Materials and methods for detecting senescent cells

Abstract

Provided herein are methods of detecting senescent cells using bioresponsive magnetic resonance imaging (MRI) contrast agents.

Description

[0001] MATERIALS AND METHODS FOR DETECTING SENESCENT CELLS

[0002] PRIORITY STATEMENT

[0003] This application claims priority to U.S. Provisional Application No. 63 / 562,340, filed March 7, 2024, the entire contents of which are incorporated herein by reference for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant numbers NS 115571 and CA268112 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD

[0007] Provided herein are methods of detecting senescent cells using a bioresponsive magnetic resonance imaging (MRI) contrast agent.

[0008] BACKGROUND

[0009] Senescent cells are key mediators of a wide range of diseases and conditions, including musculoskeletal (MSK) diseases, cardiovascular diseases, diabetes, neurological diseases, liver disease, kidney disease, pulmonary disease, autoimmune disease, among others. Moreover, senescence is associated with cancer and senolytic therapies have been used to treat cancers after classical chemotherapy or irradiation. It was recently discovered that SARS-CoV-2 can evoke virus-induced senescence. As such, cell senescence plays a pivotal role in numerous disease states. However, clinically relevant methods for detecting senescent cells are lacking. Thus, there is a clear need for biomarkers that can detect senescent cells with clinical imaging tools, including for use in diagnostics and monitoring of senolytic therapies.

[0010] SUMMARY

[0011] In some aspects, provided herein are methods of detecting senescent cells in a subject or a sample obtained therefrom. In some embodiments, the method comprises administering to the subject or the sample a bioresponsive magnetic resonance imaging (MRI) complex and detecting a signal from the complex. In some embodiments, the MRI complex comprises: an MRI contrast agent; a blocking substrate cleavable by P-galactosidase; and a self-immolative linker attaching the blocking substrate to the MRI contrast agent. In some embodiments, cleavage of the blocking substrate by P-galactosidase activates the MRI contrast agent to generate a signal detectable by MRI. In some embodiments, detection of a signal equal to or above a threshold value indicates the presence of senescent cells in the subject. In some embodiments, no detectable signal (e.g., a signal no greater than a background level of signal) indicates the absence of senescent cells in the subject or a relatively low amount of senescent cells in the subject. In some embodiments, the detectable signal is generated within 1 hour of cleavage of the blocking substrate.

[0012] In some embodiments, the self-immolative linker provides a functional group that coordinatively saturates the MRI contrast agent (e.g., the Gd(III) iron of the MRI contrast agent), thereby preventing a water access to the MRI contrast agent resulting in no detectable signal in the absence of P-galactosidase. “No detectable signal” indicates that the level of signal generated by the contrast agent is no greater than a background level of signal. Cleavage of the blocking substrate by P-galactosidase activates the MRI contrast agent by initiating a spontaneous electron cascade of the self-immolative linker that results in removal of the functional group from the MRI contrast agent. In some embodiments, the functional group is attached to the self-immolative linker by a spacer comprising at least 4 carbons. In some embodiments, the spacer consists of 4, 5, 6, 7, or 8 methylene groups. In some embodiments, the spacer is attached to an amine present in the self-immolative linker. In some embodiments, the functional group is a single carboxylate.

[0013] In some embodiments, the complex exhibits at least a 50% increase (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) in relaxivity in vivo following cleavage of the blocking substrate. In some embodiments, the complex exhibits at least an 80% increase in relaxivity in vivo following cleavage of the blocking substrate. In some embodiments, the complex exhibits at least a 50% increase, at least a 60% increase, at least a 70% increase, at least an 80%, or at least a 90% increase in vivo within I hour of cleavage of the blocking substrate.

[0014] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the complex is administered to the subject parenterally. In some embodiments, the subject has or is suspected of having disease or condition. In some embodiments, the disease or condition is a musculoskeletal disease (e.g., osteoporosis, osteoarthritis, rheumatoid arthritis), cardiovascular disease (e.g., atherosclerosis, hypertension), diabetes (type 1 diabetes, type 2 diabetes), liver disease (e.g., liver fibrosis), kidney disease, age- related macular degeneration, pulmonary disease (e.g., chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, asthma), neurological disease (c.g., Alzheimer’s disease, Parkinson’s disease, other dementias), autoimmune disease (e.g., systemic lupus erythematosus, multiple sclerosis), cancer, viral infection (e.g., coronavirus infection), or skin aging. In some embodiments, the musculoskeletal disease is osteoarthritis, rheumatoid arthritis, or osteoporosis. In some embodiments, the neurological disease is Alzheimer’s Disease or Parkinson’s Disease. In some embodiments, the viral infection is infection with a coronavirus. In some embodiments, detection of a detectable signal indicates the presence of senescent cells in the subject which indicates or aids in determining that the subject has the disease or condition. For example, in some embodiments detection of a detectable signal indicates that the subject has the musculoskeletal disease (e.g., osteoporosis, osteoarthritis, rheumatoid arthritis), cardiovascular disease (e.g., atherosclerosis, hypertension), diabetes (type 1 diabetes, type 2 diabetes), liver disease (e.g., liver fibrosis), kidney disease, age-related macular degeneration, pulmonary disease (e.g., chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, asthma), neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, other dementias), autoimmune disease (e.g., systemic lupus erythematosus, multiple sclerosis), cancer, viral infection (e.g., coronavirus infection), or skin aging. In some embodiments, the subject has received at least one dose of a senolytic therapy. In some embodiments, the subject has received at least one dose of a senolytic therapy and the method is performed to determine responsiveness of the subject to the at least one dose of the senolytic therapy. In some embodiments, a detectable signal indicates the presence of senescent cells, which indicates that the subject is not responsive to the at least one dose of the senolytic therapy. In some embodiments, no detectable signal (e.g., only a background level of signal) indicates that the senescent cells have been effectively ablated by the senolytic therapy, indicating that he subject is responsive to the at least one dose of the senolytic therapy.

[0015] In some aspects, provided herein are methods of determining responsiveness to a senolytic therapy in a subject. In some embodiments, the method comprises detecting a level or amount of senescent cells in the subject after the subject has received at least one dose of the senolytic therapy. In some embodiments, detecting the level or amount of senescent cells in the subject comprises administering to the subject or to a sample obtained from the subject a bioresponsive magnetic resonance imaging (MRI) complex and detecting a signal from the complex. In some embodiments, the MRI complex comprises: an MRI contrast agent, a blocking substrate cleavable by 0-galactosidase, and a self-immolative linker attaching the blocking substrate to the MRI contrast agent. In some embodiments, cleavage of the blocking substrate by fl-galactosidasc activates the MRI contrast agent to generate a signal detectable by MRI. In some embodiments, the detectable signal is generated within 1 hour of cleavage of the blocking substrate.

[0016] In some embodiments, a detectable signal equal to or above a baseline level indicates that the subject is not responsive to the at least one dose of the senolytic therapy. In some embodiments, the method comprises increasing the dose of the senolytic therapy, providing an alternative senolytic therapy to the subject, or both increasing the dose of the senolytic therapy and providing an alternative senolytic therapy to the subject indicated to be not responsive. In some embodiments, detection of a signal less than a baseline level indicates that the subject is responsive to the at least one dose of the senolytic therapy.

[0017] In some embodiments, the baseline level is a level determined prior to the at least one dose of the senolytic therapy. For example, in some embodiments the method comprising comparing the detectable signal after at least one dose of the senolytic therapy to a baseline level of signal determined prior to the at least one dose of the senolytic therapy, and determining whether the subject is responsive or not responsive to the senolytic therapy based on this comparison. A detectable signal equal to or above the baseline level indicates that the subject is not responsive to the at least one dose of the senolytic therapy, as the amount of senescent cells (which corresponds to the level of signal on the MR image) has not decreased from the baseline amount of senescent cells determined prior to the at least one dose of the senolytic therapy. A detectable signal less than the baseline level indicates that the subject is responsive to the at least one dose of the senolytic therapy, as the amount of senescent cells has decreased from the baseline amount of senescent cells determined prior to the at least one dose of the senolytic therapy.

[0018] In some embodiments, the method further comprises detecting the baseline level in the subject prior to the subject receiving the at least one dose of the senolytic therapy. The baseline level is thus indicative of the level or amount of senescent cells in the subject prior to receiving the at least one dose of the senolytic therapy. In some embodiments, detecting the baseline level comprises administering to the subject or a sample obtained from the subject the bioresponsive MRI complex and detecting a signal from the complex. In some embodiments, the method comprises comparing the level or amount of senescent cells detected in the subject after the at least one dose of the senolytic therapy to the baseline level or amount. In some embodiments, a decrease in the level or amount of senescent cells from the baseline level indicates the subject is responsive to the at least one dose of the scnolytic therapy. In some embodiments, no change or an increase in the level or amount from the baseline level indicates the subject is not responsive to the at least one dose of the senolytic therapy. In some embodiments, the method comprises increasing the dose of the senolytic therapy, providing an alternative senolytic therapy to the subject, or both increasing the dose of the senolytic therapy and providing an alternative senolytic therapy to the subject indicated to be not responsive.

[0019] In some embodiments, the self-immolative linker provides a functional group that coordinatively saturates the Gd(III) ion of the MRI contrast agent, thereby preventing a water access to the Gd(III) ion resulting in no detectable signal in the absence of P-galactosidase. “No detectable signal” indicates that the level of signal generated by the contrast agent is no greater than a background level. Cleavage of the blocking substrate by P-galactosidase activates the MRI contrast agent by initiating a spontaneous electron cascade of the self-immolative linker that results in removal of the functional group from the MRI contrast agent. In some embodiments, the functional group is attached to the self-immolative linker by a spacer comprising at least 4 carbons. In some embodiments, the spacer consists of 4, 5, 6, 7, or 8 methylene groups. In some embodiments, the spacer is attached to an amine present in the self-immolative linker. In some embodiments, the functional group is a single carboxylate.

[0020] In some embodiments, the complex exhibits at least a 50% increase (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) in relaxivity in vivo following cleavage of the blocking substrate. In some embodiments, the complex exhibits at least an 80% increase in relaxivity in vivo following cleavage of the blocking substrate. In some embodiments, the complex exhibits at least a 50% increase, at least a 60% increase, at least a 70% increase, at least an 80%, or at least a 90% increase in vivo within 1 hour of cleavage of the blocking substrate.

[0021] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the complex is administered to the subject parenterally. In some embodiments, the subject has or is suspected of having a disease or condition. In some embodiments, the disease or condition is a musculoskeletal disease (e.g., osteoporosis, osteoarthritis, rheumatoid arthritis), cardiovascular disease (e.g., atherosclerosis, hypertension), diabetes, liver disease (e.g., liver fibrosis), kidney disease, age-related macular degeneration, pulmonary disease (e.g., chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, asthma), neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, other dementias), cancer, viral infection (e.g., coronavirus infection), or skin aging. In some embodiments, the at least one dose of the senolytic therapy is provided to the subject to treat the disease or condition. For example, in some embodiments the at least one dose of the senolytic therapy is provide to the subject to treat the musculoskeletal disease (e.g., osteoporosis, osteoarthritis, rheumatoid arthritis), cardiovascular disease (e.g., atherosclerosis, hypertension), diabetes, liver disease (e.g., liver fibrosis), kidney disease, age-related macular degeneration, pulmonary disease (e.g., chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, asthma), neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, other dementias), cancer, viral infection (e.g., coronavirus infection), or skin aging. In some embodiments, the musculoskeletal disease is osteoarthritis, rheumatoid arthritis, or osteoporosis. In some embodiments, the neurological disease is Alzheimer’s Disease or Parkinson’s Disease. In some embodiments, the viral infection is infection with a coronavirus.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a schematic showing the structure and mechanism of an exemplary self- immolative magnetic resonance (MR) agent incorporating a coordinating carboxylate arm (blue) as an exemplary functional group. Gd(III) coordination by this functional group effectively prohibits water access to Gd(III), creating an inactive, or dark, agent by MR imaging. Hydrolysis of the glycoside by P-gal results in an electron cascade (red) that provides an open coordination site for water to bind to Gd(III).

[0024] FIGS. 2A-2D show determination of the optimal concentration of the P-gal responsive Gd- chelate for MRI detection of P-gal Gd-chelate activation. FIG. 2A shows representative axial Ti- weighted MR images of test tubes with increasing concentrations of the P-gal responsive Gd- chelate, with and without the addition of 10U P-gal enzyme at pH 6.0. FIG. 2B shows mean Ti relaxation times of increasing concentrations of the P-gal responsive Gd-chelate with and without the addition of 10U P-gal enzyme. FIG. 2C shows corresponding Ti maps color-coded to depict Ti-shortening visually. FIG. 2D shows mean Ri relaxation rates of increasing concentrations of the P-gal responsive Gd-chelate with and without the addition of 10U P-gal enzyme.

[0025] FIGS. 3A-3J show senescence of MSCs detected by P-gal responsive Gd-chelate. FIG. 3A shows confocal microscopy of senescent and viable mesenchymal stromal cells (MSC) after incubation with CellEvent™ Senescence Green Probe. Green fluorescence indicates P-gal expression. FIG. 3B shows relative number of P-gal positive green, fluorescent cells (%) for senescent and viable MSC on fluorescence microscopy with CellEvent™ Senescence Green staining. FIG. 3C shows IL-6 and IL-8 levels of control and senescent cells, as measured with ELISA. FIG. 3D shows western blots depicting p53 and Lamin Bl expression in control and senescent cells along with loading control GAPDH. FIG. 3E shows band intensities of p53 and FIG. 3F shows band intensities of laminin B 1 quantified and normalized to GAPDH with ImageJ software. FIG. 3G shows CCK-8 assay to indicate viability of control and senescent cells with and without incubation with the P-gal responsive Gd-chelate. FIG. 3H shows representative axial Ti-weighted MR images acquired on a 3T MRI of test tubes with senescent cells or viable controls after incubation with different concentrations of the P-gal responsive Gd-chelate. Corresponding Ti maps are color-coded to depict Ti -shortening. FIG. 31 shows quantitative Ri relaxation rates, derived from Ti maps, of control and senescent MSCs after incubation with 0.25 mM and 0.5 mM P-gal responsive Gd-chelate. FIG. 3J shows ICP-OES measurements which demonstrated no significant difference in Gd concentration of test tubes with P-gal probe with added viable or senescent MSC, confirming that the concentration of the probes did not change significantly after addition of cells. Therefore, changes in MRI signal are due to activation of the probe. Data are displayed as means and standard deviations of ten samples per experimental group. *p< 0.05.

[0026] FIGS. 4A-4C show senescent cells in porcine explants detected by P-gal responsive Gd- chelate. FIG. 4A shows a representative sagittal Ti-weighted MR image acquired on a 3T MRI of a distal femur specimen with implants of senescent cells (green arrow) and viable cells (orange arrow) in full-thickness cartilage defects and corresponding Ti map. FIG. 4B shows corresponding mean Ri relaxation rates of senescent cell implants and controls. FIG. 4C shows mean SignaLto- noise ratios of senescent cell implants and controls. All data are displayed as means and standard deviations of ten implants per group. *p< 0.05.

[0027] FIGS. 5A-5J show detection of senescent cells with the P-gal responsive Gd-chelate and MRI. FIG. 5A shows an intraoperative photo of cartilage defects in the distal femur of a live pig with implants of senescent cells (green arrow) and viable cells (orange arrow). FIG. 5B shows a representative sagittal Ti-weighted MR image acquired on a 3T MRI of the same knee joint after intra-articular injection of the P-gal responsive Gd-chelate. Senescent cells (green arrow) demonstrate stronger Ti-signal compared to viable cells (orange arrow). FIG. 5C shows corresponding Ti map demonstrates Ti-shortening in senescent cells (green arrow) compared to viable cells (orange arrow). FIG. 5D shows corresponding mean Ri relaxation rates of senescent cell implants and viable cell implants. FIG. 5E shows mean signal-to-noise ratios (SNR) of senescent cell implants and viable cell implants. FIG. 5F shows a photo of excised distal femur shows cartilage defects with implants of senescent cells (green arrow) and viable cells (orange arrow). FIG. 5G shows representative sagittal Ti -weighted MR image of the explant acquired on a 3T MRI with senescent cells (green arrow) and viable cells (orange arrow) in full-thickness cartilage defects. FIG. 5H shows corresponding Ti map demonstrates Ti-shortening in senescent cells (green arrow) and not viable cells (orange arrow). FIG. 51 shows mean Ri relaxation rates of implants with senescent cells and control cells. FIG. 5J shows SNR values for implants with senescent cells and control cells. Data are displayed as means and standard deviations of eight implants per experimental group. *p< 0.05, **p<0.005

[0028] FIGS. 6A-D show histological comparison of viable and senescent cell implants in pig knee cartilage. After MRI, the pig knee joints were excised and processed for histology. Fluorescence images of cartilage defects with implants of senescent cells and viable control cells, stained with (FIG. 6A) pl6 (2x magnification), (FIG. 6B) p21 (2x magnification), and (FIG. 6C) P-gal (2x magnification). FIG. 6D shows fluorescence intensity of senescent cells and viable controls on respective stains. All data are displayed as mean data and standard deviations of eight implants per group. *p< 0.05.

[0029] FIG. 7 shows Ti relaxation time of the P-gal responsive Gd-probe with and without the presence of P-gal enzyme. Nine test tubes with 0.5 mM P-gal responsive Gd-chelate were prepared with addition of 10 mM phosphate buffered saline (black squares) or with addition of 17 U of P- galactosidase (red squares). All samples underwent Ti relaxation time measurements at pH 7.4, 37 °C and 1.7 Tesla, at 10, 20, 30, 40, 60, 80, 120, and 150 minutes after incubation with either PBS or P-galactosidase. The graph shows shortened Ti-relaxation times for the P-gal responsive Gd- probe incubated with P-galactosidase, apparently as a function of enzymatic cleavage. A plateau of Ti relaxation time shortening was noted at approximately 2 hours.

[0030] FIGS. 8A-8B show R2 relaxation rate of the P-gal responsive Gd-probe with and without the presence of P-gal enzyme. FIG. 8A shows representative axial T -weighted MR images of test tubes with increasing concentrations of the P-gal responsive Gd-chelate, with and without the addition of 10U P-gal enzyme and corresponding To maps are color-coded. FIG. 8B shows mean R2 relaxation rates of increasing concentrations of the P-gal responsive Gd-chelate with and without the addition of 10U P-gal enzyme.

[0031] FIGS. 9A-9C show MRI of a representative pig knee joint before and at 2 hours after intraarticular injection of the P-gal responsive Gd-chelate. FIG. 9A shows sagittal Ti-weighted MR image of a knee joint prior to intra-articular injection of the P-gal responsive Gd-chelate. Note that the patella was dislocated laterally directly after implantation of viable (red) and senescent (green) mesenchymal stromal cells. FIG. 9B shows a representative sagittal Ti-weighted MR image of the same knee joint at 2 hours after intra-articular injection of the P-gal responsive Gd-chelate. Ti- hyperintense contrast agent is noted in the knee joint. There is a stronger Ti- signal in the senescent cell implant (green arrow) compared to the viable cell implant (red arrow), apparently indicating contrast agent activation. FIG. 9C shows corresponding signal-to-noise ratios (SNR) of senescent cell implants and viable cell implants before and after injection of the P-gal responsive Gd-chelate.

[0032] FIGS. 10A-10B show measurements of n and n relaxivities of the P-gal responsive Gd- chelate with and without the presence of P-gal enzyme at 1.41T. FIG. 10A shows Ri relaxation rates of test tubes with increasing concentrations of the P-gal responsive Gd-chelate with (red dots) and without (blue dots) addition of 17U of P-gal enzyme, measured at 1.4T and 37°C. The slope of the Ri-versus-concentration curves was used to calculate the n relaxivity at 1.4T and 37°C. FIG. 10B shows R2 relaxation rates of test tubes with increasing concentrations of the P-gal responsive Gd-chelate with (red dots) and without (blue dots) addition of 17U of P-gal enzyme, measured at 1.4T and 37°C. The slope of the R2-versus-concentration curves was used to calculate the r2 relaxivity at 1.4T and 37°C.

[0033] FIGS. 11A-11B show histological staining of mesenchymal stem cells (MSCs) markers in viable and senescent cell implants in pig knee cartilage. After MRI, the pig knee joints were excised and processed for histology. FIG. 11 A shows fluorescence images of cartilage defects with implants of senescent cells and viable control cells, stained with CD90 (2x magnification), CD 105 (2x magnification), and CD73 (2x magnification). FIG. 11B shows fluorescence intensity of senescent cells and viable controls on respective stains. All data are displayed as mean data and standard deviations of four implants per group. *p< 0.05.

[0034] FIG. 12 shows histological staining to determine fibrosis in viable and senescent cell implants in pig knee cartilage. After MRI, the pig knee joints were excised and processed for histology. Representative images of cartilage defects with implants of senescent cells and viable control cells stained with Masson’s Trichromc staining (2x magnification).

[0035] FIG. 13 shows histological staining to determine vascularity and presence of macrophages in viable and senescent cell implants in pig knee cartilage. After MRI, the pig knee joints were excised and processed for histology. Representative fluorescence images of cartilage defects with implants of senescent cells and viable control cells, stained with F4 / 80, a macrophage marker (2x magnification), and CD31 , a marker for endothelial cells indicating vascularity (2x magnification), All data are displayed as mean data and standard deviations of four implants per group. *p< 0.05.

[0036] FIGS. 14A-14G shows MR imaging of a human hip specimen. FIG. 14A shows a hip radiograph of the specimen. FIG. 14B shows a proton density weighted image of the specimen. FIG. 14C shows the excised specimen. FIG. 14D shows a T1 weighted conventional spin echo (Tl-SE) image. FIG. 14E shows a T1 weighted conventional spin echo (Tl-SE) image in the presence of the P-gal responsive Gd-chelate. FIG. 14F shows a T1 weighted heat map from FIG. 14D and FIG. 14G shows a T1 weighted heat map image from FIG. 14E (in the presence of the P- gal responsive Gd-chelate). Taken together, these results demonstrate successful MR imaging of senescent cells in a human hip specimen.

[0037] FIGS. 15A-1 J show MR imaging of a human knee specimen. FIG. 15 A shows an X-ray of the specimen. FIG. 15B shows the excised specimen. FIG. 15C shows a proton density weighted (PDW) image of the specimen. FIG. 15D shows a T1 weighted conventional spin echo (Tl-SE) image. FIG. 15E shows a T1 weighted conventional spin echo (Tl-SE) image in the presence of the P-gal responsive Gd-chelate. FIG. 15F shows a T1 weighted heat map from FIG. 15D and FIG. 15G shows a T1 weighted heat map image from FIG. 15E (in the presence of the P- gal responsive Gd-chelate). FIG. 15H shows the signal to noise ratio (SNR) for the P-gal responsive Gd-chelate before and after injection of the P-gal responsive Gd-chelate. A significant enhancement in the signal to noise ratio was seen after injection of P-gal responsive Gd-chelate. FIG. 151 shows the R1 relaxation rate before and after injection of the P-gal responsive Gd-chelate. A significant enhancement in R1 relaxation rate was seen after injection of P-gal responsive Gd- chelate. FIG. 15J shows the R1 relaxation rate vs. grade of osteoarthritis, demonstrating an enhancement in the R1 relaxation rate that correlates with the grade of osteoarthritis in a given specimen. Taken together, these results demonstrate successful MR imaging of senescent cells in a human knee specimen. FIGS. 16A-6H show MR imaging of human joints compared to Gadavist. FIG. 16A and FIG. 16E show proton density weighted (PDW) images of the specimen. FIG. 16B and FIG. 16F show T2 weighted heat maps of the specimen. FIG. 16C and FIG. 16G show T1 weighted heat maps of the specimen. FIG. 16D shows a T1 weighted heat map of the specimen in the presence of the gadolinium-based contrast agent Gadavist. FIG. 16E shows a T1 weighted heat map of the specimen in the presence of the P-gal responsive Gd-chelate. Gadavist was not able to detect senescent cells by MRI. In contrast, the P-gal responsive Gd-chelate successfully detected senescent cells by MRI in human joints.

[0038] DETAILED DESCRIPTION

[0039] In some aspects, provided herein are MRI contrast agents and methods of use thereof for detecting senescent cells. In some embodiments, methods of detecting senescent cells involve use of a class of agents that 1) respond to an in vivo metabolic event such as enzyme cleavage and 2) translate this response to an image signal detectable by MR. These agents are referred to herein as bioresponsive MRI contrast agents. These agents comprise a contrast agent that is activated by an enzyme. For example, the contrast agent may be blocked (e.g., “dark”) prior to a metabolic event (e.g., enzymatic cleavage) and activated (e.g., “bright”) following the metabolic event. The contrast agents provided herein are shown to detect senescent cells in both animal models and human specimen with clinical standard MRI technology. The agents can be used to identifying patients who might benefit from senolytic therapies, monitor response to senolytic therapies, an / or assess the efficacy of combination therapies.

[0040] In some aspects, provided herein is a clinically relevant method of identifying senescent cells using a bioresponsive MRI contrast agent engineered for the detection of P-gal activity. Specifically, a P-gal responsive Gd-chelate was used. In this agent, the coordination sphere of Gd (III) is saturated before enzyme reaction, preventing the interaction with water protons, thereby resulting in relatively low signal (e.g., only a background level of signal) on Tl-weighted MRI scans. Upon exposure to P-gal, the P-galactosyl moiety is cleaved, leading to a spontaneous electron cascade that opens the coordination site of Gd (III) for the interaction with water protons, which results in an increased bright (hyperintense) MRI signal on Tl-weighted MRI scans. 1. Definitions

[0041] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only and is not intended to limit the scope of the embodiments described herein.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

[0043] As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a peptide amphiphile” is a reference to one or more peptide amphiphiles and equivalents thereof known to those skilled in the art, and so forth.

[0044] As used herein, the terms “comprise”, “include”, and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of’ and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and / or “consisting essentially of’ embodiments, which may alternatively be claimed or described using such language.

[0045] The term “antibody” includes antibody fragments, as are known in the art, including Fab Fab , single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. In some embodiments, antibody targeting moieties are humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.

[0046] The term “bioresponsive” as used herein in reference to a MRI complex or contrast agent refers to a complex or contrast agent that is activated in response to a biological event. The term “activated” indicates that the MRI complex or contrast agent changes from a state of producing a background level of signal (e.g., an “off’ state or a “dark” state) to producing a detectable signal (e.g., an “on” state or a “bright” state) significantly higher than the background level. In some embodiments, the biological event is enzymatic cleavage of a moiety that otherwise blocks the contrast agent, thereby preventing a detectable signal in the absence of the enzyme. For example, the MRI contrast agent may be activated (e.g., exposed) following enzymatic cleavage of the blocking substrate.

[0047] As used herein, the term “macrocycle” refers to a molecule containing a twelve or more membered ring. Common examples of macrocycles that may be used in accordance with the disclosed contrast agents are described herein, including EDTA, DTPA, DOTA, D03A, and the like.

[0048] As used herein, the term “magnetic resonance imaging (MRI) device” or “MRI” incorporates all devices capable of magnetic resonance imaging or equivalents. The methods of the invention can be practiced using any such device, or variation of a magnetic resonance imaging (MRI) device or equivalent, or in conjunction with any known MRI methodology. For example, in magnetic resonance methods and apparatuses, a static magnetic field is applied to a tissue or a body under investigation in order to define an equilibrium axis of magnetic alignment in a region of interest. A radio frequency field is then applied to that region in a direction orthogonal to the static magnetic field direction in order to excite magnetic resonance in the region. Magnetic field gradients are applied to spatially encode the signals. The resulting signals are detected by radiofrequency coils placed adjacent to the tissue or area of the body of interest. See, e.g., U.S. Pat. Nos. 6,144,202; 6,128,52f2; 6,127,775; 6,119,032; 6,111,410; 5,555,251; 5,455,512; 5,450,010, each of which is herein incorporated by reference in its entirety. MRI and supporting devices are manufactured by, e.g., Bruker Medical GMBH; Caprius; Esoate Biomedica; Fonar; GE Medical Systems (GEMS); Hitachi Medical Systems America; Intermagnetics General Corporation; Lunar Corporation; MagneVu; Marconi Medicals; Philips Medical Systems; Shimadzu; Siemens; Toshiba America Medical Systems; and Varian; including imaging systems, by, e.g., Silicon Graphics.

[0049] As used herein, the terms “paramagnetic metal ion”, “paramagnetic ion” or “metal ion” refer to a metal ion that is magnetized parallel or antiparallel to a magnetic field to an extent proportional to the field. Generally, these are metal ions that have unpaired electrons. Examples of suitable paramagnetic metal ions, include, but are not limited to, gadolinium III (Gd+3 or Gd(III)), iron III (Fe+3 or Fe(III)), manganese II (Mnt2 or Mn(II)), yttrium III (Yt+3 or Yt(III)), dysprosium (Dy+3 or Dy(III)), and chromium (Cr(III) or Cr+3). In a preferred embodiment the paramagnetic ion is the lanthanide atom Gd(III), due to its high magnetic moment (u2=63 BM2), a symmetric electronic ground state (S8), and its current approval for diagnostic use in humans. As used herein, the term “superparamagnetic” refers to a form of magnetism which appears in sufficiently small particles. Superparamagnetic materials have a larger magnetic susceptibility than paramagnetic materials. In some embodiments, superparamagnetic materials comprise iron, such as iron oxide or iron platinum particles.

[0050] The term “self-immolative” as used herein (e.g., in relation to a linker) refers to an entity that undergoes a cascade of reactions in response to an external stimuli. For example, a self- immolative linker as described herein undergoes a series of disassembly reactions in response to cleavage of the blocking substrate. Thus, a self-immolative linker will be removed from an MRI complex following a cascade of reactions initiated by enzymatic cleavage of the blocking substrate, leaving behind byproducts of the self-immolative reaction, the free blocking substrate, and the free (e.g., bright) contrast agent.

[0051] As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment or diagnostic procedure. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

[0052] As used herein, the term “targeting moiety” refers to any suitable moiety that serves to target or direct the complex to a desired area. For example, the targeting moiety may refer to a functional group which serves to target or direct the complex to a particular location, cell type, diseased tissue, or target molecule within a subject. As used herein, “relaxivity” is the degree to which a magnetic resonance contrast agent can enhance the longitudinal or transverse water relaxation rate constant (Ri = 1 / Ti or R2 = I / T2, respectively, normalized to concentration of the contrast agent. R is the rate constant, T is the relaxation time and 1 and 2 denote longitudinal and transverse, respectively. Longitudinal and transverse relaxivity are denoted n and n, respectively and have the units of M’1. Gadolinium- based contrast agents are generally referred to as Ti agents, because they have a larger effect on tissue Ti than on To. Thus, administration of contrast agents that reduce tissue Ti result in positive image contrast in Ti-weighted images. Because Gd agents are most often used for Ti relaxation measurements, longitudinal relaxivity is often simply referred to as “relaxivity” herein.

[0053] As used herein, the term MRI “signal” generally refers to the image contrast in specific areas of an MR image (e.g. a Ti-weighted MR image, a T2- weighted MR image) compared to the surrounding areas. Enhanced contrast (e.g., enhanced signal) in specific areas of an image denote areas where the bioresponsive contrast agent has been activated. Thus, in one preferred embodiment of the invention, activation of the contrast agent results in increased contrast in Ti- weighted images. In other embodiments of the invention, activation of the contrast agent will result in increased contrast in T2-weighted images. In further embodiments of the invention, activation of the contrast agent may result in decreased contrast in the Ti -weighted or T2-weighted images.

[0054] As used herein, the term “threshold level” or “background level” are used interchangeably and refer a relatively low contrast level that is characteristic of areas of an MR image where the bioresponsive contrast is not activated. Those skilled in the art will recognize that the threshold value can be determined in multiple ways. By way of example, in some embodiments the threshold vale may simply be the average contrast level of all areas of the image (on an area-by-area basis with an arbitrarily chosen area size). In some embodiments, the contrast of an area may be compared statistically to the immediately surrounding areas as a threshold.

[0055] In some embodiments, the standard deviation of the noise level of the pixels in an area of the image is determined. The signal-to-noise (S / N) ratio is then determined for any area of interest in that image as the ratio of the average signal level for that area to the standard deviation of the noise level. In some embodiments, the area for the noise level determination is a low-contrast area at the comer or edge of the image. Alternatively, the noise level can be determined from an area of the image that corresponds anatomically to an area of the subject or tissue that shows low contrast or that is not suspected to contain senescent cells. The S / N ratio or SNR for the area of high signal area of interest in the image can be reported as a single number that characterizes the amount over level of senescence in the subject at that anatomical area. Alternatively, the SNR can be determined for every pixel in an image and used to generate an alternative image that emphasizes the senescence signal compared to the background level of the surrounding areas.

[0056] In another embodiment, the threshold is determined on a time basis, where the threshold level contrast is determined from an earlier or later MR image of the same subject or tissue. In a preferred embodiment, a Ti-weighted “heat map” is generated which creates a color image from the contrast image showing different colors and intensities in areas of the image having significantly different contrast from the surrounding areas. Those skilled in the art understand the term threshold and its use in the context of MR imaging and the use of and generation of Ti- weighted MR images and MRI heat maps.

[0057] The phrase “generate a signal detectable by MRI” or “generating a signal detectable by MRI” refers to a change (e.g., increase) in signal compared to a background level or threshold level that occurs as a result of activation of the contrast agent. In some embodiments, cleavage of the blocking substrate by a target enzyme (e.g., beta-galactosidase) initiates a spontaneous electron cascade of the self-immolative linker that results in removal of the functional group from the contrast agent such that water can access the contrast agent, thereby producing a change (e.g. an increase) in the signal from the contrast agent compared to the threshold / background level of signal. In some embodiments, the signal from the contrast agent is compared to a threshold level of signal determined mathematically from the same image (e.g., by averaging the signal over the entire image or averaging the signal in an area of the image where the target enzyme is not active or less active). In another embodiment, artificial intelligence is used to analyze the MR image(s) to determine a threshold level of signal. In a further embodiment, a first MR image is taken before the administration of the contrast agent and a second MR image is taken after the administration. In this case, the threshold level of signal is determined from the first image and the level of signal from the second image is compared to that threshold to determine the presence of senescent cells. In some embodiments, the image is an array of many pixels and the signal level of each pixel is individually treated mathematically or by using artificial intelligence to generate an image indicative of the presence or absence of senescent cells. To “generate a signal detectable by MRI” or “generating a signal detectable by MRI” is used herein to describe a change (e.g., increase) in signal compared to thrcshold / background level.

[0058] 2. Methods of Detecting Senescent Cells

[0059] Senescent cells contribute to many diseases and conditions. Senescent cells can promote tumorigenesis by creating a pro-inflammatory environment that supports cancer cell growth and survival. The accumulation of senescent cells in the cardiovascular system contributes to atherosclerosis, hypertension, and other cardiovascular conditions by promoting inflammation and vascular dysfunction. Senescent cells in adipose tissue and other organs can impair insulin signaling and glucose metabolism, leading to insulin resistance and diabetes. Senescent cells in bone tissue can disrupt the balance between bone formation and resorption, leading to decreased bone density and increased fracture risk. The presence of senescent cells in joint tissues contributes to the degradation of cartilage and the development of osteoarthritis through the secretion of inflammatory mediators. Senescent cells in the brain can exacerbate neuroinflammation and neuronal dysfunction, contributing to cognitive decline and the progression of Alzheimer’s disease and other dementias. Senescent cells in the brain can lead to the aggregation of misfolded proteins and the formation of Lewy bodies, leading to Parkinson’ s Disease. Senescent cells in the liver can promote fibrosis by secreting factors that stimulate the proliferation of fibroblasts and the deposition of extracellular matrix. Senescent cells in the kidneys can contribute to chronic kidney disease by promoting inflammation and fibrosis. Senescent cells in the retina can contribute to the development of age-related macular degeneration by promoting inflammation and tissue damage. Senescent cells in the lungs can contribute to chronic obstructive pulmonary disease (COPD), asthma, and idiopathic pulmonary fibrosis (IPF) by promoting inflammation and tissue remodeling. Senescent cells secrete a variety of proinflammatory molecules that create a pro- inflammatory environment that can exacerbate autoimmune responses, leading to autoimmune disease. Senescent cells in the skin can contribute to the aging process by promoting the breakdown of collagen and other extracellular matrix components, leading to wrinkles and loss of skin elasticity. Senescent cells have been implicated in the severe inflammatory response seen in older adults with COVID-19, known as the cytokine storm. As such, senescent cells are associated with a multitude of disease states, including musculoskeletal disease (e.g., osteoporosis, osteoarthritis, rheumatoid arthritis), cardiovascular disease (e.g., atherosclerosis, hypertension), diabetes (type 1 diabetes, type 2 diabetes), liver disease (e.g., liver fibrosis), kidney disease, age- related macular degeneration, pulmonary disease (e.g., chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, asthma), neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, other dementias), autoimmune disease (e.g., systemic lupus erythematosus, multiple sclerosis), cancer, viral infection (e.g., coronavirus infection), and skin aging.

[0060] The accumulation of senescent cells and their associated SASP is a significant factor in the pathogenesis of the above diseases and conditions. Targeting senescent cells, such as by using senolytic therapies that selectively ablate senolytic cells, or modulating SASP to mitigate detrimental effects using senomorphic therapies, represents a promising therapeutic strategy for mitigating the adverse effects of aging and improving health span. For example, senolytic therapies may be useful for treating any one of more of the above diseases or conditions. However, safe, effective, non-invasive methods for imaging senescent cells are lacking. The use of PET and radioligands that bind cell surface biomarkers require the use of radiochemicals and are therefore considered unsafe unless extreme care is taken in their use. The instant disclosure addresses this need and provides a safe and effective, non-invasive method for imaging senescent cells, including for use in diagnosing a disease or condition or monitoring efficacy of senolytic therapies.

[0061] Osteoarthritis (OA), the most common chronic joint disease, is characterized by joint pain, limited motion, and joint stiffness. While the typical course of OA involves slow, progressive joint damage over many years, some patients experience unpredictable bursts of rapid progression. Recent evidence implicates senescent cells in cartilage and synovium as key players in OA development, highlighting their potential as therapeutic targets. Senescent cells undergo replicative arrest and develop a senescence-associated secretory phenotype (SASP), characterized by the secretion of chemokines, inflammatory cytokines and matrix-degrading proteases. These mediators contribute to cartilage degradation and eventual joint destruction, leading to OA. Various senolytic therapies, such as dasatinib, quercetin, navitoclax, and metformin, have entered clinical trials, demonstrating promising results in slowing the progression of OA and improving clinical symptoms. However, despite the rapid development of senolytic therapies for OA treatment, no imaging technology is currently capable of detecting senescent cells in vivo or in patients. A clinically translatable in vivo imaging biomarker for senescent cells could help identify candidates for senolytic therapies and monitor their response to these therapies. Magnetic resonance imaging (MRI), often enhanced with gadolinium (Gd) chelates as MRI contrast agents is the standard clinical imaging modality for diagnosing degenerative arthritis in patients with OA. However, MRI primarily detects downstream sequelae of disease progression, such as osteophytes, cartilage loss, and subchondral cysts. These findings reflect structural changes, not directly capturing the underlying molecular drivers of disease progression. There is currently no diagnostic test available that can detect and quantify cellular senescence in vivo in human articular joints. Thus far, cellular senescence in the joint synovium, articular cartilage, and bone / bone marrow has been diagnosed based on SA-P-gal activity, cell cycle restricted proteins, and production of reactive oxygen species, among others, using immunohistochemistry, flow cytometry, fluorescence imaging, or reporter gene imaging approaches. However, none of these previously applied biomarkers is clinically translatable.

[0062] The present disclosure overcomes these shortcomings and addresses the need for clinically relevant methods of identifying senescent cells. Specifically, a P-gal responsive Gd-chelate was used as a non-invasive MRI method for detecting P-gal expressing senescent cells in vivo.

[0063] In some aspects, provided herein are methods of detecting senescent cells using bioresponsive MRI complexes. The term “senescent cells” as used herein refers to cells that no longer undergo division and that secrete chemokines, cytokines, growth factors, and proteases collectively known as the senescence-associated secretory phenotype (SASP). As described above, these mediators, comprising inflammatory cytokines and matrix-degrading enzymes, contribute to a variety of diseases and conditions. For example, senescent cells and the mediators produced thereby contribute to musculoskeletal diseases including osteoarthritis, rheumatoid arthritis, osteoporosis, and others. For example, senescent cells excrete mediators that contribute to cartilage degradation and eventual joint destruction, leading to osteoarthritis. Accordingly, the methods of detecting senescent cells described herein may be used to detect musculoskeletal disease in a subject. For example, the methods of detecting senescent cells described may be used to detect musculoskeletal diseases including osteoarthritis, rheumatoid arthritis, and / or osteoporosis in a subject. In some embodiments, the methods herein achieve early detection of musculoskeletal disease, e.g., before symptoms of musculoskeletal disease are prominent.

[0064] Senescent cells are also a hallmark characteristic of neurological disease, including Alzheimer’s disease and Parkinson’s disease. Accordingly, the methods of detecting senescent cells described herein can be used to detect neurological disease in a subject, such as to detect Alzheimer’s disease or detect Parkinson’s disease in a subject. Cell senescence also plays a role in cancer progression. For example, senescence of immune cells, in particular T-cclls, promotes tumor growth. Chemotherapy and / or irradiation for treatment of cancer can cause cancer cells to enter into therapy-induced senesce. These senescent cancer cells can continue to release continue to release of pro-inflammatory senescence associated proteins which promote growth of bystander cells and facilitate metastasis. Accordingly, in some embodiments the methods of detecting senescent cells described herein enable detection of cancer and / or enable evaluation of risk of metastasis of a cancer that has been treated (e.g., with irradiation, with chemotherapy). Cell senescence may also contribute to susceptibility to severe symptoms and / or chronic illness following viral infection, such as infection with SARS-CoV-2. As such, in some embodiments the methods of detecting senescent cells described are useful for identifying risk of severe infection and / or chronic illness following exposure to a virus, such as SARS-CoV-2.

[0065] The methods of detecting senescent cells provided herein can be used to detect a disease or condition associated with senescent cells in a subject. In some embodiments, the disease or condition is selected from musculoskeletal disease (e.g., osteoporosis, osteoarthritis, rheumatoid arthritis), cardiovascular disease (e.g., atherosclerosis, hypertension), diabetes (type 1 diabetes, type 2 diabetes), liver disease (e.g., liver fibrosis), kidney disease, age-related macular degeneration, pulmonary disease (e.g., chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, asthma), neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, other dementias), autoimmune disease (e.g., systemic lupus erythematosus, multiple sclerosis), cancer, viral infection (e.g., coronavirus infection), and skin aging. The methods herein may further comprise providing an appropriate disease therapy to the subject based upon the detection of senescent cells. For example, the method may comprise providing an appropriate disease therapy, such as providing senolytic therapy to the subject, when senescent cells are detected (e.g., when the presence of senescent cells is determined, when the amount of senescent cells is above a threshold value)

[0066] In some embodiments, the methods of detecting senescent cells comprise administering to the subject or the sample obtained therefrom a bioresponsive MRI complex, and detecting a signal from the complex, wherein the sample obtained from the subject is a biopsy sample or blood, serum, plasma, or cerebrospinal fluid sample or tissue sample. The term “detecting” is inclusive of both quantitative and qualitative detection. In some embodiments, “detecting” senescent cells refers to determining the presence or absence of senescent cells. In some embodiments, “detecting” senescent cells refers to determining the presence of senescent cells. In some embodiments, “detecting” senescent cells refers to determining the absence of senescent cells. In some embodiments, “detecting” senescent cells refers to quantitative detection, e.g., e.g., determining the amount or number of senescent cells. In some embodiments “detecting” senescent cells includes evaluating a signal from an MRI complex provided herein. In some embodiments, a signal equal to or above a threshold value indicates the presence of senescent cells or is used to quantify the number or amount of senescent cells in a subject. In some embodiments, a signal below a threshold value, such as a signal not significantly greater than a background level, is indicative of the absence of senescent cells or is indicative of a relatively low level or amount of senescent cells in the subject. Any of the above embodiments are included in the term “detecting” senescent cells.

[0067] In some embodiments, the methods of detecting senescent cells comprise administering to the subject or the sample obtained therefrom a bioresponsive MRI complex, and detecting a signal from the complex. In some embodiments, a detectable signal indicates the presence of senescent cells in the subject, whereas no detectable signal (e.g., e.g., a signal not greater than a background level) indicates the lack of senescent cells in the subject. In some embodiments, detection of a signal (e.g., e.g., by MRI) equal to or above a threshold value indicates the presence of senescent cells in the subject. As described above, in some embodiments detection of a signal equal to or above a threshold value (which indicates the presence of senescent cells) indicates that the subject has a disease or condition associated with senescent cells. Suitable diseases and conditions are described herein, including musculoskeletal disease (e.g., e.g., osteoporosis, osteoarthritis, rheumatoid arthritis), cardiovascular disease (e.g., e.g., atherosclerosis, hypertension), diabetes (type I diabetes, type 2 diabetes), liver disease (e.g., e.g., liver fibrosis), kidney disease, age-related macular degeneration, pulmonary disease (e.g., e.g., chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, asthma), neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, other dementias), autoimmune disease (e.g., systemic lupus erythematosus, multiple sclerosis), cancer, viral infection (e.g., coronavirus infection), or skin aging. In some embodiments, methods of detecting senescent cells are used to evaluate responsiveness of a subject to scnolytic therapy. The term “scnolytic” refers to a therapy that selectively removes / ablates / destroys scnolytic cells. In some embodiments, the senolytic therapy is a FOXO4-related peptide, a BCL-2 inhibitor, a Src inhibitor, a USP7 inhibitor, senescencespecific killing compound 1 (SSK1), a kidney-type glutaminase 1 (GLS1) inhibitor, a cardiac glycoside, an HSP90 inhibitor, or a combination thereof. Exemplary senolytic therapies include, for example, dasatinib, quercetin, venetoclax, navitoclax, fisetin, 17-DMAG, IPI504, UBX0101, F0X04-DRI peptide, cardiotonic steroids, GMK, SSK1, metformin, or combinations thereof. In some embodiments, methods of detecting senescent cells are used to track or evaluate response to a senolytic therapy in a subject. In some embodiments, evaluating response to a senolytic therapy in a subject comprising detecting a level or amount of senescent cells in the subject after the subject has received at least one dose of a senolytic therapy. In some embodiments, detection of a signal equal to or above a baseline level indicates that the subject is not responsive to the at least one dose of the senolytic therapy. In some embodiments, the method comprises increasing the dose of the senolytic therapy, providing an alternative senolytic therapy to the subject, or both increasing the dose of the senolytic therapy and providing an alternative senolytic therapy to the subject indicated to be not responsive. In some embodiments, detection of a signal less than a baseline level indicates that the subject is responsive to the at least one dose of the senolytic therapy.

[0068] In some embodiments, the baseline level is determined prior to the subject receiving the at least one dose of the senolytic therapy. In some embodiments, the baseline level is determined by administering to the subject or to a sample obtained from the subject a bioresponsive magnetic resonance imaging (MRI) complex described herein and detecting a signal from the complex. In some embodiments, the method of evaluating response to a senolytic therapy in a subject comprises detecting the baseline level or amount of senescent cells in the subject and detecting a level or amount of senescent cells in the subject after at least one dose of senolytic therapy has been provided to the subject. In some embodiments, the baseline level or amount of senescent cells is detected prior to the subject receiving at least one dose of the senolytic therapy. In some embodiments, the baseline level or amount of senescent cells is detected before the subject has received any doses of senolytic therapy. In some embodiments, the baseline level or amount of senescent cells is detected in between doses of the senolytic therapy. For example, in some embodiments the baseline level is detected after a given dose of the senolytic therapy and a level or amount of senescent cells is subsequently detected after a different, later dose of the senolytic therapy. In some embodiments, the level or amount of senescent cells after the later dose of the senolytic therapy is compared to the level or amount of senescent cells after the earlier dose of the senolytic therapy (e.g. is compared to the baseline level) to determine responsiveness of the subject to the senolytic therapy.

[0069] In some embodiments, the method comprises comparing the level or amount of senescent cells after the subject has received the at least one dose of the senolytic therapy to the baseline level or amount. In some embodiments, detection of a signal greater than the baseline signal after the subject has received a senolytic therapy indicates that senolytic therapy has not sufficiently ablated senescent cells in the subject (e.g., in the subject having a disease or condition characterized by cell senescence, for which the senolytic therapy was provided to treat the disease or condition). In other embodiments, detection of a signal below the baseline signal after the subject has received the senolytic therapy indicates that the senolytic therapy has effectively ablated senescent cells in the subject. The senolytic therapy may be provided to the subject to treat the disease or condition associated with senescent cells, and the methods provided herein used to track efficacy of the senolytic therapy in the subject.

[0070] The method may further comprise providing senolytic therapy to the subject or modifying the senolytic therapy being provided to the subject based upon the detection of senescent cells. For example, the method may comprise providing an alternative senolytic therapy to the subject, increasing the dose of the senolytic therapy, or both increasing the dose of the senolytic therapy and providing an alternative senolytic therapy to the subject, when senescent cells are detected after the subject has received a senolytic therapy. For example, the method may comprise providing an alternative senolytic therapy to the subject, increasing the dose of the senolytic therapy, or both increasing the dose of the senolytic therapy and providing an alternative senolytic therapy to the subject, when a level or amount of senescent cells in the subject after receiving at least one dose of senolytic therapy is equal to or above the baseline level or amount of senescent cells detected in the subject prior to the at least one dose of senolytic therapy. “Increasing the dose” of the senolytic therapy may involve providing to the subject the senolytic therapy at an increased dosing frequency and / or in a higher dosage amount. As another example, the method may decreasing the dose of the senolytic therapy or ceasing administration of the senolytic therapy when senescent cells are not detected or when the amount of senescent cells is determined to be below the threshold value.

[0071] In some embodiments, the bioresponsive MRI complex comprises an MRI contrast agent and a blocking substrate. In some embodiments, the blocking substrate is cleavable by a target enzyme. In some embodiments, the blocking substrate is cleavable by the enzyme P-galactosidase. In some embodiments, the blocking substrate is attached to the contrast agent by a self-immolative linker. In some embodiments, the self-immolative linker provides a functional group that coordinatively saturates the contrast agent in the absence of the target enzyme. In some embodiments, cleavage of the blocking substrate induces a spontaneous electron cascade of the self-immolative linker that releases the functional group from the contrast agent, thereby activating the contrast agent to produce a detectable signal. In the absence of the enzyme p-galactosidase, the blocking substrate blocks the contrast agent, keeping the agent “dark” or inactive (e.g., producing only a background level of signal). Following enzymatic removal of the blocking substrate (e.g., by P-galactosidase cleavage) the contrast agent is “bright” or activated (e.g., producing a signal significantly greater than the background level). Accordingly, the MRI complexes described herein allow for conditional activation of the contrast agent within the complex. Accordingly, the MRI complexes and contrast agents described herein are referred to as “conditionally activated” or “bioresponsive”.

[0072] Any suitable contrast agent may be used in the MRI complexes described herein. In some embodiments, the contrast agent is a paramagnetic contrast agent. Exemplary paramagnetic contrast agents suitable for use in the present compositions include, for example, stable free radicals, such as, for example, stable nitroxides, as well as compounds comprising transition, lanthanide and actinide elements. The contrast agent be in the form of a salt (e.g., inorganic or organic salt) or may be covalently or non-covalently bound to complexing agents, including lipophilic derivatives thereof, or to polypeptide-containing macromolecules. Preferable transition, lanthanide and actinide elements include, for example, Gd(III), Mn(II), Cu(II), Cr(III), Fe(II), Fe(III), Co(II), Er(II), Ni(II), Eu(III) and Dy(III). For example, paramagnetic contrast agents may comprise dysprosium (DY3+), lanthanide metals (e.g., gadolinium), or transition metals (e.g., manganese). In some embodiments, the contrast agent is a gadolinium-based contrast agent. Any suitable gadolinium-based contrast agent may be used. For example, the contrast agent may be gadoterate, gadodiamide, gadobenate, gadopentetate, gadoteridol, gadoversetamide, gadobutrol gadopentetic acid dimeglumine, gadofosveset, gadocoletic acid, gadomelitol, gadomer 17, gadoxctic acid, etc. In some embodiments, the contrast agent is a supcrparamagnctic contrast agent. For example, the contrast agent may be an iron oxide contrast agent (e.g., superparamagnetic iron oxide, ultrasmall superparamagnetic iron oxide). As another example, the contrast agent may be an iron platinum contrast agent (e.g., superparamagnetic iron platinum).

[0073] In some embodiments, the contrast agent may be complexed, for example, through covalent or noncovalent association, to one or more complexing agents, including lipophilic derivatives thereof, or to polypeptide-containing macromolecules. Such complexes are also referred to herein as chelates. Preferable complexing agents for the present invention include, for example, diethylenetriaminepentaacetic acid (DTPA), ethylene-diaminetetraacetic acid (EDTA), 1,4,7,10- tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA), 1,4,7, 10- tetraazacy clododecane- N,N',N"-triacetic acid (DOTA), 3,6,9-triaza-12-oxa-3,6,9-tricarboxymethylene-10-carboxy-13- phenyl-trideca noic acid (B- 19036), hydroxybenzylethylenediamine diacetic acid (HBED), N,N'- bis(pyridoxy)-5-phosphate)ethylene diamine, N,N '-diacetate (DPDP), 1,4,7-triazacyclononane- N,N',N"-triacetic acid (NOTA), 1,4,8, l l-tetraazacyclotetradecane-N,N',N'',N'"-tetraacetic acid (TETA), kryptands (macrocyclic complexes), and desferrioxamine. More preferably, the complexing agents are EDTA, DTPA, DOTA, DO3A and kryptands, most preferably DTPA. Preferable lipophilic complexes include alkylated derivatives of the complexing agents EDTA, DOTA, for example, N,N'-bis-(carboxydecylamidomethyl-N-2,3-dihydroxypropyl)- ethylenediamine-N,N '-diacetate (EDTA-DDP); N,N'-bis-(carboxy-octadecylamido-methyl-N- 2,3-dihydroxypropyl)-ethylenediamine-N,N'-diacetate (EDTA-ODP); N,N'-Bis(carboxy- laurylamidomethyl-N-2,3-dihydroxypropyl)ethylenediamine-N,N'-diacetate (EDTA-LDP); and the like, including those described in U.S. Pat. No. 5,312,617, the disclosures of which are hereby incorporated herein by reference, in their entirety. Preferable polypeptide-containing macromolecules include, for example, albumin, collagen, polyarginine, polylysine, polyhistidine, gamma-globulin and beta-globulin, or any polypeptide sequence.

[0074] Suitable complexes (e.g., chelates) therefore include, but are not limited to: Mn(I)-DTPA, Mn(II)-EDTA, Mn(II)-DOTA, Mn(II)-DO3A, Mn(II)-kryptands, Gd(III)-DTPA, Gd(III)-DOTA, Gd(III)-DO3A, Gd(III) -kryptands, Cr(II)-EDTA, Cu(I)-EDTA, or iron-desferrioxamine, especially Mn(II)-DTPA or Gd(III)-DTPA. In particular embodiments, the contrast agent comprises a gadolinium chelate. In some embodiments, the gadolinium chelate may comprise an ionic and hydrophilic chelate (e.g., Gd- DTPA, Gd-DOTA, Gd-poly aspartate). In some embodiments, the gadolinium chelate may comprise a nonionic hydrophilic chelate (e.g., Gd-DTPA-BMA, the macrocyclic chelate analog of Gd-DOTA referred to as Gd-HP-DO3A). In some embodiments, the gadolinium chelate may comprise an ionic lipophilic chelate (e.g., Gd-BOPTA, GD-EOB-DPTA). Suitable gadolinium chelates are described in the accompanying examples. In some embodiments, the gadolinium chelate is a macrocyclic chelate.

[0075] The MRI complexes described herein further comprise a blocking substrate. The blocking substrate prevents activation of the contrast agent in the absence of 0-galactosidase activity. The blocking substrate may comprise any suitable moiety cleavable by P-galactosidase. In some embodiments, the blocking substrate comprises a sugar moiety. The sugar moiety may be a monosaccharide, a disaccharide, or a polysaccharide. In some embodiments, the sugar moiety comprises a monosaccharide. Suitable sugar moieties include, for example, moieties containing glucose, fructose, galactose, mannose, ribose or derivatives thereof. In some embodiments, the blocking substrate comprises galactose or a derivative thereof (e.g., -galactose). The blocking substrate (e.g., galactose or a derivative thereof) is removable by p-galactosidase, thus exposing the contrast agent. Accordingly, the contrast agent may be activated in response to enzymatic cleavage by p-galactosidase.

[0076] In some embodiments, the MRI complex further comprises a linker. In some embodiments, the linker conjugates the contrast agent to the blocking substrate. Accordingly, the linker length may be optimized to minimize the MRI signal prior to probe activation (e.g., prior to cleavage of the blocking substrate. For example, the linker may conjugate the macrocyclic contrast agent chelate to the blocking substrate. In some embodiments, the linker is self-immolative in response to cleavage (e.g., hydrolysis) of the blocking substrate. For example, the linker may be self- immolative in response to enzymatic cleavage of the galactose containing moiety. The linker may also include groups to provide desired steric, solubility, and / or biocompatibility properties to the contrast agent. Exemplary groups that may be included in the linker include, but are not limited to, alkyl and aryl groups, including substituted alkyl and aryl groups and heteroalkyl (particularly oxo groups) and heteroaryl groups, including alkyl amine groups. Exemplary groups include p- aminobenzyl, substituted p-aminobenzyl, diphenyl and substituted diphenyl, alkyl furan such as benzylfuran, carboxy, and straight chain alkyl groups of 1 to 10 carbons in length. In some embodiments, groups include p-aminobenzyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, acetic acid, propionic acid, aminobutyl, p-alkyl phenols, 4-alkylimidazole, carbonyls, OH, COOH, glycols, etc.

[0077] In some embodiments, the self-immolative linker provides a functional group that coordinatively saturates the MRI contrast agent, thereby preventing a water access to the MRI contrast agent resulting in no detectable signal in the absence of P-galactosidase. In some embodiments, cleavage of the blocking substrate by P-galactosidase activates the MRI contrast agent by initiating a spontaneous electron cascade of the self-immolative linker that results in removal of the functional group from the MRI contrast agent. Removal of the functional group from the MRI contrast agent results in water gaining access to the contrast agent, thereby activating the MRI contrast agent to produce a detectable signal. In some embodiments, the detectable signal is generated within 1 hour of cleavage of the blocking substrate. For example, in some embodiments the detectable signal is generated within 1 hour, within 50 minutes, within 40 minutes, within 30 minutes, or within 20 minutes following cleavage of the blocking substrate. This mechanism is shown in the schematic of figure 1.

[0078] In some embodiments, the complex exhibits at least a 50% increase in relaxivity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) in vivo following cleavage of the blocking substrate. In some embodiments, the complex exhibits at least an 80% increase in relaxivity in vivo following cleavage of the blocking substrate. In some embodiments, the complex exhibits at least a 50%, 60%, 70%, 80%, or 90% increase in relaxivity in vivo within 1 hour (e.g., within 60 minutes, within 50 minutes, within 40 minutes, within 30 minutes, within 20 minutes) following cleavage of the blocking substrate.

[0079] In some embodiments, the functional group is attached to the self-immolative linker by a spacer comprising at least 4 carbons. For example, in some embodiments the spacer comprises at least 4 carbons, at least 5 carbons, at least 6 carbons, at least 7 carbons, at least 8 carbons, at least 9 carbons, or about 10 carbons. In some embodiments, the spacer comprises 4, 5, 6, 7, or 8 methylene groups. In some embodiments, the spacer consists of 4, 5, 6, 7, or 8 methylene groups. In some embodiments, the spacer is attached to an amine present in the self-immolative linker. In some embodiments, the functional group is a single carboxylate. In some embodiments, the MRI complex may further comprise one or more additional components that provide a desired functionality. For example, the complex may optionally comprise a targeting moiety. Preferred targeting moieties are those that allow concentration of the agents in a particular localization. For example, antibodies, cell surface receptor ligands and hormones, lipids, sugars and dextrans, alcohols, bile acids, fatty acids, amino acids, peptides and nucleic acids may all be attached to the MRI complex to localize or target the contrast agent to a particular site. In some embodiments, the targeting moiety allows targeting of the MRI agents of the invention to a particular tissue, the surface of a cell or a subcellular localization. In some embodiments, the targeting moiety is a peptide. In some embodiments, the targeting moiety is an antibody.

[0080] An exemplary MRI contrast agent for use in the presently described methods of detecting senescent cells is shown in FIG. 1. Exemplary MRI contrast agents for use in the presently claimed method, including the contrast agent shown in FIG. 1 and suitable methods of producing bioresponsive contrast agents, are described in U.S. Patent Publication No. US20210030896A1 the entire contents of which are incorporated herein by reference. As described in US20210030896A1, optimization of the bioresponsive MRI contrast agent was performed using three versions of the self-immolative contrast agent containing zero, one, or two coordinating acetates, and the complex with a single coordinating acetate to Gd(III) had a significantly lower observed relaxivity (i.e., dark in an MR image). Accordingly, in some embodiments the functional group is a single coordinating carboxylate. In some embodiments, the single coordinating carboxylate is attached to an amine present in the linker. Further, as described in US20210030896A1, using computational analysis on a related series of agents, the length of the spacer was varied (where n = 1, 3, 5, and 7 methylene units) and complexes where the length of the spacer was 5 methylene units had the highest change in relaxivity (90%) when exposed to fl- gal (i.e., dark to bright). Coordination of Gd(III) by this functionality significantly reduces water access prior to enzyme activation, thereby affording a “dark” agent (q ~ 0). Hydrolysis of the glycosidic bond by 0-gal restored agent efficiency following self-immolation with subsequent water binding to Gd(III) turning the agent “bright” (q ~ 1).

[0081] The MRI complexes described herein may incorporated into a pharmaceutical composition. For example, the MRI complex may be incorporated into a composition for delivery to a subject. The composition may further comprise one or more excipients, dependent on the intended mode of administration. In embodiments of the present invention, the composition can be introduced into a biological structure disposed in a subject. The mode of administration to a sample or subject can determine the sites and / or cells in the organism to which an agent will be delivered. In some embodiments, compositions can be injected into a subject parenterally (e.g., by injection, including subcutaneous, intraarterial, intravenous, intradermal, intramuscular, intraarticular, intrathecal, etc.). In some embodiments, the compositions can be injected intrathecally. Accordingly, a suitable composition may be administered in the form of a sterile, aqueous solution; such a solution can contain other solutes, including, but not limited to, salts or glucose in quantities that will make the solution isotonic.

[0082] When a contrast enhancement agent of the present invention is administered to humans, the prescribing physician will ultimately determine the appropriate dosage for a given human subject, and this can be expected to vary according to the weight, age and response of the individual as well as the nature and severity of the patient's condition. In some embodiments, the MRI complexes of the present invention may also be co-administered with one or more additional imaging, diagnostic, or therapeutic agents.

[0083] EXPERIMENTAL

[0084] Example 1

[0085] Senescent cells promote osteoarthritis progression through secretion of inflammatory mediators. Preclinical studies have identified senescence-associated beta-galactosidase (P-gal) as a biomarker of senescence, but in vivo detection remains challenging. Here, a P-gal responsive gadolinium (Gd) chelate was used for non-invasive in vivo detection of senescent cells with standard clinical magnetic resonance imaging (MRI) technology. In vitro studies showed that senescent mesenchymal stromal cells (MSCs) exhibited significant MRI signal enhancement upon incubation with the P-gal responsive Gd-chelate probe compared to viable control cells. In vivo, intraarticular injection of the probe into pig knee joints revealed its retention and activation within senescent cells in cartilage defects, evidenced by a significant increase in Ri relaxation rate. MRI- based senescent cell detection holds promise for identifying patients amenable to senolytic therapies, tailoring treatment plans, and monitoring therapy response in real time. The P-Gal-activated MR probe use herein is composed of gadolinium (Gd III) conjugated to a hydrophilic P-Gal substrate. Before enzyme reaction, the coordination sphere of Gd is saturated by a functional group provided by the self-immolative linker, preventing the interaction with water protons and resulting in low signal on T1 -weighted MRI scans. After exposure to P- gal, the P-galactosyl moiety is cleaved, an electron cascade (red) provides an open coordination site for water to bind to Gd (III) resulting in an increased (hyperintense) MRI signal on Tl- weighted MR scans. Viable or senescent mesenchymal stromal cells were implanted in cartilage defects of the distal femur of pig knee joints. The P-gal responsive Gd-chelate was injected into the knee joint, leading to activation of the P-gal sensitive Gd-chelate in senescent cell transplants and not viable cell transplanted, as measured by MRI.

[0086] RESULTS

[0087] Optimal concentration of P-gal responsive Gd-chelate

[0088] Effective detection of the activation of the P-gal responsive Gd-chelate occurs within a narrow concentration window. Excessive agent leads to overwhelming non-activated signal, while low concentrations limit the detectable activation signal. Fig. 2 shows the Ti-weighted MR contrast effect of P-gal responsive Gd-chelate without and with P-gal enzyme. From Ti MR phantom images and Ti mapping (Fig. 2A, 2C), the P-gal responsive Gd-chelate after incubating P-gal enzyme shows enhanced Ti contrast effect and shorter Ti relaxation time (Fig. 2B). Additionally, rl relaxivity of P-gal responsive Gd-chelate after incubating with P-gal enzyme was higher (rl= 4.9mM-1s'1) compared to P-gal responsive Gd-chelate without incubation with P-gal (rl= 3.3 mM1s'1) shown in Fig. 2D. Additionally, To-weighted MR contrast effect of P-gal responsive Gd-chelate without and with P-gal enzyme (Fig. 8 A) indicates that r2 relaxivity of P- gal responsive Gd-chelate after incubating with P-gal enzyme was higher (r2= 5.0 mM'1s'1) compared to P-gal responsive Gd-chelate without incubation with P-gal (r2= 3.2 mM'1s'1) shown in Fig. 8B.

[0089] MRI differentiation of senescent and viable cells with the P-gal responsive Gd-chelate

[0090] To assess the ability of the P-gal responsive Gd-chelate to discriminate viable and senescent mesenchymal stromal cells (MSCs), MSC were incubated with the chelate, followed by MRI analysis. Triplicate samples of 1 x 107primary MSCs were incubated with doxorubicin, a senescence inducer. 89+2.1 % of these cells were P-gal positive compared to 13+3.2% in untreated controls (p = 0.025) (Fig. 3A, 3B). The SASP phenotype of doxorubicin-cxposcd cells was further confirmed by an increase in the expression levels of IL-6 (control: 490±75 pg / mL, senescence: 2,700+160 pg / mL p = 0.025) and IL-8 (control: 1100+46 pg / mL; senescence 2,600+240 pg / mL, p = 0.025) (Fig. 3C). Additional markers which indicate cell cycle arrest were used to confirm doxorubicin-induced senescence, as seen by increased p53 (3.43 fold change, p=0.014, Fig. 3D, 3E), increased p21 (2.08 fold change, p=0.014, Fig. 3D, 3E) and reduced Lamin Bl (0.68 fold change, p=0.014, 3D, 3E) protein expression in senescent cells compared to control cells, as determined by western blotting (Fig. 3D, 3E). Viable and senescence MSCs treated with P-gal responsive Gd-chelate had no effect on the viability, as determined by CCK-8 assay (Fig. 3G). Senescent MSCs exposed to P-gal responsive Gd-chelate at 0.5 mM and 0.25 mM exhibited a mean Ri relaxation rate of 3.7+0.43 s’1and 3.1+0.51 s’1, respectively (Fig. 3H). Viable MSCs exposed to P-gal responsive Gd-chelate at the 0.5 mM and 0.25 mM concentrations showed a mean Ri relaxation rate of 2.0 + 0.73 s’1and 1.6 + 3.1 s’1, respectively. At 0.5mM concentration, senescent cells demonstrated stronger MRI signal on Ti- weighted MRI images and significantly longer Ri relaxation rates compared to viable controls (N=9; 0.25 mM, p = 0.084; 0.5 mM, p = 0.013) (Fig. 31). ICP-OES measurements of the same cell samples demonstrated no significant difference in Gd concentration of control and senescent cell samples exposed to P-gal responsive Gd-chelate (Fig. 3J), indicating that the increased Ti-signal of senescent cells is due to activation of the P-gal responsive Gd-chelate.

[0091] P-gal responsive Gd-chelate detects senescent cells in pig knee specimens with MRI

[0092] To assess the ability of P-gal responsive Gd-chelate to detect senescent cells against the typical anatomical background tissue of an articular joint, control and senescent cells were incubated with the chelate and implanted into cartilage defects of pig knee specimen, followed by MRI. Tr-weighted spin-echo (Ti-SE) sequences provided anatomical details, while a Ti-weighted RARE variable TR saturation recovery sequence was used to generate Ti maps for quantitative Ri relaxation rate measurements. Implants of senescent MSCs exhibited a more pronounced hyperintense signal on Tl-weighted MR images compared to their viable counterparts (Fig. 4A). The mean Ri relaxation rate of senescent cell implants (1.6 + 0.80 s’1) was significantly longer than the Ri relaxation rate of viable cell implants (0.97 + 0.27 s’1; p = 0.007) (Fig. 4B). Accordingly, the signal-to-noise ratio (SNR) of senescent cell implants (SNR: 11 ±2.1 ) was significantly higher compared to viable controls (SNR: 8.5±1.8, p=0.032) (Fig. 4C).

[0093] P-gal responsive Gd-chelate detects senescent cells in vivo with MRI

[0094] The ability of the P-g l responsive Gd-chelate to detect senescence in vivo was next evaluated in the knee joints of live pigs using MRI. IxlO7senescent and IxlO7control cells were implanted into full-thickness cartilage defects of eight distal femurs of four Yucatan minipigs (Fig. 5 A). 24 hours later, 5 ml of 2.5 mM -gal responsive Gd-chelate was injected intraarticularly into each knee joint. The higher concentration compared to in vitro studies was chosen to account for the dilution of the probe in joint fluid. Ti-weighted MRI scans after intra-articular injection of the P-gal responsive Gd-chelate revealed stronger MRI signal in cartilage defects containing senescent cells compared to defects with control cells (Fig. 5B, 5C). The Ri relaxation rate calculated from the Ti maps indicated significant activation of the P-gal responsive Gd-chelate in senescent cell implants (1.5+0.61 s'1) compared to controls (0.95+0.26 s'1, p=0.01) (Fig. 5D). Accordingly, the signal-to-noise ratio (SNR) of senescent cells (SNR: 410+120) was significantly higher compared to that of control viable cells (SNR: 230±47; p=0.00l ) (Fig. 5E). To further demonstrate the specific activation of senescent cells loaded in the cartilage defects, MRI of the knee joint was performed prior to intra-articular injection of the P-gal responsive Gd-chelate and no bright signal in the cartilage defect loaded with senescent cells was observed, in contrast to the bright signal in the sagittal Ti-weighted MR image of the same knee joint at 2 hours after intra-articular injection of the P-gal responsive Gd-chelate (FIG. 9).

[0095] After sacrificing the animal, the knee joints were excised (Fig. 5F) and underwent ex vivo MR imaging. Similar to the results in live pigs, implants of senescent MSCs exhibited a more pronounced hyperintense signal on Ti -weighted MR images compared to their viable counterparts (Fig. 5G, 5H). The Ri relaxation rate of senescent cell implants (2.7 + 0.75 s'1) was significantly higher compared to viable cell implants (1.5 + 0.42 s'1; p = 0.002) (Fig. 51). Furthermore, the signal-to-noise ratio (SNR) of the MRI signal was significantly higher for senescent cells (11 ±2.0) compared to controls (8.5±1.7; p=0.018) (Fig. 5 J). Taken together, results indicate that the P-gal responsive Gd-chelate can be used as an MR probe for imaging senescent cells in articular joints.

[0096] Validation of senescence MR imaging with immunocytochemistry To further validate the imaging technique with established methods for detecting senescent cells, histological analysis of all pig knee specimens was performed. Immunocytochemistry (ICC) was employed to visualize additional senescence markers, including pl 6, p21, and P-gal. Senescent cell implants in cartilage defects expressed significantly higher levels of p!6 (Control: 277.00 ±51.06, Senescence: 3000± 290) (Fig. 6A, 6D), p21 (Control: 170±25, Senescence: 2800 ± 27) (Fig. 6B, 6D) and P-gal (Control: 200+25.31, Senescence: 2900+ 280) (Fig. 6C, 6D) compared to control implants (p=0.025). This sustained expression of senescence markers indicates that implanted cells retained their characteristic features throughout the duration of the experiment. Additional staining was performed to confirm the presence of MSCs in the cartilage defects one day after implantation of the cells and that the differences in R1 relaxation times are due to the distinct differences between control and senescent cells and not influx of macrophages or variance in vascularity. The retention of MSCs in the cartilage defects of pig knees on day 2 was confirmed by the expression of CD73, CD90 and CD 105 in the cartilage defects loaded with control and senescent cells (Fig. 11). CD73, CD90 and CD 105 are established surface markers of mesenchymal stem cells. Further, trichrome staining which stains the collagen fibers blue, indicated no fibrosis in the cartilage defects (Fig. 12). F4 / 80 staining indicates no difference in the influx of macrophages in the defects loaded with control and senescent cells (Fig 13A). F4 / 80 is an established cell-surface marker for macrophages. CD31 staining indicates no vascularity in the defects loaded with control and senescent cells (Fig. 13B).

[0097] Additional experiments were conducted using human joint specimens to evaluate the ability of the P-gal responsive Gd-chelate described herein to detect senescent cells in such human joint specimens. Results are presented in FIG. 14, FIG. 15, and FIG. 16. The results therein demonstrate successful detection of senescent cells in human hips (FIG. 14), human knees (FIG. 15), and that this ability to detect senescent cells was seen for the P-gal responsive Gd-chelate but was not seen for the commercially available contrast agent Gadavist, which has the following structure:

[0098]

[0099] DISCUSSION

[0100] This study demonstrated that a P-gal responsive Gd-chelate can detect senescent cells non- invasively using MRI in vitro, in knee explants, and in vivo, in a live pig model. The senescence induction model yielded a robust senescence phenotype, as confirmed by established biomarkers, including increased levels of senescence-associated P-gal (SA-P-gal), IL-6, and IL-8.

[0101] Other imaging techniques such as bioluminescence imaging or fluorescence imaging to have been used to attempt to detect SA-P-gal in vitro and in vivo. Yang L. et al. used a methylene blue (MB) fluorophore coupled to P-galactose (MB-Pgal) to detect senescent HeLa cells and mouse embryonic fibroblast (MEF) cells in vitro. Upon exposure to P-gal, MB-P-gal released free methylene blue (MB), generating near-infrared (NIR) fluorescence detectable with optical imaging techniques (Yang, L. et al. An activatable NIR probe for the detection and elimination of senescent cells. Anal. Chem. 94, 5425-5431 (2022)). Redy-Keisar, O. et al. described the GQCy7 compound, which undergoes a chemical reaction where the glycosidic bond is hydrolyzed when exposed to P- gal. The release of QCy7 resulted in an extended ^-conjugated system that underwent an internal charge transfer process, turning the fluorescence "on." Mice injected with a solution of GQCy7 that had been incubated with P-gal showed an increased near-infrared fluorescent (NIR- fluorescent) signal (Redy-Keisar, O., Kisin-Finfer, E., Ferber, S., Satchi-Fainaro, R. & Shabat, D. Synthesis and use of QCy7-derived modular probes for the detection and imaging of biologically relevant analytes. Nat. Protoc. 9, 27-36 (2014)). Wehrman, T. et al. developed a bioluminescent imaging approach that allowed for noninvasive in vivo imaging of P-galactosidase activity. In their study, in vitro and in vivo experiments were performed to test Lugal, a caged luciferin-galactoside conjugate, which is cleaved by P-galactosidase to produce luminescence (Wehrman, T. S., von Degenfeld, G., Krutzik, P. O., Nolan, G. P. & Blau, H. M. Luminescent imaging of P-galactosidase activity in living subjects using sequential reporter-enzyme luminescence. Nat. Methods 3, 295- 301 (2006)). Lee HW et al. developed a fluorescent biomarker, which showed a blue-to-yellow color change in response to P-gal. They used this florescent probe to detect senescence-associated P-gal in murine fibroblast skin cells and rat skin tissue in vitro using two-photon microscopy (Chang, E. Y. et al. SSR white paper: guidelines for utilization and performance of direct MR arthrography. Skeletal Radiol. (2023) doi:10.1007 / s00256-023-04420-6). However, for these approaches reliance on florescence or optical imaging restricts their application to preclinical models due to the limited tissue penetration of the applied imaging biomarker. This hinders clinical translation. In contrast, the bioactivatable probe described herein is used with MR imaging, the gold standard for clinical imaging of OA in knee joints. The MR contrast effect of Gd(III) can be modulated through various physical parameters, such as the number of bound water molecules (q), the rate of molecular tumbling (rr), and the rate of water exchange (rm). The use of a pendant carboxyl ligand herein to saturate the coordination sphere of Gd (III) leads to a significant signal difference between the inactive ('off) and active ('on') versions of the probe, enabling the effective detection of senescent cells in vitro and in vivo. A major advantage of the P-gal responsive Gd-chelate is that it can be applied repeatedly in the same subject, to evaluate changes in senescent cell distribution or quantity over time. The imaging technique is not associated with any irradiation and exposure and can easily be integrated with clinical standard MRI protocols.

[0102] Provided herein is a P-gal responsive Gd-chelate for detecting senescent cells on MR images. This Gd-chelate effectively identified senescent cells ex vivo, in joint specimen and in vivo, in a large animal model of arthritis. The P-gal responsive Gd-chelate herein can be used to select patients with high burden of senescent cells, prescribe tailored treatment strategies, and monitor response to these therapies, ultimately decelerating disease progression and enhancing quality of life for patients with OA and other age-related disorders. The P-gal responsive Gd- chelate can be used in diagnosing senescence linked to natural aging and degenerative joint diseases. The P-gal responsive Gd-chelate provides a clinically translatable biomarker for monitoring the effectiveness of new therapies designed to eliminate senescent cells (senolytics). METHODS

[0103] Characterization of P-gal responsive Gd-chelate

[0104] The P-gal responsive biomarker is composed of a Gd-chelate and a pendant coordinating carboxylate unit connected via a six-carbon (C6) linker to an aniline moiety (Figure 1). Prior to enzyme activation, the Gd (III) coordination sphere is fully occupied, blocking interaction with water protons. This translates to a weak signal on Ti-weighted MRI scans. Exposure to P-gal triggers cleavage of the P-galactosyl moiety, releasing the functional group and exposing a coordination site on Gd (III). This newly accessible site permits interaction with water protons, resulting in a strong signal enhancement on Ti-weighted MRI scans.

[0105] The chemical structure of the P-gal responsive Gd-chelate is shown in Figure 1. It has a molecular weight of 986 Dalton. The n relaxivity of the probe in the absence of P-gal is 3.2 mM"1s’1at 1.41T and 37°C and the n relaxivity of the probe in the presence of P-gal is 3.91 mM"1s"1at 1.41T and 37°C (FIG. 10A). The relaxivity of the probe in the absence of P-gal is 3.16 mM"1s"1at 1.41T and 37°C and the n relaxivity of the probe in the presence of P-gal is 4.08 mM"1s"1at 1.41T and 37°C (FIG. 10B). The relaxivity at 1.41T was measured by scanning increasing concentrations of 0.23, 0.33, 0.47, 0.65, and 0.84 mM of the P-gal responsive Gd-chelate, with and without addition of 17U P-gal at pH 6.0.

[0106] The n relaxivity of the probe in the absence of P-gal is 3.9 mM"1s"1at 3T and 37°C and the n relaxivity of the probe in the presence of P-gal is 4.7 mM"1s"1at 3T and 25°C. The relaxivity was measured by scanning increasing concentrations of 0.125, 0.25, 0.5, 0.625, 0.75, 1 mM of the P-gal responsive Gd-chelate, with and without addition of 10U P-gal at pH 6.0. The relaxivity at 3T was measured by standard linear fitting of the Ri relaxation rate measurements at different concentrations of the P-gal responsive Gd-chelate. Simple linear regression analysis was used for linear fitting of the relaxation rate versus concentrations and calculated by y=alpha+ beta*x.

[0107] Induction of senescence in MSCs, as confirmed by ELISA and P-gal assays

[0108] Bone marrow-derived MSCs were isolated from 10 ml of bone marrow fluid aspirated from the iliac crest of donor pigs. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 1% penicillin- streptomycin (pen-strep) and 10% fetal bovine serum (FBS) at 5% CO2 and 37°C. The media was replenished every 24-48 h. After reaching -90% confluency, adherent MSCs were further expanded, used for experiments, or stocked at -195°C. MSCs were used at a low passage number (p = 3 or p = 4) to avoid spontaneous senescence.

[0109] Triplicate samples of IxlO7MSCs in 12-well plates in DMEM media were exposed to 400nM doxorubicin on day 1 and day 3, followed by analysis for senescence on day 5. Triplicate samples of IxlO7MSCs in DMEM media served as non-exposed controls. Control and senescent cells were washed with phosphate buffered saline (PBS) twice and fixed with 2% paraformaldehyde in PBS for 10 min at room temperature. The fixative solution was removed by washing the cells thrice with 1% bovine serum albumin (BSA) in PBS. Cells were stained for senescence detection using the Senescence Green Probe from the CellEvent Senescence Green Detection Kit (C10851, Invitrogen). Briefly, the probe was warmed to 37°C for 10 minutes in the dark before being added to the cells for a 2-hour incubation at 37 °C without CO2 and under dark conditions. Following incubation, cells were washed three times with PBS and then imaged using a fluorescent microscope (Keyence, BZ-X710, Cupertino, CA).

[0110] In addition, the supernatants were collected for ELISA analysis. Commercially available kits were used to measure IL-6 (RAB0310, Sigma, St. Louis, MA) and IL-8 (RAB0321, Sigma, St. Louis, MA) levels in the supernatants, as per the manufacturer’s instructions.

[0111] Western blotting

[0112] Lysates from Control and senescent cells were subjected to SDS-PAGE on a 10% or 4- 20% precast gel (Invitrogen, Carlsbad, CA) run at constant voltage (225 V) and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membrane was blocked for 1 h at room temperature in blocking buffer [Tris-buffered saline + Tween 20 (TBST) with 1% BSA] to reduce nonspecific binding. The membranes were then incubated with the respective primary antibodies overnight. All primary antibodies used were either validated by the supplier or in laboratory. The primary antibodies used were LaminB (no. 13435, 1:1,000 dilution; Cell Signaling), p21 (no. abl88224, 1:1,000 dilution; Abeam), and p53 (no. 9282, 1:1,000 dilution; Cell Signaling); GAPDH (1:10,000 dilution, no. 5174, Cell Signaling). After four 10-min washes with TBST, membranes were incubated for 1 h with the respective species-specific horseradish peroxidase- conjugated secondary antibodies (Bio-Rad, Hercules, CA) in TBST containing 1% BSA. The membranes were rinsed again four times with TBST for 10 min each time, and bands were detected using Supersignal luminol enhancer (Perbio Science UK, Cheshire, UK) followed by exposure to blue light-sensitive X-ray film (Hyperfilm; Amersham Biosciences, Little Chalfont, UK). Equal protein loading was verified by reprobing membranes with anti-P-actin antibody. The relative intensities of protein bands (relative density units) were quantified by scanning densitometry using ImageJ software (Molecular Dynamics, Sunnyvale, CA).

[0113] Determining the optimal incubation time and concentration of the P-gal responsive Gd- chelate

[0114] To determine the optimal concentration of the Gd-chelate for the detection of P-gal- mediated MRI signal changes, decreasing concentrations of 1 mM, 0.75 mM, 0.625 mM, 0.5 mM, 0.25 mM, and 0.125 mM P-gal responsive Gd-chelate were suspended in PBS (25°C, pH 7.4). In a pilot study, the initial concentration was doubled from 0.125 to 0.25, 0.5 and 1 mM. Since 0.25 and 0.5 provided the best results, the experiment was repeated with additional concentrations between 0.5 and 1 mM. One series of samples was left untreated, and a second series was incubated with 10 units of P-gal enzyme (Sigma-Aldrich, St. Louis, MO) at 37°C for 8 h. To determine the optimal incubation time of the Gd-chelate for the detection of P-gal-mediated MRI signal changes, nine test tubes with 0.5 mM P-gal responsive Gd-chelate were prepared with addition of 10 mM phosphate buffered saline or with addition of 17 U of P-galactosidase. All samples underwent Ti relaxation time measurements at pH 7.4, 37 °C and 1.7 Tesla, at 10, 20, 30, 40, 60, 80, 120, and 150 minutes after incubation with either PBS or -galactosidase (FIG. 7). To minimize air-induced artifacts, triplicate samples of the aforementioned solutions in Eppendorf tubes were submerged in water baths before acquiring MRI images. All samples underwent MRI on a 3T MRI scanner (Bruker BioSpec, Billerica, MA) using a Tl-weighted sequence (TR = 12115 ms, TE = 24 ms, FOV = 40 mm x 20 mm, matrix = 96 x 64, slice thickness = 1 min.GLB image size = 96 mm x 96 mm); inversion recovery sequence (TI = 35-10000 ms, TR = 11050-21015 ms, TE = 24 ms, FOV = 40 mm x 20 mm, matrix = 96 x 64, slice thickness = 1 mm, image size = 96 mm x 96 mm) and T2-weighted multi -slice multi-echo (MSME): TR: 3000 ms, TE: 132 ms, slice thickness = 1 mm, FOV = 35 mm x 35 mm, matrix = 160 x 160.

[0115] MRI differentiation of senescent and viable cells with the P-gal responsive Gd-chelate

[0116] Triplicate samples of 1X107MSCS in T-75 flasks in DMEM media were exposed to 400nM doxorubicin on day 1 and day 3, followed by analysis for senescence on day 5. Triplicate samples of IxlO7MSCs in T-75 flasks in DMEM media served as non-exposed controls. Control and senescent cells were washed with phosphate buffered saline (PBS) twice and trypsinized to obtain cell suspensions. Cell suspensions of triplicate samples of 1 x 107control and senescent cells were incubated with 0.5 mM or 0.25 mM P-gal responsive Gd-chelate in PBS at 37°C for 8h. After the incubation with the P-gal responsive Gd-chelate, the cells were pipetted well to resuspend the cells to obtain a suspension for MRI. All samples underwent MRI on a 3T MRI scanner (Bruker BioSpec, Billerica, MA), using a Ti-weighted fast SE sequence for anatomical orientation (repetition time [TR] = 700, time to echo [TE] =12 ms, 80 mm x 40 mm field of view [FOV], 192 x 192-pixel matrix, 12 averages) and a variable TR rapid acquisition with relaxation enhancement (RARE) sequence (TE = 10 ms, TR = 40, 75, 150, 300, 500, 700, 1,000, 1,500, and 2,000 ms, RARE factor = 2, 80 mm x 40 mm FOV, 500 x 250 pixel matrix, 2 averages) for generating Ti- maps and measuring Ri relaxation rates.

[0117] Cell Viability Assay

[0118] To assess the impact of the incubation of the cells with 0.25mM and 0.5mM -gal responsive Gd-chelate for 24 hours, a viability assay was performed using a commercially available cell Counting Kit 8 (WST-8) (ab228554, Abeam, Cambridge, UK). The cells were seeded in 96-well plate and incubated with 0.5 mM or 0.25 mM P-gal responsive Gd-chelate at 37°C for 8 h. lOplAvell of WST-8 solution was added to each well and the plate was incubated at dark, 37°C for 3 hours. The absorbance was measured at 460nm.

[0119] Inductively coupled plasma optical emission spectroscopy (ICP-OES)

[0120] The concentration of Gd (III) in above mentioned test tubes with control and senescence cells (which had been incubated with 0.5 mM and 0.25 mM P-gal responsive Gd-chelate) was determined by ICP-OES. Each sample was diluted to 1 mL with concentrated nitric and water for ICP-OES analysis. Gd were measured on a VG Elemental PQExcell spectrometer that was standardized with eight concentrations spanning 0-50 ppb Gd (III). In (III) (1 ppb) was used as an internal standard. Ex vivo detection of senescent cells in pig knee specimens

[0121] Ten knee joints of five Yucatan minipigs (2 male, 3 female) were acquired postmortem from the Veterinary Service Center at Stanford Medicine as a donation in accordance with the 3R principles (Reduce, Refine, Replace). The specimen originated from animals that had undergone other experiments at the facility that did not affect the knee joints. Two 5 mm full thickness cartilage defects were created in the distal femur of each knee specimen. To evaluate the ability of the P-gal responsive Gd-chelate to differentiate senescent and viable cells in arthritic joints, 1 x 107control and senescent cells were incubated with 0.25 mM P-gal responsive Gd-chelate at 37°C for 8 h, implanted into the cartilage defects the specimen was scanned on a 3T MRI scanner (3T GE MR 750, GE Healthcare, Boston, MA), using a Ti-map sequence (TR = 40, 75, 150, 300, 500, 700, and 2,000 ms, TE = 10 ms, FOV 80 mm x 40 mm, matrix 500 x 250 pixel, 2 averages) and Ti weighted fast SE sequence (TR = 700 ms, TE = 10 ms, RARE factor = 2, 80 mm x 40 mm FOV, 500 x 250 sampling matrix, and 2 averages).

[0122] In vivo detection of senescent cells with MRI and the P-gal responsive Gd-chelate

[0123] All animal studies were approved by the institution’s Administrative Panel on Laboratory Animal Care (APLAC 29859) and were conducted under the supervision of the Stanford Veterinary Service team. Four 4-12-month-old female Yucatan minipigs were obtained from S&S Farms Selma, CA). Following delivery to the facility, all animals underwent a 4-day quarantine and stabilization period. Study animals were housed individually indoors in an AAALAC- accredited facility with a 12 / 12-h light / dark cycle, controlled temperature (68-76°F), humidity (30-70%), and ventilation (10-15 air changes hourly). All animals were housed in pens adjacent to pens housing other pigs and were allowed to ambulate at all times. Animals were fed twice daily and had free access to drinking water ad libitum. Environmental enrichment with toys and food rewards was provided on a regular basis. Prior to anesthesia for surgery, imaging and euthanasia, food was withheld for 12 hours.

[0124] All animals underwent bilateral knee surgeries to create full-thickness cartilage defects, followed by MRI on the following day. All procedures were performed under general anesthesia. For surgery, animals were sedated with tiletamine and zolazepam (Telazol; Zoetis, Kalamazoo, MI; 2-8 mg i.m. / kg), intubated, and maintained under general anesthesia with 1 % to 3% isoflurane. An eye lubricant was applied to protect the eyes. Animals were monitored continuously, and health status was documented at least every 15 min for heart rate, respiration rate, body temperature, ventilation, oxygenation, and depth of anesthesia by eliciting reflex responses. For each pig, the stifle area was shaved, cleaned with betadine and chlorhexidine, and draped. Sciatic and femoral nerve blocks were performed under ultrasound guidance. Pigs were placed into the supine position on a heating mat and secured to the surgical table. A surgical incision was performed on the medial aspect of the joint, allowing for lateral dislocation of the patella and access to the knee joint.

[0125] Sixteen 5 mm wide full-thickness cartilage defects were created in eight distal head of femur (two defects per knee joint) with a sterile biopsy punch and curette. IxlO7senescent MSCs and IxlO7viable MSCs were implanted into the cartilage defects and secured with fibrin glue (Evicel; Ethicon Inc., Somerville, NJ, USA). In each distal femur, one defect contained senescent MSCs and the other contained viable MSCs. The incision was closed in layers using Vicryl 2-0 and 3-0 (Ethicon Inc.). Upon recovery, animals were allowed unrestricted weight bearing. Animals were monitored for signs of pain and distress and analgesia was provided accordingly.

[0126] The next day, animals were anesthetized with isoflurane and underwent intraarticular injection of 2.5 mM of P-gal responsive Gd-chelate intraarticularly into each knee joint. The higher concentration compared to in vitro studies was chosen to account for the dilution of the probe in joint fluid. At two hours after intraarticular injection of the P-gal responsive Gd-chelate, MRI was performed on a 3T clinical scanner ( 3T GE MR 750, GE Healthcare, Boston, MA) under isofluorane anesthesia, using a knee surface coil and a variable TR saturation recovery sequence (TR = 40, 75, 150, 300, 500, 700, and 2,000 ms, TE = 14 ms, FOV 17 cm) and Ti weighted fast SE sequence (TR = 600 ms, TE = 10.07 ms, FOV 17 cm, slice thickness = 2mm, spacing = 0.2 and 2 averages). A pilot imaging study was done by performing MRI of a pig knee joint before the intra- articular injection of the P-gal responsive Gd-chelate.

[0127] After completion of MRI, the anesthetized animals were euthanized. A commercially available euthanasia solution (Euthasol, Virbac Corp, Westlake, TX, USA) was administered intravenously under deep anesthesia at 1 mL per 4.5 kg of body weight. Death was confirmed by complete cessation of heartbeat by cardiac auscultation. Following this confirmation, samples of the stifles were excised for postmortem MRI and histopathological assessment. The knee joints were excised and underwent a postmortem scan on a 3T preclinical MRI scanner (Bruker, BioSpec 3T MRI, Billerica, MA, USA), using a knee surface coil and with the same MR parameters used to scan the explants, which were described above. MR image data analyses

[0128] MRI scans of contrast agent samples, cell samples, joint specimen, live pigs and explants were analyzed by generating Ti-maps with the FIJI software (Bethesda, Maryland). The Ti-maps were created by fitting the MRI signal at increasing TRs to the formula Sn= S0(l-EXP(-TRn / Ti)) where the Sn: longitudinal magnetization at time n, So: equilibrium longitudinal magnetization, TR: repetition time and Ti: spin-lattice relaxation time. Ti-relaxation times of contrast agent samples, viable and senescent cells were measured in vitro, ex vivo, and in vivo on the Ti-maps through operator-defined regions of interest (ROI). Ri-relaxation rates were calculated as Ri=l / Ti. Signal intensity of the cell implants and the background noise was measured on anatomical Ti- weighted SE images through ROIs and calculated the Signal-to-Noise Ratio (SNR) = SIceii implant / noise.

[0129] Validation of senescence MR imaging biomarker with immunocytochemistry

[0130] The excised pig knees were fixed in 10% neutral buffered formalin, followed by decalcification in Formical-2000 for one week. Subsequently, tissues were paraffin-embedded and sectioned into 10-pm slides using a cryostat microtome (Leica CM1800). For immunocytochemistry (ICC), slides underwent deparaffinization by sequential immersions in xylene (5 min), 100% ethanol (2 min), and 95% ethanol (1 min), followed by rehydration in water. The samples were fixed with 4% paraformaldehyde for 10 min and then washed in triplicate with PBS. Following fixation, tissue sections were permeabilized with 0.25% Triton X-100 in PBS (PBST) for 10 minutes at room temperature. Subsequently, sections were blocked with 1% BSA in PBST for 30 min at room temperature, then washed and incubated overnight with primary monoclonal antibodies (mAb) pl6 mAb (ab51243, Abeam), p21 mAb (abl88224, Abeam), and P- gal mAb (ab 203749, Abeam), F4 / 80 (ab6640, Abeam, Cambridge, UK), CD31 (3528T Cell Signaling), CD73 (13160T Cell Signaling), CD105 (14606S Cell Signaling) and CD90 (138O1S Cell Signaling) at 4°C. After washing, sections were incubated with secondary antibody (Alexa Fluor 488 goat anti-rat IgG, Al 1006, Invitrogen) for 2 hours at room temperature in the dark. Finally, sections were washed and mounted for fluorescence microscopy (Keyence BZ-X710). Histopathologic images of distal femur cartilage were obtained using a defined experimental protocol on the fluorescence microscope (Keyence, Itasca). The mean fluorescence intensity of the green senescent cell was calculated from the sum of the values of all the pixels in the tissue of interest divided by the number of pixels (% of staincd / total area) using ImagcJ software in a 2x field of view25.

[0131] Statistical analysis

[0132] The wilcoxon ranksum test was applied to all analyses except the two pig studies, where clustered wicoxon ranksum test was conducted. All analyses were done using Stata (Stata 18.0, College Station, TX) and R (R 4.3.2, R Core Team (2023)). p<0.05 indicates statistically significant findings.

[0133] Example 2

[0134] Senescence and COVID-19

[0135] SARS-CoV-2 can evoke virus-induced senescence and amplify the production of senescence-associated proinflammatory cytokines in pre-existing senescent cells, which may explain the increased susceptibility of elderly individuals to COVID-related complications. SARS- CoV-2 triggered and SARS-CoV-2 amplified senescence may give rise to chronic inflammatory microenvironments in various organs, including the lung, leading to lung fibrosis, the heart, leading to heart failure and the brain, leading to neurocognitive problems including the so called “long COVID” phenomenon. Similar effects can be also triggered by a variety of other viruses, such as Retroviridae, Polyomaviridae, Paramyxoviridae, Parvoviridae, Rhabdoviridae and Coronaviridae families. Targeting senescent cells provides a novel opportunity for delaying, preventing, alleviating or treating severe and chronic forms of COVID- 19.

[0136] COVID-19 infection can also lead to persistent myocardial damage and cardiomyopathy. Senolytics may be used to enhance recovery of cardiac function. With regards to effects on the brain, long-COVID, a subtype of Post- Acute Sequelae of SARS-CoV-2 Infection, is characterized by increasing symptoms of hyposmia, sleep disturbances, and cognitive impairment, affecting nearly a quarter of patients with COVID- 19. Transcriptomic data from deceased COVID- 19 patients suggest an association between cognitive decline and molecular aging signatures in the brain. Several senolytics, including dasatinib plus quercetin can permeate the blood-brain barrier after oral administration and reduce neurocognitive deficits in mouse models of other neurocognitive disorders, such as Alzheimers disease. Therefore, senolytic therapies have become an interesting candidate for treatment of patients with long-COVID. However, clinically relevant methods for monitoring efficacy of such senolytic therapies are lacking.

[0137] The bioresponsive MR complexes (e.g., contrast agents) provided herein can be used to identify senolytic cells. For example, the bioresponsive MR complexes provided herein can be used to identify senolytic cells in a subject and thus identify the subject as at risk of having severe symptoms, secondary sequelae from, or chronic illness with COVID- 19. For example, the bioresponsive MR complex can be provided to a subject known to have or suspected of having an infection with a coronavirus (e.g., SARS-CoV-2) and a signal from the contrast agent can be detected to indicate the presence, level, or amount of senescent cells. A signal equal to or above a threshold value indicates that the presence or amount of senescent cells is sufficient such that the subject is at risk of having severe symptoms, secondary sequelae from, or chronic illness resulting from the infection, facilitating the determination of an adequate treatment plan for the subject.

[0138] The methods provided herein provide a non-invasive imaging technique for the detection of senescent cells, which can be used to monitor efficacy of senolytic drugs. Given that multiple senolytic drugs are currently being investigated for the treatment of COVID-related late effect, the contrast agents provided herein are particularly useful for evaluating efficacy of senolytic therapy for treatment of CO VID- 19 and various symptoms thereof, including chronic infection, myocardial damage, and cognitive decline. For example, the bioresponsive MR complexes (e.g., contrast agents) provided herein can be provided to a subject having received a senolytic therapy for treatment of infection with coronavirus to monitor responsiveness of the subject to the senolytic therapy. A decrease in signal from the complex (e.g., a decrease from baseline, a decrease from an earlier timepoint) indicates that the senolytic therapy is effective (e.g., has cleared at least a portion of senolytic cells from the subject). In contrast, no decrease in signal from the complex (e.g., a signal equal to or above a threshold, or a signal equal to or above a signal obtained from an earlier time point) indicates that the senolytic therapy is not effective. This may indicate that the subject would benefit from an increased dose of the senolytic therapy, a different senolytic therapy, or a combination therapy approach. Example 3

[0139] Senescence and Cancer

[0140] Cellular senescence is characterized by the emergence of a senescence-associated secretory phenotype (SASP), a complex signaling network that detrimentally impacts tissue homeostasis and contributes to disease progression. SASP factors impede tissue regeneration and orchestrate a pro-inflammatory milieu, thereby playing a pivotal role in cancer initiation, therapy resistance, and dissemination (e.g., metastasis).

[0141] Aging is a primary risk factor for cancer development, at least in part because cells and cells in various tissues throughout the body develop a cell cycle arrest and start to produce pro- inflammatory mediators, which cause chronic inflammation and supports immune complex deposition, vascular damage, and ultimately tumor development. Immunosenescence (senescence of immune cells, particularly T cells) is a complex process, generally thought to promote tumor growth. Furthermore, senescent cells in various tissues throughout the body secrete pro- inflammatory mediators and can produce defective daughter cells that may progress into dysplastic cells over time. The bioresponsive MR complexes (e.g., contrast agents) provided herein can be used to identify senolytic cells and thus identify risk of cancer developing, progressing, and / or metastasizing in a subject.

[0142] Senolyic therapies can be effective in preventing cancers. Accordingly, several senolytic drugs are currently being developed and tested in clinical trials. Senolytics are drugs or dietary supplements that specifically induce programmed cell death of senescent cells. Eliminating tissueresident senescent cells may substantially improve lifespan. Furthermore, senolytic therapies can also effectively treat existing cancers as a secondary therapy after classical chemotherapy or irradiation. Chemotherapy and radiation induces therapy -induced senescence in cancer cells, a desired effect to stop tumor growth. However, cancer cells that develop therapy-induced senescence enter a dormant state rather than cell death. These senescent cells in treated tumors continue to release of pro -inflammatory senescence associated proteins which promote growth of bystander cells and may facilitate the formation of metastasis. In addition, the senescent cells can return to actively growing cells at a later time, giving rise to tumor recurrences and metastases. Senolytic drugs can selectively clear senescent cells from tumors and thereby, stop treatment resistance, tumor recurrence and metastasis. The bioresponsive MR complexes (e.g., contrast agents) provided herein can be provided to a subject having received a scnolytic therapy for treatment of cancer to monitor efficacy of the senolytic therapy. A decrease in signal from the complex (e.g., a decrease from baseline, a decrease from an earlier timepoint) indicates that the senolytic therapy is effective (e.g., has cleared at least a portion of senolytic cells from the subject). In contrast, no decrease in signal from the complex (e.g., a signal equal to or above a threshold, or a signal equal to or above a signal obtained from an earlier time point) indicates that the senolytic therapy not is effective. This may indicate that the subject would benefit from a different senolytic therapy, or a combination therapy approach.

Claims

CLAIMSWhat is claimed is:

1. A method of detecting senescent cells in a subject or a sample obtained therefrom, the method comprising administering to the subject or the sample a bioresponsive magnetic resonance imaging (MRI) complex and detecting a signal from the complex, wherein the MRI complex comprises: a) an MRI contrast agent; b) a blocking substrate cleavable by P-galactosidase; and c) a self-immolative linker attaching the blocking substrate to the MRI contrast agent, wherein cleavage of the blocking substrate by P-galactosidase activates the MRI contrast agent to generate a signal detectable by MRI, and wherein detection of a signal above a threshold value indicates the presence of senescent cells in the subject or the sample.

2. The method of claim 1, wherein the self-immolative linker provides a functional group that coordinatively saturates the MRI contrast agent, thereby preventing a water access to the MRI contrast agent resulting in no detectable signal in the absence of P-galactosidase, and wherein cleavage of the blocking substrate by P- galactosidase activates the MRI contrast agent by initiating a spontaneous electron cascade of the self-immolative linker that results in removal of the functional group from the MRI contrast agent.

3. The method of claim 2, wherein the functional group is attached to the self- immolative linker by a spacer comprising at least 4 carbons.

4. The method of claim 3, wherein the spacer consists of 4, 5, 6, 7, or 8 methylene groups.

5. The method of claim 3 or claim 4, wherein the spacer is attached to an amine present in the sclf-immolativc linker.

6. The method of any one of claims 2-5, wherein the functional group is a single carboxylate.

7. The method of any one of claims 1-6, wherein the complex exhibits at least a 50% increase in relaxivity in vivo following cleavage of the blocking substrate.

8. The method of any one of claims 1-7, wherein the complex exhibits at least an 80% increase in relaxivity in vivo following cleavage of the blocking substrate.

9. The method of any one of claims 1-8, wherein the detectable signal is generated within 1 hour following cleavage of the blocking substrate.

10. The method of any one of claims 1-9, wherein the contrast agent comprises gadolinium.

11. The method of claim 10, wherein the contrast agent comprises a macrocyclic gadolinium chelate.

12. The method of any one of claims 1-11, wherein the blocking substrate comprises P-galactose.

13. The method of any one of claims 1-12, wherein the subject is a mammal.

14. The method of claim 13, wherein the subject is a human.

15. The method of any one of claims 1-14, wherein the complex is administered to the subject parenterally.

16. The method of any one of claims 1- 15, wherein the subject has or is suspected of having a musculoskeletal disease, cardiovascular disease, diabetes, liver disease, kidney disease, age-related macular degeneration, pulmonary disease, neurological disease, autoimmune disease, cancer, viral infection, or skin aging.

17. The method of claim 16, wherein the musculoskeletal disease is osteoporosis, osteoarthritis, or rheumatoid arthritis.

18. The method of any one of claims 15-17, wherein the subject has received at least one dose of a senolytic therapy prior to detecting senescent cells in the subject.

19. A method of determining responsiveness to a senolytic therapy in a subject, the method comprising detecting a level or amount of senescent cells in the subject after the subject has received at least one dose of the senolytic therapy, wherein said detecting comprises administering to the subject or to a sample obtained from the subject a bioresponsive magnetic resonance imaging (MRI) complex and detecting a signal from the complex, wherein the MRI complex comprises: a) an MRI contrast agent; b) a blocking substrate cleavable by 0-galactosidase; and c) a self-immolative linker attaching the blocking substrate to the MRI contrast agent, wherein cleavage of the blocking substrate by P-galactosidase activates the MRI contrast agent to changes a signal detectable by MRI.

20. The method of claim 19, wherein detection of a signal equal to or above a baseline level indicates that the subject is not responsive to the at least one dose of the senolytic therapy, or wherein detection of a signal less than the baseline level indicates that the subject is responsive to the at least one dose of the senolytic therapy.

21. The method of claim 20, wherein the baseline level is determined prior to the at least one dose of the scnolytic therapy.

22. The method of claim 20 or claim 21, further comprising increasing the dose of the senolytic therapy, providing an alternative senolytic therapy to the subject, or both increasing the dose of the senolytic therapy and providing an alternative senolytic therapy to the subject indicated to be not responsive.

23. The method of any one of claims 20-22, further comprising detecting the baseline level or amount of senescent cells in the subject prior to the subject receiving the at least one dose of the senolytic therapy, wherein detecting the baseline level or amount comprises administering to the subject or a sample obtained from the subject the bioresponsive MRI complex and detecting a signal from the complex.

24. The method of any one of claims 19-23, wherein the self-immolative linker provides a functional group that coordinatively saturates the MRI contrast agent, thereby preventing a water access to the MRI contrast agent resulting in no detectable signal in the absence of P-galactosidase, and wherein cleavage of the blocking substrate by P-galactosidase activates the MRI contrast agent by initiating a spontaneous electron cascade of the self-immolative linker that results in removal of the functional group from the MRI contrast agent.

25. The method of claim 24, wherein the functional group is attached to the self- immolative linker by a spacer comprising at least 4 carbons.

26. The method of claim 25, wherein the spacer consists of 4, 5, 6, 7, or 8 methylene groups.

27. The method of claim 25 or claim 26, wherein the spacer is attached to an amine present in the self-immolative linker.

28. The method of any one of claims 24-27, wherein the functional group is a single carboxylate.

29. The method of any one of claims 19-28, wherein the complex exhibits at least a 50% increase in relaxivity in vivo following cleavage of the blocking substrate.

30. The method of any one of claims 19-29, wherein the complex exhibits at least an 80% increase in relaxivity in vivo following cleavage of the blocking substrate.

31. The method of any one of claims 19-30, wherein the detectable signal is generated within 1 hour following cleavage of the blocking substrate.

32. The method of any one of claims 19-31, wherein the contrast agent comprises gadolinium.

33. The method of claim 32, wherein the contrast agent comprises a macrocyclic gadolinium chelate.

34. The method of any one of claims 19-33, wherein the blocking substrate comprises P-galactose.

35. The method of any one of claims 19-34, wherein the subject is a mammal.

36. The method of claim 35, wherein the subject is a human.

37. The method of any one of claims 19-36, wherein the complex is administered to the subject parenterally.

38. The method of any one of claims 19-37, wherein the at least one dose of the senolytic therapy is provided to the subject to treat a musculoskeletal disease,cardiovascular disease, diabetes, liver disease, kidney disease, age-related macular degeneration, pulmonary disease, neurological disease, autoimmune disease, cancer, viral infection, or skin aging in the subject.

39. The method of claim 38, wherein the musculoskeletal disease is osteoarthritis, rheumatoid arthritis, or osteoporosis.

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