A SINGLE CELL MASS CYTOMETRY PLATFORM TO MAP THE EFFECTS OF CANDIDATE AGENTS ON CARTILAGE

BACKGROUND

[0001 ] Cartilage repair remains an unmet medical need with no disease-modifying drug available for degenerative diseases like osteoarthritis (OA), for which clinical care is limited to pain management or total joint replacement. Although multiple pathways and targets have been considered for OA treatment, the rate of drug failure in clinical trials is high. Notably, OA is a complex disease with a known interplay of metabolic, epigenetic, genetic, and cellular factors that influence its etiology. Drug discovery efforts are further confounded by the late detection of the disease and ambiguity about the causal and early events in OA pathogenesis. Another critical knowledge gap is the lack of understanding of the heterogeneity between patients and its molecular underpinnings.

[0002] The recent advent and accessibility of single-cell technologies, both transcriptional and proteomic, have made it possible to achieve a high-resolution understanding of the composition, architecture, and functioning of tissues by parsing out the contribution of single cells in tissues in health and disease.

[0003] The present disclosure provides a single cell platform directed to osteoarthritis drug discovery.

SUMMARY

[0004] Compositions and methods are provided for determining the effect of an agent on cartilage, using single cell flow cytometry for analysis of chondrocytes following treatment with an agent of interest. In some embodiments, mass cytometry analysis is performed on a population of chondrocytes cultured in vitro with an agent of interest, e.g. analyzed by time-of- flight (cyTOF). In some embodiments the chondrocytes are human cells.

[0005] A population of chondrocytes obtained from a human cartilage sample may be analyzed for a plurality of markers, e.g. at least 5, at least 10, at least 15, at least 20, at least 25 markers, and may comprise 30 markers, or more. In some embodiments a marker is one or more of SOX9; CD24; Idu; Ki67; Hif2a; PNFκB(S29); iNOS; CD126; CD121 B; CD121A; CD120B; P-SAPK/JNK (T183/Y185); p-SMAD1/5(S463/465); pStat3(pY705); TET1 ; SOD2; Runxl ; Runx2; CD106; CD73; Notch-1 ; Stro-1 ; CD171 ; CD105; CD33; CD49E; CD146; TLR4; TLR2; and p16ink4a.

[0006] In some embodiments the cells are clustered into discrete subpopulations based on the presence and level of expression of the plurality of markers, including the clusters as shown in Table 2, where the number of discrete clusters may be at least 5, at least 10, at least 15, at least 20, at least 25 or more. Analysis may include, without limitation, changes in a cell sub-population size, changes in expression of markers of interest, changes in phosphorylation of markers, and the like. In some embodiments, cells expressing low levels of SOX9 are included in the clustering analysis. In some embodiments, expression of p16ink4a is included in the clustering analysis.

[0007] Sub-populations (clusters) of interest may comprise cartilage-progenitor cells (CPC) subtypes, including a CD105 ⁺ Notch-1 ⁺ Stro1 ⁺ , high p16 ^ink4a chondroprogenitor population (CPC III). An inflammation amplifying (Inf-A) population was characterized by the co-expression of two cytokine receptors, IL1 R1 (CD121A) and TNFRII (CD120B) and an inflammation dampening (Inf- D) subpopulation characterized by the expression of CD24. The Inf-A, Inf-D, and CPC III populations respectively, all belonging to the SOX9 ^medium group. Five different clusters that expressed high levels of p16 ^ink4a -clusters were identified, of which, clusters 6, 16 and 17 belong to the SOX9 ^medium group while clusters 11 and 24 belong to the SOX9 ^high group. No p16 ^ink4a expressing senescent subpopulations were detected in SOX9 ^low or SOX9 ^low-medium groups.

[0008] In some embodiments an agent is a candidate agent where the effect of the agent on chondrocyte populations is determined, where the effect is determined on the change in the distribution of cells into clustered chondrocyte subpopulations. A principal component analysis may be used to plot the distribution of cells from an individual based on cluster abundance and marker expression profiles in each sample with or without drug treatment. The change resulting from drug treatment may be compared to controls, e.g. a positive control and a negative control to determine if the change is indicative of responsiveness. In some embodiments the cells are obtained from an individual with osteoarthritis. In some embodiments, analysis is patient-specific, where the effect of a candidate agent on cells obtained from an individual patient is assessed.

[0009] In some embodiments a patient sample is analyzed for responsiveness to a known drug. In such embodiments, analysis is patient-specific, where the effect of the known drug is determined on cells obtained from the individual patient. In such embodiments, the patient may have osteoarthritis. The response of chondrocytes to a drug is determined by staining with a plurality of markers, as disclosed herein, followed by mass cytometry analysis. The presence and level of expression of the plurality of markers is used to cluster the cells. The clustering is used to compare the patient sample response to a reference response, and the individual patient is determined to be a responder or non-responder to the agent. In some embodiments an analysis of the results is provided to the individual. In some embodiments an individual patient is treated with the known drug after being determined to be a responder.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures. [001 1 ] FIGS. 1A-1 G. Stratification of OA chondrocytes based on SOX9 expression. (A) Schematic outlining the experimental design for profiling OA chondrocytes. Briefly, OA chondrocytes were isolated from patient samples (n=6) and cultured, followed by staining with metal-conjugated antibodies and data acquisition using cyTOF. (B) UMAP of 25 clusters identified by unsupervised hierarchical clustering by FlowSOM. (C) UMAP plot of SOX9 expression across all clusters. (D) Bar plot showing the abundance of the four groups stratified by SOX9 expression. Each point represents an individual patient. Data represent mean ± standard deviation. (E) Heatmap with hierarchical clustering with Euclidean distance measurement for stratification of clusters based on SOX9 expression. (F) UMAP representation of the SOX9 ^low , sOX9 ^{low medium} , SOX9 ^medium and SOX9 ^high groups. (G) Heatmap for the median expression of various panel markers in the groups. Marker columns were z-scored for standardization.

[0012] FIGS. 2A-2D. Identification of inflammatory and senescent populations in the OA chondrocyte landscape. (A) UMAP demarcating Inf-A (cluster 19), Inf-D (cluster 23), and CPCIII (cluster 16) populations in OA chondrocytes (n=6). (B) Heatmap shows median expression of markers used to precisely identify CPCIII (CD105 ⁺ Notch-1 ⁺ Stro1 ⁺ ), Inf A (Hif2a ⁺ CD121 A ⁺ CD120B ⁺ pNF-κB ⁺ pJNK ⁺ pSMAD1/5 ⁺ ) and Inf-D (CD24 ⁺ ) clusters. (C) UMAP projection showing p16 ^ink4a expressing senescent clusters (SnC). (D) UMAP of the median expression of p16 ^ink4a in OA chondrocytes (n=6). The area demarcated by dashed line denotes the juxtaposition of the p16 ^ink4a clusters on the p16 ^ink4a expression map.

[0013] FIGS. 3A-3G. Effect of NF-κB inhibition on the OA chondrocyte landscape. (A) Heatmap of median p-NF-κB expression in all FlowSOM-identified clusters in DMSO-treated (control) and BMS-treated OA chondrocytes (n=6 patient samples), p- NF-κB expression in each cluster was column-wise z-scored for standardization. Rectangles denote significantly different clusters between control and BMS-treated samples measured by paired t-test (p<0.05). (B) UMAP projections of Inf-A (cluster 19), Inf-D (cluster 23), and CPCIII (cluster 16) populations, along with p16 ^ink4a expressing senescent clusters: SnC I (cluster 6), SnC II (cluster 1 1 ), SnC III (cluster 17) and SnC IV (cluster 24) in control and BMS-treated samples. Stars (*) indicate significantly depleted clusters in BMS-treated samples. (C) UMAP represents median p- NF-κB expression across OA landscape in control and BMS-treated samples (n=6). (D), (F) UMAP of significantly depleted or expanded clusters upon BMS treatment. (E), (G) Percent abundance of significantly depleted or expanded clusters in paired samples (n=6) . Statistical significance was calculated by paired t-test (n=6) at 95% confidence level and represented by adjusted p values.

[0014] FIGS. 4A-4D. NF-κB pathway inhibition targets p16 ^ink4a expressing senescent cells.

(A) UMAP of Inf-A, Inf-D and Snc CPC III clusters in control and BMS-treated samples (n=6). Dashed circle identifies SnC CPC III cluster (cluster 16) that is depleted upon BMS treatment.

(B) Heatmap of median expression of markers that are significantly different in the SnC CPC III cluster between control and BMS-treated samples (paired t-test, p<0.05). (C) UMAP depicting the differential abundance of p16 ^ink4a expressing clusters in the combined control and BMS- treated samples (n=6). Dashed line encircles clusters significantly depleted upon BMS treatment (paired t-test, p<0.05). (D) UMAP of the median expression of p16 ^ink4a in the combined control and BMS-treated samples (n=6).

[0015] FIGS. 5A-5F. Effect of Kartogenin treatment on the OA chondrocyte landscape. (A) UMAP of differentially abundant clusters in the combined control and Kartogenin-treated samples (n=6). (B) Percent abundance of significantly different clusters in paired samples with control or Kartogenin treatment. Statistical significance was calculated by paired t-test at 95% confidence level and represented by exact p values. (C) UMAP of p16 ^ink4a expressing clusters in control and Kartogenin samples. Dashed line encircling SnC I cluster represents significant depletion following Kartogenin treatment. (D) UMAP of the median expression of p16 ^ink4a in the combined control and Kartogenin-treated samples (n=6). (E) Mean expression of Runxl and Runx2 in paired samples (n=6) with control or Kartogenin treatment. Statistical significance was calculated by paired t-test at 95% confidence level and represented by adjusted p values. (F) UMAP illustrating Inf-A, Inf-D and Snc CPCIII clusters in control and Kartogenin treated groups.

[0016] FIGS. 6A-6D. Delineating ‘responders’ and ‘non-responders’ to OA drugs using CyTOF analyses (A) Heatmap with hierarchical clustering of OA chondrocytes treated with control, BMS, or Kartogenin based on the expression profile of panel markers in each sample. (B) Heatmap with hierarchical clustering of OA chondrocytes treated with control, BMS or Kartogenin based on the percent abundance of clusters in each sample. (C) Principal Component Analysis (PCA) plot the distribution of patients based on cluster abundance and marker expression profiles in each patient sample with or without drug treatment. (D) Schematic outlining a platform for CyTOF based profiling and analyses to identify responders and non-responders to a drug candidate in patient cohorts.

[0017] FIG. 7. Median expression profile of p16 ^ink4a clusters. Heatmap of the median expression of markers in the four p16 ^ink4a clusters, namely SnC I, SnC II, SnC III, SnC CPC III, and Snc IV in combined DMSO-treated samples. The median intensities were z-scored row-wise (per marker) for standardization.

[0018] FIGS. 8A-8E. Effect of NF-κB pathway inhibition on iNOS and Hif2a expression. Heatmap of the median expression of (A) iNOS and (B) Hif2a in clusters that were significantly different in their expression between DMSO-treated controls and BMS-treated samples. The significant difference in the mean expression of iNOS or Hif2a was calculated by paired t-test at 95% confidence level. Venn diagram representing clusters where the expression of (C) NF-κB and iNOS, (D) NF-κB and Hif2a, and (E) NF-κB, iNOS and Hif2a were commonly downregulated in BMS-treated samples when compared with DMSO-treated controls.

[0019] FIGS. 9A-9D. Effect of drugs on the OA secretory output. OA chondrocytes harvested from the surgical waste of 5 additional patients undergoing total knee replacement surgeries (n = 5) were treated with BMS-345541 (25 μM), kartogenin (25 μM). or DMSO for 48 hours. Spent medium was used for 80-plex autoantibody assay by Luminex. (A and C) Fold change in the raw mean fluorescence intensity (MFI) of all analytes in samples treated with BMS-345541 (A) or kartogenin (C) normalized to respective DMSO-treated samples. Statistical significance was measured by pairwise t test with post hoc Benjamini-Hochberg test. Significantly different (P < 0.05) analytes between drug treatment and DMSO controls are denoted in red, and log adjusted P values are provided (inset). (B and D) Raw MFI values of select SASP-associated analytes (IL-1 α, IL-1 β, IL-6, TNF-α, TNF-β, and VEGF) are represented in paired samples (η= 5) for BMS-345541 (B) and kartogenin (D) treatments.

[0020] FIGS. 10A-10B, FlowSOM identified the OA chondrocyte landscape. (A) UMAP of ail 25 clusters identified by FlowSOM in individual DMSO, BMS-345541 and Kartogenin treated samples (n=6 per treatment). Each UMAP is a representation of 8043 cells. (B) Proportional representation of all clusters as a percent of total cells in each sample across treatment groups.

[0021] FIGS. 11A-11C. Drug effects on OA chondrocyte clusters stratified by SOX9 expression. (A) UMAP of all clusters juxtaposed below with the median expression of SOX9 in the clusters in concatenated (n=6) DMSO, BMS-345541 and Kartogenin treated samples. (B) Frequency of SOX9low, SOX9low-medium, SOX9medium and SOX9high clusters in drug-treated groups. Bar graphs represent mean ± standard deviation. Statistical significance was calculated by repeated measures one-way ANOVA with Bonferroni correction and significant differences were represented by adjusted p values. (C) Clustered dot plot represents the frequency as a percent of total cells in each cluster (dot size) and the median SOX9 expression in each cluster (dot color) in individual samples.

[0022] FIG. 12. Drug treatment effects on the secretory profile of OA chondrocytes. Heatmap represents the raw MFI of 80 analytes (scaled row-wise) measured by multiplex autoantibody assay by Luminex in all DMSO, BMS-345541 and Kartogenin treated samples. Hierarchical clustering of rows as a measure of Euclidean distance was performed to visualize treatment effects on analytes. Analytes that are dampened in BMS-345541 treatment group compared to DMSO or Kartogenin treatment groups cluster together and are indicated by rectangle box in red.

DETAILED DESCRIPTION

[0023] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0024] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0026] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0027] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0028] Cartilage is a hyperhydrated structure with water comprising 70% to 80% of its weight. The remaining 20% to 30% comprises type II collagen and proteoglycan. Collagen usually accounts for 70% of the dry weight of cartilage. Proteoglycans are composed of a central protein core from which long chains of polysaccharides extend. These polysaccharides, called glycosaminoglycans, include: chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate. Cartilage has a characteristic structural organization consisting of chondrocytes dispersed within an endogenously produced and secreted extracellular matrix. The cavities in the matrix which contain the chondrocytes are called cartilage lacunae. Unlike bone, cartilage is neither innervated nor penetrated by either the vascular or lymphatic systems.

[0029] Three types of cartilage are present in mammals and include: hyaline cartilage; fibrocartilage and elastic cartilage. Hyaline cartilage consists of a gristly mass having a firm, elastic consistency, is translucent and is pearly blue in color. Hyaline cartilage is predominantly found on the articulating surfaces of articulating joints. It is found also in epiphyseal plates, costal cartilage, tracheal cartilage, bronchial cartilage and nasal cartilage. Fibrocartilage is essentially the same as hyaline cartilage except that it contains fibrils of type I collagen that add tensile strength to the cartilage. The collagenous fibers are arranged in bundles, with the cartilage cells located between the bundles. Fibrocartilage is found commonly in the annulus fibrosis of the invertebral disc, tendinous and ligamentous insertions, menisci, the symphysis pubis, and insertions of joint capsules. Elastic cartilage also is similar to hyaline cartilage except that it contains fibers of elastin. It is more opaque than hyaline cartilage and is more flexible and pliant. These characteristics are defined in part by the elastic fibers embedded in the cartilage matrix. Typically, elastic cartilage is present in the pinna of the ears, the epiglottis, and the larynx.

[0030] The surfaces of articulating bones in mammalian joints are covered with articular cartilage. The articular cartilage prevents direct contact of the opposing bone surfaces and permits the near frictionless movement of the articulating bones relative to one another. Two types of articular cartilage defects are commonly observed in mammals and include full-thickness and partial-thickness defects. The two-types of defects differ not only in the extent of physical damage but also in the nature of repair response each type of lesion elicits.

[0031] Full-thickness articular cartilage defects include damage to the articular cartilage, the underlying subchondral bone tissue, and the calcified layer of cartilage located between the articular cartilage and the subchondral bone. Full-thickness defects typically arise during severe trauma of the joint or during the late stages of degenerative joint diseases, for example, during osteoarthritis. Since the subchondral bone tissue is both innervated and vascularized, damage to this tissue is often painful. The repair reaction induced by damage to the subchondral bone usually results in the formation of fibrocartilage at the site of the full-thickness defect. Fibrocartilage, however, lacks the biomechanical properties of articular cartilage and fails to persist in the joint on a long term basis.

[0032] Partial-thickness articular cartilage defects are restricted to the cartilage tissue itself. These defects usually include fissures or clefts in the articulating surface of the cartilage. Partialthickness defects are caused by mechanical arrangements of the joint which in turn induce wearing of the cartilage tissue within the joint. In the absence of innervation and vasculature, partial-thickness defects do not elicit repair responses and therefore tend not to heal. Although painless, partial-thickness defects often degenerate into full-thickness defects.

[0033] Osteoarthritis (OA). OA affects nearly 27 million people in the United States, accounting for 25% of visits to primary care physicians, and half of all prescriptions for non-steroidal antiinflammatory drugs (NSAIDs). It is a chronic arthropathy characterized by disruption and potential loss of joint cartilage along with other joint changes, including bone remodeling such as bone hypertrophy (osteophyte formation), subchondral sclerosis, and formation of subchondral cysts. OA results in the degradation of joints, including degradation of articular cartilage and subchondral bone, resulting in mechanical abnormalities and joint dysfunction. Symptoms may include joint pain, tenderness, stiffness, sometimes an effusion, and impaired joint function. A variety of causes can initiate processes leading to loss of cartilage in OA.

[0034] OA may begin with joint damage caused by trauma to the joint; mechanical injury to the meniscus, articular cartilage, a joint ligament, or other joint structure; defects in cartilage matrix components; and the like. Mechanical stress on joints may underlie the development of OA in many individuals, with the sources of such mechanical stress being many and varied, including misalignment of bones as a result of congenital or pathogenic causes; mechanical injury; overweight; loss of strength in muscles supporting joints; and impairment of peripheral nerves, leading to sudden or dyscoordinated movements that overstress joints.

[0035] In synovial joints there are at least two movable bony surfaces that are surrounded by the synovial membrane, which secretes synovial fluid, a transparent alkaline viscid fluid that fills the joint cavity, and articular cartilage, which is interposed between the articulating bony surfaces. The earliest gross pathologic finding in OA is softening of the articular cartilage in habitually loaded areas of the joint surface. This softening or swelling of the articular cartilage is frequently accompanied by loss of proteoglycans from the cartilage matrix. As OA progresses, the integrity of the cartilage surface is lost and the articular cartilage thins, with vertical clefts extending into the depth of the cartilage in a process called fibrillation. Joint motion may cause fibrillated cartilage to shed segments and thereby expose the bone underneath (subchondral bone). In OA, the subchondral bone is remodeled, featuring subchondral sclerosis, subchondral cycts, and ectopic bone comprising osteophytes. The osteophytes (bone spurs) form at the joint margins, and the subchondral cysts may be filled with synovial fluid. The remodeling of subchondral bone increases the mechanical strain and stresses on both the overlying articular cartilage and the subchondral bone, leading to further damage of both the cartilage and subchondral bone.

[0036] The tissue damage stimulates chondrocytes to attempt repair by increasing their production of proteoglycans and collagen. However, efforts at repair also stimulate the enzymes that degrade cartilage, as well as inflammatory cytokines, which are normally present in only small amounts. Inflammatory mediators trigger an inflammatory cycle that further stimulates the chondrocytes and synovial lining cells, eventually breaking down the cartilage. Chondrocytes undergo programmed cell death (apoptosis) in OA joints.

[0037] OA is characterized by low-grade infiltration of inflammatory cells, primarily macrophages, but also B cells and T cells. These cells, again primarily macrophages, are capable of producing inflammatory cytokines and matrix metalloproteases (MMPs) in the OA joint. However, when stimulated by inflammatory cytokines, such as IL-1 and TNF, tissue-resident cells within the joint, including synovial fibroblasts and chondrocytes, can produce additional inflammatory cytokines, including IL-6, as well as multiple MMPs.

[0038] OA should be suspected in patients with gradual onset of joint symptoms and signs, particularly in older adults, usually beginning with one or a few joints. Pain can be the earliest symptom, sometimes described as a deep ache. Pain is usually worsened by weight bearing and relieved by rest but can eventually become constant. Joint stiffness in OA is associated with awakening or inactivity. If OA is suspected, plain x-rays should be taken of the most symptomatic joints. X-rays generally reveal marginal osteophytes, narrowing of the joint space, increased density of the subchondral bone, subchondral cyst formation, bony remodeling, and joint effusions. Standing x-rays of knees are more sensitive in detecting joint-space narrowing. Magnetic resonance imaging (MRI) can be used to detect cartilage degeneration, and several MRI-based based scoring systems exist for characterizing the severity of OA (Hunter et al, PM R. 2012 May;4(5 Suppl):S68-74).

[0039] OA commonly affects the hands, feet, spine, and the large weight-bearing joints, such as the hips and knees, although in theory any joint in the body can be affected. As OA progresses, the affected joints appear larger, are stiff and painful, and usually feel better with gentle use but worse with excessive or prolonged use. Treatment generally involves a combination of exercise, lifestyle modification, and analgesics. If pain becomes debilitating, joint-replacement surgery may be used to improve quality of life.

[0040] In addition to affecting humans, OA and joint degeneration also frequently impacts animals, including dogs, cats, horses, and other animals in which it can causes significant joint pain and dysfunction. Osteoarthritis (OA) is the most common form of arthritis in dogs, affecting approximately a quarter of the population. It is a chronic joint disease characterized by loss of joint cartilage, thickening of the joint capsule and new bone formation around the joint (osteophytosis) and ultimately leading to pain and limb dysfunction. The majority of OA in dogs occur secondarily to developmental orthopedic disease, such as cranial cruciate ligament disease, hip dysplasia, elbow dysplasia, OCD, patella (knee cap) dislocation. In a small subset of dogs, OA occurs with no obvious primary causes and can be related to genetic and age. Other contributing factors to OA in dogs include body weight, obesity, gender, exercise, and diet.

[0041] Among the agents proposed to modify disease in OA, such as doxycycline (presumably through its ability to inhibit MMPs), bisphosphonates (presumably through their ability to inhibit osteoclast activation), and licofelone (presumably through its ability to inhibit the cyclooxygenase and lipoxegenase pathways), none have been shown to afford robust chondroprotection as defined by slowing of cartilage breakdown. Among the agents that have demonstrated partial efficacy in controlling OA-associated pain are analgesics such as acetaminophen and antiinflammatories such as NSAIDs, opiates, intra-articular corticosteroids, and hyaluronic acid derivatives injected into the joint. These agents have not been demonstrated to prevent cartilage loss or slow the loss of joint function.

[0042] Established or advanced OA can be defined radiographically as KL grade >=3 or as MRI evidence of extensive, complete, or near-complete loss of articular cartilage. Other evidence of joint failure can be determined by direct or arthroscopic visualization of extensive, complete, or near-complete loss of joint space or cartilage, by biomechanical assessment of inability to maintain functional joint integrity, or by clinical assessment of joint failure, as evidenced by inability to perform full range of motion or to maintain normal joint function. Patients with advanced OA frequently experience joint pain. On physical examination, patients with advanced OA can have bony enlargement, small effusions, crepitus, and malalignment of the synovial joints. Examples of semiquantitative MRI scoring systems that can be used to classify the severity of OA include: WORMS (Whole-Organ Magnetic Resonance Imaging Score; Peterfy CG, et al. Osteoarthritis Cartilage 2004;12:177-190); KOSS (Knee Osteoarthritis Scoring System; Kornaat PR, et al. Skeletal Radiol 2005;34:95-102); BLOKS (Boston Leeds Osteoarthritis Knee Score; Hunter DJ, et al. Ann Rheum Dis 2008;67:206-211 ); MOAKS (MRI Osteoarthritis Knee Score; Hunter DJ, et al. Osteoarthritis Cartilage. 2011 ;19(8) :990- 1002); HOAMS (Hip Osteoarthritis MRI Score; Roemer FW, et al. Osteoarthritis Cartilage. 2011 ;19(8):946-62); OHOA (Oslo Hand Osteoarthritis MRI Score). Advanced OA can result in significant joint pain and loss of mobility owing to joint dysfunction.

[0043] Mass cytometry. Elemental mass spectrometry-based flow cytometry (mass cytometry) is a method to characterize single cells or particles with elemental metal isotope-labeled binding reagents. Because there are many stable metal isotopes available, and little overlap between measurement channels, dozens of molecules (parameters) can be readily measured. An example of a mass cytometer used to read the metal tags is an inductively-coupled plasma mass spectrometer (ICP-MS). In a typical workflow (similar to fluorescence based cytometry), cells are first incubated with antibodies/affinity binders conjugated to pure isotopes and subsequently the cell suspension is injected as a single cell stream into the mass cytometer. Single cell droplets are generated via nebulization and are carried by an argon gas stream into a .about.7500 degrees Kelvin plasma where each single cell is completely atomized and ionized. Thereby generated metal ions are then directed into a time-of-flight (TOF) mass spectrometer and the mass over charge ratio and number of metal ions is measured per cell and thereby the abundance of the target epitope/molecules.

[0044] As used herein, the term "elemental analysis" refers to a method by which the presence and/or abundance of elements of a sample are evaluated. "Capacitively coupled plasma" (CCP) means a source of ionization in which a plasma is established by capacitive coupling of radiofrequency energy at atmospheric pressure or at a reduced pressure (typically between 1 and 500 Torr) in a graphite or quartz tube.

[0045] "Mass spectrometer" means an instrument for producing ions in a gas and analyzing them according to their mass/charge ratio. "Microwave induced plasma" (MIP) means a source of atomization and ionization in which a plasma is established in an inert gas (typically nitrogen, argon or helium) by the coupling of microwave energy. The frequency of excitation force is in the GHz range. "Glow discharge" (GD) means a source of ionization in which a discharge is established in a low pressure gas (typically between 0.01 and 10 Torr), typically argon, nitrogen or air, by a direct current (or less commonly radiofrequency) potential between electrodes. "Graphite furnace" means a spectrometer system that includes a vaporization and atomization source comprised of a heated graphite tube. Spectroscopic detection of elements within the furnace may be performed by optical absorption or emission, or the sample may be transported from the furnace to a plasma source (e.g. inductively coupled plasma) for excitation and determination by optical or mass spectrometry.

[0046] In some embodiments the methods utilize ICP-MS. In some embodiments the ICP-MS is performed with solution analysis, for example ELAN DRC II, Perkin-Elmer. In other embodiments the analysis is performed with a mass cytometer (e.g. CyTOF, DVS Sciences), which uses a nebulizer to administer a suspension of cells, beads, or other particles in a single-particle stream to an ICP-MS chamber, thereby yielding single particle/cell data similar to a flow cytometer. Alternatively the analysis is performed by an elemental analysis-driven imaging system (e.g. laser ablation ICP-MS). Devices for such analytic methods are known in the art.

[0047] The term "flow cytometry" as used herein refers to a method and a process whereby cells within a sample can be detected and identified when transversing past a detector within an apparatus containing a detecting source and a flowing apparatus.

[0048] An active agent can be administered by any suitable means, including topical, oral, parenteral, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration. An agent can be administered in any manner which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. Sustained release administration is also specifically included in the disclosure, by such means as depot injections or erodible implants.

[0049] As noted above, an agent can be formulated with an a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application). A suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art. An "effective amount" refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.

[0050] An agent can be administered as a pharmaceutical composition comprising a pharmaceutically acceptable excipient. The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0051] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem and Argonaut Technology.

[0052] Compounds useful for co-administration with the active agents of the invention can also be made by methods known to one of ordinary skill in the art. As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatises that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C. may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.

[0053] The active agents of the invention and/or the compounds administered therewith are incorporated into a variety of formulations for therapeutic administration. In one aspect, the agents are formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and are formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the active agents and/or other compounds can be achieved in various ways, usually by oral administration. The active agents and/or other compounds may be systemic after administration or may be localized by virtue of the formulation, or by the use of an implant that acts to retain the active dose at the site of implantation.

[0054] In pharmaceutical dosage forms, the active agents and/or other compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds. The agents may be combined, as previously described, to provide a cocktail of activities. The following methods and excipients are exemplary and are not to be construed as limiting the invention.

[0055] For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0056] Formulations are typically provided in a unit dosage form, where the term "unit dosage form," refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular complex employed and the effect to be achieved, and the pharmacodynamics associated with each complex in the host.

[0057] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are commercially available. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available. Any compound useful in the methods and compositions of the invention can be provided as a pharmaceutically acceptable base addition salt. "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0058] Depending on the patient and condition being treated and on the administration route, the active agent may be administered in dosages of 0.01 mg to 500 mg /kg body weight per day, e.g. about 20 mg/day for an average person. Dosages will be appropriately adjusted for pediatric formulation.

[0059] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0060] Compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97- 119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0061] Toxicity of the active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in further optimizing and/or defining a therapeutic dosage range and/or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

[0062] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0063] The term "sequence identity," as used herein in reference to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. When a subunit position in both of the molecules is occupied by the same monomeric subunit (e.g., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, the sequences are aligned so that the highest order match is obtained. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990).

[0064] By "protein variant" or "variant protein" or "variant polypeptide" herein is meant a protein that differs from a wild-type protein by virtue of at least one amino acid modification. The parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or may be a modified version of a WT polypeptide. Variant polypeptide may refer to the polypeptide itself, a composition comprising the polypeptide, or the amino sequence that encodes it. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g. from about one to about ten amino acid modifications, and preferably from about one to about five amino acid modifications compared to the parent.

[0065] By "parent polypeptide", "parent protein", "precursor polypeptide", or "precursor protein" as used herein is meant an unmodified polypeptide that is subsequently modified to generate a variant. A parent polypeptide may be a wild-type (or native) polypeptide, or a variant or engineered version of a wild-type polypeptide. Parent polypeptide may refer to the polypeptide itself, compositions that comprise the parent polypeptide, or the amino acid sequence that encodes it.

[0066] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma- carboxyglutamate, and O-phosphoserine. “Amino acid analogs” refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0067] Amino acid modifications disclosed herein may include amino acid substitutions, deletions and insertions, particularly amino acid substitutions. Variant proteins may also include conservative modifications and substitutions at other positions of the cytokine and/or receptor (e.g., positions other than those involved in the affinity engineering). Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978), and by Argos in EMBO J., 8:779-785 (1989). For example, amino acids belonging to one of the following groups represent conservative changes: Group I: Ala, Pro, Gly, Gin, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Vai, lie, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu. Further, amino acid substitutions with a designated amino acid may be replaced with a conservative change.

[0068] The term “isolated” refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term refers to preparations where the isolated protein is sufficiently pure to be administered as a therapeutic composition, or at least 70% to 80% (w/w) pure, more preferably, at least 80%-90% (w/w) pure, even more preferably, 90-95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure. A “separated” compound refers to a compound that is removed from at least 90% of at least one component of a sample from which the compound was obtained. Any compound described herein can be provided as an isolated or separated compound.

[0069] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and/or being treated. In some embodiments, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having a disease. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, etc.

[0070] The term “sample” with reference to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as diseased cells. The definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. A “biological sample” includes a sample obtained from a patient’s diseased cell, e.g., a sample comprising polynucleotides and/or polypeptides that is obtained from a patient’s diseased cell (e.g., a cell lysate or other cell extract comprising polynucleotides and/or polypeptides); and a sample comprising diseased cells from a patient. A biological sample comprising a diseased cell from a patient can also include non-diseased cells.

[0071 ] Cells for use in the methods as described above may be collected from a cartilage sample from a subject or a donor, and may optionally may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation. An appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.

[0072] The collected and optionally enriched cell population may be used immediately or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0073] Cells can be obtained from cartilage tissue by digestion to release individual chondrocytes, which can be expanded and manipulated in culture, e.g. by contacting with a candidate or known agent for treating osteoarthritis. These cells are then collected and fixed in preparation for cyTOF, followed by standard staining and analysis protocols, see, for example, Grandi et al. (2021 ) Bio Protoc. 11 (14): e4086, herein specifically incorporated by reference. Cartilage is shaved off the bone using a scalpel, and 1 -2 mm cartilage pieces placed in standard tissue culture treated plates in complete media overnight. The sample is digested overnight, e.g. with collagenase. The cells are then collected and washed. Cells can be maintained in culture for several months, preferably at high densities. [0074] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition in a subject, individual, or patient.

[0075] The term “prognosis” is used herein to refer to the prediction of the likelihood of death or disease progression, including recurrence, spread, and drug resistance, in a subject, individual, or patient. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning, the likelihood of a subject, individual, or patient experiencing a particular event or clinical outcome. In one example, a physician may attempt to predict the likelihood that a patient will survive. Prognosis can also refer to identifying whether an individual is a responder or non-responder to a candidate agent.

[0076] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect on or in a subject, individual, or patient. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of effecting a partial or complete cure for a disease and/or symptoms of the disease. “Treatment,” as used herein, may include treatment of OA in a mammal, particularly in a human, and includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease or its symptoms, i.e., causing regression of the disease or its symptoms.

[0077] Treating may refer to any indicia of success in the treatment or amelioration or prevention of a disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of an agent to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with disease or other diseases. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0078] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., to delay or minimize the growth and spread of oesteoarthritis. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease. [0079] As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

Methods

[0080] The methods of the invention are to provide an analysis of the response of chondrocytes to an agent of interest, which may be a candidate drug for treatment of a disease of cartilage, a known drug, where the cells are tested for responsiveness, and the like. The cells are profiled for the presence and level of analytes, e.g. by mass cytometry. As used herein, analytes refers to quantifiable components of cells or biological material, particularly components that can be accurately measured. An analyte can be any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. Proteins are of particular interest. Some variability may be expected and a range of values may be obtained using standard statistical methods with a common statistical method used to provide single values.

[0081 ] Individuals may be monitored for treatment and/or selected for therapy by determining the phenotype of the chondrocytes, e.g. the expression levels of proteins selected from at least 5, at least 10, at least 15, at least 20, at least 25 markers, and may comprise 30 markers, or more selected from SOX9; CD24; Idu; Ki67; Hif2a; PNFκB(S29) ; iNOS; CD126; CD121 B; CD121A; CD120B; P-SAPK/JNK (T183/Y185); p-SMAD1/5(S463/465); pStat3(pY705); TET1 ; SOD2; Runxl ; Runx2; CD106; CD73; Notch-1 ; Stro-1 ; CD171 ; CD105; CD33; CD49E; CD146; TLR4; TLR2; and p16ink4a. In some embodiments the cells are clustered into discrete subpopulations based on the presence and level of expression of the plurality of markers, including the clusters as shown in Table 2, where the number of discrete clusters may be at least 5, at least 10, at least 15, at least 20, at least 25 or more. Analysis may include, without limitation, changes in a cell sub-population size, changes in expression of markers of interest, changes in phosphorylation of markers, and the like. In some embodiments, cells expressing low levels of SOX9 are included in the clustering analysis. In some embodiments, expression of p16ink4a is included in the clustering analysis.

[0082] In some embodiments a specific change in a chondrocyte population, e.g. percentage of cells in the population, growth, viability, protein expression, protein phosphorylation, etc. is correlated with response to an agent. Assessment responsiveness allows improved care by indicating where therapeutic action may be required, at an early stage of disease. Where an individual is analyzed and found to be responsive to a therapy, treatment may be continued, or the patient allowed to recover following a suitable response. Where an individual is treated and found to not be responsive, an alternative therapy is administered.

[0083] In some embodiments an individual is assessed for responsiveness to a therapeutic agent by culturing a population of chondrocytes with the agent. Following a period of time sufficient for response, e.g. at 12 hours, at least 18 hours, at least 24 hours, up to 3 weeks or more, up to 2 weeks, up to 10 days, up to 7 days, up to 5 days, up to 3 days, up to 48 hours, up to 24 hours, the cell population can be analyzed, or frozen for future analysis.

[0084] Multiple samples may be obtained and analyzed from an individual over time. Multiple samples may also be obtained and analyzed over a patient cohort group, for example in the context of clinical trials.

[0085] Analytes of interest include cytoplasmic, cell surface or secreted biomolecules, frequently biopolymers, e.g. polypeptides, polysaccharides, polynucleotides, lipids, etc. In some embodiments, analytes include specific epitopes. Epitopes are frequently identified using specific monoclonal antibodies or receptor probes. In some cases the molecular entities comprising the epitope are from two or more substances and comprise a defined structure; examples include combinatorially determined epitopes associated with heterodimeric proteins. An analyte may be detection of a specifically modified protein or oligosaccharide, e.g. a phosphorylated protein, such as a STAT transcriptional protein; or sulfated oligosaccharide, or such as the carbohydrate structure Sialyl Lewis x, a selectin ligand. The presence of the active conformation of a receptor may comprise one analyte while an inactive conformation of a receptor may comprise another, e.g. the active and inactive forms of heterodimeric integrin.

[0086] Analytes of interest include polypeptides, and the epitope that is being quantitated by a primary amino acid epitope, an epitope formed by protein secondary or tertiary structure, an epitope formed by two or more interacting polypeptides, or an epitope that results from posttranslational modification of a polypeptide.

[0087] The term "specific binding member" as used herein refers to a member of a specific binding pair, i.e. two molecules, usually two different molecules, where one of the molecules through chemical or physical means specifically binds to the other molecule. For the purposes of the present invention, one of the molecules is an analyte as defined above, and generally the specific binding member is labeled for detection of fluorescence or elemental analysis, as known in the art.

[0088] The complementary members of a specific binding pair are sometimes referred to as a ligand and receptor; or receptor and counter-receptor. Specific binding indicates that the agent can distinguish a target antigen, or epitope within it, from other non-target antigens. It is specific in the sense that it can be used to detect a target antigen above background noise ("non-specific binding"). For example, a specific binding partner can detect a specific sequence or a topological conformation. A specific sequence can be a defined order of amino acids or a defined chemical moiety (e.g., where an antibody recognizes a phosphotyrosine or a particular carbohydrate configuration, etc.) which occurs in the target antigen. The term "antigen" is issued broadly, to indicate any agent which elicits an immune response in the body. An antigen can have one or more epitopes.

[0089] Binding pairs of interest include antigen and antibody specific binding pairs, complementary nucleic acids, peptide-MHC-antigen complexes and T cell receptor pairs, biotin and avidin or streptavidin; carbohydrates and lectins; complementary nucleotide sequences; peptide ligands and receptor; effector and receptor molecules; hormones and hormone binding protein; enzyme cofactors and enzymes; enzyme inhibitors and enzymes; and the like. The specific binding pairs may include analogs, derivatives and fragments of the original specific binding member. For example, an antibody directed to a protein antigen may also recognize peptide fragments, chemically synthesized peptidomimetics, labeled protein, derivatized protein, etc. so long as an epitope is present.

[0090] Immunological specific binding pairs include antigens and antigen specific antibodies; and T cell antigen receptors, and their cognate MHC-peptide conjugates. Suitable antigens may be haptens, proteins, peptides, carbohydrates, etc. Recombinant DNA methods or peptide synthesis may be used to produce chimeric, truncated, or single chain analogs of either member of the binding pair, where chimeric proteins may provide mixture(s) or fragment(s) thereof, or a mixture of an antibody and other specific binding members. Antibodies and T cell receptors may be monoclonal or polyclonal, and may be produced by transgenic animals, immunized animals, immortalized human or animal B-cells, cells transfected with DNA vectors encoding the antibody or T cell receptor, etc. The details of the preparation of antibodies and their suitability for use as specific binding members are well-known to those skilled in the art.

[0091 ] A nucleic acid based binding partner such as an oligonucleotide can be used to recognize and bind DNA or RNA based analytes. The term "polynucleotide" as used herein may refer to peptide nucleic acids, locked nucleic acids, modified nucleic acids, and the like as known in the art. The polynucleotide can be DNA, RNA, LNA or PNA, although it is not so limited. It can also be a combination of one or more of these elements and/or can comprise other nucleic acid mimics. [0092] Binding partners can be primary or secondary. Primary binding partners are those bound to the analyte of interest. Secondary binding partners are those that bind to the primary binding partner.

[0093] In one embodiment analysis is performed on a mass cytometer, in which cells are introduced into a fluidic system and introduced into the mass cytometer one cell at a time. In one embodiment, cells are carried in a liquid suspension and sprayed into a plasma source by means of a nebulizer. In another embodiment, the cells may be hydrodynamically focused one cell at a time through a flow cell using a sheath fluid. In particular embodiments, the cells may be compartmentalized in the flow cell by introduction of an immiscible barrier, e.g., using a gas (e.g., air or nitrogen) or oil, such that the cell is physically separated from other cells that are passing through the flow cell. The cells may be compartmentalized prior to or during introduction of the cell into the flow cell by introducing an immiscible material (e.g., air or oil) into the flow path.

[0094] The general principles of mass cytometry, including methods by which single cell suspensions can be made, methods by which cells can be labeled, methods for atomizing particles and methods for performing elemental analysis on particles, as well as hardware that can be employed in mass cytometry, including flow cells, ionization chambers, reagents, mass spectrometers and computer control systems are known and are reviewed in a variety of publications including, but not limited to Bandura et al Analytical Chemistry 2009 81 6813-6822), Tanner et al (Pure Appl. Chem 2008 80: 2627-2641 ), U.S. Pat. No. 7,479,630 (Method and apparatus for flow cytometry linked with elemental analysis) and U.S. Pat. No. 7,135,296 (Elemental analysis of tagged biologically active materials); and published U.S. patent application 20080046194, for example, which publications are incorporated by reference herein for disclosure of those methods and hardware.

[0095] The results of such analysis may be compared to results obtained from reference compounds and cells, positive controls, concentration curves, controls, etc. The comparison of results is accomplished by the use of suitable deduction protocols, Al systems, statistical comparisons, etc.

[0096] In particular embodiments, the method described above may be employed in a multiplex assay in which a heterogeneous population of cells is labeled with a plurality of distinguishably labeled binding agents (e.g., a number of different antibodies). After the population of cells is labeled, the cells are introduced into the flow cell, and individually analyzed using the method described above, where the viable cells are distinguished from non-viable cells by the presence of platinum derived from the viability reagent.

[0097] The analyte distribution pattern may be generated from a cell sample using any convenient protocol. The readout may be a mean, average, median or the variance or other statistically or mathematically-derived value associated with the measurement. The readout information may be further refined by direct comparison with the corresponding reference or control pattern. A pattern may be evaluated on a number of points: to determine if there is a statistically significant change at any point in the data matrix; whether the change is an increase or decrease in prevalence of an isoform; and the like. The absolute values will display a variability that is inherent in live biological systems.

[0098] In some embodiments the cells that are analyzed by mass cytometry are clustered into discrete subpopulations based on the presence and level of expression of the plurality of markers. Clustering allows for assignment of cells into different groups, and provides a means of determining how the population of cells changes in response to the presence of a candidate agent.

[0099] In some embodiments the clustering step utilizes an algorithm for hierarchical clustering. Clustering algorithms group similar objects such that the objects in the same group are more similar to each other than the objects in the other groups. The group of similar objects is called a cluster. In some embodiments the analysis is hierarchical cluster analysis or HCA, which is an unsupervised clustering algorithm that involves creating clusters that have predominant ordering from top to bottom. The algorithm groups similar objects, where the endpoint is a set of clusters or groups, where each cluster is distinct from each other cluster, and the objects within each cluster are broadly similar to each other. This clustering technique is divided into two types: Agglomerative Hierarchical Clustering; and Divisive Hierarchical Clustering. The Agglomerative Hierarchical Clustering is the most common type of hierarchical clustering used to group objects in clusters based on their similarity. It's a “bottom-up” approach: each observation starts in its own cluster, and pairs of clusters are merged as one moves up the hierarchy. There are several ways to measure the distance between clusters in order to decide the rules for clustering, and they are often called Linkage Methods. Some of the common linkage methods are: Complete- linkage: the distance between two clusters is defined as the longest distance between two points in each cluster; and Single-linkage: the distance between two clusters is defined as the shortest distance between two points in each cluster. This linkage may be used to detect high values in your dataset which may be outliers as they will be merged at the end. Average-linkage: the distance between two clusters is defined as the average distance between each point in one cluster to every point in the other cluster. Centroid-linkage: finds the centroid of cluster 1 and centroid of cluster 2, and then calculates the distance between the two before merging.

[00100] Clusters can be sorted into a dendrogram, which is a type of tree diagram showing hierarchical relationships between different sets of data. A dendrogram contains the memory of hierarchical clustering algorithm.

[00101] Divisive or DIANA (Divisive ANAIysis Clustering) is a top-down clustering method where all of the observations are assigned to a single cluster and then partitioned to two least similar clusters, proceeding recursively on each cluster until there is one cluster for each observation.

[00102] Commercially available software packages can be used for the hierarchical clustering, e.g. FlowSOM, UMAP, etc. FlowSOM is an algorithm that speeds time to analysis and quality of clustering with Self-Organizing Maps (SOMs) that can reveal how all markers are behaving on all cells, and can detect subsets that might otherwise be missed. It clusters cells (or other observations) based on chosen clustering channels (or markers/features), generates a SOM of clusters, produces a Minimum Spanning Tree (MST) of the clusters, and assigns each cluster to a metacluster, effectively grouping them into a population. The FlowSOM algorithm outputs SOMs and MSTs showing population abundances and marker expression in various formats including pie charts, star plots, and channel-colored plots. See, for example, Van Gassen et al. (2015), FlowSOM: Using self-organizing maps for visualization and interpretation of cytometry data.

[00103] In some embodiments the analysis is performed with UMAP. UMAP is an algorithm for dimension reduction based on manifold learning techniques and ideas from topological data analysis. UMAP uses simplicial complexes as a means to construct topological spaces out of simple combinatorial components. This allows one to reduce the complexities of dealing with the continuous geometry of topological spaces to the task of relatively simple combinatorics and counting. The algorithm constructs a simplicial complex by constructing a fuzzy topological representation, then optimizing the low dimensional representation to have as close a fuzzy topological representation as possible as measured by cross entropy.

[00104] In certain embodiments, the obtained clustering pattern is compared to a single reference/control profile to obtain information regarding the phenotype of the cells being assayed. In yet other embodiments, the obtained protein distribution pattern is compared to two or more different reference/control profiles to obtain more in depth information regarding the phenotype of the cells. For example, the obtained protein distribution pattern may be compared to a positive and negative reference profile to obtain confirmed information regarding whether a cell has the phenotype of interest.

[00105] An algorithm that will discriminate robustly between groups of cells in different classifications with respect to responsiveness to therapy, and controls for confounding variables and evaluating potential interactions may be used for identifying candidate drugs.

[00106] The protein expression pattern is determined by the methods described above. The quantitative data thus obtained is can be subjected to an analytic classification process. In such a process, the raw data is manipulated according to an algorithm, where the algorithm has been pre-defined by a training set of data, for example as described in the examples provided herein. An algorithm may utilize the training set of data provided herein, or may utilize the guidelines provided herein to generate an algorithm with a different set of data.

[00107] Classification can be made according to predictive modeling methods that set a threshold for determining the probability that a sample belongs to a given class, i.e. responsive, non- responsive, etc. The probability preferably is at least 50%, or at least 60% or at least 70% or at least 80% or higher. Classifications also may be made by determining whether a comparison between an obtained protein distribution pattern and a reference protein distribution pattern yields a statistically significant difference. If such a comparison is not statistically significantly different from the reference protein distribution pattern, then the sample from which the protein distribution pattern was obtained is classified as belonging to the reference protein distribution pattern class.

[00108] The predictive ability of a model may be evaluated according to its ability to provide a quality metric, e.g. AUG or accuracy, of a particular value, or range of values. In some embodiments, a desired quality threshold is a predictive model that will classify a sample with an accuracy of at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95, or higher. As an alternative measure, a desired quality threshold may refer to a predictive model that will classify a sample with an AUG (area under the curve) of at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, or higher.

[00109] As is known in the art, the relative sensitivity and specificity of a predictive model can be “tuned” to favor either the selectivity metric or the sensitivity metric, where the two metrics have an inverse relationship. The limits in a model as described above can be adjusted to provide a selected sensitivity or specificity level, depending on the particular requirements of the test being performed. One or both of sensitivity and specificity may be at least about at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, or higher.

[00110] The raw data may be initially analyzed by measuring the values for each marker, usually in triplicate or in multiple triplicates. The data may be manipulated, for example, raw data may be transformed using standard curves, and the average of triplicate measurements used to calculate the average and standard deviation for each patient. These values may be transformed before being used in the models, e.g. log-transformed, Box-Cox transformed (see Box and Cox (1964) J. Royal Stat. Soc., Series B, 26:211 —246), etc. The data are then input into a predictive model, which will classify the sample according to the state. The resulting information may be transmitted to a patient or health professional.

[00111] Candidate agents of interest are biologically active agents that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. An important aspect of the invention is to evaluate candidate drugs, select therapeutic antibodies and protein-based therapeutics, with preferred biological response functions. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. [00112] Included are pharmacologically active drugs, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. Exemplary of pharmaceutical agents suitable for this invention are those described in, "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous System; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference. Also included are toxins, and biological and chemical warfare agents, for example see Somani, S. M. (Ed.), "Chemical Warfare Agents," Academic Press, New York, 1992).

[00113] Candidate agents include all of the classes of molecules described above, and may further comprise samples of unknown content. Of interest are complex mixtures of naturally occurring compounds derived from natural sources such as plants. While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed. Samples of interest include environmental samples, e.g. ground water, sea water, mining waste, etc.; biological samples, e.g. lysates prepared from crops, tissue samples, etc.; manufacturing samples, e.g. time course during preparation of pharmaceuticals; as well as libraries of compounds prepared for analysis; and the like. Samples of interest include compounds being assessed for potential therapeutic value, i.e. drug candidates.

[00114] The term samples also includes the fluids described above to which additional components have been added, for example components that affect the ionic strength, pH, total protein concentration, etc. In addition, the samples may be treated to achieve at least partial fractionation or concentration. Biological samples may be stored if care is taken to reduce degradation of the compound, e.g. under nitrogen, frozen, or a combination thereof. The volume of sample used is sufficient to allow for measurable detection, usually from about 0.1 .mu.I to 1 ml of a biological sample is sufficient.

[00115] Compounds, including candidate agents, are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[00116] Agents are screened for biological activity by adding the agent to the chondrocyte cultures disclosed herein. The change in parameter readout in response to the agent is measured, desirably normalized, and clustered.

[00117] The agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.

[00118] Preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself.

[00119] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.

[00120] Various methods can be utilized for quantifying the presence of the selected markers, particularly mass cytometry, as disclosed herein. Mass cytometry may be used to quantitate parameters such as the presence of cell surface proteins or conformational or posttranslational modification thereof; intracellular or secreted protein, where permeabilization allows antibody (or probe) access, and the like. The readouts of selected parameters are capable of being read simultaneously, or in sequence during a single analysis, as for example through the use of itotope labeled antibodies to proteins.

[00121 ] Also provided are databases and computer systems for analysis. Databases can typically comprise distribution pattern information from various conditions, such as responses of cells to a variety of treatments. The results and databases thereof may be provided in a variety of media to facilitate their use.

[00122] "Media" can refer to a manufacture that contains the distribution pattern information; and methods of analysis as described above. The databases and comparative algorithms can be recorded on computer readable media, e.g. any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD- ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. One of skill in the art can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising a recording of the present database information. "Recorded" refers to a process for storing information on computer readable medium, using any such methods as known in the art. Any convenient data storage structure may be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g. word processing text file, database format, etc.

[00123] As used herein, "a computer-based system" refers to the hardware means, software means, and data storage means used to analyze the information provided herein. The minimum hardware of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in analysis. The data storage means may comprise any manufacture comprising a recording of the present information as described above, or a memory access means that can access such a manufacture.

[00124] A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test expression repertoire.

[00125] The data analysis may be implemented in hardware or software, or a combination of both. In one embodiment, a machine-readable storage medium is provided, the medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a any of the datasets and data comparisons of this invention. Such data may be used for a variety of purposes, such as drug discovery, analysis of interactions between cellular components, and the like. In some embodiments, the analysis is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device. Program code is applied to input data to perform the functions described above and generate output information. The output information is applied to one or more output devices, in known fashion. The computer may be, for example, a personal computer, microcomputer, or workstation of conventional design.

[00126] Each program can be implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. In any case, the language may be a compiled or interpreted language. Each such computer program can be stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein. The system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.

[00127] A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems. One format for an output tests datasets possessing varying degrees of similarity to a trusted repertoire. Such presentation provides a skilled artisan with a ranking of similarities and identifies the degree of similarity contained in the test repertoire.

[00128] Further provided herein is a method of storing and/or transmitting, via computer, data collected by the methods disclosed herein. Any computer or computer accessory including, but not limited to software and storage devices, can be utilized to practice the present invention. Data can be input into a computer by a user either directly or indirectly. Additionally, any of the devices which can be used to perform or analyze NIA can be linked to a computer, such that the data is transferred to a computer and/or computer-compatible storage device. Data can be stored on a computer or suitable storage device (e.g., CD). Data can also be sent from a computer to another computer or data collection point via methods well known in the art (e.g., the internet, ground mail, air mail). Thus, data collected by the methods described herein can be collected at any point or geographical location and sent to any other geographical location.

[00129] Also provided are reagents and kits thereof for practicing one or more of the above- described methods. The subject reagents and kits thereof may vary greatly. Reagents of interest include reagents specifically designed for use in production of the above described protein distribution patterns associated with chondrocytes and their responsiveness to therapy.

EXPERIMENTAL

[00130] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1

A single cell mass cytometry platform to map the effects of preclinical drugs on cartilage homeostasis

[00131] No disease-modifying drug exists for osteoarthritis (OA). Despite success in animal models, candidate drugs continue to fail in clinical trials owing to the unmapped interpatient heterogeneity and disease complexity. We used a single-cell platform based on cytometry by time-of-flight (cyTOF) to precisely outline the effects of candidate drugs on human OA chondrocytes. OA chondrocytes harvested from patients undergoing total knee arthroplasty were treated with 2 drugs, an NF-κB pathway inhibitor, BMS-345541 , and a chondroinductive small molecule, kartogenin, that showed preclinical success in animal models for OA. cyTOF conducted with 30 metal isotope-labeled antibodies parsed the effects of the drugs on inflammatory, senescent, and chondroprogenitor cell populations. The NF-κB pathway inhibition decreased the expression of p-NF-κB, HIF2A, and inducible NOS in multiple chondrocyte clusters and significantly depleted 4 p16 ^ink4a -expressing senescent populations, including NOTCH1 ⁺ STRO1 ⁺ chondroprogenitor cells. While kartogenin also affected select p16 ^ink4a - expressing senescent clusters, there was a less discernible effect on chondroprogenitor cell populations. Overall, BMS-345541 elicited a uniform drug response in all patients, while only a few responded to kartogenin. These studies demonstrate that a single-cell cyTOF-based drug screening platform can provide insights into patient response assessment and patient stratification.

[00132] Osteoarthritic chondrocytes were harvested from patients undergoing total knee arthroplasty and treated with two preclinically successful drugs for OA, an NF-κB pathway inhibitor, BMS-345541 and a stem cell modulator, Kartogenin. The cells were stained with 30 metal isotope- labeled antibodies and cyTOF acquired single-cell data was analyzed by unsupervised hierarchical clustering by FlowSOM to identify inflammatory, senescent and chondroprogenitor populations.

[00133] The single cell analyses parsed out the effects of the drugs on chondrocyte subpopulations and their redistribution. The NF-κB pathway inhibitor significantly decreased NF- κB signaling in most but not all chondrocyte clusters along with Hif2a and iNOS expression. Interestingly, NF-κB inhibition significantly depleted four p16 ^ink4a expressing senescent populations including Notch1 ⁺ Stro-1 ⁺ chondroprogenitor cells and reshaped the OA landscape. While both drugs led to depletion or expansion of multiple cell populations, Kartogenin had a modest effect in comparison to BMS-345541. Interestingly, neither drug affected the previously identified inflammation amplifying (Inf-A) population, marked by IL1 R1 and TNFRII. Based on population densities and marker expression profiles, NF-κB pathway inhibition by BMS-345541 elicited a uniform drug response in all, while Kartogenin treatment affected only a subset of patient samples.

[00134] Single cell cyTOF-based screening platform can provide insightful predictive analyses of the uniformity of drug response as well as to stratify putative responders and non-responders. Such approaches can accelerate precision medicine-based drug discovery efforts for OA.

[00135] Using a single-cell mass cytometry approach, we recently built a cellular atlas for human cartilage in healthy and OA patients wherein we defined a set of regenerative chondroprogenitor cell populations as well as new cartilage-intrinsic immunomodulatory populations. Our cytometry by time-of-flight (cyTOF) analyses identified and delineated 3 distinct chondroprogenitor cell (CPC) subtypes: CPC I (CD105 ^lo ), which were enriched in normal chondrocytes; CPC II, which were present in both normal and OA chondrocytes; and CPC III, which were enriched in OA chondrocytes (inflamed CD105 ^hi ). Additionally, we identified an inflammation-amplifying population termed Inf-A, defined by elevated levels of IL1 R1 (CD121A) and TNFRII (CD120B) receptors. TNFRII has been shown to have antiinflammatory effects as opposed to TNFRI in some cell types. Chondrocytes coexpressing IL1 R1 (CD121A) and TNFRII (CD120B), however, showed active signaling through SMAD1/5 and JNK pathways, and inhibition of either of these pathways dampened secretion of inflammatory cytokines. Another functional, inflammation- dampening population, Inf-D, was characterized by the expression of CD24, a molecule we had previously identified as a negative regulator of NF-κB-mediated inflammation. Activation of Inf-D in combination with Inf-A inhibition affected multiple cytokines for an immunomodulatory effect on OA cartilage secretome.

[00136] Given this understanding of the cartilage landscape, we have now utilized the power of cyTOF to test how a given drug affects different chondrocyte subpopulations and their cross-talk to get a high-resolution snapshot rather than just the cumulative effect of the total population. There exists a huge knowledge gap between studies in rodent or large animal models and the ultimate clinical trials in humans. A single cell-based screening platform for human patient samples, with its high resolution, provides insightful analyses for effective screening approaches for OA therapeutics before clinical trials.

METHODS

[00137] Isolation, culture, and drug treatment of OA chondrocytes. OA cartilage tissue was procured from the surgical waste of patients (n = 6, >56 years of age, and a mix of male and female) undergoing total knee replacement who had radiographic end-stage OA, under an approved Institutional Review Board protocol (IRB35067). Cartilage was shaved off and chondrocytes were isolated and cultured as previously described. Briefly, to harvest chondrocytes from cartilage, the cartilage tissue was first dissected into smaller pieces and incubated in a dissociation medium (25 pg/mL collagenase II and 25 μg/mL collagenase IV in DMEM-F12 medium with 10% FBS) overnight in a CO ₂ incubator. After overnight treatment with the collagenases, the undigested tissue and debris were separated from the cells using a 70 pm cell strainer. The cells were centrifuged, washed twice with fresh DMEM-F12 medium, and plated in 150 mm tissue culture dishes. OA cells (n = 6 patient samples) at passage 2 were seeded at high density in 100 mm plates and treated with control (DMSO) or drug the next day for 48 hours. Drug doses were determined based on prior literature and validation: 25 μM NF-κB inhibitor BMS- 345541 (Sigma-Aldrich B9935) and 25 μM kartogenin (Sigma-Aldrich SML0370).

[00138] Cell staining and cyTOF. OA cells were stained with 25 μM Idu for 15 min at 37°C, followed by 0.5 μM cisplatin for 5 min at room temperature (RT), fixed, washed, and frozen. For staining, cells were thawed on ice and barcoded using the Cell-ID 20-plex Pd Barcoding Kit (Fluidigm) and labeled with antibodies conjugated with metal isotopes (Table 1 ) as previously described. Cells were measured using the cyTOF 2 (Fluidigm) and injected using the super sampler. To normalize signal over time during data acquisition, stained cells were resuspended in 10% EU beads (Fluidigm) in water before runtime.

Table 1. List of antibody markers categorized into chondrocyte, cell cycle, inflammation, signaling, chondroprogenitor (CPC) or TLRs and senescence related marker types with corresponding metal isotope conjugates identified by unique mass numbers.

Table 2

Table of high confidence cluster identities. Categorization of clusters based on their median SOX9 expression, and empirical identities, and annotation of select clusters using the markers included in the cyTOF panel are shown. [00139] FlowSOM analysis and visualization. Unsupervised hierarchical clustering by FlowSOM was performed on all live cells (cisplatin negative) per sample in Cytobank using all surface markers (CD24, CD126, CD121A, CD121 B, CD120B, CD106, CD73, CD171 , CD105, CD33, CD49E, CD146) in addition to SOX9, TET1 , and Hif2a. The selection of markers for clustering is approximate to the methodology in previous study where surface markers with SOX9 and Hif2a were used so that previously defined subpopulations, particularly Inf-A, Inf-D and CPC clusters are adequately distinguishable. For consistency, 25 clusters and 10 metaclusters with 10 iterations were used as pre-determined input parameters as calibrated and utilized in previous study to identify subpopulations of interest with sufficient resolution. Downsampling was avoided and therefore, all cells per sample were included in creating self-organizing maps (SOMs) for cluster generation. Abundances of clusters were represented as a percentage of total cells analyzed per sample. UMAP projection was performed using Cytobank’s online platform to visualize FlowSOM generated clusters using 8043 cells per sample, pheatmap package was used to generate heatmaps in RStudio software. Principal Component Analysis (PCA) was performed using Factoextra package in Rstudio based on all clusters and median expression of all markers in individual patient samples.

[00140] Multiplex autoantibody assay. OA chondrocytes from 5 new patient donors (different from those used in cyTOF experiment) were collected and treated with DMSO, BMS-345541 (25 μM), or kartogenin (25 μM) for 48 hours. Cell culture supernatants from all samples (DMSO, n = 5; BMS-345541 , n = 5; kartogenin, n = 5) were collected and centrifuged at 10,000g for 10 minutes at 4°C to remove cell debris. The supernatants were snap-frozen in liquid nitrogen and submitted to the Human Immune Monitoring Center at Stanford University for multiplex autoantibody assay via 80-plex Luminex assay custom combination from EMD Millipore panel. Duplicate measurements for each analyte were recorded. Raw MFI values were averaged for each analyte and used in data analysis. Data visualization in the form of a heatmap of the average MFI readings for all 80 analytes across samples was conducted via the pheatmap package in R software.

[00141] Statistical analysis. GraphPad Prism software was used for paired 2-tailed t tests for DMSO- and drug-treated samples corrected for multiple hypotheses by Bonferroni correction and reported as adjusted Rvalues. In heatmaps, clustering was performed row- or column-wise using the Euclidean distance method for distance matrices. For Luminex assay, a pairwise t test was performed between BMS-4345541 or kartogenin and respective DMSO-treated samples with post hoc Benjamini-Hochberg test to avoid type 1 errors and represented as -log adjusted P values for significantly different analytes. All Rvalues less than 0.05 were considered significant.

RESULTS [00142] Characteristics of Sox9 ^low/medium cell populations in OA cartilage. As described previously, we utilized cyTOF, a mass-spectrometry based high dimensional method for single-cell detection of isotope-labeled antibodies to stain OA chondrocytes (FIG. 1 A). Our previous panel of 33- markers for profiling chondrocytes included cell surface receptors, adhesion molecules, signaling mediators and cell cycle and transcription factors. For these studies, we have retained most of the previous markers like the cytokine receptors, IL1 R1 (CD121 A) and TNFRII (CD120B) that defined an inflammation amplifying population (Inf-A), CD24 that charcaterized an inflammation dampening (Inf-D) subpopulation and multiple cartilage-progenitor cell (CPC) markers like CD105, CD73, CD106, Notch-1 and Stro-1 (Total 30 markers, Table 1 ). We have refined the panel by excluding redundant markers and including markers like p16 ^ink4a that has been reported to identify a senescent population in OA cartilage and Toll-like receptors, TLR2 and 4 that play roles in innate immunity.

[00143] Additionally, in our previous study, we had discarded cells with low expression of Sox9 (-10-13% of the total chondrocyte population). With the rationale that these cells can comprise putative stem/progenitor cells or rare infiltrating stromal or immune cells and should be characterized, we have now included these SOX9 ^low/medium cells in our analyses using Sox9 as a stratification marker for their identification. FlowSOM was utilized for identifying clusters or subpopulations in the OA chondrocytes (n=6 patients) (FIG.1 B-C). For cluster input parameters for FlowSOM, we utilized the parameters that were optimized in our previous study i.e. a cluster number of 25 and a metacluster number of 10 in the Cytobank platform. The optimal cluster and metacluster numbers (25 and 10 respectively) were selected after calibration and deemed sufficient in resolving the clusters (each cluster should have non-zero and greater than 1000 events across all samples) and identifying distinct subpopulations. Using these parameters, we were able to easily define the chondrocyte sub-populations identified in our previous study i.e inflammation-amplifying (Inf-A), inflammation-dampening (Inf-D) and chondroprogenitor population (CPCs) in the present dataset. Based only on the median expression levels of SOX9, all clusters across all 6 OA patient samples were stratified into 4 groups using unsupervised hierarchical clustering: SOX9 ^low , sOX9 ^{low medium} , SOX9 ^medium , SOX9 ^high (FIG. 1 D-F). The SOX9 ^low group consisted of four clusters (4, 5, 9 and 10) and comprised only 2.6% of the total live cells (FIG. 1 D). The SOX9 ^{mid low} group consisted of five clusters (1 , 3, 8, 13 and 22) and comprised 10.7% of the total live cells. Together, these cells that were excluded from analyses in our previous study comprised 13.3% of cells, hence were overall a small fraction of the OA chondrocytes. Interestingly, the clusters in the SOX9 ^low group were distinct from all other groups in having quite low median expression levels for the markers for chondroprogenitor cells (CPC) or inflammatory populations, suggesting that these are either OA chondrocytes with low inflammation or chondrocytes undergoing dedifferentiation (FIG. 1 G). In contrast, clusters in the SOX9 ^{mid- low} group were like the previously profiled SOX9 ^medium and SOX9 ^high groups in consisting of clusters that had either CPC-like phenotype (like CD105 ⁺ cluster 13) or had high inflammation (like cluster 22 with the expression of IL1 β receptors, CD121A and CD121 B). The S0X9 ^medium group was the largest, comprising 71.5% of total live cells while the SOX9 ^high group comprised the rest 15%. Interestingly, SOX9 ^high clusters (11 ,20,21 ,24) appeared highly inflamed with a higher overall expression of Hif2, NF-κB, iNOS, the IL6 receptor CD126 and IL1 β receptor CD121 B. The SOX9 ^medium group on the other hand is mixed, consisting of clusters with relatively lower levels of inflammation (clusters 2, 7 and 15) along with clusters showing higher levels of NF-κB, iNOS and IL6 receptor CD126 (clusters 6, 12 and 16).

[00144] p16 ^ink4a expressing senescent cell populations in OA chondrocytes are distinct from Inf-A cells. Our previously reported sub-populations in OA cartilage were easily identified in this study. Briefly, an inflammation-amplifying population was termed Inf-A that was defined by elevated levels of ILi β (CD121A) and TNFaRII (CD120B) receptors. TNF RII has been shown to have pro-inflammatory effects as opposed to TNFRI in some cell types (Bluml, et al, Arthritis Rheum, 2010). Chondrocytes co-expressing IL1 β (CD121A) and TNFaRII (CD120B) however showed active signaling through SMAD1/5 and JNK pathways, and inhibition of either of these pathways dampened secretion of inflammatory cytokines (Sci adv ref). An inflammation dampening population, Inf-D was defined by high CD24 expression and a CD105 ⁺ Notch-1 ⁺ Stro1 ⁺ chondroprogenitor population was termed CPC III. In this study, clusters 19, 23, and 16 were identified to be the Inf-A, Inf-D, and CPC III populations respectively, all belonging to the SOX9 ^medium group (FIG. 2A-B).

[00145] p16 ^ink4a was previously identified to mark a functional senescent population in OA cartilage that upon ablation or inhibition showed a decrease in senescence-associated secretory phenotype (SASP) and delayed OA in a mouse model (Jeon et al. Nat Med, 2017). We therefore investigated p16 ^ink4a expression, and found it to be quite widespread in OA chondrocytes and not restricted to a small population. FlowSOM identified five different clusters that expressed high levels of p16 ^ink4a -clusters 6, 11 , 16, 17, and 24 (FIG. 2C,D). Out of these, clusters 6, 16 and 17 belong to the SOX9 ^medium group while clusters 11 and 24 belong to the SOX9 ^high group. Interestingly, no p16 ^ink4a expressing senescent subpopulations were detected in SOX9 ^low or SOX9 ^{low medium} groups. It was also interesting that the Inf-A population was distinct from the senescent populations identified by FlowSOM, although a few Inf-A cells did express p16 ^ink4a . This observation suggests that Inf-A cells provide distinct contributions to the inflammatory micro- environment of cartilage in addition to the senescence-associated secretory phenotype (SASP) by p16 ^ink4a expressing cells. We also noted that the CPC III population (cluster 16) showed high p16 ^ink4a and was one of the senescent clusters (heretofore referred to as SnC CPCIII). An independent study had postulated that chondroprogenitor cells can undergo senescence in OA cartilage. SnC CPC III cells also expressed CD33 and TLR2 (FIG. 7), markers that are also coexpressed with p16 ^ink4a in clusters 6 and 11 (labeled SnC I and SnC II). The other two clusters, 17 and 24 (labeled SnC III and SnC IV) showed higher expression of CD105 and p16 ^ink4a but not of CD33, TLR2, Notch-1 or Strol (FIG. 6). [00146] NF-κB inhibition greatly alters the OA landscape depleting p16 ^ink4a expressing senescent clusters. The NF-κB pathway is known to play a significant role in the function of inflammatory cytokines including IL1 β and TNFa in OA pathogenesis and drugs targeting this pathway have been extensively investigated. Since activation of inhibitor of κB (IκB) kinase is essential for NF- κB activation, we used a known selective inhibitor of IkB kinase (BMS 345541 ) to test the precise effect of NF-κB inhibition. This inhibitor BMS 345541 has already been shown to be effective in inhibiting OA pathogenesis in a preclinical mouse model of OA.

[00147] In our previous study, we had been able to stratify OA chondrocytes from different patients into three subtypes-Group A, B and C where the group A was the most frequent. In order for a high-resolution mapping of the effect of a pre-clinical drug, we chose 6 patient samples that were available in sufficient numbers for drug treatments an all samples were from Group A patients. Chondrocytes were treated with this NF-κB pathway inhibitor (BMS) or control (DMSO) for 48 hours, fixed, stained and profiled by cyTOF as previously described. On average, there was variable inhibition of p-NF-κB (phospho serine 29) in different clusters of individual OA patients with only 10 of the clusters demonstrating a significant downregulation (FIG. 3A). Notably, p-NF- κB expression was significantly reduced in Inf-A (cluster19), Inf-D (cluster23) and SnC CPCIII (cluster16) clusters along with in SnC I (cluster 6) (FIG. 3B,C). The expression of inflammatory iNOS and Hif2a, directly regulated by NF-κB, was also significantly reduced in select clusters, with all three markers being downregulated only in Inf-A and Inf-D clusters (FIG. 8).

[00148] A shift in the density of chondrocyte clusters was noticeable in BMS-treated samples (FIG. 3D-F). Clusters of the SOX9 ^high group (clusters 1 1 , 21 and 24) and select clusters of the SOX9 ^medium group (clusters 6, 7, 12 and16) were significantly depleted in cell numbers (FIG. 3D- E). In contrast, the clusters in SOX9 ^low groups and select clusters of the SOX9 ^{mid low} group (Clusters 3, 8, and 13) were significantly expanded (FIG. 3 F,G). Another major effect of p-NF- κB inhibition was a significant depletion of the senescent Notch-1 ⁺ /Stro1 ⁺ CPC III population across all patient samples (FIG. 4A,B). No significant changes were observed in the abundance of Inf-A or Inf-D populations. Among the p16 ^ink4a clusters, however, there was a significant depletion of all clusters following BMS treatment except for SnC III (FIG. 4C,D). Thus, p-NF-κB inhibition drastically reduced inflammation and affected the abundances of multiple cell populations.

[00149] Kartogenin has a modest effect distinct from NF-κB pathway inhibition. Next, we examined the effect of a regenerative preclinical drug, kartogenin, on the OA landscape. In contrast to NF-κB pathway inhibition by BMS-345541 , kartogenin treatment had a modest effect on the OA subpopulations (FIG 5A). Only 5 clusters showed a significant change in abundance (FIG 5B). One of the p16 ^ink4a -expressing clusters in the SOX9 ^lo/mid group, SnC I (cluster 6), was significantly depleted (FIG. 5, C and D). Other significantly depleted clusters included cluster 3 of the SOX9 ^lo/mid group and clusters 7 and 14 of the SOX9 ^mid group. Cluster 5 of the SOX9 ^lo group, consisting of low-inflammation chondrocytes, was the only cluster that showed significant expansion. RUNX2 expression was significantly downregulated in SnC I (cluster 6) among others (clusters 12, 20, 21 , and 25) (FIG. 5E). Mechanistically, kartogenin was shown to regulate the nuclear shuttling of core-binding factor β (CBF|3), which binds to RUNX family members (RUNX1 , 2, and 3) to form a transcriptional complex that regulates multiple genes besides autoregulating the RUNX members themselves. Consistent with this mechanism, kartogenin treatment led to a significant increase in RUNX1 in clusters 2 and 15, while both RUNX1 and RUNX2 were increased in clusters 12 and 21. Excluding the SnC I population, none of the clusters where RUNX1 and/or RUNX2 were downregulated demonstrated a change in population density. Kartogenin did not affect the density of Inf-A, Inf-D, or SnC CPC III populations (FIG. 5F). In the clusters grouped based on the median expression of SOX9 (FIG. 1 , C-F), NF-κB pathway inhibition by BMS-345541 significantly depleted the frequencies of the SOX9 ^mid and SOX9 ^hi groups, whereas treatment of OA chondrocytes with kartogenin had no significant effect in comparison with respective DMSO-treated controls. Notably, both BMS-345541 and kartogenin increased the frequency of the SOX9 ^lo group. The median expression of SOX9 in individual clusters belonging to all groups was maintained in kartogenin-treated samples to levels comparable to those in DMSO-treated controls. In contrast, the SOX9 expression profile in BMS- 345541 -treated samples was dissimilar to that in DMSO- and kartogenin-treated counterparts, with lower median SOX9 expression levels in clusters belonging to the SOX9 ^mid and SOX9 ^hi groups, owing to the significant depletion in their frequencies.

[00150] NF-κB inhibition modulates the secretome of OA chondrocytes to a greater degree than kartogenin. As demonstrated by cyTOF, NF-κB pathway inhibition and kartogenin treatment had distinct effects on the OA landscape. To examine whether the drug treatments had a similar functional impact on the soluble mediators in the OA microenvironment, we sought to compare the effect of NF-κB pathway inhibition by BMS-345541 and kartogenin on the inflammatory secretome of OA chondrocytes. An 80-plex autoantibody assay (Luminex) comprising cytokines, chemokines, and growth factors was chosen to assess the effect of the drug treatments on the OA chondrocyte secretome.

[00151] To this end, OA chondrocytes were harvested from the surgical waste of 5 additional patients undergoing total knee replacement surgeries (n = 5 biological samples) and were treated with BMS-345541 , kartogenin, or DMSO (vehicle control) for 48 hours, similar to the experimental conditions used for cyTOF study. Multiplex autoantibody assay by Luminex on the culture supernatants of the treatment groups revealed substantial dissimilarity in the secretory profile of BMS-345541-treated OA chondrocytes compared with kartogenin treatment and DMSO-treated controls. Notably, the levels of a plurality of secreted factors were decreased upon NF-κB pathway inhibition by BMS-345541 (FIG. 6A). Significant fold decreases were observed in the levels of FMS-related tyrosine kinase 3 ligand (FLT3L), platelet-derived growth factor-AA (PDGF- AA), soluble FAS (sFAS), plasminogen activator inhibitor-1 (PAl1), and IL-10 when normalized to respective DMSO-treated controls. FLT3L, PDGF-AA, and sFAS are known to be accumulated in the synovial fluid in OA patients. PAI1 has pleiotropic roles dependent on tissue context such that it inhibits MMP activity under normal conditions whereas it increases ECM protein turnover, leading to fibrosis, under pathological conditions. IL-10 is an antiinflammatory cytokine that regulates TNF-α-mediated effects in OA cartilage. However, local fluctuations in IL-10 are transient but TNF-α expression levels can persist in OA; hence the downward trend in the levels of TNF-α and TNF-β in most patient samples implies positive anabolic regulation by BMS-345541 treatment (FIG. 6B). Among the significantly elevated cytokines in BMS-345541 -treated samples, the levels of macrophage migration inhibitory factor (MIF) were substantially higher compared with the levels observed in IL-20, -33, and -3. Contradictory roles have been defined for MIF in OA, including induction of proinflammatory cytokines such as IL-1 β as well as enhanced cell proliferation and antiapoptotic pathways. The levels of cytokines implicated in senescence- associated secretory phenotype (SASP) such as IL-1 β, IL-1 a, IL-6, TNF-α, TNF-β, and VEGF did not change significantly upon BMS-345541 treatment, although the levels of IL-6 and VEGF were lowered in a majority of patient samples (FIG. 6B). This response is likely due to the fact that BMS-345541 is not a senolytic and affected only a few senescent subpopulations. The secretory profile of kartogenin-treated samples did not deviate much from that of the respective DMSO- treated controls (FIG. 6C). Notable exceptions were the significantly elevated levels of epidermal growth factor (EGF) and a modest increase in the death signaling receptor FasL. As with BMS- 34551 , the levels of select SASP-associated cytokines were variable between patient samples in kartogenin-treated samples (FIG. 6D).

[00152] Delineating responders and non-responders. A high-resolution platform like cyTOF can help provide precise insight into the patient-drug response. Using the response of the patients from the two drugs BMS and Kartogenin, we sought to assess whether these drugs had a uniform effect on the patient-derived OA chondrocytes. Analyzing the effects of BMS treatment on the 25 distinct subpopulations in the patient chondrocytes, it was clear that this drug had a uniform effect. Unsupervised hierarchical clustering showed that the DMSO and BMS-treated OA chondrocytes fall into two distinct groups both when accounting for cluster abundances (FIG. 6A) and median expression of all markers (FIG. 6B). On the other hand, Kartogenin treatment led to a heterogeneous response among the patients even in this small cohort. Only two patients were responders while for the other patients, the control and Kartogenin treated samples clustered together (FIG. 6A,B). The PGA plot mirrored the same findings (FIG. 6C).

[00153] A high-resolution cellular atlas of OA articular cartilage is useful for understanding disease pathology and for devising new therapeutic strategies. We had previously identified multiple populations of cartilage progenitor cells (CPC) and two rare chondrocyte subpopulations, Inf-A and Inf-D, that amplified or dampened inflammation. In this study, we have also utilized p16 ^ink4a , which was previously identified to mark a functional senescent population in OA cartilage. Ablation or inhibition of p16 ^ink4a expressing cells showed a decrease in senescence-associated secretory phenotype (SASP) and delayed OA in a mouse model (Jeon et al. Nat Med, 2017). We have identified five distinct clusters of p16 ^ink4a expressing senescent cells - SnC I, II, III, and IV along with the CPC III population that was earlier characterized by the high expression of Notch- 1 and Strol . A recent study had also validated that progenitor cells tend to become senescent in OA cartilage. Besides CPCIII, SnC III and IV were also identified to be senescent CPCs based on the high expression of both CD105 and p16 ^ink4a . Notably, the Inf-A population was found to be distinct from the major p16 ^ink4a expressing senescent populations identified, although a few Inf-A cells showed p16 ^ink4a expression. These observations highlight the complex nature of the inflammatory micro-environment of cartilage, wherein multiple, small cell populations appear to be at play. Additionally, p16 ^ink4a is just one of the senescent markers and a comprehensive analyses of other markers including p21 , p53, uPAR. Incidentally, three of the SnC clusters (besides SnC III and IV) showed a co-expression of TLR2 and CD33 (Siglec 3) with p16 ^ink4a . TLR2 has been previously observed to regulate oncogene-induced senescence and p16 ^ink4a function. The role of TLRs in addition to the other players like p21 , p53 and uPAR in cartilage- specific senescent cells and the multiple contributions to the senescence-associated secretory phenotype (SASP) will be interesting to explore in future studies.

[00154] Another refinement to our previous analyses was that we did not exclude SOX9 ^low and SOX9 ^{low medium} groups of cells that comprised 2.6% and 10.7% of the total cells. The SOX9 ^low group clusters showed low expression levels for the chondroprogenitor and inflammatory markers in the panel. These cells could be OA chondrocytes with low inflammation or dedifferentiated chondrocytes. Although collagen 1 is a characteristic marker for dedifferentiated chondrocytes and collagen 10 for hypertrophic chondrocytes, these ECM markers did not stain well in isolated chondrocytes. It was, therefore, difficult to confirm the exact identity of this small population of cells. However, both the drug treatments specifically expand these cells, while decreasing SnC cells.

[00155] Assaying population-level gene or protein expression changes elicited by drug treatments provide only limited understanding of the effects of a drug on the cellular subpopulations, their crosstalk and the cellular landscape that emerges after the drug treatment. For this study, we chose two model drug candidates- BMS and Kartogenin- that had already been shown to be effective in the modulation of OA pathogenesis in animal models. While BMS selectively inhibits IkB kinase in the NF-κB pathway, Kartogenin was originally identified in a screen to expand mesenchymal stem cells and has since been shown in multiple studies to be a pro-chondrogenic modulator of OA progression in an animal model. The mode of action of these two drugs is distinct: while BMS dampens inflammation, Kartogenin is pro-regenerative. Using these two drugs, we were able to identify the precise populations affected in OA chondrocytes. Interestingly, the targeted pathways were modulated only in selective populations, for example only about half the clusters showed significant downregulation of NF-κB signaling upon BMS treatment or RUNX 1/2 upregulation upon Kartogenin treatment. While NF-κB inhibition drastically changed the overall landscape of the OA chondrocytes affecting multiple populations, the effect of Kartogenin was modest.

[00156] Interestingly, BMS not only affected overall NF-κB signaling and inflammation as expected, but also reduced the abundance of p16 ^ink4a expressing senescent populations including the Notch-1 and Strol expressing SnC CPC III population. Kartogenin also reduced the frequency of SnC I but did not affect SnC CPCIII or the other p16 ^ink4a expressing SnC populations. Neither BMS nor Kartogenin affected the inflammation amplifying, Inf-A or inflammation dampening, Inf-D populations. An unexpected observation was that Kartogenin treatment did not lead to the expansion of any chondroprogenitor cells. Instead, both Kartogenin and BMS led to an expansion of clusters in the SOX9 ^low group that does not show any CPC markers.

[00157] Our results show that cyTOF based analyses provide a powerful platform to test whether a drug elicits a uniform or heterogeneous response in OA patients. The subset of patient samples tested here stratified together in CyTOF analyses in the absence of drugs i.e. belonging to the most abundant group A patients, and hence were a homogenous set of patients. In testing the two model drugs, it was however clear that BMS treatment caused a uniform effect on all OA patients tested while there were only two patients that responded to Kartogenin even in this small cohort (n=6). It was therefore apparent that with the use of cyTOF based studies, patients can be stratified into responders versus non-responders based on a particular candidate drug providing a proof-of-concept for this platform (schematic in FIG. 6D). In the future, establishing an OA chondrocyte biobank for rapid in vitro preclinical drug screening can identify drugs that are likely to fail in clinical trials due to the heterogeneity in patient response.

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[00182] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.