of China (82505287), China Postdoctoral Science Foundation (2025M773882), Provincial TCM Science and Technology Development Plan Projects (QN202503).

Glossary

Abbreviations

D + Q

Dasatinib plus Quercetin

BLM

bleomycin

DOX

doxorubicin

SA-β-Gal

senescence-associated β-galactosidase

SASP

senescence-associated secretory phenotype

TIS

therapy-induced senescence

The data supporting the findings of this study are available within the paper and its Supporting Information files. Raw data are available from the corresponding author upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.6c01028.

  • Schemes S1 and S2; Figures S1−S29 (PDF)

#.

L.D., Y.Z., and N.W. contributed equally to this work. L.J. originated the idea and supervised the study. L.D. synthesized the probe and conducted spectroscopic experiments. L.D., Y.Z., and N.W. conducted fluorescence imaging experiments. L.D., N.W., and Z.L. conducted the animal experiments. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

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Associated Data

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

Supplementary Materials

au6c01028_si_001.pdf (873.2KB, pdf)

Data Availability Statement

The data supporting the findings of this study are available within the paper and its Supporting Information files. Raw data are available from the corresponding author upon reasonable request.

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