Senescent cell surface markers

Abstract

Senescent cells, in some aspects, are associated with age-related decline in health and may be the cause of certain diseases. Embodiments include biomarkers characteristic of senescent cells. The biomarkers are used in the diagnosis and treatment of age-related diseases and disorders. Embodiments also include methods of distinguishing senescent cells from non-senescent cells based on the presence or absence of one or more biomarkers. Senescent cells can be selectively labeled to detect disease in a subject, devise treatments, and / or determine the effectiveness of senolytic agents. Embodiments also include methods of removing senescent cells from a patient or affected tissue. The methods can target senescent cells by inhibiting lysosomal exocytosis.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 305,227, filed January 31, 2022, and U.S. Provisional Patent Application No. 63 / 395,289, filed August 4, 2022, the contents of which are incorporated herein by reference.

[0002] The present invention relates to diagnostics, and more particularly to methods for diagnosing and treating age-related diseases and disorders. [Background technology]

[0003] Aging can be defined as the process of growing older. In humans, aging represents the accumulation of changes over time and can encompass physical, psychological, and social changes. Old age is the greatest risk factor for many chronic diseases. More than 90% of adults aged 65 years and older will experience at least one chronic disease, such as cancer, diabetes, and cardiovascular disease. Aging phenotypes and pathologies, including a variety of age-related diseases and disorders, are causally linked to the accumulation of senescent cell burden with age.

[0004] Senescent cells are characterized by irreversible cell cycle arrest of proliferation-competent cells, morphological and metabolic changes, altered gene expression, chromatin reorganization, and a unique proinflammatory senescence-associated secretory phenotype (SASP). Senescent cells in older individuals contribute to chronic inflammation and damage to surrounding tissues. It has been demonstrated that genetically engineered elimination of senescent cells in transgenic mouse models can prevent or delay tissue dysfunction, ameliorate age-related pathologies, and extend healthspan. This suggests that elimination of senescent cell burden in older individuals is worth further investigation as a targeted therapeutic target for the treatment and prevention of diseases of aging. Senolytic drugs have also shown promising results in mouse and human cell culture models.

[0005] Recent efforts have focused on methods to identify senescent cells. The ability to distinguish senescent from non-senescent cells can lead to a variety of therapeutic and diagnostic approaches. The identification and validation of senescent cell surface markers represents an innovative and powerful advance in the study of cellular senescence. It can reveal promising targets for selective elimination of senescent cells via immunotherapy, live image labeling, drug targeting, and differentiation of senescent from non-senescent cells. However, the lack of robust and universal biomarkers signaling senescence and ageing has hindered efforts to develop therapeutics. Without reliable markers, it is difficult to accurately quantify the senescence burden or evaluate the efficacy of seno therapeutics.

[0006] Studies have identified intracellular molecules and secreted proteins that are characteristic of senescent cells. Although several proteins have been identified as upregulated on the surface of senescent cells, none of these have been proven to be specific to senescent cells with a high degree of confidence, especially in vivo. For example, several surface proteins have been identified in senescent cell culture models. However, these experiments were performed in cancer cell lines, and senescence was induced by overexpression of p16 or p21, rather than traditional senescence models (e.g., induction of persistent DNA damage or activation of oncogenes). In another study, Kim et al. identified DDP4 as a potential marker of senescence (Kim et al., Genes Dev 31:1529-1534, 2017). However, the widespread surface expression of DPP4 on various immune cells limits its application as a biomarker in vivo. Furthermore, increased expression of SCAM4 or oxidized vimentin at the cell surface of senescent cells and in the senescence-associated secretory phenotype (SASP) has been demonstrated, although surface expression of these molecules has yet to be confirmed.

[0007] Thus, there is a need for reliable biomarkers for identifying senescent cells. The present invention includes protein biomarkers that can identify senescent cells with high specificity and reliability. Also included are methods for identifying senescent cells, diagnosing associated diseases, and treating age-related diseases and conditions by selective elimination of senescent cells. Summary of the Invention

[0008] The invention described and claimed herein has many attributes and embodiments, including but not limited to those described, illustrated, or referenced in this brief summary. The invention described and claimed herein is not limited to the features or embodiments identified in this summary, which is included for illustrative purposes only and is not intended to be limiting.

[0009] As described herein, the applicants have identified novel surface markers of senescent cells. Thus, embodiments include methods for identifying senescent biomarkers. The applicants have identified 18 proteins that are increased on the surface of senescent cells by both doxorubicin and radiation treatment. Five specific proteins have been further studied and validated as indicators of cellular senescence.

[0010] Embodiments also include proteins (i.e., biomarkers) that have been identified as universal surface markers of senescent cells.

[0011] Embodiments also include the use of certain biomarkers for the diagnosis, prognosis, theranosis and / or prediction of aging-related diseases and disorders.

[0012] Embodiments also include methods of diagnosing / identifying an aging-related disease or disorder using one or more biomarkers.

[0013] Embodiments also include methods of treating aging-related diseases and disorders using senolytic agents.

[0014] Embodiments include methods of distinguishing between non-senescent and senescent cells. The methods may include comparing the levels of one or more biomarkers from non-senescent and senescent cells. The methods may include selectively labeling senescent cells and / or selectively removing senescent cells from non-senescent cells.

[0015] Embodiments also include methods of detecting a disease or determining a prognosis of a disease. The method may include (a) detecting the level of one or more biomarkers in a sample from a test subject, (b) identifying the disease or determining a prognosis of the disease based on elevated expression levels of the one or more biomarkers in the sample, and (c) treating the subject with a senolytic agent to prevent or ameliorate the disease. In certain aspects, the disease is a senescence-related disease or disorder.

[0016] Embodiments also include a method of detecting a disease in a subject. The method may include (a) detecting the level of one or more biomarkers in a sample from the subject, (b) identifying the disease based on elevated levels of the one or more biomarkers, and (c) administering a therapeutic amount of a senolytic agent based on the identification of the disease. In some aspects, the disease is a senescence-related disease or disorder. In some aspects, the senolytic agent inhibits lysosomal exocytosis.

[0017] The embodiments also include a method of detecting or diagnosing a disease or determining a prognosis of a subject having a disease. The method may include the steps of: (a) measuring an expression level of at least one biomarker in a test sample from a subject; (b) receiving by a computer the expression level of at least one biomarker in the test sample; (c) comparing the expression level of at least one biomarker in the test sample with the level in a base sample for the same at least one biomarker; (d) receiving a result of comparing the expression levels of at least one biomarker in the test sample measured in a) and in the base sample measured in c); (e) diagnosing the disease or determining a prognosis of the disease based on a change in expression of at least one biomarker in the test sample compared to the base sample as determined by a computer; and (f) treating the subject for the disease based on the diagnosis or prognosis. In some aspects, the disease is a senescence-related disease or disorder. In some aspects, a senolytic agent is used for treatment. In some aspects, the senolytic agent inhibits lysosomal exocytosis. The senolytic agent can be, for example, vacorin-1 or apilimod.

[0018] In embodiments, the biomarkers include one or more of CO8A1, CATC, LYAG, IBP7, TPP1. In embodiments, the biomarkers include one or more of CO8A1, VCAM1, CATC, LYAG, PTGIS, IBP7, TPP1, CA2D1, MA2B1, CAVN2, FBLN1, LYOX, PCP, DPP4, GLCM, HEXB, PCYOX, and GGALNS (as identified in Table 4). In embodiments, the biomarkers include one or more of the proteins listed in Table 2. In embodiments, the biomarkers include one or more of the proteins listed in Table 3.

[0019] In embodiments, the biomarkers include one or more proteins selected from TPP1, LYAG, VCAM1, GARS, CATC, NCEH1, CSPG4, ELOV1, CO8A1, IBP7, PTGIS, MA2B1, CAH2, GLCM, S10A6, DPP4, LYOX, PCP, HACD3, SDCB1, MGST3, EZRI, LEG3, FBLN1, PCYOX, HEXB, GALNS, ESYT1, ACTN4 and DHCR7 (listed in Table 5).

[0020] In embodiments, the biomarkers include one or more proteins selected from LYAG, VCAM1, FBLN1, GALNS, CATC, IBP7, LRC15, CREL1, EZRI, CAVN2, NCEH1, PCYOX, SDCB1, MA2B1, TPP1, HEXB, PCP, PTGIS, CSPG4, SYNPO, CD248, FKB11, GOT1B, DPP4, CO8A1, ITA1, LYOX, GLCM, TOR1A and PHB2 (listed in Table 6).

[0021] The embodiments also include a method of diagnosing a disease (i.e., a senescence-related disease or disorder) or determining a prognosis of a test subject having a disease. The method may include a) measuring the expression level of one or more biomarkers from a subject having a disease; b) measuring the expression level of one or more biomarkers from a healthy subject; c) comparing the expression level of one or more biomarkers from a sample from a subject having a disease with the levels in a sample from a healthy subject; d) identifying biomarkers having an altered level of expression from a sample from a subject having a disease; and e) diagnosing the disease or determining a prognosis of the disease in the test subject by comparing the levels of the biomarkers in the test subject from the healthy subject and the subject having the disease. The method may include treating the disease (e.g., with a senolytic agent). The method also includes creating a biomarker fingerprint from the biomarkers having an altered level of expression.

[0022] Embodiments also include diagnostic kits for diagnosing a disease (i.e., a senescence-associated disease or disorder). The kits can be used to identify senescent cells based on the presence of one or more biomarkers.

[0023] Embodiments also include methods for removing senescent cells for diagnostic and / or therapeutic purposes.

[0024] Embodiments also include methods for targeted delivery of senolytic or senolytic drugs to senescent cells.

[0025] Embodiments also include methods of identifying senescent cells for targeted therapy.

[0026] The embodiments also include a method of regulating the activity of one or more lysosomal proteins or a method of regulating the expression of one or more lysosomal proteins. The lysosomal proteins may be involved in lysosomal exocytosis. The one or more lysosomal proteins may be, for example, COLINA1, LOX, PBLN1, PTGIS, VCAM1, CACNA2D1, IGFBF3, GALN8, PCYOX1, SDPFR, DPP4, PRCP, CT8C, TPP1, NAN281, HEXB, CAA, NAN281, and / or GBA.

[0027] Embodiments also include methods of removing senescent cells from diseased tissue in a subject. The methods can target senescent cells by inhibiting lysosomal exocytosis.

[0028] Embodiments also include methods for treating a senescence-associated disease or disorder comprising administering a senolytic agent (e.g., a small molecule), where the senolytic agent selectively kills senescent cells over non-senescent cells. In embodiments, the senolytic agent is baquolin-1 or apilimod.

[0029] Embodiments also include methods of treating a senescence-associated disease or disorder by measuring the expression levels of one or more biomarkers and administering a senolytic agent to selectively kill senescent cells. The senolytic agent can target senescent cells by inhibiting lysosomal exocytosis.

[0030] In certain aspects, the methods described herein can delay the onset or progression of age-associated diseases or conditions by identifying and selectively removing senescent cells over non-senescent cells.

[0031] The embodiments include methods of using drug conjugates to kill senescent cells. The conjugates may include (i) a senescent cell targeting agent configured to, in use, specifically target and bind at least one senescent cell biomarker described herein, and (ii) a cytotoxic agent that kills the bound senescent cells. In some aspects, the targeting agent is an antibody or antigen-binding fragment thereof, an aptamer, a plastic antibody, or a small molecule. In some aspects, the cytotoxic agent is a senolytic agent, a radioisotope, a toxin, or a toxic peptide. In some aspects, the senolytic agent is (a) an inhibitor of a Bcl-2 anti-apoptotic protein family member, (b) an MDM2 inhibitor, or (c) an Akt-specific inhibitor.

[0032] Embodiments include methods of using biomarkers to determine the effectiveness of a senolytic agent. In some aspects, the level of a marker is measured in a subject before and after administration of the agent. The levels can then be compared. Reduced levels reflect senolytic activity.

[0033] The accompanying drawings illustrate aspects of the present invention. [Brief description of the drawings]

[0034] [Figure 1A]A group of images of SAβ-Gal stained senescent and non-senescent IMR-90 cells. Non-senescent cells (NS) were compared with cells treated with doxorubicin (S-Doxo) and X-ray irradiation (S-IR). [Figure 1B] 1 is a graphical depiction of quantification of EdU incorporation in non-senescent (NS) and senescent (S-Doxo and IR treated) IMR-90 cells. [Figure 1C] 1 is a graphic depiction of mRNA expression of cell cycle regulators p16 and p21 in non-senescent and senescent IMR-90 cells. [Figure 1D] 1 is a graphic depiction of cellular lamin B1 mRNA expression in non-senescent and senescent IMR-90 cells. [Figure 1E] 1 is a graphic depiction of mRNA expression of SASP factors IL-6, IL-8, and IL-1α. [Figure 1F] Figure 1 shows a panel of images of immunofluorescence assays performed to detect the expression of γ-H2AX and HMGB1 in non-senescent and senescent IMR-90 cells. Cells were stained with HMGB1 (red) and γ-H2AX (green). Nuclei were stained with Hoechst (blue). Non-senescent cells (NS) were compared to cells treated with 300 nM doxorubicin (S-Doxo) and 10 Gy X-rays (S-IR). [Figure 2A] 1 is a flowchart of the procedure for Surfaceome-TriCEPS. [Figure 2B] 1 is a volcano plot showing cell surface protein abundance after treatment of cells with doxorubicin. [Figure 2C] 1 is a volcano plot showing cell surface protein abundance after treating cells with radiation. [Figure 2D] FIG. 11 is a Venn diagram of differentially expressed proteins upon doxorubicin treatment and irradiation compared to control cells. [Figure 3A] 1 is a blot showing protein expression (CATC, LYAG, IBP7, TPP1, p16 INK4A, and β-actin) in non-senescent (NS) and senescent (S) IMR-90 fibroblasts. [Figure 3B] 1 is a blot showing protein expression of proteins (CATC, LYAG, IBP7, TPP1, p16INK4A, and β-actin) in non-senescent (NS) and senescent (S) primary endothelial cells. [Figure 3C] Western blot comparing protein expression (CO8A1, IBP7, TPP1, Na / KATPase) in membrane and cytosolic fractions of non-senescent (NS) and senescent (S) IMR-90 fibroblasts. [Figure 3D] Western blot comparing protein expression (CATC, LYAG, and Na / KATPase) in membrane and cytosolic fractions of non-senescent (NS) and senescent (S) IMR-90 fibroblasts. [Figure 3E] 13 is a panel of images of immunofluorescence assay of TPP1 expression in non-senescent (NS) and senescent (Sen) IMR-90 cells. [Figure 4A] Analysis of enriched ontology clusters among 22 proteins identified as statistically enriched terms that were hierarchically clustered based on kappa statistical similarity between their gene membership. A 0.3 kappa score was applied as a threshold for generating term clusters. [Figure 4B] Flow cytometry analysis of LAMP-1 surface expression in NS (blue) and S (red) compared to isotype control (grey). [Figure 4C] Immunoblot of TFEB expression in non-senescent (NS) and senescent (S) IMR-90 fibroblasts. [Figure 4D] Immunoblots of TFEB expression in NS versus S primary endothelial cells are shown. [Figure 4E] Quantification of NS vs. S fibroblasts treated with the PIKfyve inhibitor, baquolin-1. [Figure 4F]Quantification of NS vs. S fibroblasts treated with apilimod from three independent experiments. Statistical analysis was performed using an unpaired t-test comparing NS vs. S cells at a drug concentration of 1 μM after 48 hours of treatment. [Figure 4G] Representative light microscopy images of selective cell death of senescent cells after 48 hours of apilimod treatment (1 μM) are shown. [Figure 4H] Bar graph of quantification of selective cell death of senescent cells. Three fields were quantified per well (n=3) and a total of 5997 and 1293 fibroblasts were counted for NS and S conditions, respectively. Error bars represent ±SEM. Statistical analysis was performed using unpaired t-test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Diagram 5] 1 shows the results of a validation study of senescent cell surface markers by flow cytometry.

[0035] definition Reference herein to "one embodiment / aspect" or "embodiment / aspect" means that a particular feature, structure, or characteristic described in connection with the embodiment / aspect is included in at least one embodiment / aspect of the present disclosure. The use of the phrase "in one embodiment / aspect" or "in another embodiment / aspect" in various places herein does not necessarily all refer to the same embodiment / aspect, nor does it mean that separate or alternative embodiments / aspects are mutually exclusive with other embodiments / aspects. Furthermore, various features are described that may be exhibited by some embodiments / aspects and not exhibited by other embodiments / aspects. Similarly, various requirements are described that may be requirements for some embodiments / aspects but not other embodiments / aspects. Embodiments and aspects may be used interchangeably in some cases.

[0036] The terms used in this specification generally have their ordinary meanings in the art, within the context of the disclosure, and in the specific context in which each term is used.Specific terms used to describe the disclosure are discussed below or elsewhere in the specification to provide additional guidance to practitioners regarding the description of the disclosure.It will be understood that the same thing can be said in multiple ways.

[0037] Thus, alternative language and synonyms may be used for any one or more of the terms discussed herein. Also, there is no particular importance to whether a term is detailed or discussed herein. Synonyms are provided for certain terms. The description of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any term discussed herein, is illustrative only and is not intended to further limit the scope and meaning of the disclosure or any exemplified term. Similarly, the disclosure is not limited to the various embodiments described herein.

[0038] Without intending to further limit the scope of the present disclosure, examples of instruments, devices, methods and their related results according to the embodiments of the present disclosure are given below. Subjects and subtitles may be used in the examples for the convenience of the reader, but this does not limit the scope of the disclosure in any way. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of conflict, the present specification, including definitions, shall prevail.

[0039] The term "senescence" refers to the gradual decline of functional properties in an organism. Cellular senescence is often defined as the stress-induced permanent cell cycle arrest of previously replication-competent cells. The effects of senescent cells can be considered beneficial or detrimental with respect to host physiology and disease, but in some situations, senescent cells affect disease states in complex ways that both promote and hinder certain conditions.

[0040] The term "aging-related disease or disorder" refers to a disease associated with aging, and may include, for example, atherosclerosis, osteoarthritis, osteoporosis, hypertension, arthritis, cataracts, cancer, Alzheimer's disease, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis. Other diseases associated with aging or aging (including age-related conditions) include gray hair, sarcopenia, obesity, neurogenesis, fibrosis, and glaucoma.

[0041] The terms "theranosis" or "theranostics" generally refer to a process used to tailor treatment for a patient. It is the use of diagnostic tests to identify patients who are more suitable for any one drug, drug(s), medicine, or therapeutic agent, or to determine how well any drug(s), medicine(s), or therapeutic agent(s) is working.

[0042] Other diseases related to aging or senescence include cardiovascular diseases (e.g., atherosclerosis, angina, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress resistance, cardiac fibrosis, cerebral aneurysm, and stroke). Aging-related diseases or disorders can also be inflammatory or autoimmune diseases or disorders (e.g., osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease, or kyphosis). Aging-related diseases or disorders can also be neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, mild cognitive impairment, or motor neuron dysfunction). Aging-related diseases or disorders can also be metabolic diseases (e.g., diabetes, diabetic ulcers, metabolic syndrome, or obesity). The aging-related disease or disorder may also be a pulmonary disease (e.g., pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis, or age-related decline in lung function). The aging-related disease or disorder may also be an ocular disease or disorder (e.g., macular degeneration, glaucoma, cataract, presbyopia, or vision loss). The aging-related disease or disorder may be an age-related disorder, which may be renal disease, renal failure, frailty, hearing loss, muscle fatigue, skin disease, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucosal fibrosis, or sarcopenia. The aging-related disease or disorder may also be a skin disease or disorder (e.g., eczema, psoriasis, pigmentation, birthmark, rash, atopic dermatitis, urticaria, photosensitivity, or photoaging-related disease or disorder).

[0043] The term "senescence-associated beta-galactosidase", "SA-beta-gal" or "SABG" refers to a hypothetical hydrolase that catalyzes the hydrolysis of beta-galactosides to monosaccharides exclusively in senescent cells. Senescence-associated beta-galactosidase, together with p16Ink4A, can be used as a biomarker of cellular senescence.

[0044] The term "senolytic" or "senolytic agent" refers to a therapeutic agent, such as a small molecule, that can selectively or preferentially induce the death of senescent cells. Senolytic agents may kill senescent cells by inducing (activating, stimulating, or removing inhibition of) apoptotic pathways that lead to cell death. Senolytic agents may be effective in treating aging-related diseases or disorders. Drugs such as dasatinib, quercetin, fisetin, and navitoclax have potential senolytic activity.

[0045] The term "biomarker" generally refers to a DNA, RNA, protein, carbohydrate, or glycolipid-based molecular marker, the expression or presence of which in a subject's sample can be detected by standard methods (or the methods disclosed herein) and predicts or prognosticates an effective response or susceptibility of a mammalian subject having a disease. A biomarker may be present in a test sample but absent in a control sample, absent in a test sample but present in a control sample, or the amount of a biomarker may differ between a test sample and a control sample. For example, a protein biomarker may be present in such a sample but absent in a control sample, or a particular biomarker may be seropositive in a sample but seronegative in a control sample. Also, the expression of such a biomarker may be determined to be higher than that observed from a control sample. The terms "marker" and "biomarker" are used interchangeably herein.

[0046] An "upregulated" biomarker generally refers to an increase in the level of expression in response to a given treatment or condition. A "downregulated" biomarker generally refers to a decrease in the level of expression of a biomarker in response to a given treatment or condition. In some circumstances, the level of a biomarker may remain unchanged during a given treatment or condition. A biomarker from a patient sample may be "upregulated", i.e., its level may increase, for example, by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 500%, about 1,000%, about 5,000% or more, compared to a reference level. Alternatively, a biomarker may be "downregulated," i.e., the level may be decreased by, for example, about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 2%, about 1% or less, compared to a reference level.

[0047] The expression levels of the described biomarkers can be assessed by any of a variety of known methods for detecting expression of transcribed nucleic acids or proteins, non-limiting examples of which include immunological methods for detecting secreted, cell surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

[0048] In one embodiment, expression of the biomarker set is assessed using antibodies (radiolabeled, chromophore-labeled, fluorescently-labeled, or enzyme-labeled antibodies), antibody derivatives (e.g., antibodies conjugated to a substrate or to a protein or ligand of a protein-ligand pair (e.g., biotin-streptavidin)), or antibody fragments (e.g., single chain antibodies, hypervariable domains of isolated antibodies, etc.) that specifically bind to the biomarker protein or a fragment thereof, including the biomarker protein that has undergone all or some of the post-translational modifications (e.g., glycosylation, phosphorylation, methylation, etc.) that it normally undergoes in tumor cells.

[0049] The amount of biomarkers can be measured in a test sample and compared to a "normal control level" using techniques such as reference limits, discrimination limits, risk definition thresholds, etc. to define cut-off points and outliers for disease. Normal control levels refer to the levels of one or more biomarkers or combined biomarker indicators typically found in subjects who are not affected by disease (or susceptible to disease, such as LOAD). Such normal control levels and cut-off points can vary depending on whether a biomarker is used alone or in a procedure to combine with other biomarkers into an indicator. Alternatively, normal control levels can be a database of biomarker patterns from previously tested subjects who have not experienced disease over a clinically relevant period of time.

[0050] After selection of a set of biomarkers, a formula for the calculation of a risk score can be developed using well-known techniques such as cross-correlation, principal component analysis (PCA), factor rotation, logistic regression (LogReg), linear discriminant analysis (LDA), Eigengene linear discriminant analysis (ELDA), support vector machine (SVM), random forest (RF), recursive partitioning tree (RPART), related decision tree classification techniques, shrunken centroids (SC, Shrunken Centroids), StepAIC, K nearest neighbors, boosting, decision trees, neural networks, Bayesian networks, support vector machines, hidden Markov models, linear regression or classification algorithms, non-linear regression or classification algorithms, variant analysis (ANOVA), hierarchical analysis or clustering algorithms; hierarchical algorithms using decision trees; kernel-based machine algorithms such as kernel partial least squares algorithm, kernel matching pursuit algorithm, kernel Fisher discriminant analysis algorithm, kernel principal component analysis algorithm, or other mathematical and statistical methods. A selected population of individuals is used, where historical information is available regarding the values ​​of the biomarkers in that population and their clinical outcomes. To calculate a risk score for a particular individual, biomarker values ​​are obtained from one or more samples collected from that individual and used as input data.

[0051] Tests that measure biomarkers and biomarker panels can be implemented in a variety of diagnostic testing systems. Diagnostic testing systems are devices that typically include a means for obtaining test results from a biological sample. Examples of such means include modules that automate testing (e.g., chemistry, immunology, nucleic acid detection assays). Some diagnostic testing systems are designed to handle multiple biological samples and can be programmed to perform the same or different tests on each sample. Diagnostic testing systems typically include a means for collecting, storing, and / or tracking the test results of each sample, usually in a data structure or database. Examples include well-known physical and electronic data storage devices (e.g., hard drives, flash memory, magnetic tape, paper printouts, etc.). It is also typical for diagnostic testing systems to include a means for reporting test results. Examples of reporting means include a visual display, a link to a data structure or database, a printer, etc. The reporting means can be a data link for transmitting test results to an external device, such as a data structure, a database, a visual display, a printer, etc.

[0052] The term "area under the curve" or "AUC" refers to the area under the curve of a receiver operating characteristic (ROC) curve, both of which are well known in the art. AUC measurements are useful for comparing the accuracy of classifiers across a range of data. A classifier with a larger AUC has a higher ability to correctly classify unknowns between two groups of interest (such as affected samples and normal or control samples). ROC curves are useful for plotting the performance of a particular feature (e.g., any of the biomarkers described herein, and / or any item of additional biomedical information) in distinguishing between two populations (e.g., cases with disease and controls without disease). Typically, feature data across a population (e.g., cases and controls) are sorted in ascending order based on the value of a single feature. Then, for each value of that feature, the true positive rate and false positive rate of the data are calculated. The true positive rate is determined by counting the number of cases that exceed the value of that feature and dividing it by the total number of cases. The false positive rate is determined by counting the number of controls that exceed the value of that feature and dividing it by the total number of controls. This definition refers to a scenario in which a feature is elevated in cases compared to controls, but it also applies to scenarios in which a feature is lower in cases compared to controls (in such scenarios, samples below the value of the feature are counted). ROC curves can be generated not only for single features, but also for other single outputs, for example, combinations of two or more features can be mathematically combined (e.g., added, subtracted, multiplied, etc.) to provide a single total value, which can be plotted on a ROC curve. Furthermore, any combination of multiple features whose combination derives a single output value can be plotted on a ROC curve. These combinations of features can constitute a test. A ROC curve is a plot of the true positive rate (sensitivity) of a test against the false positive rate (1-specificity) of the test.

[0053] The terms "detecting" or "determining" with respect to a biomarker value include the use of both the equipment needed to observe and record a signal corresponding to the biomarker value, and the material(s) needed to generate that signal. In various embodiments, the biomarker value is detected using any suitable method, such as fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic wave, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection, nuclear magnetic resonance, quantum dots, and the like.

[0054] The term "fingerprint," "disease fingerprint," or "biomarker signature" refers to the presence or pattern of biomarkers that have elevated or decreased levels in subjects with a disease. Fingerprints can be generated by comparing diseased and healthy subjects and can be used for screening / diagnosis of disease.

[0055] The term "treat" or "treatment" refers to one or more of: (1) inhibiting a disease, condition or disorder in an individual experiencing or exhibiting a pathology or symptom of the disease, condition or disorder (i.e., preventing further progression of the pathology and / or symptoms), and (2) ameliorating a disease, condition or disorder in an individual experiencing or exhibiting a pathology or symptom of the disease, condition or disorder (i.e., reversing the pathology and / or symptoms), e.g., reducing the severity of the disease.

[0056] The term "administration" refers to the introduction of any amount of a given substance into a patient by a particular suitable method. The compositions disclosed herein may be administered via any of the common routes, such as, for example, inhalation, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, or rectal administration, so long as it can reach the desired tissue.

[0057] The term "subject" refers to a person susceptible to, suspected of having, or diagnosed with a disease (e.g., a disease associated with aging), but includes, without limitation, any subject treated with the therapeutic methods described herein.

[0058] The term "TriCEPS" refers to a ligand-based receptor capture (LRC) technology for identifying cell surface receptors and off-targets on live cells for a variety of orphan ligands, such as peptides and proteins. The "TriCEPS" molecule comprises a chemoproteomic reagent with three moieties: a) a moiety that binds ligands containing amino groups, b) a second moiety that binds glycosylated receptors on live cells, and c) a biotin tag to purify receptor peptides for identification by quantitative mass spectrometry (see, e.g., Frei et al., Nature Biotechnology 30:997-1001, 2012).

[0059] The term "antibody-dependent cellular cytotoxicity," "ADCC," or "antibody-dependent cell-mediated cytotoxicity" refers to a mechanism of cell-mediated immune defense whereby effector cells of the immune system actively lyse target cells whose membrane surface antigens have been bound by specific antibodies. This is one of the mechanisms by which antibodies, as part of the humoral immune response, act to limit and contain infection.

[0060] The term "lysosome" refers to membrane-bounded organelles found in many animal cells. They are spherical vesicles that contain hydrolytic enzymes that can degrade many types of biomolecules. The lumen of lysosomes has a low pH (approximately 4.5-5.0), which is optimal for the enzymes involved in hydrolysis. The process of hydrolytic digestion can be described in four steps. In the first step, materials enter the food vacuole through the plasma membrane (i.e., endocytosis). In the next step, hydrolytic enzymes become active as the food vacuole moves away from the plasma membrane. In the third step, the lysosome fuses with the food vacuole and the hydrolytic enzymes enter the food vacuole. In the final step, the hydrolytic enzymes digest the food particle. Lysosomes are also involved in various cellular processes, including secretion, plasma membrane repair, apoptosis, cell signaling, and energy metabolism.

[0061] Lysosomes act as the cell's waste disposal system by digesting used materials in the cytoplasm, both from inside and outside the cell. Material from outside the cell is taken up by endocytosis, while material from inside the cell is digested by autophagy.

[0062] The term "endocytosis" refers to the cellular process by which materials are taken into cells. Internalized materials are surrounded by plasma membrane regions that then separate within the cell to form vesicles containing the ingested material. The endocytic pathway of mammalian cells has distinct membrane compartments that internalize molecules from the plasma membrane and recycle them back to the surface (in early endosomes and recycling endosomes) or sort and degrade them (in late endosomes and lysosomes).

[0063] The term "exocytosis" refers to a form of active and bulk transport by which cells transport molecules (e.g., neurotransmitters and proteins) out of the cell. Lysosomal exocytosis is the main output pathway of lysosomes and is important for several cellular processes, including plasma membrane repair, secretion and transmitter release, neurite outgrowth, and particle uptake in macrophages. Upon stimulation, lysosomes migrate from the perinuclear and cytoplasmic regions to the plasma membrane along microtubules. After docking, lysosomes fuse directly with the plasma membrane to release the lysosomal contents into the extracellular space.

[0064] The term "lysosome-associated membrane protein 1" or "LAMP-1" also refers to the protein, known as lysosome-associated membrane glycoprotein 1 and CD107a (cluster of differentiation antigen 107a), which in humans is encoded by the LAMP1 gene. The human LAMP1 gene is located in region 3, band 4 of the long arm (q) of chromosome 13 (13q34). The LAMP-1 glycoprotein is a type I transmembrane protein that is expressed at high or moderate levels in at least 76 different normal tissue cell types. It resides primarily across the lysosomal membrane and functions to provide carbohydrate ligands for selectins.

[0065] The term "five-position phosphoinositide kinase containing FYVE finger" or "PIKfyve" is a lipid kinase that phosphorylates phosphatidylinositol-3-phosphate (PI(3)P) to generate PI(3,5)P2. PIKfyve plays an important role in the endosomal and lysosomal systems, regulating membrane homeostasis, endosomal trafficking, and autophagy (see, e.g., Toxicol. Appl. Pharmacol. 383, 114771; 2019).

[0066] The term "apilimod" refers to an inhibitor of the lipid kinase enzyme PIKfyve.

[0067] The term "baquolin-1" refers to a cell-permeable, water-soluble triazine-based compound. Baquolin-1 is a potent, cell-permeable lysosomal exocytosis inhibitor. Studies have revealed that baquolin-1 induced rapid homotypic fusion of endosomes and lysosomes to form large, swollen structures, but it did not disrupt the cytoskeleton network of cells.

[0068] The term "Gene Ontology term enrichment" refers to a technique for interpreting gene sets utilizing the Gene Ontology (GO) classification system, which assigns genes to a set of predefined bins according to their functional properties.

[0069] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are understood as approximations according to common practice in the art. The term "about" as used herein may include a variation of (+) or (-) 1%, 5%, 10%, 15%, or 20% of the recited amount, depending on the context. It is also to be understood that, although not always expressly indicated, the reagents described herein are merely exemplary, and equivalents thereof are known in the art.

[0070] Many known and useful compounds and the like are listed in Remington's Pharmaceutical Sciences (13th Ed), Mack Publishing Company, Easton, PA, a standard reference for various types of administration. As used herein, the term "formulation(s)" refers to a combination of at least one active ingredient and one or more other ingredients, also commonly called excipients, which may be independently active or inactive. The term "formulation" may or may not refer to a pharma- ceutically acceptable composition for administration to humans or animals, and may include compositions that are useful intermediates for storage or research purposes.

[0071] Other technical terms used herein have their ordinary meaning in the art in which they are used, as exemplified by various technical dictionaries. The specific values ​​and configurations discussed in these non-limiting examples may vary and are cited merely to describe at least one embodiment and are not intended to limit the scope thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the claimed subject technology. Additional features and advantages of the subject technology will be set forth in the following description, and in part will be obvious from the description, or may be learned by practice of the subject technology. The advantages of the subject technology will be realized and attained by the structure particularly pointed out in the description and claims hereof.

[0073] Some types of stress factors can induce distinct senescent phenotypes characterized by intracellular molecules or secreted proteins. However, the surface structure of senescent cells remains largely unknown. The applicant has identified universal surface markers that are uniquely present in senescent cells. Thus, the present invention is based on the discovery that senescence can be reliably identified based on the expression of specific protein biomarkers. Biomarker expression can include both up- and down-regulated levels, but the biomarkers identified herein are up-regulated.

[0074] Doxorubicin and radiation were used to induce cellular senescence in human IMR-90 fetal lung fibroblasts. Applicants identified distinct proteins that were significantly increased on the surface of senescent cells compared to non-senescent control cells. A total of 64 statistically significant differentially abundant proteins were common to both doxorubicin-treated (S-Doxo) and irradiated (S-IR) senescent cells. Of these proteins, 22 common proteins were differentially present in both cells, as described below. These proteins were further validated and five proteins were shortlisted based on increased protein expression by performing Western blotting and immunofluorescence assays. Thus, embodiments include proteins that are universal surface markers for senescent cells. In one embodiment, the protein biomarkers include one or more proteins shown in Table 1. In one embodiment, the protein biomarkers include one or more proteins shown in Table 2. In one embodiment, the protein biomarkers include one or more proteins shown in Table 3. In one embodiment, the protein biomarkers include one or more proteins shown in Table 4. In one embodiment, the protein biomarkers include one or more proteins shown in Table 5. In one embodiment, the protein biomarkers include one or more proteins shown in Table 6. [Table 1]

[0075] Embodiments also include methods of using the biomarkers for the diagnosis, prognosis, and / or treatment of aging. The methods described herein may include the combined measurement of at least one protein / peptide biomarker and / or fragment of a protein biomarker from human serum, plasma, blood derivatives, or blood itself.

[0076] TriCEPS To identify novel surface markers of senescent cells, the TriCEPS technology was used to capture and enrich surface proteins of senescent cells. Figure 2A is a flow chart of the steps of the Surfacesome-TriCEPS method. Senescent cell surface glycoproteins were oxidized and then covalently attached (i.e., labeled) to the NHS ester of the TriCEPS v.3.0 reagent. The tagged glycoproteins were then purified, identified, and quantified by mass spectrometry-based proteomics. Table 2 lists the surface proteins that were upregulated in cells treated with doxorubicin. Similarly, Table 3 lists the surface proteins that were upregulated in cells treated with radiation. Proteins common to both treatments (i.e., proteins found in both doxorubicin and radiation treatments) are shown in Table 4.

[0077] FIG. 2B is a series of volcano plots showing the abundance of approximately 50 surface proteins upregulated by doxorubicin treatment (Bi) and 35 surface proteins upregulated by radiation (FIG. 2C) when compared to non-senescent cells. Proteins with adjusted p-values ​​less than 0.01 were defined as "high confidence" proteins. Proteins with statistical significance less than 0.01 but with a fold change difference of more than 4 were defined as "medium confidence" differentially expressed. Table 5 lists surface proteins identified from cells treated with doxorubicin (S-Doxo) that had adjusted p-values ​​less than 0.01. Table 6 lists surface proteins identified from cells treated with radiation (S-IR) that had adjusted p-values ​​less than 0.01.

[0078] Figure 2D shows a Venn diagram of proteins found to be differentially abundant upon doxorubicin treatment and irradiation compared to control cells. To investigate the differentially abundant proteins that were common to both doxorubicin-treated and irradiated senescent cells, 64 statistically significant differentially abundant proteins were analyzed. 22 common proteins were identified that were differentially abundant in cells treated with doxorubicin and irradiation.

[0079] Identification of novel surface markers for senescent cells has many important advantages, including: 1. Biomarkers of senescent cells; 2. Removal of senescent cells from ex vivo cell therapy and tissue engineered products; 3. Removal of senescent cells for therapeutic purposes; 4. Removal of senescent cells for diagnostic purposes; 5. Development of targeted dyes for senescent cells using senescent cell surface markers; 6. Targeted delivery of senolytic or senostatic agents, or other drugs or therapeutics; 7. Enhanced cellular cytotoxicity against senescent cells (e.g., via antibody-dependent cellular cytotoxicity or ADCC); 8. Development of immunotherapeutic approaches to inhibit killing of senescent cells by CAR-modified T cells, NK cells, or gd T cells, macrophages, and dendritic cells.

[0080] Lysosomal proteins Published literature has provided evidence that organelles that deliver cellular material to lysosomes for degradation (e.g., autophagosomes and endosomes) can redirect their destination from fusion with lysosomes to fusion with the plasma membrane for extracellular release, a phenomenon known as lysosomal exocytosis (see, e.g., Buratta et al., Int J Mol Sci. 2020 Apr 8; 21(7): 2576). Lysosomal exocytosis is also known to play an important role in plasma membrane repair in all cell types (see, e.g., Samie et al., J Lipid Res. 2014 Jun; 55(6): 995-1009). This process helps restore plasma membrane integrity after injury, because lysosomes located near the wound site can rapidly migrate and fuse with the plasma membrane, efficiently resealing the injury and remodeling the tissue.

[0081] Applicants have identified lysosomal proteins that are increased on the surface of senescent cells compared to non-senescent control cells. The proteins are identified in the Figure. Thus, embodiments include methods of modulating the activity of one or more lysosomal proteins to treat aging-related diseases or disorders.

[0082] Working Example The following non-limiting examples are provided for illustrative purposes only, to facilitate a more complete understanding of the representative embodiments contemplated herein.These examples are intended to be merely a subset of all possible situations in which the components of the formulation can be combined.Therefore, these examples should not be construed as limiting any of the embodiments described herein, including those related to the type and amount of the components of the formulation, and / or its method and use.

[0083] method cell culture IMR-90 fibroblasts (ATCC, USA: Cat. No. CCL-186) were maintained at 37°C in humidified air containing 5% CO2 and 3% O2. Fibroblasts were used at population doubling levels (PDL) 30-47 and maintained in Dulbecco's Modified Eagle Medium (DMEM) complete medium (Corning; Cat. No. 10-013-CV) supplemented with 10% fetal bovine serum (FBS) (Millipore Sigma, USA; Cat. No. F4135) and 1X penicillin-streptomycin (Corning; Cat. No. 30-001-CI). Cumulative PDL was calculated using the following formula:

number

[0084] Induction of aging. Human IMR-90 fibroblasts (ATCC, USA) were treated with 300 nM doxorubicin hydrochloride (Millipore Sigma, USA; Cat. No. 504042) in DMEM complete medium for 24 h or treated with ionizing radiation (10 Gy X-rays) and maintained in culture. Primary human endothelial cells were treated with 250 nM doxorubicin in promo cell basal medium supplemented with growth medium MV2 supplement pack for 24 h and maintained in culture. Senescent cells were used 7–13 days after treatment.

[0085] Senescence-associated β-galactosidase staining Senescence burden in IMR-90 cells was determined by performing a senescence-associated β-galactosidase (SA-β-gal) activity assay as previously reported (Laberge et al. 2015) using a Senescence Detection Kit (BioVision; Cat. no. K320) according to the manufacturer's instructions. For the assay, fibroblasts were plated (8x10 cells / well) one day before senescence induction in 6-well cell culture plates (Greiner Bio-One; Cat. no. 657160) containing 2.5 ml of DMEM complete medium per well. 4 Non-senescent cells were also plated (5x10 cells / well) in 6-well cell culture plates 4 days before SA-β-gal staining. 4Cells / well). The medium was replaced with low serum medium 24 hours before staining. Staining was performed 13 days after doxorubicin treatment of IMR-90 cells. During staining, cells were incubated at 37°C in the absence of CO2 for 24 hours and then visualized and imaged by bright field microscopy.

[0086] Real-time quantitative PCR Non-senescent fibroblasts were plated (1.5 × 10 cells) in T-25 cell culture flasks (Cellstar; catalog no. 690160) containing 4 ml / flask of DMEM complete medium 4 days before cell harvest. 4 For senescent cells, fibroblasts were plated (2.5x10 cells / well) in T-25 cell culture flasks one day before induction of senescence. 4 / well). Senescent cell pellets were collected 14 days after doxorubicin treatment or radiation. The medium was replaced with low serum medium 24 hours before collecting all cell pellets. Cell pellets were stored at -80°C before RNA isolation. Total RNA was isolated from cell pellets using Quick-RNA MiniPrep (Zymo Research; Cat. No. R1055) according to the manufacturer's protocol. One microgram of total RNA per sample was reverse transcribed using PrimeScript RT Master Mix (Takara; Cat. No. RR036B) and cDNA was analyzed by real-time qPCR using TaqMan Fast Advanced Master MixApplied Biosystems; Cat. No. 4444557) (StepOnePlus™ Real-Time PCR System). Gene expression analysis was performed using Applied Biosystems TaqMan Gene Expression single-tube assays. All reactions were performed in triplicate and the relative expression levels of each gene were normalized to actin. Relative expression of mRNA was determined using the comparative threshold (Ct) method by normalizing target cDNA Ct values ​​to those of actin.

[0087] Analysis of cell proliferation. Cell proliferation was measured by EdU incorporation in dividing cells using the Click-IT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 dye (Thermo Scientific; Catalog No. C10337) according to the manufacturer's instructions. Non-senescent fibroblasts were plated (2x10 4 For senescent cells, fibroblasts were plated (1x10 4 / well). Staining was performed 7 days after induction of senescence. EdU incorporation was visualized and imaged using a fluorescent microscope. The percentage of EdU-positive cells was quantified by counting the total number of cells and EdU-positive cells across 12 microscopic fields per assay.

[0088] Immunofluorescence (IF) Fibroblasts were plated (1–2 × 10 cells) in black 96-well plates with square wells and clear flat bottom (Ibidi; Cat. No. 89626) containing 250 μl / well of DMEM complete medium for senescence induction 1 day before doxorubicin treatment. 4 / well). Non-senescent cells were plated (1–2x10 5 / well). All staining for immunofluorescence was performed 7 days after induction of senescence in IMR-90 cells. Cells were fixed with 200 μl / well of 4% paraformaldehyde in 1XPBS (Thermo Scientific; Cat. No. AAJ19943K2) for 15 min at room temperature, carefully rinsed with 1XPBS (Corning; Cat. No. 21-031-CV), and then permeabilized with 300 μl / well of 0.5% Triton X-100 for 10 min at room temperature. Cells were then rinsed once with 1XPBS and incubated with 250 μl / well of anti-γH2AX[pSer139] (Novus Biologicals; Cat. No. NB100-74435) and anti-HMGB1 (abcam; Cat. No. ab18256) antibodies diluted in 5% BSA (Research Products International; Cat. No. A30075) in 1XPBS overnight at 4°C. Cells were then washed five times with 1XPBS and incubated with 250 μl / well of Alexa Fluor 488 goat anti-mouse antibody (Invitrogen; Catalog No. A11029), Alexa Fluor 546 goat anti-rabbit antibody (Invitrogen; Catalog No. A11010), and Hoechst33342, trihydrochloride, trihydrate (Invitrogen; Catalog No. H3570) diluted in 5% BSA for 20 minutes at room temperature in the dark. Cells were washed five times with 1XPBS, after which 200 μl of 1XPBS was added to each well before images were acquired on a Molecular Devices Image Express Micro (Molecular Devices, San Jose, CA, USA). Seven images were acquired per well, with six wells captured per condition (NS or S).

[0089] Immunoblotting and cell membrane isolation For analysis of whole cell lysates, cells were lysed in RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, and 1 μg / ml leupeptin) containing 1× protease inhibitor cocktail (Cell Signaling Technology; Cat. No. 5871). The cell suspension was incubated on ice for 10 min and then centrifuged at 4° C. for 15 min to remove cell debris from the lysate. To extract cell membrane proteins, cell lysates were prepared using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Scientific; Cat. No. 89842). Protein concentrations were quantified using the BCA protein assay (Thermo Scientific; Cat. No. 23227). Equal amounts of protein were separated by SDS / PAGE and transferred to PVDF membranes using a semi-dry transfer apparatus (Bio-Rad). Membranes were incubated with primary antibodies overnight at 4°C, followed by secondary antibodies for 1 h at room temperature. Blots were developed using Pierce ECL Western Blotting substrate (Thermo Scientific; Cat. No. 32209). The following antibodies were used for immunoblotting analysis: COL9A1 (Sigmal Aldrich, USA; HPA053107), cathepsin C (D-6) (Santa Cruz; sc-74590), GAA (Sigma-Aldrich, USA; HPA029126), IGFBP7 (Abcam; ab171085), TPP1 clone 2E12 (Millipore Sigma, US; MABN1806), and Na / K ATPase (Invitrogen; MA5-32184). Other primary antibodies used included anti-TFEB (Santa Cruz Biotechnology; catalog number sc-166736), anti-p16 (Santa Cruz; catalog number 56330), and anti-β-actin (Cell Signaling; catalog number 4967).The following secondary antibodies were included: anti-rabbit IgG H&L(HRP) (Abcam; Cat. No. ab6802) and anti-mouse IgG H&L(HRP) (ThermoScientific; Cat. No. G-21040).

[0090] TriCEPS™-based Ligand Receptor Capture (LRC-TriCEPS). Dualsystems Biotech AG, TriCEPS™ V.3.0 (Cat. No. P0529), a kit for capturing proteins on the cell surface of live cells, was used using the manufacturer's instructions to identify surface proteins enriched in IMR-90 cells aged by treatment with doxorubicin or exposure to ionizing radiation as previously described. 6 Three separate 50 mL tubes of NS, Sen(IR), and Sen(Doxo) containing IMR-90 cells were scraped into PBS (pH 6.5) to detach the cells. The cells were then washed and cooled to 4°C. All remaining steps were performed at 4°C. Sodium metaperiodate (1.5 mM) was added to the cell suspension for gentle oxidation of cell surface proteins, and the cells were incubated at 4°C in the dark for 15 min with gentle rotation. The cells were then washed twice at 300xg for 5 min and resuspended in 20 ml of surfacesome buffer. For surfacesome labeling, quenched TriCEPSv.3.0 was added to the oxidized cells and incubated in the dark for 90 min with gentle agitation on a rotator. The samples were centrifuged at 1200g for 5 min at 4°C. Cell pellets were stored at -80°C before being sent to Dualsystems, where samples were purified using solid-phase chromatography, rigorously washed to remove non-specific interactions, reduced, alkylated and digested with trypsin. Tryptic peptides were then collected for LC-MS / MS analysis. Experiments were performed in biochemical triplicate to perform statistical analysis.

[0091] mass spectrometry Surfacesome-TriCEPS samples were analyzed in triplicate on a Thermo Orbitrap Elite spectrometer equipped with an electrospray ion source. Tryptic peptides were measured using a 15 cm C18 packed column with an 80 min gradient in data-dependent acquisition mode (TOP20).

[0092] Data analysis PROGENESIS® software was used for alignment of raw files and feature detection. Comet search engine was used for spectral identification. Trans proteomic pipeline was used for statistical validation of putative identification and protein inference. For protein inference, relative quantification of samples was performed based on ion extraction intensity, and differential protein abundance was examined using a statistical ANOVA model followed by multiple testing correction. This model assumes that the measurement error follows a Gaussian distribution, considers each feature as a repeat of protein abundance, and explicitly accounts for this redundancy. It examines the differential abundance of each protein in all pairwise comparisons and reports its p-value. The p-values ​​are then adjusted for multiple comparisons to control the false discovery rate (FDR) across experiments. The adjusted p-values ​​(q-values) obtained for each protein are plotted against the magnitude of fold enrichment between the two experimental conditions.

[0093] Quantitative differences between variations are shown as volcano plots. Volcano plots combine the magnitude of change with a measure of statistical significance from a statistical test (in this case adjusted p-values ​​from an ANOVA model) and allow for rapid visual identification of proteins that show changes that are also statistically significant. The x-axis represents the average ratio of fold changes (log2 scale). The y-axis represents the statistical significance p-value of the fold change ratio for each protein (-log 10 Proteins that are differentially abundant in one of the samples are plotted to the left or right of the origin on the X-axis, indicating the sample in which that protein is enriched.

[0094] Flow cytometry The cells were resuspended in 100 μl of PBS. The cells were then incubated with an APC-conjugated anti-human CD3 antibody (MiltenyiBiotec; Cat# 130-113-135). LAMP-1 antibody (BioLegend; Cat# 328601) was incubated with the cells for 30 minutes on ice. The cells were washed with 1 ml of ice-cold PBS and resuspended in 100 μl of ice-cold PBS. APC-conjugated anti-mouse IgG (H+L) secondary antibody (Thermo Scientific; Cat# A-865) was incubated with the cells for 30 minutes on ice. The cells were then washed with 1 ml of cold PBS and resuspended in 100 μl of ice-cold PBS. Data were collected by a flow cytometer (DB Accuri C6). Cell viability was determined by PI staining and live cells were gated for downstream analysis. Data were analyzed using Flowlogic software (Miltenyi Biotech, Germany).

[0095] Crystal violet staining Cell viability after 48 h treatment with apilimod (1 μM) or baquolin-1 (1 μM) was estimated by crystal violet. Cells in 6-well cell culture plates (Greiner Bio-One; Cat. No. 657160) were washed twice with PBS, incubated with 4% paraformaldehyde for 15 min at room temperature, and then stained with 0.25% crystal violet for 10 min. Cells were washed with deionized water and three images were taken per well.

[0096] Impedance Measurement For background value measurements, 50 μl of complete medium was added to E-Plates96 (Agilent). Cells were seeded at a density of 10,000 cells / well in a total of 200 μl of medium per well. Cell adhesion was monitored using a RTCA MP (Agilent) instrument and RTCA software (Agilent). Medium was replaced with medium containing either DMSO, vacolin-1, or apilimod. Cells treated with 0.2% Triton X-100 were used as a 100% dead cell positive control for the cytotoxicity assay. After drug addition, impedance measurements were recorded every 15 min. All experiments were performed in at least five replicates for each dose. The impedance change is expressed as a cell index (CI) value. This value is derived from the relative impedance change corresponding to the cell coverage of the electrode sensor and normalized to the baseline impedance value of medium alone. To analyze the acquired data, CI values ​​were exported and the percentage of attached cell surface was calculated in comparison to positive control and DMSO treated cells.

[0097] Diagnostic methods using biomarkers One or more of the biomarkers can be used in the method of diagnosing disease (e.g., aging-related disease or disorder) or in the method of determining the prognosis of the test subject having the disease.Thus, one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and / or 22 biomarkers can be used in the method of diagnosing disease or in the method of determining the prognosis of the test subject having the disease.Thus, at least one biomarker or a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and / or 22 biomarkers can be used in the method of diagnosing disease or in the method of determining the prognosis of the test subject having the disease.

[0098] Thus, no more than one biomarker or a combination of no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and / or 22 biomarkers may be used in the method of diagnosing a disease or determining the prognosis of a test subject having a disease. Thus, about one biomarker or a combination of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and / or 22 biomarkers are used in the method of diagnosing a disease or determining the prognosis of a test subject having a disease.

[0099] In the first step of the method, the expression level of one or more proteins is measured in a plasma sample from a subject with a disease.In one embodiment, the expression level of one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and / or 22 biomarkers is used to generate a footprint or signature for the subsequent diagnosis of the patient.In one embodiment, the expression level of at least one biomarker or a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and / or 22 biomarkers is used to generate a footprint or signature for the subsequent diagnosis of the patient.

[0100] In one embodiment, the expression level of no more than one biomarker, or a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and / or 22 or fewer biomarkers, is used to generate a footprint or signature for subsequent diagnosis of the patient. In one embodiment, the expression level of about one biomarker, or a combination of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and / or 22 biomarkers, is used to generate a footprint or signature for subsequent diagnosis of the patient.

[0101] The expression level of the same protein is then measured in plasma, blood or tissue samples from healthy subjects. This is used as a control. The sample from healthy patients can then be compared to identify proteins with altered expression levels in plasma samples from subjects with disease. Biomarker fingerprints or signatures can be made from proteins with altered expression levels. This can be used to diagnose disease or determine disease prognosis in test subjects by comparing the protein levels from the plasma of test subjects. Conventional statistical analysis can be used to determine, for example, confidence levels.

[0102] Diagnostic kits for screening for diseases The following examples are based on the above construction. The embodiments of the present invention can be compiled into a diagnostic kit for diagnosing a disease, such as an aging-related disease or disorder. The kit can identify one or more target cells that have a biomarker of the disease in the plasma from a test subject.

[0103] The kit may include reagents that can be used to identify variations in the expression levels of one or more proteins in a sample from a test subject. The expression levels of the proteins can be used to compare / analyze the test samples with fingerprints that indicate the presence of disease.

[0104] In certain embodiments, the present disclosure provides a kit for diagnosing aging-related diseases.The kit can include one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and / or 22 biomarkers disclosed herein.Those skilled in the art will understand that the number of biomarkers can be changed without departing from the nature of the present disclosure, and therefore other combinations of biomarkers are also included in the present disclosure.Those skilled in the art will know which one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and / or 22 biomarkers to use based on the symptoms of patients suffering from the disease.

[0105] In certain embodiments, the kit comprises one biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and / or 22 biomarkers disclosed herein. The kit may further optionally comprise instructions for use. The kit may further optionally comprise (e.g., comprise, consist essentially of, consist of) a tube, applicator, vial, or other storage container that contains the biomarkers and / or a vial that contains one or more of the biomarkers. In one embodiment, each biomarker is in its own tube, applicator, vial, or storage container, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and / or 22 biomarkers are in a tube, applicator, vial, or storage container.

[0106] Regardless of its type, a kit will typically include one or more containers having deposited therein, preferably appropriately dispensed, a single biomarker or a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and / or 22 biomarkers. The kit components may be packaged either in aqueous medium or in lyophilized form.

[0107] Example 1 Genotoxic stress-induced model of aging To identify novel senescent cell surface markers, IMR-90 human fetal lung fibroblasts, a commonly used cell culture model of senescence, were used. Senescence burden in IMR-90 cells was determined by observing senescence-associated β-galactosidase (SA-β-gal) activity, which can be used to measure increased activity of lysosomal β-galactosidase, a hallmark of senescent cells (see, e.g., Itahana et al., Methods Mol Biol 371:21-31,2007). Strong induction of lysosomal beta-galactosidase was observed in IMR-90 cells treated with either doxorubicin (300 nM) or 10 Gy X-rays when compared to quiescent cells 13 days after treatment, as shown graphically in Figure 1A.

[0108] Another characteristic of senescent cells is the loss of cell proliferation. A significant decrease in the number of proliferating cells was observed 7 days after treatment with doxorubicin or radiation, as measured by EdU cell proliferation assay, as shown in Figure 1B. In both doxorubicin-treated (S-Doxo) and irradiated (S-IR) IMR-90 cells, the non-senescent control cells showed greater proliferative activity than the senescent cells.

[0109] Cells were plated in 8-well chamber slides and counted on day 7 after treatment. Four fields were quantified per well (n=3), resulting in a total of 1276, 672, and 1102 cells counted for NS, S(Doxo), and S(IR) cells, respectively.

[0110] The loss of proliferation was further confirmed by measuring increased expression of cell cycle checkpoint markers such as p16INK4A and p21CIP1A in doxorubicin-treated and irradiated fibroblasts compared to non-senescent fibroblasts (see, e.g., Baker et al., Nature 530:184-189, 2016). The results are graphically depicted in Figure 1C.

[0111] Since senescent cells undergo significant morphological changes, including changes in the nuclear lamina, such as decreased expression of lamin B1, the applicants measured the mRNA expression of nuclear lamin B1. The results show a significant decrease in the expression of lamin B1 in both doxorubicin-treated and irradiated senescent IMR-90 cells compared to quiescent control cells, as shown in Figure 1D. The chromatin changes caused by the loss of lamin B1 lead to increased expression of p53 and p38 MAPK, which is causally related to the robust increase in SASP expression (see, e.g., Freund et al., Mol Biol Cell 23:2066-2075, 2012; Lopes-Paciencia et al., Cytokine 117:15-22, 2019). Applicants demonstrated expression of several prototypical SASP factors and observed strong induction of IL-6, IL-8, and IL-1a by qRT-PCR analysis as shown in FIG. 1E. Furthermore, previous studies have shown that senescent cells exhibit loss of HMGB1, a non-histone nuclear DNA binding protein (Davalos et al., J Cell Biol 201:613-629, 2013). As shown in FIG. 1F, we confirmed nuclear loss of HMGB1 from both doxorubicin-treated and irradiated senescent cells. Furthermore, as shown in FIG. 1F, we demonstrated persistent DNA damage foci in senescent cells as measured by gH2AX staining in senescent cells compared to quiescent cells. Collectively, these data demonstrate that the genotoxic stress-induced model of senescence successfully induced a strong senescent phenotype in IMR-90 fibroblasts.

[0112] Example 2 Identification of senescent cell surface markers Cellular responses are generally mediated through interactions with proteins on the cell surface. Progress has been made in elucidating the molecular mechanisms involved in cellular senescence. However, characterization of the surface of senescent cells is lacking. Several reports have suggested the identification of senescent cell surface markers. However, further analysis has shown that these markers are not universal and their surface expression is not specific to senescent cells (e.g., Frescas et al., Proc Natl Acad Sci USA 114:E1668-E1677, 2017).

[0113] Example 3 Surfacesome characterization of senescent IMR-90 fibroblasts After the Surfacesome-TriCEPS experiment, about 50 surface proteins were identified as differentially abundant in senescent cells (S-Doxo) compared to non-senescent (NS) cells using the protocol described above. Meanwhile, the abundance of approximately 35 surface proteins was affected in senescent cells after irradiation. The differentially abundant proteins were assigned to two groups: high confidence and medium confidence. High confidence differential expression is defined as proteins with an adjusted p-value less than 0.01 (-Log10(adjusted p-value)>2). Medium confidence differentially expressed proteins are defined as proteins with a fold change difference greater than 4 but with statistical significance less than 0.01. These results are shown in volcano plots (Figure 2B, Figure 2C) showing the comparison between NS and senescent cells (S-Doxo or S-IR).

[0114] Table 2 lists surface proteins identified from doxorubicin-treated (S-Doxo) cells that had an adjusted p-value of less than 0.01. Table 3 lists surface proteins identified from radiation-treated (S-IR) cells that had an adjusted p-value of less than 0.01. To investigate differentially abundant proteins common to both doxorubicin-treated and irradiated senescent cells, a total of 64 statistically significant differentially abundant proteins were analyzed. 22 common proteins were identified as differentially abundant in both, as shown in Figure 2B and Figure 2C.

[0115] From the 22 proteins that were common between the two variations, the applicants focused on 18 proteins that were upregulated in both conditions (S-Doxo, S-IR) compared to NS cells. High-confidence candidates that were at least 2.2 log2 times higher in senescent cells compared to NS controls were selected. The list was further narrowed by selecting those that were not expressed on the surface of biological tissue samples according to Protein Atlas (see, for example, www.proteinatlas.org). Thus, five proteins were finalized: CO8A1, CATC, LYAG, IBP7, and TPP1.

[0116] Evaluating the protein expression of these candidates, increased protein expression of CATC, LYAG, and TPP1 was verified in whole cell lysates of both senescent IMR-90 fibroblasts (Figure 3A) and primary endothelial cells (Figure 3B) compared to non-senescent (NS) control cells. Western blot analysis was then performed on membrane and cytoplasmic fractions of senescent (S) and non-senescent (NS) fibroblasts. Increased expression of CATC, LYAG, and TPP1 in the membrane fraction of senescent cells compared to NS cells was observed. + / K +ATPase was used to monitor cell fractionation (Figure 3C). These results were further supported by immunofluorescence data that showed increased expression of TPP1 on the surface of senescent IMR-90 fibroblasts and senescent endothelial cells when compared to non-senescent controls (Figure 3D).

[0117] Example 3 Senescent cells exploit lysosomal exocytosis for survival Analysis of 22 differentially expressed surface proteins common to both doxorubicin-treated (300 nM, 24 h) and irradiated (20 Gy) cells when compared with nonsenescent (NS) control cells revealed that several lysosomal proteins were disproportionately abundant on the surface of senescent cells (Figure 1A). The underlying mechanisms by which senescent cells may present lysosomal proteins on their surface were further investigated as follows.

[0118] Given the abundance of lysosomal proteins present on the surface of senescent cells, markers of lysosomal exocytosis were validated in senescent cells. After lysosomal exocytosis, lysosomal membrane proteins such as LAMP-1 have been shown to be present on the cell surface. Thus, surface expression of LAMP-1 is often used as a marker of lysosomal exocytosis. Flow cytometry analysis of senescent IMR-90 cells revealed a marked increase in LAMP-1 surface expression compared to non-senescent control cells (Figure 1B). Transcription factor EB (TFEB) is known as a master regulator of lysosomal biogenesis and autophagy. TFEB is also known to regulate lysosomal exocytosis. Western blot analysis of senescent IMR-90 cells and primary endothelial cells showed increased expression of TFEB compared to non-senescent control cells (Figure 1C).

[0119] Senescent cells are known to employ several mechanisms to enhance their survival. For example, increased expression of pro-survival BCL family proteins has been widely studied. Indeed, BCL antagonism is the basis of many senolytic drugs (see, e.g., Zhu, et al., Aging Cell. 2016 Jun;15(3):428-35). Baquolin-1 was used to determine whether lysosomal exocytosis is a mechanism of survival for senescent cells. Baquolin-1 is a potent inhibitor of phosphatidylinositol-3-phosphate 5-kinase type III (PIKfyve), which plays a key role in lysosomal exocytosis, which fuses lysosomes with the plasma membrane. Viability assays were performed in senescent IMR-90 cells using the xCELLigence Real-Time Cell Analysis (RTCA) system, which uses label-free cell impedance to monitor cell health and viability over time. The results demonstrated that blocking baquolin-1-mediated lysosomal exocytosis selectively killed senescent IMR-90 cells in a dose-dependent manner, with the average normalized cell index after 48 h treatment with 1 μM baquolin-1 being 1.2 in NS cells and 0.4 in senescent cells (Figure 1D).

[0120] Apilimod is a clinical PIKfyve inhibitor. Apilimod was used to further validate the senescent cell ablation effect of PIKfyve inhibitors. Results demonstrated that, like baquorin-1, apilimod caused a dose-dependent decrease in senescent cell viability. Senescent cells were not viable after 48 h treatment with 1 μM and 5 μM apilimod (Figure 1D). To further characterize the potent cytotoxic effect of apilimod on non-senescent versus senescent cells, IMR-90 fibroblasts were treated with 1 μM apilimod. Light microscopy images of crystal violet stained cells after 48 h of apilimod treatment demonstrated a marked decrease in senescent cell viability with no effect on non-senescent cell viability (Figure 1E). Quantification of adherent cells revealed 92% non-senescent cell viability versus only 17% viable cells in the senescent state (Figure 1E). Notably, the decrease in viable apilimod-treated non-senescent cells 48 hours after treatment is likely due in part to a decrease in proliferation but not viability of apilimod-treated cells.

[0121] Example 4 Validation of senescent cell surface markers by flow cytometry IMR90 cells were treated with doxorubicin (DOX, 300 nM) for 10 days to induce senescence. Senescent cells (S-Doxo) and non-senescent cells (NS) were harvested and stained with primary and APC-labeled secondary antibodies for 30 min at 4°C. Cells were analyzed by flow cytometry. Cells were stained with anti-TPP1 antibody (clone 2E12, EMD Millipore). The MFI of the isotype control (gray line) is 1154, the MFI of non-senescent cells (blue line) is 1134, and the MFI of senescent cells (red line) is 3251. The data demonstrated that Lamp-1 and TPP1 are presented on the surface of senescent cells. The results are shown in Figure 5.

[0122] Example 5 Therapeutic elimination of senescent cells In this example, a patient (55 year old male) visits his internal medicine physician with signs and symptoms of atherosclerosis and hypertension, and the medical practitioner believes that targeted removal of the patient's senescent cells may improve the patient's cardiovascular disease.

[0123] Tissue and blood samples demonstrate elevated levels of the following proteins: CO8A1, CATC, LYAG, IBP7, and TPP1. These markers indicate the presence of senescent cells at levels higher than normal for a patient of that age.

[0124] The patient is also administered (intravenously) a drug conjugate to kill the senescent cells. The conjugate includes an antibody (that recognizes CO8A1) and a cytotoxic agent (i.e., a toxic peptide). The patient is also administered a senolytic agent. The patient's blood pressure gradually improves. The healthcare provider continues to monitor the atherosclerosis and hypertension.

[0125] How to use Embodiments include methods of diagnosing a disease (i.e., a senescence-associated disease or disorder) or determining a prognosis of a test subject having a disease. The method includes measuring expression levels of one or more biomarkers to identify those with altered expression levels. Embodiments also include therapeutic methods for treating a senescence-associated disease or disorder. In one embodiment, the method includes administering a pharmaceutical formulation comprising a therapeutic agent that selectively kills senescent cells (i.e., selectively kills senescent cells over non-senescent cells). The treatment regimen may include administering the pharmaceutical formulation for a time and in an amount sufficient to selectively kill senescent cells.

[0126] Compositions according to the embodiments described herein have desirable properties, such as desirable solubility, viscosity, injectability, and stability. Lyophilates according to the embodiments described herein also have desirable properties, such as desirable recovery, stability, and reconstitution.

[0127] In one embodiment, the pH of the pharmaceutical formulation is at least about 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, or 9.

[0128] In one embodiment, the pH of the pharmaceutical formulation is about 1 to about 14, about 2 to about 14, about 3 to about 14, about 4 to about 14, about 5 to about 14, about 6 to about 14, about 7 to about 14, about 8 to about 14, about 9 to about 14, about 10 to about 14, about 11 to about 14, about 12 to about 14, about 13 to about 14, about 1 to about 10, about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, about 8 to about 10, about 9 to about 10, about 1 to about 11, about 2 to about 11, about 3 to about 11, about 4 to about 11, about 5 to about 11, about 6 to about 11, about 7 to about 11, about 8 to about 11, about 9 to about 11, about 10 to about 11, about 1 to about 12, about 2 to about 12, about 3 to about 12, about 4 to about 12, about 5 to about 12 , about 6 to about 12, about 7 to about 12, about 8 to about 12, about 9 to about 12, about 10 to about 12, about 11 to about 12, about 1 to about 13, about 2 to about 13, about 3 to about 13, about 4 to about 13, about 5 to about 13, about 6 to about 13, about 7 to about 13, about 8 to about 13, about 9 to about 13, about 10 to about 13, about 11 to about 13, about 12 to about 13, about 1 to about 9, about 2 about 9, about 3 to about 9, about 4 to about 9, about 5 to about 9, about 6 to about 8, about 6 to about 7, about 6 to about 9, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 4 to about 9, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 3 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 3 to about 4, about 7 to about 8, about 7 to about 9, about 7 to about 10.

[0129] Certain embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will be appropriately adopted by those skilled in the art, and the inventors intend for the invention to be carried out in ways other than as specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations is encompassed by the present invention, unless otherwise indicated herein or clearly contradicted by context.

[0130] Grouping of alternative embodiments, elements, or steps of the invention should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or exclusion occurs, the specification is deemed to include the group as amended and thus fulfills the description of all Markush groups used in the appended claims.

[0131] Unless otherwise indicated, all numerical values ​​expressing features, items, quantities, parameters, characteristics, terms, etc. used in the specification and claims should be understood to be modified in all instances by the term "about". As used herein, the term "about" means that the feature, item, quantity, parameter, characteristic, or term so qualified encompasses a range of plus or minus 10 percent above and below the value of the described feature, item, quantity, parameter, characteristic, or term. Thus, unless otherwise indicated, the numerical parameters described in this specification and the appended claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values ​​setting forth the broad scope of the invention are approximations, the numerical ranges and values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical range or value inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of numerical ranges herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value falling within the range, and unless otherwise stated herein, each separate value in a numerical range is incorporated herein as if it were individually recited herein.

[0132] The terms "a," "an," "the," and similar references used in the context of describing the present invention (particularly in the context of the claims below) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better clarify the invention and does not otherwise pose a limitation on the scope of the invention as claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0133] Certain embodiments disclosed herein may be further limited in the claims using the language "consisting of" or "consisting essentially of." When used in the claims, the transitional term "consisting of," whether added at the time of filing or by amendment, does not include any element, step, or ingredient not specified in the claim. The transitional term "consisting essentially of" limits the claim to those materials or steps specified and that do not materially affect the basic and novel characteristic(s). The embodiments of the invention so claimed are essentially or explicitly described and embodied herein.

[0134] Grouping of alternative embodiments, elements, or steps of the invention should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or exclusion occurs, the specification is deemed to include the group as amended and thus fulfills the description of all Markush groups used in the appended claims.

[0135] All patents, patent publications, and other publications cited and identified herein are individually and expressly incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that may be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements regarding dates or representations regarding the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0136] Finally, although aspects of the present specification are emphasized by reference to specific embodiments, it should be understood that those skilled in the art will readily recognize that these disclosed embodiments merely exemplify the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to the specific methodology, protocols, and / or reagents, etc. described herein. Thus, various modifications or changes to the disclosed subject matter, or alternative configurations, can be made in accordance with the teachings of the specification without departing from the spirit of the specification. Finally, the terms used in the specification are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the invention, which is defined solely by the claims. Thus, the present invention is not limited to what has been precisely shown and described. [Table 2] TIFF2025504704000005.tif205159 [Table 3] [Table 4] [Table 5] TIFF2025504704000009.tif49159 [Table 6] TIFF2025504704000011.tif43159

Claims

1. 1. A method for distinguishing between non-senescent and senescent cells, the method comprising comparing the levels of one or more biomarkers from said non-senescent and senescent cells.

2. 2. The method of claim 1, wherein the one or more biomarkers consist of proteins selected from CO8A1, CATC, LYAG, IBP7, and TPP1.

3. 2. The method of claim 1, wherein the one or more biomarkers consist of proteins selected from CO8A1, VCAM1, CATC, LYAG, PTGIS, IBP7, TPP1, CA2D1, MA2B1, CAVN2, FBLN1, LYOX, PCP, DPP4, GLCM, HEXB, PCYOX, and GGALNS.

4. 2. The method of claim 1, wherein the one or more biomarkers consist of proteins selected from TPP1, LYAG, VCAM1, GARS, CATC, NCEH1, CSPG4, ELOV1, CO8A1, IBP7, PTGIS, MA2B1, CAH2, GLCM, S10A6, DPP4, LYOX, PCP, HACD3, SDCB1, MGST3, EZRI, LEG3, FBLN1, PCYOX, HEXB, GALNS, ESYT1, ACTN4 and DHCR7.

5. 2. The method of claim 1, wherein the one or more biomarkers consist of proteins selected from LYAG, VCAM1, FBLN1, GALNS, CATC, IBP7, LRC15, CREL1, EZRI, CAVN2, NCEH1, PCYOX, SDCB1, MA2B1, TPP1, HEXB, PCP, PTGIS, CSPG4, SYNPO, CD248, FKB11, GOT1B, DPP4, CO8A1, ITA1, LYOX, GLCM, TOR1A, and PHB2.

6. 10. The method of claim 1, further comprising selectively removing senescent cells from non-senescent cells.

7. 10. The method of claim 1, further comprising selectively labeling senescent cells.

8. 1. A method for detecting or determining the prognosis of a disease, comprising: a) detecting the level of one or more biomarkers in a sample from a test subject; b) identifying the disease or determining a prognosis of the disease based on an elevated level of expression of the one or more biomarkers in the sample; and c) treating said subject with a senolytic agent to prevent or ameliorate said disease; Including, The method, wherein the disease is an aging-related disease or disorder.

9. 9. The method of claim 8, wherein the one or more biomarkers consist of proteins selected from CO8A1, CATC, LYAG, IBP7, and TPP1.

10. 2. The method of claim 1, wherein the one or more biomarkers consist of proteins selected from CO8A1, VCAM1, CATC, LYAG, PTGIS, IBP7, TPP1, CA2D1, MA2B1, CAVN2, FBLN1, LYOX, PCP, DPP4, GLCM, HEXB, PCYOX, and GGALNS.

11. 9. The method of claim 8, wherein the one or more biomarkers consist of proteins selected from TPP1, LYAG, VCAM1, GARS, CATC, NCEH1, CSPG4, ELOV1, CO8A1, IBP7, PTGIS, MA2B1, CAH2, GLCM, S10A6, DPP4, LYOX, PCP, HACD3, SDCB1, MGST3, EZRI, LEG3, FBLN1, PCYOX, HEXB, GALNS, ESYT1, ACTN4 and DHCR7.

12. 9. The method of claim 8, wherein the one or more biomarkers consist of proteins selected from LYAG, VCAM1, FBLN1, GALNS, CATC, IBP7, LRC15, CREL1, EZRI, CAVN2, NCEH1, PCYOX, SDCB1, MA2B1, TPP1, HEXB, PCP, PTGIS, CSPG4, SYNPO, CD248, FKB11, GOT1B, DPP4, CO8A1, ITA1, LYOX, GLCM, TOR1A and PHB2.

13. 9. The method of claim 8, wherein the senolytic agent inhibits lysosomal exocytosis.

14. 14. The method of claim 13, wherein the aging-related disease or disorder is one or more of atherosclerosis, osteoarthritis, osteoporosis, hypertension, arthritis, cataracts, cancer, Alzheimer's disease, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, hair graying, sarcopenia, obesity, neurogenesis, fibrosis, and glaucoma.

15. 1. A method for detecting a disease in a subject, comprising: (a) detecting the level of one or more biomarkers in a sample from the subject; (b) identifying a disease based on elevated levels of said one or more biomarkers; (c) administering a therapeutic amount of a senolytic agent based on the identity of the disease; A method comprising:

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